Báo cáo khoa học: POSTER PRESENTATIONS P1 Functional Genomics, Proteomics and Bioinformatics

Poster Presentations

Abstracts

POSTER PRESENTATIONS

P1 Functional Genomics, Proteomics and Bioinformatics

P1–1 The influence of the HERV-K LTR on KIAA1245 subfamily gene expression N. Abrarova, E. Stoukacheva, T. Vinogradova and E. Sverdlov Laboratory of structure and function of human genes, Institute of Bioorganic Chemistry, Moscow, RUSSIA

200 and 400 bp in length (Mid200

carbonate as a phosphate binder for the treatment of hyperphos- phatemia. Lanthanides3+ compounds have also been investi- gated for their anti-cancer potential. Clinical reports suggested that several compounds such as cerium (III) iodide posed cyto- toxic effect, however the biochemical mechanism of their action is still unclear (1–3). Therefore, we aim our attention on study of interactions DNA with inorganic salts of cerium (III), lanthanum (III) and gadolinium (III). Raman spectroscopy was employed to characterize the perturbations to DNA conformation induced in DNA by three inorganic salts of the above mentioned lantha- nides (2). All studied lanthanides coordinated to N7 of two neighbouring guanine bases, as this nitrogen does not form H bonds with other bases, in the same or in opposite DNA strands. The results of the present work demonstrate that Raman spec- troscopy represents a suitable tool to provide insights into struc- tural factors involved in the mechanisms underlying antitumor effects of drugs. Acknowledgement: This work was supported by GA CR 522/ 07/0692. References: 1. Fricker SP. Chem. Soc. Rev. 2006; 35(6): 524–533. 2. Vrana O et al., J. Struct. Biol. 2007; 159(1): 1–8. 3. Babula P et al., Curr. Pharm. Anal. 2009; 5(1): 47–68.

P1–3 Real-time polymerase chain reaction with electrochemical detection for detection pandemic viruses V. Adam1, D. Huska1, S. Krizkova1, J. Hubalek2 and R. Kizek1 1Department of Chemistry and Biochemistry, Mendel University of Agriculture and Forestry in Brno, Brno, CZECH REPUBLIC, 2Department of Microelectronics, Brno University of Technology, Brno, CZECH REPUBLIC

Long terminal repeats (LTR) of endogenous retroviruses com- prise about 8% of human genome. Typical LTR contains a set of regulatory elements: promoters, enhancers, polyadenilation sites, which can take part in neighbouring genes expression regu- lation. Earlier, we have described a subfamily of closely related genes highly similar to the KIAA1245 mRNA, part of which contains a HERV-K LTR in their structure, whereas the other lacks it. Using this subfamily as a model for studying regulation of gene transcription, we showed that LTR serves as an alterna- tive promoter and possess high enhancer activity. Genes that contain LTR differ from genes lacking it in cell type specificity of transcription. We generated a series of successive 5¢- and 3¢-dele- tion mutants with a 200 bp increment, and also two middle vari- and Mid400, ants, respectively). To determine promoter activity of LTR and its fragments they were cloned into pGL3-BV vector, containing luciferase reporter gene. The full-sized LTR demonstrated high promoter activity in Tera1 and NT2/D1 cell lines. To determine the enhancer activity LTR and subfragments were cloned in pGL3-PV vector, which has SV40 promoter in its structure besides luciferase gene. Transient transfections demonstrated that Mid200 fragment of the LTR exhibits high cell type specific enhancer activity. In Tera1 cell line it was comparable to the activity of universal SV40 enhancer. This fact allows to suggest that enhancer is localized in this region of the LTR. The analysis of transcription of KIAA1245 subfamily genes have shown that LTR-lacking gene Al592309 is not transcribed in Tera1 and NT2/D1 cell lines, whereas LTR-containing genes are transcribed in these cell lines. Thus, we may suggest that insertion of the LTR in second intron of KIAA1245 genes may lead to change in cell type specificity of their transcription. This fact allows to sug- gest the role of the LTR in evolution of KIAA1245 subfamily genes.

P1–2 Study of DNA interactions with cerium (III), lanthanum (III) and gadolinium (III) ions by using of Raman spectroscopy V. Kohoutkova1, P. Babula1, R. Opatrilova1, O. Vrana2, V. Adam3, J. Zehnalek3 and R. Kizek3 1Department of Natural Drugs, University of Veterinary and Pharmaceutical Sciences, Brno, CZECH REPUBLIC, 2Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, CZECH REPUBLIC, 3Department of Chemistry and Biochemis- try, Mendel University of Agriculture and Forestry, Brno, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

The biological properties of the lanthanides, based on their simi- larity to calcium, have stimulated research into their therapeutic application. Up-to-date we have been successfully using at least two pharmaceuticals cerium nitrate as a topical cream with silver sulfadiazene for the treatment of burn wounds and lanthanum Viral infections pose a threat for man kind. Diagnostic systems focus on the direct cultural proof of identity. Almost all systems use polymerase chain reaction. Real-time polymerase chain reac- tion is based on the polymerase chain reaction and routinely used to amplify and simultaneously quantify a targeted DNA molecule. Fluorescent dyes intercalating with double-stranded DNA and modified DNA oligonucleotide probes fluorescing when hybridized with a complementary DNA are used for real time quantification of the amplicons. The process of DNA quantification has demands on robust instrument and on high cost chemicals. Electrochemical detection is low cost and sensitive alternative method to detect nucleic acids (1, 2). The main aim of this work is to propose novel electrochemical detector for monitoring PCR. Miniaturized poten- tiostat with nanotubes-based screen-printed electrodes was used for detection of specific sequence of severe acute respiratory syn- drome, avian influenza virus and Ebola virus amplified by poly- merase chain reaction. The amplicons were obtaining after 2, 4, 6, 8, 10, 15, 20, 25, 30 and 35 cycles, whereas the amount of amplified DNA was between 10 and 100 pg. The amplicons obtained even after two cycles were detectable by using the electrochemical instrument. Moreover we compared the results with agarose gel electrophoresis. The lowest detectable amount of DNA was obtained after 15 PCR cycles. Acknowledgement: This work was supported by GA AV KAN208130801.

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References: 1. Palecek E et al. Anal. Chim. Acta. 2002; 496(1): 73–83. 2. Huska D et al. Electrophoresis 2008; 29(24): 4964–4971.

Chromatin immunoprecipitation assays show significant occu- pancy by the MCE, CIITA and general transcriptional machinery at various phases of the cell cycle including mitosis in B lym- phoid cells. Mitotic maintenance of the MCE does not depend on CIITA expression and correlates with a chromatin state that is fully or partly maintained in mitotic lymphoid or non lym- phoid cells, respectively. Monitoring of newly synthesized RNA shows reduced transcriptional activity of the DRa gene in mito- sis. Constitutive expression of exogenous CIITA is sufficient to fully support mitotic expression in lymphoid but not in epithelial cells. Using an inducible system we show that CIITA synthesised in cells arrested in mitosis rescues MHCII transcription to asyn- chronous levels in lymphoid cells demonstrating full expression competence of DRa gene. These results show there are two pat- terns of mitotic deficiency of gene expression: in B lymphoid cells low but significant MHCII transcription can be fully rescued, as opposed to epithelial cells that show minimal mitotic transcrip- tion may due to enhanced chromatin condensation.

P1–4 Spatial distribution of heavy metals in healthy and melanoma animal tissues V. Adam1, O. Zitka1, D. Huska1, S. Krizkova1, J. Strnadel2, V. Horak2, T. Vaculovic3, K. Novotny3, V. Kanicky3, J. Kaiser4 and R. Kizek1 1Department of Chemistry and Biochemistry, Mendel University of Agriculture and Forestry in Brno, Brno, CZECH REPUBLIC, 2Academy of Sciences of the Czech Republic, Insti- tute of Animal Physiology and Genetics, Libechov, CZECH REPUBLIC, 3Department of Chemistry, Masaryk University, Brno, CZECH REPUBLIC, 4Institute of Physical Engineering, Brno University of Technology, Brno, CZECH REPUBLIC

P1–6 Associations between PTGS1 gene mutations and aspirin resistance in patients with congenital heart diseases I. Coskun1, Y. Atay1, A. Berdeli2, M. Mecidov1, A. Atay3, M. F. Ayik1, T. Yagdi1 and E. A. Alayunt1 1Cardiovascular surgery, Ege University, izmir, TURKEY, 2Pediatric Molecular Medicine Laboratory, Ege University, izmir, TURKEY, 3Department of Clinical Biochemistry, Ataturk Training Hospital, izmir, TURKEY

Levels of some of heavy metals are monitored and their altera- tions can be used as diagnostic markers. Nevertheless, spatial dis- tribution of the essential metals in animal tissues is still not clear. The aim of this work is detection of copper and zinc in healthy and tumour tissues of miniature pigs by using of the laser induced breakdown spectroscopy (LIBS). Concentration of essen- tial-heavy-metal-transporting protein metallothionein (MT) was determined by the Brdicka reaction. Tissue cryosections (thick- ness 40 lm) were obtained from the MeLiM strain of miniature pigs with hereditary melanoma, particularly from healthy skin, cutaneous nodular melanomas and metastases in the liver, spleen and lymph nodes. Using LIBS we measured maps of spatial dis- tribution of the essential heavy metals in cryosections and found that the maps of healthy and tumour cryosection markedly dif- fered. The highest content of MT was determined in the tumours localised on the back of animals and was nearly 500 lg of MT per gram of tissue. The MT levels determined in metastases in spleen, lungs and liver were within the range from 40 to 160 lg/g of the tissue, the average level was 110 ± 40 lg/g of the tissue. Acknowledgements: This work was supported by the GA AS CR (IAA401990701) and by the Ministry of Education, Youth and Sport CR (2B08063) and Liga proti rakovine Praha (2009). References: 1. Krizkova S et al. Sensors 2008; 8(5): 3106–3122. 2. Kaiser J et al. Spectrochim. Acta Part B 2009; 64(1): 67–73.

P1–5 Chromatin accessibility of MHCII locus and enhanceosome stability determine transcriptional competence through mitosis P. Arampatzi, M. Gialitakis, T. Makatounakis and J. Papamatheakis Department of Biology, Institute of Molecular Biology and Biotechnology, Heraklion, GREECE

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Introduction: This study was aimed to evaluate that frequency and existance of PTGS1 gene mutation which coded cyclooxy- genase-1 (COX-1) enzyme and associations with aspirin resistance in patients with congenital heart diseases (CHD) in Turkish pop- ulation. Methods: It was included 90 patients with CHD treated by aspirin after their operations. 24 healthy participants were enrolled as control group. Direct DNA coding sequence method was used for analysing PTGS1 gene mutation. PCR amplification was made by using specific oligo primers (MWG) and AmpliTaq Gold PCR kits for four exons. After capillary gel electrophoresis, nucleotide sequences of PTGS1 gene was obtained and compared with cDNA Genbank (NM_000962) and protein (NP_000953) sequences to determine single- nucleotide polymorphisms and amino acid mutations. Results: W8R missense aminoacid mutation was the most fre- quent mutation (83.3%) in exon-2. P188P sinonym aminoacid polimorphism (34%) and D189E amino acid mutation (20%) were other mutations detected in exon-6 in patient group. In con- trol group, W8R missense aminoacid mutation (83.3%) in exon- 2, Q41Q sinonim aminoacid mutation (8.3%) in exon-3, D189E missense aminoacid mutation (12.5%) in exon-6 were found. Nine new missence amino acid mutations (L13P; L15V; P18R; C58T; I45M; R53S; D189E; F200L; R239L) and eight sinonym (L23L; P39P; R53R; G44G; G62G; amino acid mutations K185K; P18P) were detected. It was observed the association between aspirin resistance and missence amino acid mutations in patients. Conclusion: Genetic variations in PTGS-1 may affect aspirin resistance in CHD. Detecting these variations may help to predict drug responce and to select optimal therapy and dosage regi- mens. This is the first study in our country dealing with PTGS1 gene mutation and polymorphism which coded COX-1 enzyme and association with aspirin resistance in CHD. During mitosis, transcription factors and RNA polymerase II are displaced from mitotic chromatin, which is condensed and tran- scription is interrupted. However specific gene regions are sug- gested to be bookmarked by TBP and histone modifications for rapid reactivation after cell division. The MHCII locus is trans- criptionally activated by the assembly of an enhanceosome (MCE) which recruits the master regulator, CIITA. We found that endogenous or GFP-fusions subunits of the MCE remain associated and dynamically exchanged on mitotic chromosomes.

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there were no significant decrease of MMP-9 levels after treat- ment in patients with the genotype -308GA and -308 AA (P = 0.147; P = 0.126). We discussed decrease of MMP-9 level in children with -308GG genotype as a possible consequence of better response to etanercept treatment compared to A allele car- riers.

P1–7 Analysis of the ID1 gene expression level and promoter methylation in patients with benign and malignant thyroid nodules A. Banaszewska1, M. Piechota1, K. Drobnik2, K. Ziemnicka2, K. Ziemnicki1 and R. Plewa1 1Department of Animal Physiology and Development, Adam Mickiewicz University, Poznan, POLAND, 2Department of Endocrinology Metabolism and Internal Disease, University of Medical Science, Poznan, POLAND

P1–9 Screening of Three Exons of the RET Proto- oncogene in Turkish Patients with Papillary Thyroid Carcinoma N. S. Bayramci1, L. Acik2, L. Y. Koc3, G. Vural4, M. Kilic5, M. Tez5, M. Koc5 and N. Ercakmak4 1Faculty of Education, Department of Science Education, Bayburt University, Bayburt, TURKEY, 2Faculty of Arts and Science, Department of Biology, Gazi University, Ankara, TURKEY, 3Faculty of Science, Department of Biology, Ankara University, Ankara, TURKEY, 4Nuclear Medicine Department, Dr. Abdurrah- man Yurtaslan Ankara Oncology Education and Research Hospi- tal, Ankara, TURKEY, 55th Surgery, Ankara Numune Education and Research Hospital, Ankara, TURKEY

Inhibitor of DNA binding (ID1) has been proposed to serve a dual function as a regulator of cell proliferation and differentia- tion in both normal and pathological conditions associated with cancer development. ID is a transcription family of factors lack- ing basic DNA binding domain, which form inactive heterodi- mers with basic helix-loop-helix transcription factors and inhibits expression of tissue specific genes and cells differentiation. Recent research shows, that ID1 mRNA level correlates with malignant thyroid carcinoma phenotype. Thus, dual role of ID1 gene in benign and malignant thyroid nodules was studied. ID1 mRNA level of 33 patients was investigated in human papillary thyroid carcinoma (PTC), non-toxic nodular goiter, toxic nodular goiter and nodular goiter with Hashimoto disease using Real time PCR and Delta Delta Ct method to estimate relative amount of ID1 mRNA. Furthermore, genomic DNA was extracted and ID1 pro- moter methylation was examined in the majority of patients spec- imens. Six of seven PTC patients samples were found to have barely detectable ID1 mRNA level. Expression of ID1 was higher in non-toxic nodular goiter and in nodular goiter with Hashimoto disease in comparison to PTC and toxic nodular goiter (P < 0.05). No hypermethyleted ID1 gene promoter was found in the majority of analyzed cases. In our study no significant association was observed between ID1 gene expression and malignant thyroid nodules. This findings indicate that ID1 could not be a marker of aggressive phenotype in PTC.

the patients exhibited mutation, 24 of

P1–8 TNF alpha -308 polymorphism and matrix metalloproteinase-9 level in children with juvenile idiopathic arthritis threated with etanercept J. Basic1, D. Pavlovic1, T. Jevtovic-Stoimenov1, J. Vojinovic2, M. Marinkovic1 and A. Veljkovic1 1Medical Faculty, Institute of Biochemistry, Nis, SERBIA, 2Clinic of Pediatrics, Clinical Centre Nis, Nis, SERBIA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Different types of tumors can develop in thyroid gland cells. Thy- roid carcinoma represents the most frequent form of cancer of the thyroid glands, with prevalence in Europe of about 3 per 100 000. PTC is the most frequent histotype of differentiated thy- roid cancer. The RET proto-oncogene, localized to chromosome subband 10q11.2, comprises 21 exons, which encodes the protein RET, a receptor tyrosine kinase (RTK) expressed in derivatives and tumors of neural crest origin. The RET proto-oncogene is involved in the genesis of papillary thyroid carcinoma. To deter- mine the mutation frequency of RET proto-oncogene in Turkish papillary thyroid carcinoma patients, we conducted this retro- spective study. Eighty-two patients with PTC were screened for mutations in exon 10, 11 and 13 using PCR and sequence analy- sis. A negative control was included in each amplification analy- sis. Twelve of them showed SNP. Twelve different mutations located in exon 11 and three different mutations in exon 13. 12.19% of the patients had mutation in exon 11. 3.65% of the patients had mutation in exon 13. Twenty-four of the patients exhibited SNP (29.26%). We found the SNP at codons 631 (1.21%), 769 (1.21%), 691 (3.65%) in exon 11 and at codons 769 (24.39%) and 763 (1.21%) in exon 13. The most frequent SNP that is found in patients with PTC (24.3%) involve codon 769 (exon 13) in the tyrosine kinase domain. The common mutation found in patients involves codon 630 (four patients) in the extracellular domain of RET encoded by exon 11, less frequently, mutation occurred codons 765, and 770 and 795 (exon 13) in the tyrosine kinase domain. The muta- tions were found in 41.46% of all thyroid carcinoma patients. The numbers of mutations were higher in exon 11 than exon 13. In our series, we detected that four patients in 82 of all PTC patients presented Cys630Ser (TGC fi AGC, 5.9%) is the most common mutation at codon 630 in exon 11. In addition to that, Cys630Ser mutation, is associated with medullary thyroid carci- noma, however in our genetic testing it was diagnosed on four patients with papillary thyroid carcinoma. Therefore, the rela- tionship between Cys630Ser variation and development of PTC should be further explored. All of the investigated polymor- phisms are silent mutations, leave out codon 691 polymorphism. We observed the codon 691 polymorphism in three patients (GGT/AGT) of all 82 PTC patients. In the 13rd exon region of the RET proto-oncogene, we found that these possible mutations, which expressed as an alteration in the form of TCC (serine) replacement with TGC (sistein) at codon 765, CGA (arginine) Juvenile idiopathic arthritis (JIA) is the most common form of persistent arthritis in children. Tumor necrosis factor-a (TNF- a) is likely to have a primary role in the pathogenesis of JIA, including matrix metalloproteinase-9 (MMP-9) production. The aim of this study was to investigate G-A -308 single nucleotide polymorphism (SNP) in the promoter region of TNF a gene, as well as to evaluate the eventual influence of etanercept (TNF receptor II-Fc fusion protein) on MMP-9 level in children with JIA. A total of 42 patients with JIA were screened for the poly- morphism using the PCR-RFLP method. Plasma MMP-9 level was determined before starting etanercept therapy and 12 months after, using Elisa kit. The genotype -308GG was present in 13 (31%) patients and the -308AA genotype was present in two (4.7%) patients. Twenty-seven (64.3%) patients were heterozy- gous. MMP-9 level in patients with the genotype -308GG was significantly decreased after 1 year of treatment with etanercept compared to the value before (P = 0.01). On the other hand,

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replacement with CAA (glutamic acid) at codon 770. The other mutation in exon 13 was at codon 770, Arg replacement with Glu. The large number of SNP at codon 769 in exon 13 with PTC patients was interesting and it should be further explored.

reductase, nitrite (nitrate

into the adaptation processes

P1–10 Metabolomic profiling of serum from obese adult females infected by Chlamydophila pneumoniae G. Bazylak1, C. Ludwig2, E. Fagadar-Cosma3 and U. L. Gunther2 1Department of Pharmaco-Bromatology & Molecular Nutrition, Faculty of Pharmacy Collegium Medicum Nicolaus Copernicus University, Bydgoszcz, POLAND, 2HWB NMR, CR UK Institute for Cancer Studies, University of Birmingham, Edbagston Birming- ham, UK, 3Department of Organic Chemistry, Institute of Chemis- try Timisoara of Romanian Academy, Timisoara, ROMANIA

transcriptomics and bioinformatics tools to consider their roles in the adaptation processes at global level. Protein compositions of four P. denitrificans strains (wild type, FnrP-, NNR- and NarR- mutants grown aerobically, semiaerobically and semiaerobically with nitrate) were analyzed using set of 36 large format 2-D PAGE gels, their statistical evaluation and MS/MS protein iden- tification of more than 500 proteins. Expression differences of key up-/down-regulated gene products were validated using qRT- PCR. Comparative proteomic analysis revealed, besides other results, differences between strains in the expression of three key denitrification enzymes reductase, nitrous oxide reductase), OmpW and UspA proteins. These find- ings are in agreement with known mechanisms and/or confirmed the positions of proposed fnr binding sites in their promoters. Detection of additional group of up-/down- regulated proteins without fnr binding sites in their promoters (e.g. SDR dehydro- genases, TonB receptors) indicates involvement of additional reg- studied. ulatory mechanisms Evidences for potential roles of transcription regulators of other families (e.g. LysR, MarR and two-domponent regulators) are discussed. Acknowledgements: We are thankful to Czech Science Foun- dation (grant No. 203/07/P0471) and to Czech Ministry of Edu- cation (grant No. MSM0021622413) for financial support.

P1–12 Analysis of proteome alteration in tomato roots after Meloidogyne hapla infection A. Obrepalska-Steplowska1, K. Nowaczyk1, M. Luczak2, M. Figlerowicz2, R. Dobosz3 and M. Budziszewska1 1Interdepartmental Laboratory of Molecular Biology, Institute of Plant Protection, Poznan, POLAND, 2Polish Academy of Sci- ences, Institute of Bioorganic Chemistry, Poznan, POLAND, 3Department of Zoology, Institute of Plant Protection, Poznan, POLAND

Chlamydophila (Chlamydia) pneumoniae is a very common path- ogen causing an acute respiratory diseases and multidirectional inflammation progress during a past and persistent infections in humans of all age. Recently, significant associations between C. pneumoniane infection and obesity indices have been observed on results of epidemiological studies in various populations from several north Europe countries. However, until now the number of NMR- or MS-based metabolomic and proteomic studies on specific biomarkers related with variety of nutrition disorders, including obese subjects, is still scarce. Thus, the goal of our studies was to find by use of the 1D- and 2D-1H-NMR proce- dures the range of characteristic, specific and unique low weight metabolites (proteins) in serum samples from wide group of over- weight and obese adult females with increased hormonal activity of adipose tissue, and which indicating past (or persistent) infec- tion by C. pneumoniae as diagnosed by introductory serological immunosorbent assay. Proposed here 1H-NMR methodology should be effective for profiling changes in the serum metabolo- me (proteome) of subjects with obesity and its associated implica- tions with bacterial infection and disorders of immunological status, thus enabling future early diagnosis, risk assessment and advanced treatment of infection related obesity in humans. Acknowledgement: Supported by the grant from EU NMR project – Contract No. RII3-026145.

P1–11 The study on Fnr-type transcription regulators functions in bacterium Paracoccus denitrificans using proteomics, transcriptomics and bioinformatics tools P. Bouchal1, I. Struharova1, E. Budinska2, O. Sedo3, T. Vyhlidalova1, Z. Zdrahal3, R. van Spanning4 and I. Kucera1 1Department of Biochemistry, Faculty of Science, Masaryk Univer- sity, Brno, CZECH REPUBLIC, 2Institute of Biostatistics and Analyses, Masaryk University, Brno, CZECH REPUBLIC, 3Department of Experimental Biology, Masaryk University, Brno, CZECH REPUBLIC, 4Department of Molecular Cell Physiology, Vrije Universiteit Faculty of Earth and Life Sciences, Amsterdam, THE NETHERLANDS

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Paracoccus denitrificans is a facultatively autotrophic soil bacte- rium which responds to decreasing level of oxygen in the environ- ment by a switch from aerobic to anaerobic (denitrifying) growth mode. In our work, we analyzed roles of fnr-type transcription regulators FnrP, NNR and NarR (with described key roles as sensors for oxygen, nitrate and nitrous oxide) using proteomics, Root-knot nematodes from the genus Meloidogyne are parasitic to many economically important crop plants and cause great losses in the crop production. Out of fifteen European species, eight were found in Poland; among them the most common and widely distributed is M. hapla, parasiting on dicotyledonous plants. During the initial stage of infection characteristic root galls form on the roots of susceptible plant hosts. As a result of interaction between parasite and the host, in the vascular cylinder of the root permanent feeding sites are formed with specialized multinucleated feeding cells (giant cells). The redifferentiation of root cells and development of metabolically active giant cells is induced by the nematode. The mechanism of giant cells forma- tion and the role of plant proteins in this processs remain unclear. Our attempt was to analyse the changes in the plant pro- teome in the early stage of M. hapla infection. Tomato plants were grown in isolated conditions and infected with M. hapla. After isolation of proteins from roots of healthy and infected plants, as well as from nematodes, two-dimensional electrophore- sis was performed. The results of experiments were compared to ensure elimination of nematode proteins from further analysis. As some qualitative and quantitative changes were observed, cho- sen proteins were analysed using mass spectrometry. The decrease of cytosolic and mitochondrial dehydrogenases of cellular respi- ration and electron transport chain was observed as well as for proteins related to cytoskeleton (actins), relevant to changes in the cell’s shape and wound response (annexin). The qualitative changes are related with proteasome endopeptidase complex, absent in infected root cells. Nematode proteins secreted during

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infection had been the subject of many recent investigations. But, as the relationship between these parasites and the host is very close, determination of proteins engaged on the both sides of the interaction can give us the insight to the mechanism of plant sub- ordination to the parasite.

concentrations (5% NaCL, -20% NaCl) and different tempera- tures (19–37(cid:2)C). Growth at these conditions was investigated and microorganisms were harvested at the end of exponential phase. Afterwards, whole cell proteins of the DSMZ 4770 were extracted and analyzed by proteomic tools. Two DE maps are prepared according to tube gel systems with NEPHGE technique in the first dimension and normal SDS-PAGE in the second dimension. Protein profiles between normal and stress conditions were determined and compared. Protein expression differences were determined by MALDI-Tof Mass Spectrometer (Waters/Mi- cromass). Proteins that involved in the osmoprotection and cold adaptation were determined.

P1–13 Determination of chromosomal regions affecting some production traits in F2 intercross chickens Z. Bulut1, E. Kurar2, Y. Ozsensoy2, M. Nizamlioglu1, M. Garip3, A. Yilmaz3, T. Caglayan3, S. Dere3, V. Kurtoglu4 and M. Dogan1 1Faculty of Veterinary Medicine, Department of Biochemistry, Sel- cuk University, Konya, TURKEY, 2Faculty of Veterinary Medi- cine, Department of Genetics, Selcuk University, Konya, TURKEY, 3Faculty of Veterinary Medicine, Department of Zoo- technics, Selcuk University, Konya, TURKEY, 4Faculty of Veteri- nary Medicine, Department of Animal Nutrition, Selcuk University, Konya, TURKEY

P1–15 Molecular typing of Staphylococcus aureus strains from ovine mastitis by pulsed-field gel electrophoresis and polymerase chain reaction A. Ciftci1, E. E. Onuk2, A. Findik1, T. Yildirim3 and M. Unlu Sogut4 1Veterinary Faculty, Microbiology, Ondokuz Mayis University, Samsun, TURKEY, 2Veterinary Faculty, Diseases and Clinical Sciences, Ondokuz Mayis University, Samsun, TURKEY, 3Science Faculty, Biology, Amasya University, Amasya, TURKEY, 4Medicine Faculty, Microbiology, Ondokuz Mayis University, Samsun, TURKEY

In this study, a F2 level Denizli X White Leghorn population (n = 441) was used to dissect chromosomal regions, which have an affect on body weight and egg yield. DNA samples were iso- lated from all F0, F1 and F2 animals using a standard phenol/ chloroform method. For chromosomal level screening, a total of 99 markers which were suitable for genome searching were ampli- fied using Polymerase Chain Reaction (PCR). The resulting PCR products were separated by capillary electrophoresis by using Beckman Coulter CEQ-8000 Genetic Analysis System and geno- types were identified at each marker loci. QTL Express Program was used for QTL data analysis. In study, Quantitative Trait Loci (QTL) were identified on chicken chromosome 1 (GGA1), GGA2, GGA4, GGA8 and sex chromosome (GGAZ) for hatch- ing weight, body weight at different ages and egg yield. There is a need for narrowing these QTL regions by typing new markers in these intervals and for identifying genes that have affect on these economically important traits.

Staphylococcus aureus is one of the most important etiological agents of ovine mastitis. In order to develop effective control mea- sures for mastitis, it is important to type S. aureus strains that have considerable genetic heterogeneity. In the current study, 47 S. aureus strains isolated from ovine mastitis were typed by poly- merase chain reaction (PCR) based on coagulase (coa) and protein A (spa) polymorphisms and by pulsed-field gel electrophoresis (PFGE). Eight different coa-types and four spa-types were identi- fied by PCR. While the most prevalent coa-type was CG2 (42.56%), the spa-types, S4 and S1, were the most commonly observed (44.68% and 38.29%, respectively). Nineteen different pulsotypes were identified, and 12 of these were represented by a single isolate. Pulsotypes J and K were predominant, and each represented nine isolates (19.14%). All isolates belonging to J and K pulsotypes were CG2. While all nine isolates belonging to J pulsotype were S4, all isolates in K pulsotype were S1. Although PFGE was found to be the best discriminatory technique for dis- tinguishing strains, coa- and spa-types were found to be in correla- tion with PFGE types and can be used for quick, preliminary epidemiologic studies for detecting strains that may cause mastitis.

P1–14 Investigation of osmoprotection and cold adaptation of halomonas halophilia DSMZ 4770 by proteomics S. Ceylan1, B. Akbulut1, D. Kazan1 and D. Kazan2 1Engineering Faculty, Bioengineering Department, Marmara University, Istanbul, TURKEY, 2TUBITAK Marmara Research Center, Genetic Engineering and Biotechnology Institute, Kocaeli, TURKEY

of moderately pathways

P1–16 Development and optimization of multiplex polymerase chain reaction for identification of Flavobacterium psychrophilum, Yersinia ruckeri and Aeromonas salmonicida subsp. salmonicida E. E. Onuk1, A. Ciftci2, A. Findik2 and Y. Durmaz3 1Veterinary Faculty, Fisheries and Fish Diseases, Ondokuz Mayis University, Samsun, TURKEY, 2Veterinary Faculty, Microbiology, Ondokuz Mayis University, Samsun, TURKEY, 3Veterinary Con- trol and Research Enstitute, Fisheries and Fish Diseases, Samsun, TURKEY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Bacterial cold water, enteric red mouth and frunculosis are the common bacterial diseases of fish worldwide. The etiologic agents Halophiles are an important group of microorganisms that can adapt to extremely saline environments. Since, they have the abil- ity to function under extreme saline conditions, halophilic micro- organisms are used at different industrial processes such as in the production of protein/enzyme stabilizer metabolites (e.g. betain, ectoin), in the removal of pollution in salinity effluents and in the production of stable enzymes. Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteins are vital parts of living organisms, as they are the main compo- nents of the physiological metabolic pathways of cells. By under- standing metabolic halophilic microorganisms it will be possible to modulate their metabolism to enhance the production of their industrially important prod- ucts. In this study osmoprotection and cold adaptation metabo- lism of moderately halophilic Halomonas halophilia was investigated. Halomonas halophilia was grown on different salt

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salmonicida subsp. and Aeromonas other proteins of E. faecalis was found as varied among these strains. It was the first study in the literature that suggested E. faecalis strains include the glycoproteins.

P1–18 RAPD-PCR analysis of the genus apodemus KAUP, 1829 (Mammalia:Rodentia) in Turkey R. Colak, G. Olgun, I. Kandemir, N. Yigit and E. Colak Faculty of Science, Biology, Ankara University, Ankara, TURKEY

these diseases are Flavobacterium psychrophilum, Yersinia of ruckeri salmonicida, respectively. In this study, a Multiplex Polymerase Chain Reac- tion (M-PCR) method which can identify these fish pathogens, Aeromonas salmonicida subsp. salmonicida, Flavobacterium psy- chrophilum and Yersinia ruckeri, simultaneously was developed and optimized. It was observed that M-PCR developed in this study, with YER8/10-Fer3/4-FP1/3 primer pairs, occured neither false specific nor nonspecific amplification. The detection limits of M-PCR method using DNA extract from dilutions of pure cultures of bacteria were detected as 35 pg for Y. ruckeri and F. Psychrophilum and 70 pg for A. salmonicida subsp. salmonicida. It was determined that 15 CFU Y. ruckeri and F. psychrophilum and 30 CFU A. salmonicida subsp. salmonicida could be detected by M-PCR developed using genomic DNA extracted from dilu- tions of suspensions. The detection limits in the presence of tissue debris were detected as 125 CFU for Y. ruckeri ve F. psychrophi- lum and 250 CFU for A. salmonicida subsp. salmonicida. In con- clusion, we decided that M-PCR method developed and optimised in this study could be used for accurate and rapid identification of these bacteria.

lowest

The aim of the present study is to survey genetic structure based on DNA markers and to make contribution to the taxonomic status, population genetics of the Genus Apodemus in Turkey. In order to analysis genetic variation in Apodemus species, a Ran- domly Amplified Polymorphic DNA (RAPD) marker system was used. A total of 82 specimens (22 A. iconicus, 26 A. flavicollis, 7 A. sylvaticus, 15 A. uralensis, 4 A. agrarius and 8 A. mystacinus) collected from 16 locations in Turkey were used. The estimates of NEI’s (1972) standart genetic identity and standart genetic dis- tance were calculated to show the genetic relationships between studied populations. All estimations were calculated with the POPGENE software. The 60 RAPD markers were tested in Apodemus, 11 ones yielded 101 polymorphic DNA bands. Genetic differentiation values, H = 0.0721 (P = 16.83%), were in A. agrarius. A. uralensis has H = 0.2303 the (P = 77.23%), being the highest value between Apodemus spe- cies. According to Nei (1972), A. agrarius and A. mystacinus are the least similar to each other (I = 0.6256), and A. iconicus and A. uralensis are the nearest to each other (I = 0.9406). Phyloge- netic relationships between Apodemus species were shown with UPGMA dendogram constructed based genetic distance values.

P1–17 Evaluation of protein profiles of Enterococcus faecalis strains in relation to antigenic glycoproteins G. Ciftci1 and H. Uysal2 1Veterinary Faculty, Biochemistry, Ondokuz Mayis University, Samsun, TURKEY, 2Veterinary Faculty, Biochemistry, Ankara University, Ankara, TURKEY

the E.

P1–19 What does it take to make an activated cortical domain within a plant cell? F. Cvrckova1, R. Bezvoda1 and V. Zarsky2 1Faculty of Science, Department of Plant Physiology, Charles University, Prague, CZECH REPUBLIC, 2Faculty of Science, Department of Plant Physiology and Laboratory of Cell Biology, Institute of Experimental Botany ASCR, Charles University, Prague, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

The aim of this research is to determine the specific antigenic gly- coproteins and comparative analysis of antigenic protein profiles of Enterococcus faecalis (E. faecalis) strains distinguished with aggregation substance (AS), gelatinase and cytolysine. For the determination of the whole cell protein profiles of E. faecalis, the Sodium Dodecyl Polyacrylamide Gel Analysis (SDS-PAGE) was faecalis strains showed protein bands performed. All between the sizes of 29 and 165 kDa. The protein bands of 44 and 83 kDa were determined as major band in E. faecalis OG1RF (gelatinase positive) and E. faecalis OG1X (pAM9058) (AS positive). On the other hand; E. faecalis OG1X (pAM944) (cytolisine positive) strain also showed a 43 kDa major band. For the antigenic characterization, the immunblot analysis was performed. All of the three strains of E. faecalis showed common antigenic protein bands which were at the size of 165, 35 ve 32 kDa. The other antigenic protein bands of E. faecalis OG1X faecalis OG1X faecalis OG1RF and E. (pAM944), E. (pAM9058) were determined as 85, 65, 55, 39 ve 29 kDa, 83, 75 ve 45 kDa and 111, 98, 83, 75, 55, 45, 29 kDa in sizes, respec- tively. The Glycan Detection Kit from Roche Diagnostics and Periodic Acid Schiff (PAS) stainings were used for the investiga- tion of glycoproteins of E. faecalis. After the PAS staining, no glycoprotein band was shown as the method has low sensitivity. When the Glycan Detection Kit used, E. faecalis OG1RF and E. faecalis OG1X (pAM9058) strains showed two glycoprotein bands which were at the size of 111 and 39 kDa. Moreover, E. faecalis OG1X (pAM944) strain was also showed some glyco- protein bands which were 111, 106 and 39 kDa in size. In conclu- sion; no difference was determined for the protein profiles among the strains of Enterococcus faecalis which have AS, gelatinase and cytolisine. The antigenic profiles of the glycoproteins and the Exocytosis is central to plant cell morphogenesis. A single cell may possess multiple plasmalemma domains performing localized exocytosis-driven expansion (‘activated cortical domains’, ACDs). Several large families of paralogous regulatory proteins might be involved in generating the diversity of ACDs within a cell, such as e.g. small GTPases of the Rop family and their interactors, su- bunits of the exocyst complex, actin-nucleating FH2 proteins, receptor-like kinases, or enzymes locally modifying membrane composition (the ‘candidate set’ genes). Rhizodermis of Arabidop- sis thaliana consists of two cell types that differ by a single ACD – trichoblasts carrying root hairs, and atrichoblasts. We used publicly available cell-type specific transcriptome data to identify genes specifically induced in trichoblasts compared to atricho- blasts (i), and to examine their relationship to over 60 ‘candidate set’ genes (ii), as well as to known genes whose mutations exhibit root hair-specific phenotypes (iii). In addition, we have searched for homologues of all three gene classes in the published soybean

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face which was also upregulated in resistant cells. Immunocyto- chemistry results confirmed microarray expression analysis. Conclusion: This report demonstrates that in docetaxel and doxorubicin resistant cells EMT process was induced indicating a possible relationship of this process and drug resistance.

supported the by root hair proteome (iv; Brechenmacher et al., 2009). We found only limited overlap among the gene classes (i–iv), with most ‘candidate set’ genes exhibiting <5· induction in trichoblasts compared to atrichoblasts and low protein abundance in root hairs; very few class iii genes were found in either the transcrip- tomic or proteomic set. Thus, unlike forward or reverse genetics, transcriptomics and proteomics alone are unlikely to identify genes involved in plant ACD specification. Acknowledgement: This work was MSM0021620858, LC06004 and LC06034 projects.

P1–21 Characterization and whole genome sequencing of Arthrobacter phenanthrenivorans, a new phenanthrene degrading bacterium C. Drainas1, A. Kallimanis1, K. Kavakiotis1, K. Mavromatis2, N. C. Kyrpides2 and A. I. Koukkou1 1Chemistry, University of Ioannina, Ioannina, GREECE, 2Genome Biology Program, DOE-Joint Genome Institute, Walnut Creek, CA, USA

P1–20 Drug resistant MCF-7 cells possessed epithelial-mesenchymal transition related gene expression pattern O. D. Iseri1, M. Demirel Kars1, F. Arpaci2, C. Atalay3, I. Pak4 and U. Gu¨ ndu¨ z1 1Department of Biological Sciences, Middle East Technical University, Ankara, TURKEY, 2Department of Oncology, Gu¨lhane Military Medical Academy, Ankara, TURKEY, 3Department of General Surgery, Ankara Oncology Hospital, Ankara, TURKEY, 4Department of Pathology, Ankara Oncology Hospital, Ankara, TURKEY

transition (EMT)

Several bacterial strains were isolated from a creosote polluted site at Perivleptos (Epirus, Greece), based on their ability to uti- lize various polycyclic aromatic hydrocarbons as a sole carbon and energy source. One of them, Sphe3, was proved to be effi- cient degrader of phenanthrene, a polycyclic aromatic hydrocar- bon (PAH). Biochemical tests and 16S rDNA analysis showed that Sphe3 belonged to the genus Arthrobacter comprising a novel species named Arthobacter phenanthrenivorans sp. nov. Whole genome sequencing of the isolated organism revealed that it had a single circular chromosome of 4.25 Mb and two circular plasmids of 190 and 94 kb, respectively. A total of 4288 genes were predicted in the Sphe3 genome. With BLAST searches using peptide fragment sequences from a purified 1-H-2-N dioxygenase activity determined by Mass Spectrometry, we identified two encoding genes with over 90% homology to each other at the nucleotide level, one (diox1) located on the 190 kb plasmid and the other (diox2) on the chromosome of the Sphe3 genome. Fur- thermore, BLAST analysis of the 190 kb plasmid revealed that it additionally contained several genes involved in phenanthrene degradation. These findings revealed novelties that confirm the extended biodiversity of PAH catabolic genes.

P1–22 Stress response to aromatic compounds in Acinetobacter radioresistens S13 P. Fattori, M. Zapponi, C. Lamberti, A. Pessione, R. Mazzoli, C. Giunta and E. Pessione University of Turin, Human and Animal Biology, Turin, ITALY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

factors Background: Epithelial-mesenchymal is a developmental process by which cells of epithelial origin lose epi- thelial characteristics, and acquire a mesenchymal phenotype. EMT, an indicator of poor prognosis, may also occur during tumor progression. Altered expression levels of Snail family tran- scription factors (Snail, Slug and Twist) are the key regulatory elements of EMT. Slug gene is one of the downstream mediators of transforming growth factor beta1 (TGFB1) signaling. Objectives: In the present study evaluation of expression of genes related to EMT was aimed in docetaxel and doxorubicin resistant MCF-7 breast carcinoma cells in order to assess their involvement in drug resistance. Methods: Resistant sublines (MCF-7/DOC and MCF-7/DOX) were developed by docetaxel and doxorubicin applications in dose increments and development of resistance was confirmed by XTT proliferation assays. RNA was isolated from sensitive and resistant cells and cDNA microarray analysis was performed using Affymetrix(cid:3) Human Genome U133 Plus 2.0 Arrays in duplicate experiments. GeneSpring GX 7.3.1 Software was used for data analysis. Immunocytochemistry was also performed to determine relevant protein expression. Results: XTT demonstrated that MCF-7/DOC and MCF-7/ DOX were resistant to docetaxel and doxorubicin, respectively. According to data analysis, estrogen receptor a was drastically downregulated in MCF-7/DOC and MCF-7/DOX. It is known as a transcriptional repressor of the Slug. Therefore, decreased ERa and increased Slug expression could cause transcriptional activation of key regulatory elements of EMT. In addition, increased expression levels of TGF beta receptor2 (TGFBR2) together with SMAD3 might have stimulated EMT in resistant cells. Furthermore, amplified epidermal growth factor signaling via epidermal growth factor receptor1 upregulation might have enhanced the pathways leading to EMT. Slug upregulation caused cadherin switch in resistant cells i.e. E-cadherin and occlu- din were downregulated, and N-cadherin was upregulated. N- cadherin associates with the fibroblast growth factor receptor1 (FGFR1) leading to increased FGFR1 expression at the cell sur- Acinetobacter radioresistens S13 is a strain selected for its ability in phenol degradation. It is also able to degrade other aromatic compounds (i.e., Benzoate) through the b-ketoadipate pathway (1). Comparative proteomics alkaline studies on bacterium grown either on phenol or benzoate as sole carbon source reveal a dif- ferent response to stress induced by these two aromatic com- pounds. Phenol is a solvent able to solubilize outer membrane lipooligosaccharides (LOS) and phosphatidyletha- lipoproteins, nolamine, damaging cell wall (2). This compound induces an over expression of two proteases (ClpX and Serine protease) and of two RNA polymerase modulator (NusA and Rho) suggesting a gene modulation based on rE regulation. Instead

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induces

P1–24 Molecular typing and methicillin resistance profile of Staphylococcus aureus strains isolated from the noses of healthy dogs A. Findik1, N. Akan2, E. E. Onuk3, D. Cakiroglu4 and A. Ciftci1 1Veterinary Faculty, Microbiology, Ondokuz Mayis University, Samsun, TURKEY, 2Veterinary Faculty, Ondokuz Mayis University, Samsun, TURKEY, 3Veterinary Faculty, Diseases and clinical sciences, Ondokuz Mayis University, Samsun, TURKEY, 4Veterinary Faculty, Internal Medicine, Ondokuz Mayis University, Samsun, TURKEY

benzoate an alternative phosphorylation-dependent stress-sensing response, maintained by a two-component regula- tory system, which consist of a membrane-embedded sensory kinase and a response regulator (3). The kinase auto-phosphory- lates a conserved histidine on the receipt of a stress-signal before transferring the phosphoryl group to an invariant aspartate in its cognate response regulator and in doing so activates its latent biological function (4). References: 1. Mazzoli R, Pessione E, Giuffrida MG, Fattori P. et al. Degra- dation of aromatic compounds by Acinetobacter radioresistens S13: growth characteristics on single substrates and mixtures. Arch. Microbiol. 2007; 188(1): 55–68.

2. Mrozik A, Piotrowska-Seget Z & Labuzek S. Cytoplasmatic bacterial membrane responses to environmental perturbations. Polish J Environ. Studies 2004; 13(5): 487–494.

3. Bekker M, Teixeira de Mattos MJ & Hellingwerf KJ. The role of two-component regulation systems in the physiology of the bacterial cell. Sci. Prog. 2006; 89: 213–242. signal transduction. 4. Mizuno T. His-Asp phosphotransfer J. Biochem. (Tokyo) 1998; 123: 555–563.

It was aimed to detect the carriage of methicillin-resistant Staph- ylococcus aureus (MRSA) strains in the nasal flora of healthy dogs and to genotype these S. aureus isolates. The swabs were taken from the nasal region of 80 dogs examined in the veteri- nary clinics for the check-up in Samsun. The methicillin resis- tance profile of 80 isolates which were identified as S. aureus were analysed phenotypically and genotypically. Agar Disc Diffu- sion Test was performed to determine the methicillin resistance phenotype of these S. aureus strains using the oxacillin antibiotic discs (5 g) and all the 80 strains were found as sensitive to methi- cillin. In Polymerase Chain Reaction (PCR) targetting nuc, mecA and fem genes, three strains (3.75%) was found to possess mecA. In the same analysis none of these strains were positive for fem gene. The PCR targetting coa and spa genes was performed for molecular typing of the S. aureus strains. Nine different coa genes and three different spa genes were determined according to the polymorphism of these genes. In conclusion, the determining of the nasal carriage of different genotypes of S. aureus in healthy dogs was considered as a basic finding for both the char- acterization of nasal isolates of S. aureus and molecular epidemi- ologic researches. In these strains, the detection of mecA gene, accepted as ‘Gold Standart’ for methicillin resistance, was evalu- ated as an important finding for community health.

P1–23 Identification of a(1,6)fucosylated proteins as biomarkers for human colorectal carcinoma L. M. Romay, S. V. Portela, R. F. Poceiro, E. G. Martı´ n and A. F. Briera Department of Biochemistry Genetics and Immunology, University of Vigo, Vigo, SPAIN

P1–25 Detection of methicillin resistance and slime factor production of Staphylococcus aureus in bovine mastitis A. Ciftci1, A. Findik1, E. E. Onuk2 and S. Savasan3 1Veterinary Faculty, Microbiology, Ondokuz Mayis University, Samsun, TURKEY, 2Veterinary Faculty, Diseases and Clinical Sciences, Ondokuz Mayis University, Samsun, TURKEY, 3Veterinary Faculty, Microbiology, Adnan Menderes University, Samsun, TURKEY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Mastitis is the most prevalent and costly disease of dairy cattle. Among the pathogens that cause mastitis, S. aureus is the most important species and the hardest to eliminate with antibiotics because of its multiple antibiotic resistance. It was aimed to detect methicillin resistant and slime producing S. aureus in the case of bovine mastitis. The triplex PCR was optimized targetting 16S rRNA, nuc and mecA for detection Staphylococcus species, S. aureus and methicillin resistance, respectively. For detection of slime producing, it was performed a PCR assay targetting icaA and icaD genes. In this study, 59 strains were detected as S. aur- eus by both conventional tests and PCR. Among the 13 S. aureus strains of them were found as methicillin resistant phenotypically and 4 (30.7%) were positive for mecA gene. Although 22 of 59 (37.2%) S. aureus isolates were slime-producing in Congo Red Agar in PCR analysis, only 15 of 59 were positive for both icaA and icaD genes. 16 of 59 strains were positive for icaA and 38 of them were positive for icaD gene. We found only two of 59 strains were positive for both methicillin resistance and slime pro- ducing, phenotypically. a(1,6)-fucose residues within the N-glycan core structures are commonly observed in glycoproteins and are often altered under pathological conditions. This type of fucosylation is product of a(1,6)fucosyltransferase [a(1,6)FT] activity and is regarded as an important manner of posttranslational modification and func- tional regulation of glycoproteins. Our previous studies showed that a(1,6)fucosyltransferase activity and expression are altered in human colorectal cancer (CRC). Therefore, the a(1,6)fucosylation of some glycoproteins might be implicated in the development and progression of colorectal carcinomas, being potential bioindi- cators for the diagnosis and prognosis or targets for CRC ther- apy. In the present study we have employed a Lens culinaris agglutinin lectin (LCA) chromatography combined with 1-DE separation and followed by MALDI-MS/MS analysis to identify distinctive core fucosylation patterns in five healthy and tumour tissue samples from CRC patients. Indeed, a lectin and western blotting methodology was performed to validate our preliminary results. We demonstrated that colorectal tumour tissues have higher levels of a(1,6)fucosylation compared to healthy ones. Besides, among the several protein bands observed after the lectin chromatography and the electrophoretic separation, we selected the bands with a higher difference of intensity between healthy and tumour specimens. Two proteins were identified as possible biomarkers for CCR, the heat shock protein gp96 precursor and the Fc binding protein receptor. The western blot analysis con- firmed the enhanced expression of heat shock protein gp96 pre- cursor and the diminution of Fc binding protein receptor levels in tumour tissues compared to healthy ones. In conclusion, these glycoproteins could be considered as candidates for future studies focused on their function in colorectal tumourigenesis and their potential clinical usefulness.

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GenBank Accession (493–498_865–870,

P1–26 Immobilization of acid phosphatase to magnetic particles and their use for dephosphorylation of proteins J. Frydlova1, L. Kubosek1, L. Kubosek2, Z. Kucerova1, M. Ticha1 and M. Ticha3 11st Faculty of Medicine, Institute of Pathophysiology and CEH, Charles University in Prague, Praha 2, CZECH REPUBLIC, 2Faculty of Chemical Technology, Department of Biological and Biochemical Sciences, University of Pardubice, Pardubice, CZECH REPUBLIC, 3Faculty of Science, Department of Biochemistry, Charles University in Prague, Praha 2, CZECH REPUBLIC

both in

517_907–916, GenBank Accession No. NM_004364.2) and CGC- GAG No. NM_004364.2). We found both these most frequent deletions in CEBPA gene in one young man (38 years old) after coronary thrombosis (heart attack). The same deletions were detected in his son (15 years old). In many cases (13 patients with AML, MDS, peripheral artery disease, heart disease, hyperlipidemia and type 2 diabetes) more than one type of these deletions was observed. These findings suggest that observed CEBPA gene dele- tions of this type are likely the result of unequal mitotic cross- over. We observed also in several patients in addition to these deletions other mutation which was created probably by mecha- nism of replication slippage in the repetitive sequence (for exam- ple 68dupl in addition to 912_929del or 311_313del in addition to cases GenBank Accession No. 44_694del, NM_04364.2). Acknowledgements: This work was supported by the Internal Grant Agency of the Ministry of Health of the Czech Republic (VZ 00023736).

P1–28 Study of thermal tolerance in Cronobacter strains J. Gajdosova1, K. Kunikova1, I. Turcovsky2, E. Kaclikova2 and H. Drahovska1 1Department of Molecular Biology, Comenius University, Bratislava, SLOVAK REPUBLIC, 2Department of Microbiology, Food Research Institute, Bratislava, SLOVAK REPUBLIC

In proteomics studies, the presence of phosphate group in a pro- tein or in a peptides obtained by proteolytic digestion is often confirmed by the comparison of mass spectra of peptides/proteins before and after the phosphatase treatment. A loss of phosphate groups results in a shift of peptide in mass spectrum. Generally, the use of enzyme immobilized to magnetic carriers has several advantages as compared with an application of soluble enzyme forms: an increased stability of enzymes, a possibility of direct use of enzyme reaction products for MALDI-TOF MS and an easy manipulation. Contrary to proteolytic enzymes, only limited information is available on properties of immobilized phosphata- ses. In the present study, acid phosphatase from potato was immobilized to glyoxal 4% agarose magnetic particles (20– 75 lm) via its free amino groups. The following properties of immobilized acid phosphatase were compared with those of the soluble enzyme: the pH dependence of the enzyme activity, the effect of the presence of cofactor (Mg2+), the effect of tempera- ture and further storage and operational stability. A reusability of the immobilized phosphatase was also examined. Both forms of acid phosphatase were used for dephosphorylation of porcine pepsin A, bovine a-casein and chicken ovalbumin. Acknowledgements: This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (grant MSM 0021620806 and project CEH LC 06044) and by the Czech Science Foundation (grant 203/09/0857).

P1–27 Deletions with the presence of two repetitive DNA sequences in CEBPA gene O. Fuchs1, A. Kostecka1, D. Provaznikova1 and J. Filkukova2 1Department of Cell Physiology, Institute of Hematology and Blood Tranfusion, Prague 2, CZECH REPUBLIC, 2Department of Molecular Genetics, Institute of Hematology and Blood Tranfusion, Prague 2, CZECH REPUBLIC

in multiple myeloma and non-Hodgkin´ s

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

C/EBPalpha (CCAAT/enhancer binding protein alpha) belongs to family of leucine zipper transcription factors and it is neces- sary for transcriptional control of granulocyte and adipocyte dif- ferentiation, glucose metabolism and lung development. C/ EBPalpha is encoded by an intronless gene that is 2783 bp long and maps to human chromosome 19q13.1. Up to now, CEBPA gene mutations were detected only in patients with acute myeloid in patients wih myelodysplastic syndrome leukemia (AML), (MDS), lymphoma patients. We detected 14 various heterozygous CEBPA gene dele- tions with the presence of two repetitions in CEBPA gene in AML patients, MDS patients, non-Hodgkin´ s lymphoma patient, ischemic heart disease, patients with peripheral artery disease, hyperlipidemia and type 2 diabetes. These mutations are charac- teristic by the loss of one from these two same repetitions on the ends of deleted sequence. Two most frequent repetitions included in these deletions in CEBPA gene are GCCAAGCAGC (508– Cronobacter spp., formerly named as Enterobacter sakazakii, is an opportunistic pathogen associated with sporadic cases of severe infections in neonates causing meningitis, necrotizing enterocolitis and sepsis. This organism is widely distributed in environment and rehydrated infant formula is the most common source of infection. Cronobacter was shown to be particularly tol- erant to osmotic stress and desiccation and some strains of this genus are also tolerant to elevated temperatures. Considering infant formula is not a sterile product, thermotolerant strains of Cronobacter spp. represent increased risk to survive during infant formula reconstitution. In our study, the genetic variability of 76 food isolates and 23 Cronobacter collection strains was measured by AFLP and 16S rRNA sequencing. Within AFLP profiles, a great variability was observed, the similarity among strains reached values 50–100%. Strains were clustered into 46 different clusters at the similarity level of 90%. Six main groups (desig- nated A-F) were clearly distinguished at the 75% similarity level; strain grouping was in concordance with their species identifica- tion and biochemical properties. Test of survival at 58(cid:2)C sepa- rated strains into two groups; D values (decreasing of bacterial counts in one order) of thermosensitive strains fell within the range 17–50 s; on the other hand eleven strains (nine Cr. sak- azakii and two to Cr. malonaticus) were assessed as thermotoler- ant because their D values reached from 100 to more than 300 s. Thermotolerant strains were also positive for PCR thermotoler- ance marker homologous to a hypothetical protein Mfla_1165 from thermotolerant bacterium Methylobacillus flagellatus KT (Williams et al. 2005). 5.5 kbp DNA fragment around thermotol- erance marker was sequenced in strain Cr. sakazakii ATCC 2954. The region possessed 94% similarity with M. flagellatus KT com- plete genome including genes Mfla_1162 – Mfla_1168. Cloning of 2950 bp long part of Cr. sakazakii thermotolerance region into pGEM plasmid resulted in 3–5 times increased survival of Escherichia coli laboratory strain at 58(cid:2)C. Our results have

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shown that this genetic region can be important in response to several stress conditions and may contribute to increased envi- ronmental resistance of Cronobacter strains.

supported the by

guish orthologues of each of the Class I formins, the situation is somewhat less clear in case of Class II proteins, consistent with gene duplications taking place even after separation of the thali- ana and lyrata lineages. Nevertheless, even one of the loci sus- pected to be pseudogenes in A. thaliana has an orthologue A. lyrata, suggesting that it might be a functional gene. Acknowledgement: This work was MSM0021620858 and LC06004 projects. Reference: 1. Grunt et al., BMC Evol. Biol. 2008; 8:115.

P1–29 Mitochondrial proteome changes during prereplicative phase of liver regeneration A. Gnoni1, R. Mangiullo1, R. Schiavone2, S. Vilella2, S. Papa1,3 and F. Zanotti1,3 1Department of Medical Biochemistry, Physics and Biology, University of Bari, ITALY, 2Department of Biological and Envi- ronmental Sciences and Technologies, Laboratory of Comparative Physiology, University of Salento, Lecce, ITALY, 3Institute of Biomembranes and Bioenergetics, CNR, Bari, ITALY

P1–31 Impaired function of mitochondrial ATP synthase depending upon 9205delTA mutation load in ATP6 gene of mtDNA K. Hejzlarova, P. Jesina, V. Kaplanova, Z. Drahota, M. Kalous and J. Houstek Department of Bioenergetics, Institute of Physiology AS CR v.v.i., Prague 4, CZECH REPUBLIC

Introduction: The liver is usually a quiescent organ, but it is able to regenerate itself and replace lost tissue, after transplanta- tion and upon the loss of cells, following chemical and viral injury, and partial hepatectomy (PH). Liver regeneration is mainly divided in two phases: the prereplicative phase, during which the liver’s energy demand increases, and the replicative phase, during which increased DNA synthesis and mitosis occurs. In the prereplicative phase (0–24 h after PH), mitochondria show, a decrease in oxidative phosphorylation capability and production of oxygen free radicals (1). We applied a proteomic approach to mitochondria isolated 6 h after PH, to characterize mitochondrial proteins that are involved in the prereplicative phase of liver regeneration. Methods: Rats were subjected to PH at 6 h and mitochondria were isolated. Mitochondria from sham-operated rats were used as controls. Mitochondrial protein expression pattern were stud- ied by 2-DE electrophoresis, and subsequent spots identification was performed by nanoLC-ESI MS/MS analysis. Results: Compared to the sham-operated control group, 1 pro- tein was up-regulated and 13 proteins were down-regulated at 6 h. We identified eight differentially expressed proteins that were associated with lipid metabolism, the OXPHOS system, biotrans- formation and other metabolic pathways. Among these, Mn- superoxide dismutase, was down-regulated 6 h after PH. SOD2 is a matrix mitochondrial enzyme that catalyzes superoxide radicals dismutation. Reference: 1. Guerrieri F et al., Free Radic. Biol. Med. 1999; 26: 34–41.

Maternally transmitted disorders of mitochondrial ATP synthase are typically caused by heteroplasmic missense point mutations in mtDNA encoded subunit 6. In contrast, unique ATP synthase disorder due to microdeletion 9205delTA in ATP6/COX3 gene is associated with altered splicing of ATP6-COX3 mRNA and diminished synthesis of subunit 6 (Fo-a). Up to now, only two patients with 9205delTA mutation have been found with very dif- ferent phenotypes and mutation load. To investigate phenotypic expression of the mutation, we have prepared transmitochondrial cybrids with varying mutation load (50–100%) and studied the relationship between heteroplasmy, mitochondrial respiration, ATP production and Fo-a subunit content. Heteroplasmy in cell lines was analyzed by PCR/RFLP using NsiI restriction endonu- clease and digitonin-permeabilised cells were used for measuring oligomycin-sensitive, ADP-stimulated oxygen consumption. In parallel, ATP production was determined in DMSO-quenched samples by a luciferin-luciferase reaction. To evaluate how the mutation load affects on biosynthesis of Fo-a subunit, patient cy- brids were analyzed by SDS-PAGE and WB using specific anti- bodies to ATP synthase subunits Fo-a and F1-alpha. We have found that the decrease in ATP production and in ADP-stimu- lated respiration as well as the loss of subunit Fo-a content exert similar threshold dependce on increasing mutation load. Pro- nounced decrease in all parameters was observed when mutation load reached about 80%. In contrast, near-linear relationship was found between ATP production, respiration and loss of Fo-a subunit. In conclusion, our results demonstrate, that similarly as ATP6 missense mutations, 9205delTA biochemical phenotype exhibits distinct threshold effect that originates from a gene-pro- tein level.

P1–30 Evolutionary dynamics of the Arabidopsis formin family M. Grunt and F. Cvrckova Faculty of Science, Plant Physiology, Charles University, Prague, CZECH REPUBLIC

P1–32 Localisation of Pcb antenna complexes in photosynthetic prokaryote Prochlorothrix hollandica M. Herbstova and F. Vacha Institute of Plant Molecular Biology, Photosynthesis, Ceske Bude- jovice, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Formins (FH2 proteins) are a family of actin-organizing proteins exhibiting interesting evolutionary dynamics, with variable domain composition in some lineages including plants. Seed plants, in particular angiosperms, exhibit an extraordinary diver- sity of formins, which can be divided into two distinct classes on the basis of sequence of the conserved actin-nucleating FH2 domain, as well as domain composition (1). The genome of the common laboratory model, Arabidopsis thaliana, encodes 11 Class I formins and up to 12 Class II formins, which appears to be especially prone to gene duplication. Since the draft genome sequence of a closely related Arabidopsis species, A. lyrata, became available recently, we have used this sequence to identify members of the A. lyrata formin family, and compared them with their A. thaliana homologues. While we could easily distin- A filamentous freshwater phytoplankton Prochlorothrix hollandic- a belongs to an unusual group of cyanobacteria called prochloro- phytes. Typical cyanobacteria organise photosynthetic light- harvesting complexes into so-called phycobilisomes where no chlorophyll is bound. Prochlorophyta lost the ability to create

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P1–34 Comparative analysis of proteome changes in contrasting flax cultivars upon cadmium exposure J. Hradilova1, P. Rehulka2, H. Rehulkova2, M. Vrbova3, M. Griga3 and B. Brzobohaty1 1Laboratory of Plant Molecular Biology, Institute of Biophysics AS CR, Mendel University of Agriculture and Forestry in Brno, Brno, CZECH REPUBLIC, 2Department of Proteomics and Glycomics, Institute of Analytical Chemistry of the AS CR, Brno, CZECH REPUBLIC, 3Plant biotechnology department, AGRITEC Ltd., Brno, CZECH REPUBLIC

phycobilisomes, but on the other hand as chloroplasts of high plants and green algae it can synthesize chlorophyll (Chl) a and b bound to the Pcb proteins (PcbA, PcbB, PcbC). They are simi- lar to the IsiA, a chl a-binding protein induced in response to an iron deficiency in many of cyanobacteria and to the CP43 protein forming the Chl a inner antenna protein of photosystem II. An aim of this work was to identify and localise Pcb antenna com- plexes in P. hollandica. The PcbA and PcbB antenna polypeptides are genetically closely related to each other, the third PcbC pro- tein is divergent from the first two. This fact can indicate their different function in association with photosynthetic complexes. The PcbA and PcbB proteins seems to function as mobile unbound antenna of both photosystems in contrast to PcbC pro- teins that may be bound to the photosystem I. Solubilization of thylakoid membranes resulted in a separation of seven zones on the sucrose density gradient. They were characterised by the absorption and fluorescence emission spectroscopy. Protein com- position was determined by SDS-PAGE and the Pcb proteins was detected by western blotting. Pigment-protein complexes were separated by colourless native electrophoresis (CN-PAGE). Subunit protein composition of complexes was resolved in a sec- ond dimension by SDS-PAGE and Pcb proteins were identified by immunoblot.

P1–33 Selection of reference genes for gene expression studies in Bombus terrestris D. Hornakova1, P. Matouskova1, J. Kindl2, I. Valterova2 and I. Pichova1 1Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences, Biochemistry, Prague 6, CZECH REPUBLIC, 2Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences, Chemistry of Natural Products, Prague 6, CZECH REPUBLIC

Cadmium (Cd) is classified as a serious pollutant due to its high toxicity and carcinogenicity, and widespread presence in environ- ment. Phytoremediation represents an effective low-cost technol- ogy for removal of pollutants from contaminated soils. Flax belongs to crops with significant phytoremediation potential. Pre- vious investigation revealed significant differences in Cd accumu- lation and tolerance among commercial flax cultivars with cv. Jitka being superior to cv. Ta´ bor in respect of tolerance to increased Cd content in soil and plant tissues. Here significant changes in expression of 14 proteins (related to disease/defence, metabolism, protein destination and storage, signal transduction, energy and cell structure) were detected by image and mass spec- trometric analysis of two-dimensionally separated proteins extracted from Cd treated cell suspension cultures derived from the two contrasting cultivars. Importantly, up-regulation by Cd of two of them, ferritin and glutamine synthetase, was found only in cv. Jitka. This might infer increased Cd detoxification in cv. Jitka via binding to ferritin, and low-molecular thiol peptides as glutamine synthetase is an important enzyme in glutathione biosynthesis. The identified proteins could turn useful in marker assisted breeding for Cd tolerance, and development of trans- genic flax lines possessing enhanced Cd accumulation and toler- ance for phytoremediation of Cd contaminated soils. Acknowledgements: This work was supported by grants 1M06030 and MSM 2678424601 (Ministry of Education of the Czech Republic), and AV0Z50040507, AV0Z50040702 and AV0Z40310501 (Academy of Sciences of the Czech Republic).

P1–35 Intergenic co-transcription, erratic alternative splicing and recruitment of transposable element sequences into transcripts of testis-specific retrogenes C.-J. Huang1, W. Y. Lin2, C. M. Chang3 and K. B. Choo2 1Department of Animal Science, School of Agriculture, Chinese Culture University, Taipei, TAIWAN, 2Taipei Veterans General Hospital, Department of Medical Research and Education, Taipei, TAIWAN, 3Graduate Institute of Biotechnology, Chinese Culture University, Taipei, TAIWAN

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Bumblebees are studied as important agricultural pollinators. They use pheromones for many life-important activities, such as mating, foraging, finding the way to food sources. In sexual com- munication males mark a territory using pheromone to attract females for mating. The marking sex pheromone is synthesized in the cephalic region of the labial gland. The pathway and enzymes involved in pheromone biosynthesis in Bombus terrestris are not known. Previous experiments showed that qualitative and quanti- tative changes in composition and amount of the labial gland secretion depend on the age of bumblebee (1). To determine, which enzymes participate in pheromone biosynthesis, in tissue restructuring and in programmed cell death of the secretory cells, we will analyze the gene expression profile in the labial gland and fat body at different points in the bumblebee’s life cycle. No ref- erence genes for qPCR quantification of gene expression in Bom- bus terrestris have been characterized. We have evaluated nine potential reference genes [arginine kinase (AK), 18S rRNA, elon- gation factor 1a (EF), b-Actin, glyceraldehyde-3P-dehydrogenase, a-tubulin, ribosomal protein L13, acidic ribosomal protein P2, and phospholipase A2 (PA2)] in the labial gland and the fat body of B. terrestris. Sequences of AK, 18S rRNA, EF were publicly accessible. Remaining six candidate gene fragments were cloned using degenerate primers. Using geNorm and NormFinder Excel based software we found that AK and PA2 genes are the most stable in both tissues from B. terrestris and can be used as refer- ence genes for gene expression profiling. Reference: 1. Sˇ obotnı´ k et al., J. Insect. Physiol. 2008; 54: 204–214. It is a puzzle why mammalian genomes are heavily burdened with highly repetitive relics of ancestral transposable elements (TEs); the biological meaning of TE in the genome remains lar- gely elusive. We have previously reported a cluster of retrogenes in the rat genome two of which, Rtdpoz-T1 and -T2, are testis- specific. In the course of investigation of the structure of T1/T2 transcripts expressed in the embryo, the exons of these retroge- nes, some duplicated as cassettes of 2–3 exons, are found to be deeply embedded within a dense minefield of up to 70% occu- pancy of LINE1 and ERV sequences. Furthermore, the retroge- nes are seemingly regulated by a single promoter and T1-T2 chimerism is also observed. To explain the novel features of the

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germ line transformation,

T1/T2 transcripts, we propose a co-transcription model in which transcripts of the downstream gene, T1, are highly complex as a result of rogue recruitment of TE remnants sequences to the untranslated regions, in particular the 5¢-UTR, due to unruly alternative splicing of some extended transcriptional read-through primary transcript(s). TE invasion of 5¢-UTR results in upstream ORFs with experimentally demonstrated deleterious effects on translation. Our findings expand the list of possible biological functions attributable to transposable elements in the mammalian genome.

from PRE-mediated silencing. It does not

silencers and prevent the spread of repressive chromatin. The detailed mechanism of insulator action is currently unknown. Methods: Plasmid construction, genetic crosses, X-ChIP. Results: We have tested the ability of the recently described Wari insulator to block the repression of the mini-white and yel- low genes by the Ubx PRE. We show that Wari insulator can block the activity ofPRE-mediated silencing. The distance between Wari insulator and a protected promoter is important for the effective blocking from PRE-mediated silencing. We found that part of Wari insulator (368 bp) which is minimal for enhancer-blocking activity could be shortened in case of the bar- rier activity. Moreover, we show with X-ChIP that proteins CP190 and Mod(mdg4)-67.2 interact with Wari insulator. In addition, we investigated that mutations in mod(mdg4)-67.2 and e(y)2 genes affect the ability of Wari insulator to protect gene expression from PRE-mediated silencing, but not its ability to block activation by enhancer. Conclusions: Wari insulator can protect yellow and mini-white genes require Mod(mdg4)-67.2 or E(y)2 for its enhancer-blocking activity, but these proteins are important for its barrier activity.

P1–36 Identification of novel cancer-associated molecules A. Chernenko1, M. Reinman2, P. R. Karhemo1, S. Goodison3, K. Takkinen2 and P. Laakkonen1 1Faculty of Medicine, Institute of Biomedicine, University of Helsinki, Helsinki, FINLAND, 2VTT Technical Research Centre of Finland, Biotechnology, Espoo, FINLAND, 3Department of Surgery, University of Florida, Jacksonville, FL, USA

P1–38 Abstract withdrawn

P1–39 A multiple sclerosis risk-associated SNP influences splicing of the myelin oligodendrocyte glycoprotein gene C. Jensen and J. Rubio Howard Florey Institute, Neurogenetics, Parkville, Vic., AUSTRALIA

Tumor metastasis is the most serious challenge for cancer treat- ment, since the metastatic dissemination of tumour, rather than primary tumours, is responsible for most cancer deaths. In our study we are aiming to discover the metastasis-promoting mole- cules and construct the antibodies against these molecules. We are using two subclones of the MDA-MB-435 human melanoma cell line as a model for the metastatic spread of cancer. One cell clone metastasizes consistently to the lungs whereas the other, equally tumorigenic, fails to metastasize to any distal site in athy- mic mice. In order to find new metastasis-associated markers we panned these cell lines with phage-displayed single-chain variable fragment (scFv) antibody library. Several clones of scFv-phages were identified to bind differently to cells with metastatic and non-metastatic phenotype, as judged by FACS analysis. The selected scFvs recognize antigens on the cell surface and some of them are able to internalize. We are currently trying to identify the antigens for selected antibodies by immunoprecipitation from the metastatic cells lysates using soluble biotinylated scFvs and scFv-expressing phages. After immunoprecipitation the putative antigens will be analyzed with MALDI-TOF mass-spectrometry. Next we are planning to study the role of the discovered mole- cules in tumour metastasis. For further studies the antibodies can be produced and purified in IgG or IgM format. The results of our study might have the direct clinical implication, since the revealed metastasis-associated molecules might be used as drug targets and the created human antibodies can be used to inhibit metastatic process in cancer patients.

P1–37 Characterization of the barrier activity of Wari insulator in D. melanogaster D. Chetverina, M. Erokhin and P. Georgiev Department of the Control of Genetic Processes, Institute of Gene Biology, Moscow, RUSSIA

Multiple sclerosis (MS) is a complex autoimmune disease charac- terised by T and B-cell mediated demyelinating lesions in the cen- tral nervous system (CNS). An auto-antigen in MS is the myelin oligodendrocyte glycoprotein (MOG), a CNS restricted protein expressed on the outer cell membrane of oligodendrocytes that can be used to induce the MS-like condition, experimental auto- immune encephalitis (EAE), in laboratory animals. There have been conflicting reports in regard to the association between com- mon variation in the MOG gene and susceptibility to MS. In this study we investigated some of the expression characteristics of MOG in post-mortem human MS and control brain tissue in order to try to elucidate the cause of an association we observed between the single nucleotide polymorphism, rs3130253G>A (V145I), and susceptibility to MS. We found that the risk-associ- ated allele, rs3130253A, was associated with a small shift in expression towards the MOG alpha1 and/or MOG alpha4 splice variants. We also demonstrated that neither rs3130253G>A (exon 3) or other common exonic variations in the MOG gene [rs282857766 (exon3, V142L) and rs3130250G>A (exon 1, S5S)] were associated with allele specific changes in overall MOG expression. In conclusion, subtle changes in MOG isoform selec- tion, but not expression levels, appear to be associated with rs3130253G>A, which in turn could influence the function of MOG and the pathogenesis of MS.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Background: Insulators are thought to isolate independent tran- scriptional units from cross-reaction with neighboring regulatory sequences specifically blocking the activity of an enhancer. Insu- lators operate in a position-dependent manner, preventing the activity of enhancers only when inserted between these regulatory elements and a promoter. It was shown that at least some of insulators have a barrier activity: they can block the activity of

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P1–40 FATT mouse: a novel leptin receptor mutant C. Jensen1, H. Chen2, M. Morris2, L. Johnson1, R. Burfoot1, K. Simpson3, G. Smyth3, K. Walder4, S. Foote3, J. Hollis5, B. Oldfield5 and J. Rubio6 1Howard Florey Institute, Neurogenetics, Parkville, Vic., AUSTRALIA, 2Department of Pharmacology, University of Melbourne, Parkville, Vic., AUSTRALIA, 3Genetics and Bioinformatics Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, Vic., AUSTRALIA, 4Metabolic Research Unit, School of Exercise and Nutrition Sciences, Deakin University, Geelong, Vic., AUSTRALIA, 5Department of Physiology, Monash University, Clayton, Vic., AUSTRALIA, 6Neurogenetics Laboratory, Howard Florey Institute, Parkville, Vic., AUSTRALIA

forms (solids, fibers, fabrics, films and gels) and they can be eas- ily fabricated giving various shapes and structures. Additionally, they are easily functionalized and, depending on the polymeric building blocks, may also be degraded in the body after a desired period. However, biomaterials used for medical implants or instruments production can cause numerous adverse effects. They may cause changes in gene expression and protein profile of the cells which were in contact with applied material. The aim of this study was to evaluate an influence of medical steel AISI 316L and the medical steel coated with poly-para-xylylene (parylene) on a cell cycle and apoptosis of human endothelial cells. The results demonstrated that interactions between cells and studied materials induce a number of changes in the gene expression profile. Acknowledgement: Supported by project No P01/0001/SPB- PSS/2008a.

P1–42 Tumor necrosis factor, tumor necrosis factor receptors type 1 and 2, lymphotoxin-a gene polymorphism in lymphoproliferative diseases in Serbian population T. Jevtovic1, G. Kocic1, D. Pavlovic1, N. Tosic2, S. Aveic2, G. Marjanovic3, L. Macukanovic-Golubovic3 and V. Djordjevic1 1Institute for Biochemistry, Medical Faculty University of Nis, Nis, SERBIA, 2Department for Molecular Biology, Institute of Molecular Genetics and Genetic Engineering, Belgrade, SERBIA, 3Institute of Hematology and Immunology, Clinical Centre Nis, Nis, SERBIA

one and TNFRII polymorphism in

A spontaneous nucleotide substitution 1279 G>A was observed in the leptin receptor (lepr) gene [NM_146146] in mice of the 129/ SvEvTac strain. Mutant mice (designated FATT) carrying two copies of the mutation (LeprFATT)became hyperphagic and obese post-weaning. In exon 7 of lepr, adjacent to the splice donor ‘GT’ consensus sequence in intron 7, the LeprFATT mutation correlated with the production of two alternatively spliced transcripts in the hypothalamus of obese mice; 1) intron 7 sequence remaining in the transcript, 2) exon 7 sequence (predicted immunoglobulin-like domain) discretely removed with the spliced variant remaining in frame. While there were a number of phenotypic similarities with other leptin signalling deficient mutant strains such as the leptin deficient ‘ob’ mouse and the leptin receptor deficient ‘db’ mouse (similarities include hyperphagia, obesity, hyperleptinaemia, insu- lin resistance, infertility and increased respiratory exchange ratio) there were also distinct differences, (principally being increased body length, and minimal changes in the lymphoid tissues, in con- trast to shorter body length and compromised lymphatic tissues in db mice). Phenotypic characteristics that differentiated the LeprFATT mutant from the ‘ob’ and ‘db’ mutants remained after backcrossing onto a C57/Bl6 genetic background. Given the unique site of the mutation, its effect on Lepr splicing and this unique combination of phenotypic characteristics it is likely that this leptin receptor mutant will prove useful in elucidating further the signalling mechanisms of the leptin receptor and their down stream consequences.

P1–41 Contact of medical steel and parylene with endothelial cells induces changes in gene expression profile H. Jerczynska1, P. Komorowski2, A. Nosal2, H. Szymanowski2, M. Gazicki-Lipman2, B. Walkowiak2 and Z. Pawlowska1 1Department of Molecular and Medical Biophysics, Medical University in Lodz, Lodz, POLAND, 2Institute of Materials Science and Engineering, Technical University of Lodz, Lodz, POLAND

lymphoproliferative patients with the to

issues in today’s biomedical

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Biomaterials can be used successfully to replace many parts of living systems in the human body. Material selection and bio- compatibility are critical implants applications. Biomaterials research is an exciting and rapidly growing field, the results of such studies should be undertaken to determine accurately the safety and performance of the implants. Currently the material of choice for thousands of medical appli- cations are polymers since they can be prepared in a variety of Tumor necrosis factor-a and lymphotoxin-a are involved in the pathogenesis of established lymphoproliferative diseases. TNF molecules bind to TNFRI and TNFRII. TNFRI is the major mediator of the TNF pro-apoptotic and prolifferative effects and TNFRII might potentiate this effects. In this study, we have analysed polymorphism in TNF gene -308 G/A, in LT-a gene +250G/A, one polymorphisms in TNFRI gene (TNFRI + 36A/ G SNP) gene (TNFRII + 676 T/G). All these polymorphisms were studied in patients with chronic lymphocytic leukemia’s (CLL), patients with non-Hodkin’s lymphoma (NHL) and in healthy controls. The present study was undertaken to investigate the genetic asso- ciation of these polymorphisms with lymproproliferative disease development. Genotyping was performed by PCR-RFLP analy- sis. The study provided evidence of the influence of TNFG/A genotype and A alleles in the susceptibility to NHL, since the association of LT-aG/G genotype with CLL was observed. High- producing TNF-a -308/LT-a +250 heterozygous haplotype is associated with high NHL incidence. No significant differences in allele frequencies of TNFR1 polymorphism were found between the patients with lymphoproliferative disease and healthy individ- uals. In a group of healthy individuals the study has revealed for the first time significantly higher TNFRI G/G genotype com- pared disease (v2 = 5.66; P = 0.017). Also, we reported the implication of TNFRII T allele in NHL pathogenesis, respectively (v2 = 10.77; P = 0.001; Mantel-Haenszel: v2 = 10.64; P = 0.0011). Our data showed that TNFRII T676G polymorphisms have an important role in NHLs pathogenesis but not in CLL patients. A/A poly- morphism in TNFRI is associated with CLL and NHL patients in Serbian population.

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changes. Protein band numbers and their optic density were high in posthibernation period. Glycoprotein band numbers and their density were also exhibited same aspect as proteins. These find- ings point out that thumb pad secretion is abounded in terms of protein content and the changes about protein and glycoprotein profiles might be related to seasonal reproductive activity. This means that they can be dependent on androgenic hormones.

P1–43 Genetic evaluation of the human oral cancer via systematic DNA resequencing approach M. C. Kao1, M. H. Yeh2, T. M. Shieh3, S. F. Chen3, M. H. Tsai4 and Y. S. Lee5 1Biological Science and Technology, China Medical University, Taichung, TAIWAN, 2National Defense Medical Center, Graduate Institute of Life Sciences, Taipei, TAIWAN, 3Department of Dental Hygiene, China Medical University, Taichung, TAIWAN, 4Department of Otolaryngology, China Medical University Hospi- tal, Taichung, TAIWAN, 5Office of Research & Development, China Medical University, Taichung, TAIWAN

P1–45 Comparison of protein and mRNA expression levels of titin isoforms in cardiac muscles of hibernating ground squirrels E. Karaduleva, I. Vikhlyantsev, J. Shumilina and Z. Podlubnaya Laboratory of Structure and Function of Muscle Proteins, Insitute of Theoretical and Experimental Biophysics, Pushchino, RUSSIA

Oral cancer is of worldwide occurrence and concerned globally, approximately 500 000 new cases of oral and pharyngeal cancers are diagnosed annually. The development of oral cancer is a mul- tistep process. It has a late onset and shows a poorly clinical out- come. Early detection of precancerous lesions or premalignant conditions exhibiting transformation potential will lead to a con- servative intervention and efficient therapy. Therefore, under- standing the molecular mechanisms of oral carcinogenesis is necessary to improve early prognosis and even implant an early detection protocol for the oral cancer. In this research, we glob- ally search for human oral cancer altered genes via a comprehen- sive Real-Time RT-PCR (qPCR) and DNA resequencing strategy. To date, we have assayed both tumor and their adjacent normal tissues from 60 oral cancer patients by qPCR and identi- fied 20 differential expression genes in human oral tumors. Fur- thermore, we used DNA resequencing approach to determine the spectrum and extent of altered somatic mutations in these 20 genes, four mutation sites were identified within three genes (MMP1, PTN and BMP3). We propose these 20 genes and the mutations may have potential as biomarkers for early diagnosis of the oral cancer. In addition, they may be used as a potential targets for anti-cancer drug design and beneficial to the clinical treatment of oral carcinomas.

squirrels was approximately

P1–44 SDS-PAGE characterization of the proteins and glycoproteins in thumb pad secretion of the frog (Rana ridibunda) E. Kaptan and S. Bolkent Faculty of Science, Department of Biology, Istanbul University, Istanbul, TURKEY

Titin is giant elastic protein of vertebrate skeletal and cardiac muscles. Titin- encoding gene located in the chromosome 2 (region 2q31) consists of 363 exons encoding 4200 kDa protein (38138 amino acid residues). Alternative splicing of titin elastic area in an I-zone of sarcomere is a basis of a variety of titin iso- forms. Cardiac titin is expressed in two isoforms: short N2B (3000 kDa) and long N2BA (3200–3400 kDa) with up to seven variants of alternative splicing. Research of protein regulation on transcriptional and translational levels is of particular interest for description of muscle functional state. In this work we carried out the investigation of changes in titin isoform composition in myocardium of ground squirrels in various stages of hibernation- al cycle. We have selected this model because hibernation is a unique model of adaptation to stress conditions, immobilization and heart function depression. The results of qRT-PCR revealed the decrease of mRNA level both for N2BA and N2B titin iso- forms in heart of hibernating ground squirrels as compared to their levels in the heart of summer active animals. Using electro- phoresis and western blotting we have shown the decrease in overall amount of titin with respect to heavy myosin chains in different heart chambers of ground squirrels upon hibernation in comparison with summer active animals (ð‡0,998). The ratio of long N2BA to N2B titin isoforms in the heart of hibernating ground two-fold increased (P < 0.0005). However this trend was not revealed for the mRNA levels of corresponding isoforms. This discrepancy in protein and mRNA levels may be considered as the posstran- scriptional regulation of titin isoforms’ expression. The overall decrease in mRNA level may be explained by repressed transcrip- tion or mRNA degradation in the cell during hibernation. To understand the mechanisms regulating titin isoform composition upon hibernation these investigations are to be extended. Acknowledgement: This work was supported by grant RFBR 07-04-00479.

P1–46 Uniform reporting standards for enzyme activity data C. Kettner Funding Department, Beilstein-Institut, Frankfurt am Main, GERMANY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Generally, frogs exhibit cyclic reproductive activity throughout 1 year. Their primary and secondary sexual characteristics show structural and biochemical changes during this cycle. Thumb pad is an androgen-dependent secondary sexual characteristic playing a role in amplexus. In this study, we focused on documenting protein and glycoprotein profile of its secretion and determining the variability among those which obtained from different sea- son. For this propose, we composed of four groups namely; active, prehibernating, hibernating and posthibernating. The secretion of thumb pad isolated from all groups and determina- tion of protein and glycoprotein concentrations were performed by method of Lowry and Dubois, respectively. Protein and glyco- proteins of secretion content were separated using sodium dode- cyl sulphate polyacrylamide gel electrophoresis. Prior to scan and analyze of the gels using densitometer, they were stained with Coomassie blue and glycoprotein detecting kit, respectively. A total of 22 bands were determined, ranging from 156 to 16 kDa protein. Of these bands, five protein (66, 32, 25, 224 and 23 kDa) were detected to be glycolyzed. In addition to these protein and they showed seasonal the secretion, glycoprotein pattern of The access to the essential background information on enzymo- logical experiments is of paramount impact for the understanding of both the experimental data themselves and their interpretation

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NPR from C. albicans could provide the critical understanding of its role in the processing of pathogenesis. Acknowledgements: This study is supported by a grant of the Korea Healthcare technology R&D Project, Ministry of Health & Welfare, A084005.

P1–48 Electrochemical and computational study of doxorubicin interactions with DNA D. Huska1, V. Adam1, J. Burda2, J. Hrabeta3, T. Eckschlager3, P. Babula4, R. Opatrilova4, L. Trnkova5, M. Stiborova6 and R. Kizek1 1Department of Chemistry and Biochemistry, Mendel University of Agriculture and Forestry in Brno, Brno, CZECH REPUBLIC, 2Department of Chemical Physics and Optics, Charles University, Prague, CZECH REPUBLIC, 3Department of Paediatric Haema- tology and Oncology, Charles University, Prague, CZECH REPUBLIC, 4Department of Natural Drugs, University of Veteri- nary and Pharmaceutical Sciences, Brno, CZECH REPUBLIC, 5Department of Chemistry, Masaryk University, Brno, CZECH REPUBLIC, 6Department of Biochemistry, Charles University, Prague, CZECH REPUBLIC

the adoption of journals was

under consideration of the broader context. However, scientists who are evaluating these data for pathway research be it as in sil- ico modellers or as experimentalists often are missing full details of the assay conditions to be able to refer the enzyme activity data presented to the materials and methods applied. Further- more, they are often are faced with the problem of a broad range of method-specific enzyme data ranges associated with individual methods found in databases or in the scientific literature. Modern experimental techniques provide apparently endless opportunities to generate huge amounts of enzyme structure and activity data. These technological advances have resulted in increased accuracy of measurement and analysis methods and given rise to large amounts of data. But this increasing rate of production exacer- bates the problem of fragmented descriptions of materials and methods which leads to incomparable and incomplete data. It appears to be perfectly obvious that these problems could be addressed by adopting a set of reporting standards. Since the need for standardization is widely recognized by the scientific community, the STRENDA Commission (1,2), founded and sup- ported by the Beilstein-Institut, proposed guidelines for reporting minimum information on both the experimental design and the obtained functional data. These guidelines resulted as checklists from consultation with the community and were embedded in the international checklists development project MIBBI (3). A piv- otal achievement of the cooperation with a number of biochemi- cal these guidelines in their instructions for authors last year. Further journals will follow these flagships in due course. It is hoped that future publications will more readily yield the sort of information that researchers hope to find. The guidelines and further aims of STRENDA will be presented in detail. References: 1. Apweiler R et al. (2005) The importance of uniformity in reporting protein-function data. TRENDS in Biochemical Sciences 30(1):11–12. 2. Alberty R et al. (2009) Standards for reporting enzyme data: the STRENDA consortium. submitted.

3. Taylor CF et al. (2008) Promoting coherent minimum report- ing guidelines for biological and biomedical investigations: the MIBBI project. Nature Biotechnology 26(8):889–896.

P1–47 Characterization of cytochrome CYP52 and NADPH-P450 reductase from Candida albicans D. Kim, H. G. Park, Y. R. Lim, C. Y. Eun and S. Han Biological Sciences, Konkuk University, Seoul, SOUTH-KOREA

Anthracyclines including doxorubicin belong to antibiotics pro- duced by Streptomyces peucetius subsp. cesius. There is compelling evidence that cellular DNA is the primary target for them. It is not surprise that cell protective mechanisms cause resistance on the treatment with these drugs. The main aim is to study intercalation of doxorubicin into DNA in vitro by using of square wave voltam- metry. The electrochemical method is further utilized for detection of doxorubicin-DNA adducts in neuroblastoma cells. Intercalated doxorubicin reduced observed dsDNA cytosine and adenine sig- nal, but also provided new signal of the cytostatic at -0.35 ± 0.12 V (n = 8). For more comprehensive understanding of the intercalation, computational experiments using design for testabil- ity approach with correction on dispersion interaction were per- formed. Several molecular structures were explored and clear preference for very stable stacked structures was observed. In addi- tion, we detected doxorubicin-DNA adducts in doxorubicin trea- ted neuroblastoma cells. We observed significant (a = 0.05) difference in DNA signals after intercalation of doxorubicin between resistant and sensitive cells. Resistant cells showed negligi- ble amount of adducts with the increasing concentration of the drug compared to resistant cells. The suppressed formation of ad- ducts can be related to presence of other mechanisms to withstand the effect of the cytostatic. Therefore we determined low-molecu- lar-mass thiols (glutathiones and its derivatives) and found the increased thiols level in resistant cells compared to sensitive ones. Acknowledgement: This work was supported by GA AV IAA401990701. References: 1. Krizkova S. et al., Electroanalysis 2007; 19(2–3): 331–338. 2. Huska D. et al., Electroanalysis 2009; 21(3–5): 487–494.

P1–49 Respiratory supercomplexes in COX-deficient mammalian mitochondria N. Kovarova1, P. Pecina1, J. Houstek1, C. Dell‘Agnello2 and M. Zeviani2 1Bioenergetics, Institute of Physiology Academy of Sciences of the Czech Republic, Prague, CZECH REPUBLIC, 2Unit of Molecular Neurogenetics, National Neurological Institute, Milan, ITALY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Candida albicans is a major pathogenic fungus that causes oppor- tunistic oral and vaginal infections in humans. It contains ten putative cytochromes P450 (CYPs) and an NADPH-P450 reduc- tase (NPR) genes coding for enzymes that appear to play impor- tant roles in fungal survival and virulence. Here, we report the characterization of CYP52, a putative alkane/fatty acid hydroxy- lase and NPR, an essential reduction partner for CYP52. The recombinant CYP52 protein was expressed and was purified. The purified protein primarily w-hydroxylated lauric acid to give 12-hydroxylauric acid, but to a lesser extent also catalyzed (w-1)- hydroxylation. The regioselectivity of CYP52 fatty acid hydroxyl- ation can be attributed to the presence of a narrow channel that restricts access of terminal atom for oxidation even though this narrow channel. As an essential enzyme for P450 reaction, the recombinant NPR protein from C. albicans was also expressed and was purified. C. albicans NPR protein showed a strong NADPH-dependent reduction activity of nitrotetrazolium blue chloride. This study of functional characterization of P450s and Respiratory chain enzyme complexes are assembled into higher structural units, respiratory supercomplexes. In mammalian mito- chondria they are mainly composed of complexes I, III, and IV.

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(SURF1-/-)

P1–51 N-linked glycosylation of the human ovarian carcinoma SKOV3 cell line E. Machado1, S. Kandzia2, P. Altevogt3, H. S. Conradt2 and J. Costa1 1ITQB, Laboratory of Glycobiology, Oeiras, PORTUGAL, 2GlycoThera, GmbH, Hannover, GERMANY, 3Division of Cellular Immunology, German Cancer Research Center, Heidelberg, GERMANY

Isolated deficiency of complex IV represents frequent cause of mitochondrial disease and we investigated how alterations of cytochrome c oxidase (COX) content influence the supercomplex assembly and occurrence in mitochondria from different tissues. We used mice model with isolated COX defect due to SURF1 gene knock-out that prevents synthesis of Surf1 assembly protein, and leads to varying degree of COX deficiency in mouse tissues. For comparison we analysed COX-deficient fi- broblasts mitochondria of a patient carying mutation in the SURF1 gene. Digitonin-solubilised protein complexes were analy- sed by 2D electrophoresis (BN-PAGE/SDS-PAGE or BN- PAGE/BN-PAGE) and Western blotting using specific antibodies to subunits of complex I, III and IV. Results of our experiments revealed presence of two types of supercomplexes, I-III2 and III2- IV, in mitochondria from liver, brain, muscle and heart from control and COX-deficient mouse. In mouse fibroblasts we also detected I-III2-IV supercomplex and the same pattern was found in human fibroblasts. In the latter case all complex I signal was present in I-III2-IV supercomplex. Decrease of COX content in mitochondria from both COX-deficient mouse and from the patient markedly decreased the assembly of COX-containing su- percomplexes, whereas the content of complex I and III in super- complexes was unchanged. Our study demonstrates significant tissue- and species-specific differences in the assembly and com- position of the respiratory supercomplexes in both control and COX-deficient mammalian mitochondria.

P1–50 Characterization of a putative threonine phosphatase in Mycobacterium bovis BCG P. C. Lee, F. M. Oh and A. A. M. Faik School of Science and Technology, Universiti Malaysia Sabah, Kota Kinabalu Sabah, MALAYSIA

Ovarian carcinoma is the leading cause of death from gynecologi- cal cancers in many Western countries. Aberrant glycosylation is an important aspect in malignant transformation, normally asso- ciated with increased expression of membrane glycoproteins, appearance of novel or truncated oligosaccharides, fucosylated glycoconjugates and abnormal types of terminal oligosaccharides containing antigenic determinants. The SKOV3 ovarian carci- noma cell line has low amounts of Lewis carbohydrate epitopes at the cell surface, synthesized by a1,3/4- fucosyltransferases (FUTs) (1). The stable overexpression of several FUTs in the SKOV3 cell line resulted in de novo synthesis of Lewis carbohy- drate determinants. Over expression of FUT4, FUT5, FUT6, FUT7 and FUT9 originated de novo expression of Lex and/or sLex epitopes, observed on the plasma membrane and intracellu- lar compartments. These findings were consistent with the prefer- ential specificity of the enzymes towards type 2 (Galb1,4GlcNAc) acceptors. Only overexpression of FUT3, with predominant a1,4- FUT activity, produced the type 1 (Galb1,3GlcNAc) related car- bohydrate sLea. The N-linked oligosaccharides from total glyco- proteins of SKOV3 cell line was further characterized by high performance anion exchange chromatography with pulsed amper- ometric detection and matrix assisted laser desorption/ionization time-of-flight mass spectrometry. The results showed that SKOV3 cells were rich in unprocessed oligomannose structures, with low peripheral sialic acid levels. Furthermore, the N-glycosylation of a recombinant secretory glycoprotein, erythropoietin, stably pro- duced from this cell line was characterized. Acknowledgement: This work will contribute to the knowledge of the glycosylation profile of human ovarian cancer cell lines. Reference: 1. Escrevente C, Machado E. et al., Int J Oncol 2006; 29: 557– 566.

P1–52 Quantification of point mutations ratio with a one-tube single nucleotide primer extension followed by reverse phase HPLC T. Majerova1, M. Ingr2, J. Dostal1 and J. Konvalinka1 1Biochemistry and Moleular Biology, Institute of Organic Chemis- try and Biochemistry, Prague, CZECH REPUBLIC, 2Faculty of Science, Biochemistry, Charles University, Prague, CZECH REPUBLIC

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SNP (single nucleotide polymorphism) quantification in DNA pools could be important for a rapid analysis of allele distribu- tion in populations, including determination of gene variants among individuals, monitoring drug resistance development and prognosis of tumor growth. Although many methods of SNP detections are available, precise SNP quantification is still trou- blesome. In this work, we demonstrate a method for accurate quantification of the SNP ratio in mixtures of two synthetic oli- gonucleotides possessing guanine (G) or adenine (A) at a defined position. This method is based on a one-tube single base primer extension with fluorescently labeled nucleotides followed by reverse phase HPLC and fluorescence detection of the primer ‘eukaryotic-like’ serine/threonine protein kinases Mycobaterial coupled with their phosphatases are involved in intracellular sur- vival and host-pathogen interaction. Understanding these kinases and phosphatases will provide clues to facilitate the development of specific protein kinase or phosphatase inhibitors, as well as drugs that modulate the activities of these kinases and phosphata- ses. We report here the cloning and characterization of a putative threonine phosphatase, Mbpp from Mycobacterium bovis BCG 1173P2. The Mbpp gene was cloned by PCR in pET42a (+) and expressed as His-tagged recombinant protein in Escherichia coli as inclusion bodies. The recombinant protein was purified by using high concentration of urea and refolded by dialysis. Mbpp showed distinct phosphatase activity toward p-nitrophenyl phosphate with optimal temperature 55(cid:2)C and pH 8.0. The phosphatase activity of Mbpp was strictly Mn2+ dependent, a characteristic that is found in other serine/threonine phosphatases. Three-dimensional struc- tural analysis revealed three Mn2+ ion binding sites at the active domain of the phosphatase. We detected strong phosphatase activ- ity toward phosphothreonine peptide but not phosphorserine or phosphotyrosine, with a Km value of 1.34 ± 0.074 mM. The phos- phatase activity was not inhibited by known protein phosphatase inhibitors such as okadaic acid and sodium orthovanadate, which are known to be specific inhibitors of eukaryotic-like phosphatases indicating Mbpp is a threonine phosphatase. Bioinformactics anal- yses revealed eleven universally conserved motifs that are well- known characteristic of PP2C and showed 99% amino acid sequence identity to Mstp, a serine/threonine phosphatase of Mycobacterium tuberculosis which is present only in slow growing mycobacterial species that might regulate cell division.

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hairpin binding allowing exonuclease

extension products. In our experiments, we addressed problems of the elimination of errors arising due to competition of two templates differing by a single base and problems with unknown concentration of the template DNA. In the presence of compet- ing template we obtained a lower yield of the extension product of interest. To solve this problem, we prepared a set of serial dilutions from the sample of interest and from a reference sample containing templates at a defined ratio. We correlated the ratio of mutations in the unknown and the reference sample. Conse- quently, we eliminated the need for the determination of absolute DNA concentration in the reference sample and in the sample of interest.

P1–53 Porential role of tumor necrosis factor alpha gene polymorphism in autoimmune disease M. Marinkovic1, T. Jevtovic-Stoimenov1, S. Stojanovic2, M. Todorovic1, J. Basic1, A. Veljkovic1, T. Cvetkovic1 and D. Pavlovic1 1Medical Faculty, Institute of Biochemistry, Nis, SERBIA, 2Medical Faculty, Clinic of Rheumatology, Nis, SERBIA

nation and RNA degradation. TherpsO mRNA encoding for the ribosomal protein S15, has been widely used as a model of study for polyadenyla- tion-mediated mRNA turnover. Escherichia coli RNase II and PNPase are two major degradative exonucleases involved in the control of mRNA stability. However, in the absence of these two exonucleases the polyadenylated rpsO mRNA is still very effi- ciently degraded. Data suggested that at least one yet unknown poly(A)-dependent ribonuclease is involved in this complex regu- lation. In this work, we demonstrate that the exonuclease RNase R is the major enzyme responsible for the poly(A)-dependent degradation of the rpsO mRNA. Moreover, RNase R is shown to have a more relevant effect on rpsO mRNA stability than PNPase, which was so far characterized as the main exonuclease involved in the poly(A)-metabolism, either acting as a free enzyme or in complex. In the absence of RNase R, the rpsO transcript presents longer poly(A) tails and has higher stability. The RNase R/polyadenylation pathway may be more general- ized, as a 3¢ rpsT mRNA fragment whose degradation is highly dependent on polyadenylation is also stabilized in an rnr single mutant. All these results highlight the importance of bacterial RNase R in the post-transcriptional control of gene expression establishing an important parallel with its eukaryotic counter- parts, Rrp44/Dis3, present in exosomes, which are also key play- ers involved in the metabolism of polyadenylated RNA.

P1–55 An in vitro fish system to unravel bone-related mechanisms of BMP2 C. Marques, V. Laize´ and M. L. Cancela CCMAR, F C M A, Faro, PORTUGAL

(73.9% versus

Introduction: Tumor necrosis factor alpha (TNF-alpha) plays a key role in immune regulation, inflammation and autoimmunity. It has been shown that this polymorphism is associated with vari- ety of inflammatory and autoimmune diseases, such as, rheuma- toid arthritis (RA) and systemic lupus erythematosus (SLE). Considering the results of association of TNF-a -308 G/A poly- morphism with RA and SLE, which are inconsistent, the aim of this study was to examine the possible association between TNF- a -308 G/A gene promoter polymorphism and RA and SLE. Materials and Methods: Polymorphism of TNF-a -308 was analyzed in RA patients (n = 76), SLE patients (n = 23) and healthy controls (n = 34). Single nucleotide polymorphism (SNP) was determined by PCR-RFLP method. The results were analyzed using chi-squared test. Results and Discussion: The observed genotype distribution in RA patients did not show significant differences compared to control group of healthy individuals. A higher frequency of het- erozygous TNF G/A was observed in the group of SLE patients- 41.2%, v2 = 5.93, compared to controls P < 0.01). Significantly lower frequency of G/G genotype was found in SLE patients compared to controls (8.7% versus 55.8%, v2 = 13.13, P < 0.001). No differences in the distribution of TNF G and TNF A alleles were observed in group of RA patients compared to control group. Contrary, there was signifi- cant difference in the TNF G and A allele between SLE patients individuals (v2 = 11.32, P < 0.001). Hence, these and control results suggest immunogenetic association involving polymor- phism of TNF-a gene, serving as paracrine and autocrine growth factors, which can contribute to the pathogenesis of SLE but not of RA.

P1–54 RNase R is the main enzyme involved in the poly(A)-dependent degradation of rpsO mRNA J. M. Andrade1, E. Hajnsdorf2, P. Re´ gnier2 and C. M. Arraiano1 1Instituto de Tecnologia Quı´mica e Biolo´gica, Universidade Nova de Lisboa, Oeiras, PORTUGAL, 2Institut de Biologie Physico-Chi- mique, CNRS UPR 9073, Paris, FRANCE

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Mechanisms of tissue mineralization are complex and controlled by several proteins involved in cell differentiation and extracellu- lar matrix synthesis. Bone morphogenetic protein 2 (BMP2) was primarily identified for its osteogenic properties and several human bone-related diseases have been associated with BMP2 defect. Although numerous studies have been performed in mam- mals to unravel BMP2 mechanisms of action, those are still not fully understood. Fish have recently emerged as a suitable model to study vertebrate biology and a suitable alternative to mamma- lian systems. We propose to develop a fish-based in vitro cell sys- tem to study bone-related mechanisms of BMP2, in particular its interaction with matrix Gla protein (MGP), a physiological inhibitor of calcification. BMP2 gene expression was profiled by real-time PCR in a variety of seabream cell lines capable of in vi- tro mineralization and shown to be differentially regulated in osteoblast- and chondrocyte-like cells. Transcriptional regulation of seabream BMP2 gene expression was later evaluated using reporter vectors containing gene promoter and shown to be under the control of various mineralization-related transcription factors, in particular osteoblast-specific factor runx2. Cellular clones, where BMP2 gene expression has been altered, have been constructed, validated by Western blot analysis using antibodies developed in mammals, then evaluated for mineralization capacity using mineralization-specific stains and for mineraliza- tion-related gene expression by real-time PCR. In future experi- ments, BMP2 regulatory network will be assessed using recently developed seabream microarray hybridized with cDNA prepared from cellular clones, and BMP2-MGP interaction will be evalu- ated in a co-culture system of clones overexpressing seabream BMP2 and/or MGP cDNAs. Results obtained within the scope of this work are preliminary but should give new and interesting insights on bone-related mechanisms of BMP2 in vertebrates. Polyadenylation is a universal post-transcriptional RNA modifi- cation. It provides a single-stranded region downstream of termi-

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P1–56 Characterization of Bv8 gene promoter and its regulation by transcription factors S. Marsango, R. Miele, M. Bonaccorsi di Patti and D. Barra Department of Biochemisty sciences ‘A. Rossi Fanelli’, Sapienza University of Rome, Rome, ITALY

hensive annotation on function, structure, splice variation and hierarchical classification of proteins from all organisms, includ- ing metagenomic projects. With 3 60 000 signatures, InterPro provides annotation for 3 80% of UniProt sequences. Novel sequences/genomes can be queried through InterProScan. Protein signatures model amino acid conservation in families or domains, then predict these classifications/features in uncharacterised pro- teins. By linking related signatures together, InterPro places them within a hierarchical classification scheme which reflects their underlying evolutionary relationships. As such, one can address issues such as the co-evolution of domains, the functional diver- gence of proteins based on domain composition and their point of divergence between different species. InterPro provides a direct comparison of protein signatures with structural data, incorpo- rating >14 000 structures from PDB with their classification in SCOP and CATH, as well as >1 650 000 homology models from SWISS-MODEL and MODBASE. Each protein signature has an abstract, references, GO mapping, taxonomy, and links to rele- vant external databases. With its multi-layered annotation, Inter- Pro is a valuable resource for biologists and bioinformaticians alike.

P1–58 Revealing of potential FoxA target genes, involved in proliferation control N. Ershov, T. Merkulova, L. Bryzgalov, M. Pakharukova, D. Oschepkov and V. Kaledin Institute of Cytology and Genetics SB RAS, Laboratory of gene expression control, Novosibirsk, RUSSIA

Bv8 is a small protein with a molecular mass close to 8 kDa secreted by frog skin (1). Homologs of Bv8 can be found in fishes, amphibians, reptiles, birds and mammals (2). Two mam- malian orthologs of Bv8, prokineticin 1 and prokineticin 2 (PKs), are the natural ligands for two G-protein-coupled receptors, PK- R1 and PK-R2. Bv8/prokineticins and their receptor expression is restricted to specific endothelial cells of steroidogenic glands, central nervous system, peripheral blood leukocytes and cells of the innate immune system. These proteins are involved in hema- topoiesis and in inflammatory/immunomodulatory processes act- ing like chemokines (3). The capacity of the immune system to respond appropriately and eradicate microbial infections depends on the production of peptide and small protein mediators, chemokines and antimicrobial peptides, many of which are induced by contact with pathogens or inflammatory stimuli (4). Recently, a high degree of similarity was evidenced among defen- sins, prokineticins and chemokines, either in structure, size, sig- nalling or biological activities (3). In mammals, the synthesis of mediators of innate immunity, like chemokines, antimicrobial peptides and cytokines, as well as effectors of adaptative immu- nity are regulated by the same mechanism that involve the activa- tion and nuclear translocation of NF-jB and NFAT (5). Here we report the organization of the amphibian Bv8 gene. Analysis of the promoter sequence permits identify several putative tran- scription factor binding sites like AP1, NF-kB and NFAT. Func- tional analysis of Bv8 promoter by one-hybrid assay in yeast indicates that these transcription factors are involved in the regu- lation of Bv8 expression. References: 1. Mollay C, Wechselberger C, Mignogna G, Negri L, Melchiorri P, Barra D & Kreil G. Eur. J. Pharmacol. 1999; 374: 189–196. 2. Kaser A, Winklmayr M, Lepperdinger G & Kreil G. EMBO Rep. 2003; 4: 469–73.

3. Monnier J, Samson M. FEBS J. 2008; 275: 4014–4021. 4. Negri L, Lattanzi R, Giannini E & Melchiorri P. Curr. Neuro- pharmacol. 2006; 4: 207–215. 5. Serfling E, et al., Int. J. Biochem. Cell. Biol. 2004; 36: 1166– 1670.

P1–57 InterPro: deciphering the function and classification of novelproteins J. McDowall and S. Hunter EMBL-EBI, Sequence Databases, Hinxton, UK

FoxA transcription factors are known to be critical to liver cell dif- ferentiation. We demonstrated a strong association between he- patocarcinogenic activity of a number of compounds and their ability to reduce FoxA DNA-binding activity in the liver. Thus rat- specific hepatocarcinogen 3¢-methyl-4-dimethylaminoazobenzene significantly reduced FoxA DNA-binding activity only in rats, while mouse-specific ortho-aminoazotoluene – only in mice. Diet- hylnitrosamine, which is highly carcinogenic for the liver of both rats and mice inhibited FoxA activity in both species, while non- carcinogenic 4¢-methyl-4-dimethylaminoazobenzene was ineffective both in rats and mice. With the aim to elucidate the mechanism of potential FoxA tumorsuppressor action we searched for their tar- get genes related to proliferation control. First we examined the published data on global gene profiling in mouse liver (a high level of FoxA) and kidneys (FoxA are absent). Then the search for FoxA binding sites was carried out by computer-assisted SIT- ECON method in regulatory regions of 40 differentially expressed genes involved in the proliferation control. 11 genes containing putative sites organized as TTTG repeats (3 to six-fold) were dis- covered. FoxA binding to these sites was confirmed by EMSA using GST-fusion protein comprising the FoxA 2 DNA-binding domain. The effect of OAT administration (leading to reduction of FoxA activity) on the expression of six genes containing confirmed FoxA binding sites was studied by real-time PCR. We found that the mRNA levels for Cul2 and CDC73 genes increased dramati- cally after OAT administration. Acknowledgement: The work was supported by a grant from the RFBR (07-04-00441).

open-source an is

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

With increasing numbers of novel sequences being identified from genome-sequencing projects, there is a dire need for elucidating functional information that goes beyond the capabilities of exper- imental work alone. Computer algorithms that take into account sequence identity, structural similarity and phylogenetic tree dis- tribution are invaluable for protein function prediction. InterPro (http://www.ebi.ac.uk/interpro/) protein resource combining ten major signature databases (PROSITE, PRINTS, PFAM, PRODOM, SMART, TIGRFAMs, PIR- SUPERFAMILY, PANTHER, GENE3D, SUPERFAMILY) into one powerful, easy-to-use diagnostic tool. InterPro capitalis- es on the strengths of each member database to provide compre-

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enzymatic and chemical probing)

P1–59 Characterization of a putative carbohydrate catabolic pathway of the megaplasmid pAO1 of Arthrobacter nicotinovorans M. Mihasan1, V. Artenie1 and R. Brandsch2 1University Alexandru Ioan Cuza, Faculty of Biology, Biochemistry and Molecular Biology Department, Iasi, ROMANIA, 2Institute for Biochemistry and Molecular Biology, Center for Biochemistry and Molecular Cell Research Albert Ludwigs University, Freiburg, GERMANY

alternatives (mutp53 proteins) are able to recognize several spe- cific DNA motifs (p53 consensus sequence for wtp53) and struc- tural DNA motifs (DNA hairpins) as well as supercoiled DNA (scDNA). In our work we gained a library of genomic fragments naturally bound by mutant p53 proteins and a library of plas- mids containing repeat DNA for non-B DNA structures forma- tion (e.g., triplex, DNA cruciform). Genomic DNA bound by mutp53 was isolated by specific chromatin imunoprecipitation (genome-wide ChIP-cloning). We combined molecular (detection by and computational approaches to identify non-canonical DNA structures (cruciforms and triplex DNA) as binding sites for p53 proteins. Electropho- retic mobility shift assay (EMSA) was used for confirmation of the specific binding of p53 proteins to DNA sequences. Our data suggest that binding of p53 proteins to non-B DNA sequences is thought to be the important molecular mechanism in human can- cerogenesis. Acknowledgements: This work was supported by EC (FP6- mutp53, QLGA-CT-2001-52001 and MERG-6-CT-2005-014875), MEYSCR (1K04119 and LC06035), GA ASCR (IAA500040701, 204/06/P369 and 204/08/1560). Further reading: M., Quante, T., Togel, L., Walter, K., Loscher, C., Tichy, V., Cincarova, L., Deppert, W., Tolstonog, G.: Modulation of gene expression in U251 glioblastoma cells by binding of mutant p53 R273H to intronic and intergenic sequences, Nucleic Acids Res. 2009; 37: 1486–1500.

The gram positive soil bacterium Arthrobacter nicotinovorans car- ries the pAO1 catabolic megaplasmid which enables it to grow on nicotine. Besides the well-characterized pathway for nicotine degradation, pAO1 carries a gene cluster of a hypothetical path- way for carbohydrate utilization. The cluster consists of ORFs of a transcriptional regulator, of a sugar ABC-transporter, and of several putative dehydrogenases and oxidoreductases. A. nicoti- novorans lacking a functional pAO1 plasmid was, as shown here, unable to degrade melibiose and inuline out of 21 carbohydrates the pAO1 orf39 gene tested. We previously established that encodes an aldehyde-dehydrogenase. Here we focused on the orf40 gene, encoding a putative oxido-reductase, in an attempt to further characterize this sugar catabolic pathway. The gene was cloned, expressed and the 45 kDa His-tagged ORF40 protein purified. The native molecular mass of 163 kDa determined by GPC indicated that the protein was a tetramer in solution. Metal content analysis showed that the enzyme binds 2Zn2+ atoms/pro- tein monomer. The enzyme contained the amino acid sequence A119GKHIFTEKP128 similar to the consensus sequence of sugar dehydrogenases. A tridimensional model of the enzyme was gen- erated by the EasyPred3D and used for in silico docking experi- ments using Chimera/Antechamber/Dock6/ZINC suite. 27 out of 29 tested sugars were found to bind to the model forming two binding clusters. One of the clusters, consisting of 23 sugars was located close to the E128KP130 motif and would indicate the puta- tive catalytic site. The binding energy values suggested the fol- lowing order of substrate preference: D-tagatose, D-psicose, L-sorbose, D-xylose. In conclusion, the ORF40 protein belongs to sugar dehydrogenases. It might be the starting point of the catabolic pathway by producing sugar aldehydes which may be oxidized in a following step to acids by the aldehyde dehydroge- nase of ORF39.

P1–61 Development of Denizli x White Leghorn F2 population for quantitative trait loci mapping M. Nizamlioglu1, M. Garip2, A. Yilmaz2, T. Caglayan2, E. Kurar3, Z. Bulut1, V. Kurtoglu4, S. Dere2, Y. Ozsensoy3 and M. Dogan1 1Faculty of Veterinary Medicine, Department of Biochemistry, Selcuk University, Konya, TURKEY, 2Faculty of Veterinary Medicine, Department of Zootechnics, Selcuk University, Konya, TURKEY, 3Faculty of Veterinary Medicine, Department of Genetics, Selcuk University, Konya, TURKEY, 4Faculty of Veterinary Medicine, Department of Animal Nutrition, Selcuk University, Konya, TURKEY

P1–60 Recognition of non-canonical DNA structures in genomic DNA sequences by p53 proteins L. Navratilova1, M. Brazdova1, V. Tichy1, M. Fojta1, M. Lexa2, I. Kyjovsky1, W. Deppert3 and E. Palecek1 1Department of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics AS CR v.v.i., Brno, CZECH REPUBLIC, 2Faculty of Informatics, Masaryk University, Brno, CZECH REPUBLIC, 3Heinrich-Pette-Institute, Tumor Virology, Hamburg, GERMANY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Development of gene maps for human and animals is critical for identification of genomic regions and gene(s) that have an effect on human and animal diseases and animal production traits. Gene level dissection of diseases and these traits allows diagnosis, protection of diseases and development of novel medical treat- ments. The genetic level knowledge also can be utilized in animal breeding programs to improve animal health, production effi- ciency and product quality. Poultry production is an important sector in agriculture for obtaining economical animal originated foods. Chicken is also accepted and extensively used as an excel- lent model organism in genetic studies. In this study, five Denizli males and eleven White Leghorn hens were mated to generate F1 population. Fourteen F1 males and 58 F1 females were crossed to generate five full-sib F2 families including 441 birds. Body weights at hatching, 3, 6, 9, 12, 16, 24, 28 and 32 weeks of age, egg yield and egg weight phenotypic data were recorded. Blood and tissue samples were collected from all F0, F1 and F2 animals and DNA samples were isolated for mapping QTL analysis. Den- izli X White Leghorn F2 population is available scientific com- munity for gene and QTL mapping studies. The human genome contains a chromosomal DNA that exists primarily as a right-handed double helix (B-DNA, canonical DNA), but DNA can also contain the formation of unusual triplexes, quadruplexes, DNA structures, such as cruciforms, unwound DNA, hairpins and others. These non-canonical DNAs (non-B DNAs) are associated with regulatory sequences of genes, recognition sites of protein and occurre in different cellular pro- cesses. Tumor suppressor protein p53 (wtp53) and its mutant

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P1–62 Posterior polymorphous corneal dystrophy – copy number, gene expression and candidate gene analyses within the PPCD1 candidate region on chromosome 20p11.2 L. Noskova1, P. Liskova2, V. Stranecky1, H. Hartmannova1, R. Ivanek3, K. Jirsova4, S. Merjava4, M. Filipec5 and S. Kmoch1 1First Faculty of Medicine, Institute of Inherited Metabolic Disor- ders, Charles University, Prague 2, CZECH REPUBLIC, 2Labo- ratory of Biology and Patology of the Eye and Department of Oftalmology, General Teaching Hospital and Charles University, Prague 2, CZECH REPUBLIC, 3Academy of Sciences, Institute of Molecular Genetics, Prague, CZECH REPUBLIC, 4Laboratory of Biology and Patology of the Eye and Ocular Tissue Bank, Gen- eral Teaching Hospital and Charles University, Prague 2, CZECH REPUBLIC, 5Department of Oftalmology, General Teaching Hospital and Charles University, Prague 2, CZECH REPUBLIC

phorylation-dephosphorylation belongs to the major cellular signaling mechanisms. Moreover, anomalous phosphorylation of proteins can be also related to development of cancer or to meta- bolic diseases. Processes involving phosphoproteins are dynamic and this fact affects their low abundance and relatively short half-life. To understand these processes, structural studies on involved phosphoproteins is desired. Despite recent advances in instruments and methods used for the characterization of phos- phoproteins, the effective separation or enrichment of phospho- peptides and phosphoproteins remains to be fully solved. The subject of the present study was to compare the efficiency of two procedures used for the separation or enrichment of phosphopep- tides from the tryptic digest of phosphoprotein: (i) sorption of phosphopeptides to different metal ions chelates immobilized to magnetic particles or (ii) sorption to titanium dioxide or zirco- nium oxide. Bovine a-casein was chosen as a model phosphopro- tein. The adsorbed phosphopeptides from the tryptic a-casein digest were analyzed by matrix-assisted laser desorption/ioniza- tion time-of-flight/time-of-flight mass spectrometry (MALDI- TOF/TOF MS). Conditions for the phosphopeptide adsorption and following desorption were optimized. Acknowledgements: This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (grant MSM 0021620806 and project CEH LC 06044) and by the Czech Science Foundation (grant 203/09/0857).

P1–64 Functional analyses of Lupinus luteus cyclophiline promoter activities K. Nuc1, P. Nuc2 and R. Slomski1 1Poznan University of Life Sciences, Biochemistry and Biotechnol- ogy, Poznan, POLAND, 2Adam Mickiewicz University, Institute of Molecular Biology and Biotechnology, Poznan, POLAND

Posterior polymorphous corneal dystrophy (PPCD) is a geneti- cally heterogeneous autosomal dominant disorder characterised by epithelisation of the endohelium and irregular thickening of Descemet’s membrane. It often leads to irreversible corneal edema and requires corneal transplantation. The genetic hetero- geneity of PPCD is currently known to be represented by three loci on chromosomes 20, 1, and 10 and several disease causing genes (COL8A2, ZEB1/TCF8) have been identified. We have pre- viously shown linkage in two Czech PPCD1 families to chromo- some 20p11.2. To further narrow the PPCD1 candidate interval we used Affymetrix Genome-Wide Human SNP Array 6.0 and genotyped family members demonstrating critical recombination events by STR analysis. Haplotype analysis narrowed the critical region to a 2.1 Mb interval delimited by markers D20S114 and rs7509232. In parallel we used the genotyping data, assesed copy number status and found no indication of microdeletions within the candidate region. We have also manufactured custom oligo- nucleotide array and analysed expression changes of all genes located within the candidate region in samples of corneal tissues obtained from patients undergoing corneal transplantation and controls. This analysis showed significantly reduced amounts of destrin (DSTN) transcript in corneal tissues of four patients com- pared to healthy control tissue. However, subsequent sequencing analysis of the coding and promotor regions of DSTN did not reveal any potential disease causing mutation. As no mutations were found by sequencing of genomic coding regions of other genes in candidate region, we decided to sequence whole 2.1 Mb candidate region by combination of NimbleGen Sequence Cap- ture array and next-gen sequencing approach.

P1–63 Sorption of phosphopeptides to metal chelate complexes immobilized to magnetic particles and to metal oxides L. Novotna1, T. Emmerova1,2, J. Vavrova1, M. Ticha1,2 and Z. Kucerova1 11st Faculty of Medicine Charles University in Prague, Institute of Pathophysiology and CEH, Praha 2, CZECH REPUBLIC, 2Faculty of Science Charles University in Prague, Department of Biochemistry, Praha 2, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Cyclophilins (CyPs) constitute a large class of ubiquitous immu- nophilins with peptidyl-prolyl cis-trans isomerase activity. It has been shown that isomerization around Xaa-Pro bonds is one of the most rate limiting steps in protein folding and this process can be accelerated by the PPIase activity of the CyPs. We have analyzed a promoter region of the cytosolic cyclophilin from Lupinus luteus (EMBL/GenBank/DDBJ AF178458, LCyp). The expression of CyP genes depends on cis elements in their pro- moter regions which promote interaction of appropriate tran- scription factors in response to different stimuli. To investigate the regulation of its expression, deletion promoter fragments were fused to the uidAint (GUS) reporter gene and the constructs were introduced into Lotus japonicus via Agrobacterium rhizogenes transformation. We have observed different patterns of the b-glu- curonidase expression in L. japonicus roots and nodules. Only the largest 1054 bp promoter fragment causes expression of the b-glucuronidase in nodules suggesting that the nodule specific cis regulating element is located in the region spanning nucleotides - 1054 to -845 indicating to 5¢AAAGAT 3¢motif. We have also analyzed the influence of different abiotic treatments on the b- glucuronidase expression level in L. luteus leaves after agroinfil- tration with Agrobacterium tumefaciens carrying analyzed pro- moter fragments. Fluorometric study of these plants revealed that LCyp promoter is responsive to plant hormones such as ABA, IAA and BAP as well as to salt, but not to cold stress. Protein phosphorylation is one of the most important posttrans- lational modifications of proteins. The system of protein phos-

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P1–66 Electrophoretic analysis of esterase and superoxide dismutase enzymes of the wheat bug, eurygaster maura (heteroptera:scutellaridae) populations in the Central Anatolia S. Tuna, G. Olgun and R. Colak Faculty of Science, Ankara University, Biology, Ankara, TURKEY

P1–65 Quantitative and temporal proteome analysis of gamma-Tocotrienol treated androgen- unresponsive prostate cancer cells delineates a novel caspase – independent programmed cell death pathway T. Roumeliotis1, A. Evdokiou2, P. Vraka3, L. Loizou4, S. Garbis1 and A. D. Odysseos4 1Center of Basic Research – Biotechnology Division, Biomedical Research Foundation of the Academy of Athens, Athens, GREECE, 2Department of Orthopaedics and Trauma, University of Adelaide, Adelaide, Australia 3Department of Chemistry, Uni- versity of Cyprus, Nicosia, CYPRUS, 4EPOS-Iasis, Biomedical Research, Nicosia, CYPRUS

and enzymatic biochemical,

In this study, a total of 194 Eurygaster maura specimens were used from 10 locations in central Anatolia. Esterase and superox- ide dismutase enzymes were studied using starch gel electrophore- sis and native-polyacrilamide gel electrophoresis (N-PAGE). A total of 16 esterase activity bands were observed. Only Est-1 locus distinguished among esterases to be thought encoding by a lot of loci. In the bug populations studied, three allele (A, B, C) were recognized in this locus. In the starch gels examined of superoxide dismutase enzymes were determined one cathodal locus. All specimens studied were homozygous for the same allele in this locus. It was determined genetic identity and genetic dis- tance of bug populations by BIOSYS-1 computer program, according to allele frequencies of two loci (EST-1 and SOD) observable. On the basis of genetic relationship of ten Eurygaster maura populations based on Nei’s Genetic Distance of two enzyme loci, ten populations were divided into two main clusters and two subgroups in each cluster; 1: Kirikkale (subgroup1), Beynam, Haymana, Afyon, Konya and Yunak (subgroup2), 2: Aksaray (subgroup1) and Ayas, Kirsehir and Beypazari (sub- group2).

P1–67 Associations between serum linolenic acid/ alpha-linolenic acid ratio and blood gene expression profiles K. S. Olsen1, C. G. Fenton2, E. Anderssen3, L. Froyland4, R. H. Paulssen2 and E. Lund1 1Institute of Community Medicine, University of Tromso, Tromso, NORWAY, 2Microarray Resource Centre, University of Tromso, Tromso, NORWAY, 3Department of Cancer Research and Molec- ular Medicine, NTNU, Trondheim, NORWAY, 4National Institute of Nutrition and Seafood Research, Bergen, NORWAY

permeability. Conclusively, In the context of a large,

Proteomic approaches have a considerable potential in the drug development process providing a meand of obtaining a detailed molecular signature of drug action, since analysis of gene expres- sion at the mRNA level alone can not adequately predict protein expression or functional states. The overall objective of this study has been to investigate the feasibility of using an LC-MS based quantitative proteomic approach for the evaluation of c-Tocotrie- nol (c-T), a potent medicinal agent based on the time dependent differential expression of proteins involved in target pathways. Combining temporal and quantitative proteomics with molecular imaging, immunoprecipitation approaches, we have revealed a new panel of proteins under c-T regulation interwoven in a novel caspase-independent programmed cell death pathway. Significantly modulated protein targets cross- sectioning redox and programmed cell death pathways through (i) heat shock proteins in the Nrf-2-mediated transcriptional control axis and (ii) the G2/M transition check point of the cell cey le pro- tein networl, converge to the MAPK cascade and activate a novel PARP-modulated caspase independent programmed cell death sig- naling pathway. Increased levels of calcium-dependent proteases (caplains) in treated cells are characteristically associated with upregulation of total PARP and apoptotic chromatin condensa- tion inducer-1. Probing against total adn cleaved PARP revealed a temporal pattern of cleavage and activation while nuclear staining disclosed chromatin condensation confirming early apoptosis. Reactive nitrogen species, inherent to the androgen-independent phenotype of DU-145 cells and additional components of the mito- chondrial axis drive peroxynitrite mediated calcium-dependent mitochondrial dysfunction and elicit features of early apoptosis with enhancement of caplain activation, whereas the anchorage dependence of these cells is also highly suggestive of anoikis-resem- bling cell death. Significant elevation of caspase-degradable La- mins A nd B2 further reflects the low degree of implication of the caspase cascade while marked upregulation of active components of the mitochondrial pathway such as VDAC I–III accenuates mitochondrial membrane this approach delineates a novel caspase independent programmed cell death signaling pathway elicited aby a natural, minimally toxic agent and defines reliable biomarkers of efficacy.

adjustment including technical for

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Nutrition is one of the major modifiable risk factors for athero- sclerosis and certain cancers. Linoleic acid (LA, 18:2n-6) and alpha-linoleic acid (ALA, 18:3n-3) are essential to humans, and modulate immune response by giving rise to pro- (LA) or anti- (ALA) inflammatory signaling molecules. Despite the well-known metabolism of fatty acids, the molecular and physiolocigal mech- anisms of these dietary risk-modifying factors need further atten- tion. representative cohort of postmenopausal Norwegian women, we have explored the associ- ation between serum LA/ALA ratio and blood gene expression profiles. Samples from 286 women were successfully analyzed for expression profiles in PAXgene-stored whole blood using ABI1700 microarrays, and for fatty acid content in citrate-buf- fered plasma using rapid gas chromatography. After data prepro- cessing variability, differentially expressed genes were identified using R software and Bioconductor packages. In women with a high LA/ALA ratio, we revealed a pro-inflammatory expression profile that includes genes related to Fc epsilon signaling, phosphatidyl inosi- tol signaling, cytokine production, arachidonic acid metabolism and generation of eicosanoids. Strikingly, we identified the increased expression of the key pro-inflammatory enzyme 5-lipox- ygenase (5-LO) in women with a high LA/ALA ratio. These pre-

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liminary results lead us to conclude that dietary fatty acids do have an effect on the blood trancriptome, and that an unfavour- able LA/ALA ratio could be related to a pro-inflammatory gene expression profile. Further analyses will be conducted to reveal the complex effects of the lipidome on the blood transcriptome.

P1–68 Proteomic approach to elucidate antimicrobial effect of Papaver polychaetum alkaloids K. Dilek1, S. A. Berna1 and U. Caglayan2 1Department of Bioengineering, Marmara University, Istanbul, TURKEY, 2Department of Pharmacognosy, Istanbul University, Istanbul, TURKEY

the untranslated regions, including mRNA’s localization and sta- bility, and translational efficiency. These functions depend on the sequence and the structure of the UTR. BRCA1 and BRCA2 are the major hereditary breast/ovarian cancer predisposing genes and their mutations increase the risk of developing cancer. Inter- pretation of sequence variants found in genetic testing is the major concern for BRCA genes, especially for risk assessment in genetic counseling. From different repositories (dbSNP, BIC, kConFab), we collected BRCA1 and BRCA2 sequence variants that were found within 5¢ and 3¢ UTRs and we analyzed, using different on-line tools like UTResource and Transterm, their potential functional significance that could be expressed by dis- rupting or creating putative regulatory elements. As it is known that the function of non-coding RNAs (ncRNAs) greatly depends on their secondary structure, we analyzed how these non-coding sequence variants could have impact on the predicted secondary structure. By analyzing changes in the predicted secondary struc- tures of the 5¢ and 3¢ UTRs from the patients with breast/ovarian cancer we tried to find out if this approach could be used for assessing clinical significance in cancer etiology.

P1–70 Phylogenetic and bioinformatic analysis of Glutathione S-Transferase Tau from Pinus brutia E. Oztetik1, M. Aydin2 and F. Kockar2 1Anadolu University, Biology, Eskisehir, TURKEY, 2Balikesir University, Biology, Balikesir, TURKEY

Although bacteria have evolved numerous mechanisms to fight against antimicrobial agents, drug-resistant pathogens are on the rise. In the past few decades, this has led many research groups to medicinal plants for a search of novel antimicrobial agents. Long before mankind discovered the existence of microbes, the idea that certain plants had healing potential was well accepted and plants were used to treat common infectious diseases. The endemic plant Papaver polychaetum, from the genus of poppies, yields berberine as its major alkaloid. Berberine is a plant alka- loid with a long history of medicinal use in both Chinese and Ay- urvedic medicine. It has demonstrated significant antimicrobial activity against different organisms including fungi and is rela- tively nontoxic to humans. However the antimicrobial mode of action of berberine is not clear. The aim of this work was to investigate the antimicrobial property of the alkaloid extract of the P. polychaetum against E. coli K12. For this purpose, proteo- mic approach was used which involved the selection of up and down regulated proteins on 2-dimensional polyacrylamide gels and their subsequent identification by MALDI-tof analysis. The common feature of the identified proteins was that they were DNA-binding. This approach enabled the elucidation of regula- tion of protein expression levels in the model microorganism E. coli K12 in the presence of the antimicrobial agent. The search for novel substances involves understanding of the molecular mechanisms of action of the drug/drug candidates and the related bacterial response. The results of this study will help to identify targets for future pro-drug design. Further reading: Musumeci R, Speciale A, Costanzo R, Annino A, Ragusa S, Rapisarda A, Pappalardo MS & Iauk L. Berberis aetnensis C. Presl. extracts: antimicrobial properties and interaction with ciprofloxacin. Int. J. Antimicrob. Agents 2003; 22: 48–53. Rios JL & Recio MC. Medicinal plants and antimicrobial activ- ity. J. Ethnopharmacol 2005; 100: 80–84.

Stermitz FR, Lorenz P, Tawara JN, Zenewicz LA & Lewis K. Synergy in a medicinal plant: antimicrobial action of berberine potentiated by 5¢-methoxyhydnocarpin, a multidrug pump inhibitor. PNAS 2000; 97(4): 1433–1437.

P1–69 Computational analysis of 5¢ and 3¢ untranslated regions of breast cancer genes BRCA1 and BRCA2 P. Ozretic, M. L. Cvok, V. Musani, M. Cretnik and S. Levanat Division of Molecular Medicine, Rudjer Boskovic Institute, Zagreb, CROATIA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Untranslated regions (UTRs) are parts of the mature mRNA located before the start codon (5¢ UTR) and after the stop codon (3¢ UTR). They are transcribed with the coding region but they are not translated. Several regulatory roles have been assigned to Glutathione S-Transferases (GSTs, EC.2.5.1.18) are widespread and complex enzyme superfamily that play important roles in detoxification and stress tolerance in plants. Plant GSTs are divided into four classes (Phi, Tau, Zeta and Theta), among which Tau is the most numerous one. It has been shown that plant Tau class GSTs could play essential roles in plant develop- ment and in buffering environmental and biotic stresses. Even, there are many studies on GSTs in plants, which have been focused generally on agricultural plants, there is no such an information considering the molecular characterization of GSTs in gymnosperms. The definition of detoxification enzymes like GSTs in perennial conifers, a large group of gymnosperms, is very important for their adaptations to several environmental stresses during their long lifespans. The present study reports the phylogenetic and bioinformatic analysis of GST gene (PbGST- Tau) from a conifer, Pinus brutia, which is widely distributed in the north-eastern Mediterranean area, including Turkey. The PbGST-Tau gene encodes a protein of 228 amino acid residues with a calculated molecular mass of 26.37 kDa. The sequence comparisons of PbGST-Tau gene to other plant GST-Tau genes (Pinus densata, Pinus tabulaeformis, Pinus yunnanennis, Oryza sa- tiva japonica, Aegilops tauschii and Ginko biloba) were performed at the amino acid level by using Bioedit programme. The phylo- genetic tree was also constructed by using Neighbor-Joining/UP- GMA method. The analyses indicated that PbGST-Tau is placed with other three GSTs in Tau class from Pinus tabulaeformis, Pi- nus densata and Pinus yunnanensis, with 10.82%, 11.45% and 11.18% sequence diversity, respectively. The current difficulties for the comprehensive characterization of GST family in conifers originates from the insufficiency of comprehensive genome infor- mation on conifers in the literature. As the described members of the GST gene family in conifers are increased, the understanding for diversity and evolution of the GST classes would also be advanced.

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P1–71 Human Accelerated Region 20 (HAR20) operates as an enhancer: functional study of a HAR20 SNP associated with diabetes and cardiovascular disease S. Pampı´ n1, L. Parnell2, J. M. Ordovas2 and J. C. Rodriguez-Rey1 1Universidad de Cantabria, Biologı´a Molecular, Santander, SPAIN, 2Tufts University, Jean Mayer USDA HNRCA, Boston MA, USA

supported by GAUK lines shows equal differentiation, then they could react differen- tially after exposure to external stress. We induced stress condi- tions by 3 various treatments- elevated incubation temperature (40(cid:2)C), oxidative stress by presence of H2O2 (500 lM) and Stau- rosporin (125 nM). But even such conditions do not altered level of apoptosis/ necrosis in lines with inhibited PrPc. Finally, we introduce the new cell culture model for study of PrPc function in erythroid differentiation. Initial observations shows that direct cytoprotection is not mode of PrPc action in studied process, but it may play role in cell cycle which is subject of undergoing research. Acknowledgements: This work was 203429, Grants of Czech Science Fundation 310/08/0878.

P1–73 Comparative exosomal and cellular genomic profiling of human colorectal cancer cells K. S. Park1, J. H. Cho2, J. H. Kim1, D. Hwang2 and Y. S. Gho1 1Department of Life Science, POSTECH, Pohang, SOUTH-KOREA, 2School of Interdisciplinary Bioscience and Bioengineering, POSTECH, Pohang, SOUTH-KOREA

Comparative genomics has made possible the discovery of human sequences which have evolved very rapidly since the recent human/non-human primate divergence. These sequences have been termed Human Accelerated Regions (HARs). Except for HAR1, which encodes a small RNA, the function of HARs is unknown. The fact that many map in non-coding sequences sug- gests that sequence differences between humans and other pri- mates drive altered the regulation of genes involved in the determination of human-specific characteristics. HAR20 is a 263 pb sequence located within the second intron of PPARCG1A (PCG1a). Changes in PCG1a expression have been associated with diabetes, and more recently a SNP (rs10028665 C>T) located within HAR20 has been associated with diabetes and car- diovascular disease. These results prompted us to investigate (i) if HAR20 is a regulatory region and (ii) if the change due to rs10028665 results in a functional change in its properties. To test if HAR20 has enhancer-like characteristics, we carried out repor- ter gene assays in HepG2 cells by cloning both variants of HAR20 into the pGL3-promoter vector. In our assays, HAR20 highly increases the promoter strength, suggesting that it has expression-regulatory properties. Moreover, C allele produces an increase of 40% in the enhancer activity compared to T allele. Subsequent EMSA analysis has shown binding differences between both variants, indicating that this SNP could alter the PCG1a expression. We conclude that HAR20 is a regulator of gene expression and that rs10028665 is at least responsible for part of the variability of PCG1a expression associated with dia- betes and cardiovascular disease.

including colorectal cancer (CRC) cells, Various cancer cells, release exosomes into the surrounding tissues and peripheral cir- culation. As exosomes are thought to be involved in tumor pro- gression, we hypothesized that CRC cells may secrete exosomes carrying mRNA that causes epigenetic reprogramming of tumor microenvironmental cells. Here, we present a global comparative exosomal and cellular transcriptomic analysis of human SW480 CRC cells. We identified 11 327 exosomal and 11 624 cellular mRNAs, and 241 and 1461 of these were overexpressed in exo- somes and in cells, respectively. The exosomal and cellular mRNAs were similarly enriched in tumorigenesis-related biologi- cal processes. Furthermore, exosome-enriched mRNAs were involved in the cell cycle, whereas cell-enriched mRNAs were involved in angiogenesis. Our study demonstrates that the exoso- mal and cellular mRNAs of human CRC cells are similar to each other at the global level, in terms of intensities and biological processes, and that exosomal mRNA may be involved in tumor progression via the horizontal transfer of mRNA to target cells. This information will help elucidate the pathological functions of tumor-derived exosomes and will aid in the development of meth- ods using exosomal mRNAs to determine the diagnosis and prognosis of CRC.

P1–72 Study of cellular prion protein (PrPc) role in erythroid differentiation in vitro M. Panigaj1, H. Glierova1, A. Simkova2 and K. Holada1 11st Faculty of Medicine, Institute of Immunology and Microbiol- ogy, Prague 2, CZECH REPUBLIC, 2Faculty of Science, Section of Biology, Prague 2, CZECH REPUBLIC

P1–74 Proteomic approach for detection of biomarkers in rheumatoid arthritis M. Park, S. Chung, T. Kim and J. Lee Department of Internal Medicine, Yonsei University College of Medicine, Seoul, SOUTH-KOREA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Despite its significant medical importance, involvement of PrPc in cell biology is still subject of discussion. Connection between PrPc/ prion pathogenesis and erythropoesis was already shown. Our pilot study of erythroid differentiation in murine erythroleu- kemia cells (MEL), indicates regulation of PrPc expression as shown by qRT-PCR and FACS analysis. Creation of stable cell line expressing shRNAmir (LP1, LP2) which downregulates PrPc (3 90% inhibition) shows similar level of total hemoglobine con- tent and normal expression pattern of AHSP and hemoglobine-a in all lines during erythroid differentiation. Interestingly we found higher expression of c-myb in LP1 and LP2 cells and its more profound downregulation after inducing to differentiation. Previous reports showed that neither physiological nor enhanced expression altered percentage of apoptotic MEL cells during dif- ferentiation. However, even opposite approach- downregulation of PrPc probably do not lead to sensitization of cells to apopto- sis. It is tempting to speculate that if under normal conditions all Background: Rheumatoid arthritis is an autoimmune disease characterized by the sequestration of various leukocyte subpopu- lations within both the developing pannus and synovial space. There is a strong demand to identify the specific antigens or pep- tides of rheumatoid synovitis for application as diagnostic bio- markers for diagnosis of RA in early stages and as potential therapeutic targets. Proteomics can be defined as the qualitative and quantitative comparison of proteomes under different condi- tions to further unravel biological processes. Objective: In this study, we aimed to identify and analyze the potential peptides expressed in the rheumatoid synovitis using proteomics-based approach.

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liquid high-performance

Methods: We enrolled synovial fluids of 10 patients with rheu- matoid arthritis and we also enrolled those of 10 patients with osteoarthritis as controls. The synovial fluids were fractionated into peptide and protein fractions; the former was subjected to one-dimensional chromatography (HPLC) and the latter fractions were analysed and identified by ion-trap mass spectrometry. Then, the latter was subjected to 2D electrophoresis and differentially displayed protein spots were identified by mass spectrometry. Results: Several protein spots were significantly up-regulated in synovial fluids from patients with rheumatoid arthritis, compared to those from patients with osteoarthritis. The up-regulated spots were identified as leucine-rich alpha2-glycoprotein, serum amy- loid A (SAA) protein precursor, fibrinogen Fragment D, and fibronectin. Conclusion: These data indicated that application of proteomics platform could facilitate analyzing and identifying protein bio- markers differentially existed in diseases.

P1–75 Proteomic analysis to identify molecular targets of low temperature sensing S. Park and M. Jang Department of Applied chemistry, Dongduk Women’s University, Seoul, KOREA

supported by grant in the regulation and maintenance of mtDNA content (PGC1, NFR1, TFAM, POLG). DNA and RNA were isolated from 26 pairs of liver and muscle tissue samples obtained at autopsy from miscarriages after informed consent, between 13th and 28th week of gestation. The mtDNA amount and gene expression levels were analyzed by the real-time PCR method using SybrGreen I. In both fetal liver and muscle tissues, mtDNA content and TFAM expression levels were increasing with onward fetal devel- (mtDNA: r = 0.50; P < 0.01; respectively r = 0.62; opment P < 0.01); (TFAM: r = 0.56; P < 0.01; respectively r = 0.61; P < 0.01). On the other hand, POLG expression level was increasing only in fetal liver tissue (r = 0.54; P < 0.01). NRF1 was unchanged in both fetal tissues and PGC1 was slightly rising between 13th and 28th week of gestation in liver tissue (r = 0.42; P < 0.05). Our results showed that POLG expression varies between two different tissues during gestation and proba- bly is not associated with mtDNA content as tightly as TFAM expression. The increase of PGC1 transcript level is statistically significant in liver tissue and we suggest that it could bear evi- dence of tissue specific expression or regulation. In fetal liver, ris- ing PGC1 expression positively affects the binding of NRF1 to its specific sites on TFAM promoter. In conclusion, this study described mainly the increasing trends of gene expression in the pathway leading from expression of PGC1 to mtDNA content changes in fetal liver and muscle tissues during second trimester of gestation. According to our results, we suppose that the mito- chondrial proliferation is growing up between 13th and 28th week of gestation and therewithal it is the first hallmark of pre- pare for postnatal adaptation on transcriptional level. Acknowledgement: This work was GAUK25755707, IGAMZ-NR9410,GACR305/08/H037.

P1–77 DNA methylation in LDL receptor and Apolipoprotein B100 genes promoter in patients with hypercholesterolemia M. Piechota1, A. Banaszewska1, E. Guzniczak2, R. Link2, G. Rosinski1, T. Siminiak2 and R. Plewa1 1Department of Animal Physiology and Development, Adam Mick- iewicz University, Poznan, POLAND, 2Cardiac and Rehabilitation Hospital Kowanowko, Poznan University of Medical Sciences, Poznan, POLAND

Transient receptor potential (TRP) channels are important for our sensory systems, responding to touch, taste, osmolarity, pain, temperature and other stimuli. The somatosensory system detects changes in ambient temperature using these channels. Intracellu- lar signaling of cold sensing in mammals is not completely under- stood, although significant progress has been made recently with the cloning of two cold-activated ion channels, TRPM8 and TRPA1. To understand the molecular basis of cold receptor- induced intracellular signaling, it is important to identify the expression or function of proteins which are modified by low temperature sensing. Little is known about the protein products produced by cells exposed to low temperature sensing since most studies have been performed at the electrophysiological effect of low temperature sensing. In the present study, we analyzed molecular modifications induced by low temperature sensing in TRPM8 expressed cells using a proteomic approach. This meth- odology provides important qualitative information on post- translational modifications to each protein and quantitative data on protein expressions in response to a particular stimulus. This information is particularly important because it provides data on early cellular events, such as, on the stimulus and signaling cas- cades triggered independently of protein neosynthesis. We identi- fied proteins that sensitively reacted to low temperature sensing by using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight-MS.

P1–76 Mitochondrial DNA content and expression of genes involved in maintenance and replication of mtDNA during human fetal development M. Pejznochova, M. Magner, M. Tesarova, H. Hansikova and J. Zeman 1st Faculty of Medicine, pediatric, Prague 2, CZECH REPUBLIC

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The DNA methylation is an epigenetic modification present in Prokaryotic and Eukaryotic genomes. The CpG alterations lead to selective utilization of gene information and are responsible for tissue specific gene expression, embryonic development, geno- mic imprinting and X-chromosome inactivation. Hypermethyla- tion of promoters is usually associated with silencing of the gene expression. Disorders of the methylation pattern may contribute to autoimmune disease development, cancer as well as metabolic and cardiovascular diseases. Atherosclerosis is a metabolic dis- ease causes by the accumulation of low-density lipoprotein cho- lesterol (LDL) in the walls of arteries. The reduce expression of LDLR or/and ApoB100 can influence on decrease the synthesis of proteins and the accumulation LDL-cholesterol in the walls and advance atherosclerosis disease. The purpose of the study was to evaluate LDLR and ApoB100 expression level and the methylation status in patients with hypercholesterolemia. The investigated group consisted of 25 patients with no mutation in LDLR exons. Expression of LDLR was significantly lower and average 24–45% then in the healthy individuals. The analysis of CpG methylation pattern within the LDLR and ApoB100 pro- moters revealed higher methylation level in 11 patients in com- parison to the healthy controls. In four patients, the methyl The inadequate efficiency of mitochondrial biogenesis leads to low energy production which may play a crucial role both in the fetal development and neonatal morbidity. The aim of our study was to characterize the changes in expression of genes involved

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all are more related to ceruloplasmin and hephaestin. Factors IX an X could not be detected what seems to be in agreement with lack of their cofactors – factors VIII and V.

group was present at seven cytosine (C) residues of LDLR, while in seven almost all cytosines were methylated of ApoB100. The elevated level of the CpG methylation in LDLR or ApoB100 pro- moters in 11 patients with hipercholesterolemia can decrease the gene expression and could be the cause of the atherosclerosis symptoms.

P1–80 Database of the secondary structures of HIV-1 genome control elements A. Potyahaylo, I. Kolomiets, M. Zarudnaya and D. Hovorun Department of Molecular and Quantum Biophysics, Institute of Molecular Biology and Genetics NAS of Ukraine, Kiev, UKRAINE

P1–78 Characterization of the non-coding MicA RNA in Escherichia coli: mutational analysis shows that 3¢ end controls stability of this sRNA V. Pobre, J. M. Andrade and C. M. Arraiano Instituto de Tecnologia Quı´mica e Biolo´gica, Universidade Nova de Lisboa, Oeiras, PORTUGAL

Multiple steps in replication cycle of human immunodeficiency virus type 1 (HIV-1) causing AIDS illness are directed by control elements located both in untranslated and translated regions of HIV-1 genome, among them are TAR, PolyA, PBS, DIS, SD, Psi, RRE and others. A high heterogeneity of HIV-1 genome sig- nificantly impedes AIDS diagnostics, and on other hand it enables to use phylogenetic approach to study mechanisms of virus replication. To assist in analyzing of phylogenetic and sec- ondary structure data we have created database of Control Ele- ments Secondary Structures of HIV-1 genome (CESSHIV-1). At present database contains secondary structures predicted by the mfold program (Zuker, 2003) of the region encompassing DIS, SD and Psi hairpins of 5¢-UTR for about 1300 HIV-1 isolates selected from NCBI (GenBank). The database has been devel- oped to assist in analyzing of frequencies of different variants of control elements, influence of mutations on the secondary struc- ture, dependencies of secondary structures of control elements on subtype and geographical region, evolution of control elements. In particular we found tolerant and intolerant combinations of base changes in DIS, SD and Psi hairpins. We present the schemes of hypothetical transitions between all frequent variants of DIS, SD and Psi hairpins via single base change. The authors believe CESSHIV-1 database to be a useful guide for a wide range of researchers and physicians engaged in HIV-1 science.

Small non-coding RNAs (sRNAs) are an expanding class of cell regulators. Changes in sRNA levels can potentially affect the genetic pathways assisted by these tiny regulatory molecules. sRNAs usually act by an antisense mechanism and bind target mRNAs through their 5¢ end, inhibiting translation and promot- ing mRNA decay. sRNA stability is mainly believed to rely on its 5¢ end as the degradation of some sRNAs was shown to be coupled with the endonucleolytic inactivation of their target mRNAs. We have previously shown that 3¢–5¢ exonucleolytic degradation is a major regulatory pathway controlling the levels of the small non-coding MicA RNA, an important regulator of outer membrane protein expression. In this study, we have now characterized the RNA determinants involved in the stability of MicA and further analysed how this may influence the expression of its targets. We constructed several plasmids harbouring either the wild-type or mutated MicAs and transformed delmicA cells. Remarkably, mutations in the 5¢ region (responsible for the base- pairing of MicA with its target mRNAs) showed a much less drastic effect rather than mutations in the 3¢ which resulted in the pronounced destabilization of this sRNA. Binding of Hfq RNA chaperone and the presence of two stable stem-loops at the 3¢ end are shown to be important determinants of sRNA stabil- ity. Moreover, we demonstrate in vivo that the most 3¢ nucleo- tides display an essential role in the establishment of MicA RNA levels, highlighting the important role of the 3¢ extremity in sRNA expression.

P1–81 Proteomic and biochemical analysis of 14-3-3-binding proteins indicates new roles of 14-3-3 proteins in apoptosis M. Pozuelo Rubio Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER), Senalizacio´n Celular 4, Sevilla, SPAIN

P1–79 The first signs of blood coagulation pathway evolution by bioinformatic studies of Branchiostoma genome M. Ponczek Department of General Biochemistry, Institute of Biochemistry, Lodz, POLAND

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

14-3-3 is a ubiquitous protein that in many cases had been involved in antiapoptotic signals in cells, promoting cell survival by association with proapoptotic proteins. Here, a proteomic and biochemical analysis was used to identify apoptotic-related 14-3- 3e-binding proteins aimed to have a comprehensive knowledge of survival functions of 14-3-3e isoform in cells. Tandem affinity purification, LC-MS/MS analysis and biochemical validation were combined to characterize 46 proteins associated with 14-3- 3e which binding status is regulated by C2-ceramide-induced apoptosis in HeLa cells. Among this pool of proteins, 16 of them were directly implicated in the apoptotic process comprising pro- teins related with regulation of intermediate filament integrity and cell shrinkage during apoptosis as Desmin or Vasodilatador- stimulated phosphoprotein (VASP), oncogenic or death promot- ers such as Nucleophosmin (B23) or Receptor interacting protein 3 (RIP3), and regulation of DNA double-strand breaks repair in early stages of apoptosis as DNA protein kinase (DNA-PKcs). The functional diversity of theses identified proteins suggests that 14-3-3e regulates the apoptotic process through new mechanisms in addition to others previously characterized. The majority of Blood clotting and fibrinolysis proteins of mammals and other higher vertebrates appear to be a result of gene duplications, divergence and domain shifting. Extrinsic pathway, important in physiologic coagulation in human, looks to be simpler in lam- preys comparing to Gnathostomata. On the other hand, intrinsic pathway, significant in pathological thrombus formation, evolved relatively early in mammals as divergence of factor XI after plasma prekallikrein duplication. The availability of sequenced Branchiostoma floridae genome enables reconstruction of early stages of blood coagulation evolution employing accessible bioin- formatic tools. Branchiostoma seems to have orthologs of pro- thrombin, but with no GLA or kringle domains. Genes coding peptides with FRED domains related to fibrinogen chains are present. Ferroxidase ‘A’ domain, typical for coagulation factors V, VIII, hephaestin and ceruloplasmin, occurs in four genes, but

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these proteins contained one or several phosphorylation-depen- dent 14-3-3-binding sites indicating a potential direct interaction with 14-3-3e. Accordingly of survival function of 14-3-3e isoform, a weak ectopic 14-3-3e expression delay cell death and knock- down of this isoform sensitized cells to C2-ceramide induced apoptosis. Finally, these biochemical and functional analysis show 14-3-3e isoform as a survival factor during C2-ceramide- induced apoptosis and characterized novel C2-ceramide regulated 14-3-3e interacting proteins related with processes that control life or death in Hela cells.

P1–82 Computational analysis of miRNA targets and CpG islands in human genes E. Rodriguez, A. Ferre´ , M. Gonzalez-Porta, M. A. Montero, E. Olle´ , E. Daura, C. Rojas, M. Mulero, M. Cabre´ , J. L. Paternain and A. Romeu Biochemistry and Biotechnology, Rovira i Virgili University, Tarragona, SPAIN

(miRNAs) are small noncoding RNAs evidence supports a potential role of MMR system in signalling the DNA damage response, in particular, has been observed the induction of hMLH1 in response to several DNA-damaging agents, including cisplatin, adriamycin and MNNG. Further- more, the stabilization of the remaining hMutL (hPMS1 and hPMS2) proteins and their nuclear compartmentalization in response to DNA damage requires hMLH1. Here, we examined the induction of hMLH1 protein in response to adryamicin- induced DNA damage. We have used the MCF7 cells, treated and untreated with adriamycin, and performed Western blot and RT-PCR assays. We found that hMLH1 and BRCA1 (molecular partner of hMLH1) were activated by adryamicin treatment in time-dependent manner. On the basis of these results specific shRNA for BRCA1 was used for silencing endogenous BRCA1 in MCF7 cells. We found that hMLH1 stabilization is mediated by BRCA1 in response to DNA damage. To further uncover a potential link between hMLH1/BRCA1, we looked for post- translational modifications of hMLH1, like Ser/Thr phosphoryla- tion. Using immunoprecipitation and 2D gel approaches we found a post-translational modification of hMLH1. Moreover, the post-translational modifications of hMLH1 protein is cur- rently under investigation by LC/MS-MS approaches.

P1–84 Functional genomic resolution of pharmacogenetic nexus between metabolic syndrome and morphogenesis M. Krupkova1, M. Janku1, L. Sedova1, L. Kazdova2, F. Liska1, D. Krenova1, V. Kren1 and O. Seda3 1First Faculty of Medicine Charles University, Institute of Biology and Medical Genetics, Prague, CZECH REPUBLIC, 2Institute for Clinical and Experimental Medicine, Department of Metabolism and Diabetes, Prague, CZECH REPUBLIC, 3CHUM, Research Centre, Montre´al, QC, CANADA

In order

MicroRNAs (single- stranded RNA molecules of about 21–23 nucleotides in length) regulate gene expression by targeting messenger RNA that (mRNA) transcripts. CpG islands are genomic regions that con- tain a high frequency of CG dinucleotides. Both, CpG islands and miRNA are involved in the epigenetic landscape, i.e., heri- table changes in gene expression caused by mechanisms other than changes in the underlying DNA sequence. CpG islands are DNA sequence features which typically occur at or near the transcription start site of genes, particularly housekeeping genes; and cytosines in such an arrangement tend to be methylated. However, miRNAs, are epigenetic factors acting at post-tran- scriptional level, through their targets in mRNAs. The aim of this work is to explore the possible relationship between miR- NA targets and CpG islands in human genes. Genomic data have been retrieved from NCBI the current Homo sapiens Build 36.3. We have used the MySQL relational database management system, MySQL and Perl scripts. First, taking into account all human genes, the overlapping percentage of gene sequence (including the flanking regions) by CpG islands were evaluate. The plot CpG-gene interactions vs number of miRNA targets, clearly shows an inverse correlation. to identify whether this behaviour plays a significant role in genes which are under an epigenetic regulation, we have analysed the inci- dence of CpG islands and miRNA targets in hypomethylated and hypermethylated in specific genes of colon cancer; and in a set of human experimentally identified imprinted genes. Project AGL2007-65678/ALI.

P1–83 DNA damage induces a post-translational modification of the mismatch protein hMLH1 F. Romeo, N. Ahmad, M. Di Sanzo, D. Scumaci, M. Saccomanno, G. Cuda, M. C. Faniello, B. Quaresima and F. S. Costanzo Experimental and Clinical Medicine, ‘Magna Graecia’ University, Catanzaro, ITALY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

DNA mismatch repair (MMR) system contributes to the mainte- nance of the genomic stability in both prokaryotes and eukary- otes, through the correction of replication errors, the suppression of recombination between non identical, but homologous sequences, and the activation of cell cycle arrest and apoptosis in response to DNA damage. Moreover, the inactivation of the linked to cancer predisposition. Recent MMR pathway is We tested the hypothesis of common genetic factors forming a nexus between morphogenesis and metabolic syndrome. We utilized metabolic and transcriptomic profiling of a unique set of rat models with previously ascertained disparity in both limb devel- opment and metabolic syndrome features. Adult male rats (n = 12/strain) of spontaneously hypertensive rat (SHR) and congenic SHR.PD-(D8Rat42-D8Arb23)/Cub (SHR-Lx) strains, differing in 1.4 Mb region of chromosome 8, were fed a high sucrose diet (HSD) for 2 weeks and subsequently treated with RA (15 mg/kg) for 16 days, while still on HSD. We contrasted metabolic (insulin sensitivity, adipokines, free fatty acids, triacyl- glycerol and cholesterol in 20 lipoprotein fractions) and transcrip- tomic (Affymetrix Rat Exon 1.0 ST, liver) profiles between SHR and SHR-Lx under standard, HSD and conditions of HSD + RA administration. We observed noticeable distinction in effect of RA between SHR and SHR-Lx strains. SHR-Lx reacted with significant impairment of glucose tolerance and less favorable distribution of cholesterol and triaylglycrols into the lipoprotein fractions compared to SHR. Significant interactions between strain and diet/RA factors were found for free fatty acid and insulin levels. Transcriptomic data corroborated the meta- bolic profile as they revealed a concerted shift in distinct path- ways between the strains in response to RA. We demonstrated interaction of retinoic acid with a 14- gene region of rat chromo- some 8, affecting concurrently the features of metabolic syndrome and, as previously shown, the limb morphogenesis.Our results sup- port the notion of interconnection of morphogenetic and metabolic

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processes, assumed in human syndromes, e.g., Bardet-Biedl or Smith-Lemli-Opitz.

P1–85 Expression and turnover of small RNAs in Salmonella typhimurium I. J. Silva, S. C. Viegas and C. M. Arraiano ITQB, Control of Gene Expression Lab, Lisboa, PORTUGAL

activity and discriminative character (i.e., different affinity to var- ious responsive elements of the p53 target genes). In some cases, p53 tdmutants showed even higher ability to transactivate some target genes than wt p53. We conclude that even a single amino acid exchange in the p53 protein can lead to diverse transactiva- tion spectra, which in turn could cause diverse biological responses. Better understanding of various functional variants of p53 can be important for development of new anticancer thera- pies. Acknowledgement: This work was supported by grants No. NR/9305-3 of IGA MZ CR and MSM 0021622415.

P1–87 New specific molecular targets for radiochemotherapy in colorectal cancer K. Snipstad1, C. G. Fenton1, J. Kjaeve2, E. Anderssen3 and R. Paulssen1 1Institute of Clinical Medicine, University of Tromso, Tromso, NORWAY, 2Department of Gastric Surgery, University Hospital of North Norway, Tromso, NORWAY, 3Institute of Cancer Research and Molecular Medicine, Norwegian University of Sci- ence and Technology, Trondheim, NORWAY

involved in the degradation of

constructed Salmonella

Eukaryotic and prokaryotic cells contain a wealth of small RNAs (sRNAs) with determinant roles in the post-transcriptional con- trol of gene expression. The mechanisms by which sRNAs modu- late gene expression are diverse and they can be differently classified according to their mode of action. In pathogenic bacte- ria, a large number of sRNAs coordinate adaptation to stress and expression of virulence genes. We have analysed the expres- sion profiles of sRNAs, conserved among many enterobacteria, in different growing conditions. This has given us valuable infor- mation about the conditions of expression of these sRNAs in Salmonella, which are probably related to their function and tar- gets in this bacterium. Namely, the higher levels of these sRNAs in growth conditions under which the expression of the Salmo- nella Pathogenicity Island (SPIs) genes is induced may indicate a relation with virulence functions. To understand the regulation by sRNAs it is fundamental to study their turnover since this may impact on the regulatory capacity of a sRNA. The specific ribonucleases (RNases) these sRNAs should be one of the main factors responsible for these typhimurium differences. We have mutants for several ribonucleases or Poly(A) Polymerase I. The processing and stability of the four sRNAs was then analyzed in the RNase mutant strains constructed and cleavage sites were mapped in order to understand the mechanism of decay. In this work we have identified some of the main RNases involved in the post-transcriptional control of each of these sRNAs. Our glo- bal results give important information about the expression, pro- cessing and turnover of these sRNAs, bringing insights into the mechanisms of sRNA function in Salmonella.

in normal

P1–86 Analysis of the p53 temperature-dependent mutants in H1299 cell line J. Slovackova1, D. Grochova1, J. Navratilova2 and J. Smardova1 1Department of Pathology, University Hospital and Faculty of Medicine Masaryk University, Brno, CZECH REPUBLIC, 2Faculty of Science Masaryk University, Institute of Experimental Biology, Brno, CZECH REPUBLIC

The preferred treatment for patients with advanced colorectal cancer is preoperative radiochemotherapy. The early side effects of this treatment have been considered to be acceptable. The aim of the study was to identify the effect on gene expression in tumour and normal rectal tissue samples before and after preop- erative radiochemotherapy. For that purpose, tissue samples from twenty patients with operable rectal adenocarinomas were used for a whole genome-based gene expression analysis, using microarray technology. A factorial experimental design allowed us to look solely at the radiation effect on tumours. This resulted in 4496 differentially expressed genes in tumour tissue with P < 0.05. In addition to known markers for radiochemotherapy, a Gene Set Enrichment Analysis (GSEA) showed that the effect on tumour tissue is specific and mostly associated with cell adhe- sion and leukocyte transendothelial migration (TEM) pathways, represented by 618 genes. Only 61 genes were differentially expressed (P < 0.05) tissue, none of which are involved in pathways described for tumour tissue. We conclude that radiochemotherapy mainly and specifically affect tumour tis- sue, and that the effect on normal tissue is not only limited, but also different from tumour tissue. The profound change of cell adhesion molecule expression in tumour tissue could either decrease the tumours invasive potential, or it could increase the risk of metastasis. Further characterization of genes involved in cell adhesion and leukocyte TEM may give new insights into the molecular responses to radiochemotherapy, and open the possi- bility for design of new tools for colorectal cancer therapy.

P1–88 The ge´ nolevures consortium: evolution of protoploid saccharomycetaceae genomes G. The Ge´ nolevures Consortium Institut de Botanique, GDR Ge´nolevures, Strasbourg, FRANCE

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

The p53 tumor suppressor plays an important role in preventing cancer development by regulating cell cycle, apoptosis and main- tenance of genome integrity. Missense mutations in the p53 gene are often associated with cancerogenesis. They can alter expres- sion profile of the p53-target genes thus resulting in different bio- logical outcomes. Using functional assay in yeast, we have collected 23 temperature-dependent (td) mutants of the p53 gene. The transactivation abilities of these p53 mutants are partially or fully restored at decreased or increased temperature. In this study, we investigated relationship between type of mutation and ability to transactivate the p53 target genes (p21, bax, RGC) in the p53-null human H1299 cell line transiently transfected with p53 td mutants. Then, we compared the p21, bax and MDM2 gene expression in H1299 cells permanently transfected with the p53 td mutants by real-time PCR. In order to pursue temperature sensitivity we have carried out both assays at 32(cid:2)C and 37(cid:2)C. We found that p53 td mutants exhibit very diverse transactivation The Ge´ nolevures Consortium (http://www.genolevures.org/) is deeply involved in the comparative genomics of Hemiascomycet- ous yeasts. An exploration of 13 hemiascomycete genomes revealed their broad evolutionary range (Souciet et al., 2009) but after complete sequencing of the genomes of a number of species (Dujon et al., 2004) it appears that the Hemiascomycetous yeasts encompass three major internal subdivisions. In a recent compar- ative genomic analysis (Souciet et al., 2009) we concentrate on

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Hep3B cells. However, neither ADAMTS-3 nor ADAMTS-8 expression was detected.

P1–90 Ubiquinone biosynthesis in Saccharomyces cerevisiae: Molecular organization of O-methylase Coq3p depends on Abc1p/Coq8p A. Tauche, U. Krause-Buchholz and G. Ro¨ del Institute of Genetics, Dresden University of Technology, Dresden, GERMANY

The redox active lipid coenzyme Q (Q) functions as an electron carrier in the mitochondrial respiratory chain. Q biosynthesis in Saccharomyces cerevisiae requires at least nine proteins (Coq1p– Coq9p), found in including Coq8p, which contains motifs eukaryotic-type protein kinases. Absence of Coq8p results in lack of Q and concomitant accumulation of the intermediate HHB. Here we show Coq8p is a soluble homomeric protein of the mito- chondrial matrix or associated with the inner mitochondrial membrane on the matrix side. As revealed by 2D-Blue Native/ SDS-PAGE analysis, Coq8p is not integral part of a Coq3p, Coq5p and Coq9p containing 1.3 MDa Q-biosynthesis complex, but – together with Coq10p – mainly associated with a complex of about 500 kDa, and only weakly or transiently associated with the 1.3 MDa complex. Deletion of COQ8 results in the release of Coq3p, but not of Coq9p from the 1.3 MDa Q-biosynthesis com- plex. This effect cannot be restored by Q6supplementation. We demonstrate further that the 1.3 MDa Q-biosynthesis complex is not associated with the complex III and IV supracomplexes of the respiratory chain. 2D-Isoelectric Focusing data indicate the Coq8p kinase activity, with Coq3p as a target. We propose a model for the role of Coq8p in the Q-biosynthetic complex.

five species, we establishe the complete and fully annotated sequences of three new species, of Saccharomycetacea (Zygosac- charomyces rouxii, Saccharomyces kluyveri and Kluyveromyces thermotolerans) that we call « protoploid » because they diverge from the S. cerevisiae lineage prior to its whole genome duplica- tion. The main points of the work are: 1. Three fully sequenced and manually annotated genomes of yeasts, selected according to previous phylogeny as part of the less extensively documented sections of Saccharomycetaceae, des- ignated ‘protoploid’ to distinguish them from the extensively studied species derived from whole genome duplication. Yeast species of various habitats and origins. 2. Analysis of genome content and composition, including genes for non-coding RNA. Discovery of an unexpected compositional heterogeneity. Discovery of a novel class transposable element. 3. Classification of entire proteomes into protein families et char- acterization of a core protein repertoire for the Saccharomyceta- ceae yeasts. 4. Recognition and analysis of ancestral duplications and numer- ous paralogs among the set of protoploid species. Analysis of conserved and species-specific paralogs, either dispersed or form- ing tandem gene arrays. 5. A new method to define gene orthologs by synteny. Multidi- mensional sequence comparisons of corresponding orthologous proteins, revealing long evolutionary distances. 6. Conservation of synteny, and first analysis of relationship between sequence divergence and loss of synteny in yeasts. Reconstruction of an ancestral genome. 7. General discussion on the common characteristics of Sacchar- omycetaceae and the evolutionary variations along each special lineage. References: 1. Souciet et al., FEBS Lett. 2000; 487: 3–12. 2. Dujon et al., Nature 2004; 430: 35–44. 3. Souciet et al., Genome Research 2009.

P1–89 The expressions of ADAMTS-1 -2 -3 and -8 in Hep3B cells F. B. Sunay1, S. Aydogan Turkoglu2 and F. Kockar2 1Faculty of Medicine, Histology and Embryology, Balikesir, TURKEY, 2Faculty of Science and Art, Biology, Balikesir, TURKEY

P1–91 Detection of enteroviruses and hepatitis A virus RNA in milk by reverse transcriptase- polymerase chain reaction G. Terzi1, H. Albayrak2, B. Siriken1, O. Cadirci1 and Z. Yazici3 1Ondokuz Mayis University Veterinary Faculty, Food Hygiene and Technology, Samsun, TURKEY, 2Samsun Veterinary Control and Directory of Research Institute, Virology, Samsun, TURKEY, 3Ondokuz Mayis University Veterinary Faculty, Virology, Samsun, TURKEY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Cell-cell and cell-matrix interactions play role not only in physio- the logical processes like embryogenesis and development of organism but also in pathological situations like tumour metasta- sis and AIDS. One factor, which controls these interactions, is proteinases. ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) proteins, firstly described in 1997, are the newest members of the proteinase family and up-to-date 19 different ADAMTS proteins have been determined. There are several studies about the expression of the different ADAMTS proteins in different tissues and cell lines. But, there is no infor- mation about the expression of these proteins in hepatocellular carcinoma cell lines. The aim of this study was to determine the expression of ADAMTS-1, -2, -3 and -8 proteins in a human hepatocellular carcinoma cell line, namely Hep3B cells. ADAM- TS-1 and -8 were included to the study because they have anti- angiogenesis effect and ADAMTS-2 and -3 were included to the study because they function in collagen synthesis and liver cirrho- sis may provide background for hepatocellular carcinoma. Total cellular RNA was prepared from Hep3B cells and subjected to RT-PCR using ADAMTS-1, -2, -3 -8 and GAPDH specific prim- ers. There was ADAMTS-1 and ADAMTS-2 expression in The importance of foodborne viral infections is increasingly recog- nized. HAV is excreted in feces and causes infection by consume contaminated water or foods. The aim of the present study is to evaluate the incidence of enteroviruses and hepatitis A virus in 50 raw cow milk samples obtained from the middle blacksea region (city and around of Samsun) of Turkey. Viral RNA was extracted from milk samples using rapid acid guanidinium thiocyanate-phe- nol-chloroform mixture as described by Chomcyznski and Sacchi (1987). The HAV primers were selected from sequences of the VP2 and VP4 capsid region. Enteroviruses primers were selected from the 5¢ non-translated region, which is the most conserved region of the genome of enteroviruses. Enteroviruses and hepatitis A virus were investigated with RT-PCR (reverse transcriptase-polymerase chain reaction). The PCR product, which was obtained after the amplification, was treated to electrophoresis at 80 V to 30 min in 2% agar gel included 0.5 lg/ml ethidium bromid. Genom frag- ments were examined under the UV transimilator at 370 bp for hepatitis A virus, at 420 bp for enteroviruses. As a conclusion, enteroviruses was detected in four samples (8%), but hepatitis A virus did not determined in cow milk.

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P1–92 A new amino acid sequence variant in NADPH- cytochrome P450 oxidoreductase found in a healthy control M. Tomkova1, C. C. Marohnic2, K. M. McCammon2, B. S. Masters2 and P. Martasek1 1Department of Paediatrics and Adolescent Medicine, Charles University in Prague First Faculty of Medicine, Prague, CZECH REPUBLIC, 2Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA

Havana, Indiana, Saintpaul, Senftenberg and Typhimurium. On the other hand, four strains of S. Agona and four strains of S. Wor- thington shared the same profile. Selected genes from salmonella bacteriophages P22, ST104, Gifsy-1, Gifsy-2, Fels-1, Fels-2 and SopEFi were detected in strains by PCR based phage typing approach. We were able to observe that presence of phage genes and their combinations were mostly serotype specific, despite being able to observe some intra-serotype variability. Taking the PCR phage typing to the level of nucleotide sequence, applying sequenc- ing analysis, we observed further improvement in discrimination power of PCR based phage typing.

P1–94 Analysis of the tellurite resistance determinant present in uropathogenic Escherichia coli L. Valkovicova, S. Vavrova, J. S. Aradska and J. Turna Department of Molecular Biology, Comenius University, Bratislava, SLOVAK REPUBLIC

grants GAUK 252000 Microsomal cytochrome P450 enzymes are involved in metabolism of drugs and xenobiotics and in catalysis of steroid biosynthesis. A single protein, NADPH-cytochrome P450 oxidoreductase (CY- POR), supplies electrons to all microsomal P450s. CYPOR is a flavoprotein, containing both FAD and FMN, that uses NADPH as electron donor. Mutations in the POR gene (encoding CYPOR) have been associated with an autosomal recessive disorder called POR deficiency. To identify CYPOR amino acid sequence varia- tions in the Czech population we sequenced POR in 37 healthy controls. We have identified a new CYPOR amino acid sequence variant P384L, which is a one-base substitution (c1151C>T) on one allele in exon 10. Wild type (WT) CYPOR and P384L variant were bacterially expressed and purified. Flavin analysis indicated- that the protein:FAD:FMN ratio of P384L was the same as WT (1 : 1 : 1). CYPOR function was quantified by measuring both NADPH-cytochrome c reduction and CYP1A2 catalyzed ethoxy- resorufin-O-dealkylase (EROD) activity. The P384L variant retained 3 60% of WT turnover in the cytochrome c reductase NADPH = 1 lM. A similar result was assay with no effect on Km obtained from the EROD assay, with 3 70% of WT activity observed for P384L. According to the location of the P384 residue in the CYPOR connecting domain, it is surprising that activity was not more severely affected by P384L substitution. It is not surpris- ing, however, that a minor loss of function from a single allele had no apparent affect on the health of the individual. Acknowledgement: Supported by 100007 and NIH grant GM081568.

P1–93 Analysis of genetic variability of Salmonella enterica strains based on prophage genes and variable number tandem repeats L. Tothova, H. Drahovska, T. Szemes, H. Al-Alami, D. Brotanova, A. Soltysova and J. Turna Department of Molecular Biology, Comenius University, Bratislava, SLOVAK REPUBLIC

Tellurium compounds have a long history as antimicrobial and therapeutic agents. Prior to the development and marketing of today’s antibiotics, they were used to treat conditions such as lep- rosy, tuberculosis, dermatitis, cystitis and eye infections. As a con- sequence, many Gram-positive and Gram-negative bacteria developed resistance to potassium tellurite. Despite the rare occur- rence of tellurite in nature, the determinant of tellurite resistance encoded by ter genes has been found in relatively wide spectrum of pathogenic microorganisms, including the emergent haemorrhagic strain Escherichia coli O157:H7. We have studied tellurite resis- tance determinant located on the previously described pTE53 plas- mid which was originally isolated from uropathogenic strain E. coli KL53. The real time PCR analysis has determined that the ter- ZABCDE genes are represented by a single RNA transcript, form- ing a single transcriptional unit that is expressed from a single promoter – predicted also by large scale bioinformatics analysis. We functionally confirmed the existence of one common function promoter for terZABCDE genes by cloning probable promoter region, also called PPRR (the potential promoter rich region) into the vector pBAD-GFPuv in the fusion with reporter gfpUV gene. The functional fusion of proposed promoter region with gfpUV resulted in the cells expressing GFP, which was observed with fluo- rimetry assay. The terW gene codifies a protein of 155 amino acids and recently, our research group demonstrated its DNA binding activity. In this work, the terW-derived protein containing a N-ter- minal His-tag was overexpressed, purified and used for in vitro DNA-binding reactions. By method EMSA, it was found that TerW binds specifically to DNA containing the potential promoter region of terZABCDE. EMSA is one of the most widely used methods to explore interactions between DNA and proteins. Ami- noacid sequence comparison and specific bioinformatic prediction suggest that TerW is related to the other DNA-binding proteins with transcriptional regulator function. However the mechanism of tellurite resistance still remains unclear, but with the promising results given above we hope to uncover its secret soon.

P1–95 Molecular modelling study of PrnB, the major proline transporter of Aspergillus nidulans Y. Vangelatos1, M. Billini2, D. Vlachakis1 and V. Sophianopoulou1 1NCSR ‘Demokritos’, Biology, Athens, GREECE, 2JGI, Production Genomics Facility, San Francisco, CA, USA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Salmonella enterica represents a leading cause of food born poison- ing universally in developed and developing countries. Rapid and reliable identification is a key step in the tracking and management of infection outbreaks. Conventional phenotype based methods, which are in use currently, are increasingly showing their limits when compared to modern molecular methods based on PCR, fragment analysis or sequencing analysis. In our study, we ana- lyzed a set of salmonella strains belonging to 21 different serotypes using three methods – MLVA (Multi Locus VNTR Analysis), PCR phage typing and sequencing based phage typing, which we selected for typing of serovar Typhimurium in our previous study. We revised our previously used VNTR markers, excluding those lacking variability or presence of markers, and used a set of seven markers in a multiplex fragment analysis assay. We observed high polymorphism in selected markers with the total number of 36 dif- ferent MLVA profiles. Inter-serovar variability was detected in strains of serovars Anatum, Bredeney, Derby, Dublin, Enteritidis, PrnB, the L-proline transporter of Aspergillus nidulans, belongs to the YAT family, one of the ten members of the Amino acid

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segments comprise 12 transmembrane

P1–97 Shear flow enhances s-nitrosylation of proteins in endothelial cells L. Wang Academia Sinica, Institute of Biomedical Sciences, Taipei, TAIWAN

Polyamine Organocation transporter superfamily. In silico analy- sis and limited biochemical evidence suggest that YAT transport- connected with ers relatively short hydrophilic loops. However, little is known on the structure-function relationships in !AT transporters. We have made use of the A. nidulans PrnB transporter to address struc- ture-function relationships by selecting, constructing and analy- sing several prnB mutations (Tavoularis et al., 2003; Vangelatos & Sophianopoulou, unpublished data). Most isolated missense mutations affecting PrnB function map in the borders of cyto- plasmic loops with transmembrane domain. In a recent study, PrnB structure-function relationships were addressed by employ- ing a Cys-scanning approach, based on the engineering of a func- tional Cys-less PrnB genetic background (Kafasla et al., 2007). The 3D structures of only few Secondary transporters have been solved to date. After obtaining them from the Protein DataBank, the structure of PrnB was established using a novel, hydropho- bicity-based, approach to conventional homology modelling tech- niques. More than one templates were chosen and structurally combined together to produce a reliable alignment. Loop search- ing and building was done by Modeller, followed by Gromacs molecular dynamics simulations, using explicit water molecules in an NVT periodic box. The viability of the PrnB homology model was evaluated by in silico scoring functions, such as Procheck and Verify3D. Currently exhaustive molecular dynamics simula- tions of PrnB are carried out within the membrane bilayer to evaluate binding of a series of substrates.

Endothelial cells (ECs) constantly exposed to blood flow increase nitric oxide (NO) production via the activation of eNOS. NO- mediated S-nitrosylation is recently identified as an important posttranslational modification that may alter signaling and/or proteins’ function. In the present study, a CyDye switch method was utilized to examine the S-nitrosylated proteins in human umbilical vein ECs exposed to flow by a flow chamber setup. After flow, free thiols in proteins were blocked by alkylation and the S-NO bond was reduced by ascorbate followed by a CyDye (Cy3 or Cy5) labeling to this reduced cysteine in proteins col- lected from respective static- or shear flow- treated ECs. The CyDye-labeled proteins were mixed and subjected to two-dimen- tional electrophoresis. The enhanced S-nitrosylation of proteins after flow treatment was reflected as an increase of Cy5-labelling proteins. More than one hundred S-nitrosoproteins were detected in static and shear-treated ECs. Among those, 12 major proteins were shown to have significantly increases in S-nitrosylation under shear flow. ECs pretreated with an eNOS inhibitor (L- NAME) followed by flow treatment decreased the flow-induced S-nitrosylation. Those S-nitrosylated proteins were heterogeneous origin. The S-nitrosylated residue in tropomyosin and vimentin were identified and localized in the hydrophobic motif. Similar S- nitrosylations were shown in ECs treated with a NO donor (SNAP). Our results demonstrate that the S-nitrosylation of endothelial proteins could be detected by this CyDye switch method coupled with 2-D DIGE. Shear flow to ECs increases S- nitrosylation in endothelial proteins. This eNOS-dependent S-nit- rosylation of proteins may be essential for the adaptation/remod- eling in ECs under flow.

P1–96 Post-transcriptional control of the sRNA MicA in Salmonella typhimurium S. C. Viegas, I. D. J. Silva, M. Saramago, S. Domingues and C. M. Arraiano ITQB, Control of Gene Expression lab, Lisboa, PORTUGAL

Its main targets

P1–98 Significance of nucleotide changes in I subunit of cytochrome oxidase gene in Polish pyrethroid-resistant populations of Melighetes aeneus P. Wieczorek1, K. Nowaczyk1, P. Wegorek2 and A. Obrepalska-Steplowska1 1Institute of Plant Protection, Interdepartmental Laboratory of Molecular Biology, Poznan, POLAND, 2Department of Zoology, Institute of Plant Protection, Poznan, POLAND

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Eukaryotic and prokaryotic cells contain a wealth of small RNAs (sRNAs) with determinant roles in the post-transcriptional con- trol of gene expression. In pathogenic bacteria, a large number of sRNAs coordinate adaptation to stress and expression of viru- lence genes. MicA is a small RNA conserved among many en- in Salmonella are the outer terobacteria. membrane protein ompA and LamB maltoporine. We have anal- ysed the expression profile of MicAin this bacterium under differ- ent growing conditions. To understand the regulation by sRNAs it is also fundamental to study their turnover since this may impact on the regulatory capacity of a sRNA. We have con- structed Salmonella typhimurium mutants for several of the main ribonucleases and Poly(A) Polymerase I. The processing and sta- bility of MicA sRNA was analyzed in these mutants. PNPase and RNase E mutations were shown to strongly influence its sta- bility. However, a mutation in the endoribonuclease III was one that mostly affected MicA decay. We have purified several of these main ribonucleases of Salmonella and we are performing in vitro activity assays to clarify its role on MicA processing and decay. We are also analysing the stability of MicA in the absence of its mRNA target(s) to investigate whether there is a coupled MicA-target regulation. Taken together, the results of this study provide initial insight into the mechanisms of sRNA decay in Salmonella, and indicate specific contributions of the RNA decay machinery components to the turnover of individual sRNAs. Pollen beetle (Melighetes aeneus F.) is a common pest of oilseed rape cultivation. It could cause great damage amounting to 80% of rape harvest by feeding onpollen flowers. Therefore, appropri- ate prevention steps have been undertaken tocontrol occurrence of this insect. Pyrethroids are insecticides commonly usedto restrict insect pests. Many populations of pollen beetle developed resistance towards the pyrethrin analogues. Resistance might be- caused by: mutations in the pyrethroid-binding site in voltage- sensitive sodium channel (knockdown resistant) or other genes involed in detoxication, for example esterases or oxidases, or by changes in their gene expression level. In our study we have investigated significance of mutational changes within partial

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P1–100 Degeneration of lipocalin-type prostaglandin D synthase in cerebrospinal fluids from patients with rheumatoid arthritis Y. Yamamoto1, H. Sato2, S. Fukasaku3, K. Takeuchi4, A. Okazaki4, K. NIshimura4, M. Izeki4 and E. Inada4 1Tokyo Metropolitan Institute for Medical Science, Cancer Thera- peutics, Tokyo, JAPAN, 2Tokyo Metropolitan Cancer and Infec- tious Diseases Center Komagome Hospital, Anesthesiology, Tokyo, JAPAN, 3Juntendo University School of Medicine, Orthopedics, Tokyo, JAPAN, 4Juntendo University School of Medicine, Anes- thesiology, Tokyo, JAPAN

sequence of cytochrome oxidase subunit I (mtCOI) gene of Polish populations of pyretroid resistant pollen beetles insects. Total DNA was isolatedfrom thirteen Polish populations of M. aeneus followed by PCR reaction with primers amplifying mtCOI gene sequence. Afterwards, SSCP analysis (single strand conformation polimorphism) was performed followed by DNA sequencing. To assess whether analyzed samples were in homo- or heterozygotic state HRM (high resolution melting analysis) was performed. The studied DNA sequences were analyzed with BioEdit, GENE- DOC and BLAST software. Then analysis of secondary structure of the mtCOI protein was done with psipred, jnet and sam and profsec software. The comparison between nucleotide sequences of mtCOI with those available in GeneBank (UK populations) has shown several point changes present within considered gene fragment. Single G849>A849 substitution was readily detected with HRM, but it does not change amino acid. Other mutations found, changed amino acids sequence, however bioinformatical modelling has localised them on the ends of helises, outside of the core of the enzyme. Therefore, we suppose that all changes observed might occur due to geographical isolation of examined populations of M. aeneus and are not significant to enzymatic abilities nor structure of the cytochrome oxidase, especially that we could not refer it to population completly sensitive to pyreth- roids, because such population was not identified in Poland. Therefore we suggest that different detoxification mechanisms, for example increased level of esterases or oxidases might be asociated with pyrethroid resistance in Polish strains of pollen beetle.

Introduction: The cause of rheumatoid arthritis (RA) pathology is still not fully known. We studied disease-specific proteins in the cerebrospinal fluid (CSF) of patients with RA by proteomic analysis. L-PGDS, a major component in the central nervous sys- tem, is a dual functional protein acting as a PGD2-synthesizing enzyme and a transporter for lipophilic ligands. Enzymatic kinet- ics was analyzed by measuring the level of PGD2, and the role of L-PGDS in RA is discussed. Methods: Proteins in CSF were assayed by isoelectric focusing and SDS-polyacrylamide gel electrophoresis. Protein spots were analyzed and identified by mass spectrometry for expression- changed spots. PGD2 and 15-deoxy-D12, 14-PGJ2 (15d-PGJ2) lev- els were measured. Results and Discussion: Differential analysis of proteins in CSF of RA and controls by two-dimensional gel electrophoresis revealed several expression-changed proteins, among which the expression of lipocalin-type prostaglandin D synthase (L-PGDS) in CSF of RA was 1.5-fold stronger than that in controls. We focused on L-PGDS, a pain-regulatory protein, and despite an apparent increase in its expression, the level of PGD2 in RA sam- ples was significantly lower than in controls. The level of 15d- PGJ2, a non-enzymatic degraded product of PGD2, was similarly low. The decrease of these products was evaluated by examining the reactivity of CSF proteins and the anti-L-PGDS antibody, which showed two primary bands: one of an L-PGDS monomer and one with a larger molecular weight. These results suggest that L-PGDS in RA patients may associate with other compo- nents and that the conformational change might be due to a decrease in L-PGDS activity.

P1–99 Identification of human blood group A/A-Leb/y and B/B-Leb/y glycotopes co-expressed from two distinct ovarian cyst glycoproteins A. M. Wu1, S. Y. Yu2, Z. Yang3 and K. H. Khoo4 1College of Medicine, Chang-Gung University, Tao Yuan, TAIWAN, 2Institute of Biochemical Sciences, National Taiwan University, Taipei, TAIWAN, 3Institute of Molecular and Cellular Biology, Chang Gung University, Tao-yuan, TAIWAN, 4Academia Sinica, Institute of Biological Chemistry, Taipei, TAIWAN

P1–101 Effects of resveratrol on oxidant/antioxdant status and sirtuin 1 and Matrix metalloproteinase-9 mRNA expression in streptozotocin-induced diabetic rat hearts A. S. Yar1, E. Alp1, D. Sahin2, N. Altan2 and S. Menevse1 1Gazi University Faculty of Medicine, Medical Biology and Genetics, Ankara, TURKEY, 2Gazi University Faculty of Medicine, Medical Biochemistry, Ankara, TURKEY

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Although the individual human blood group A and B determi- nants are well defined, their co-expression pattern on a particular glycan carrier in individuals of blood group AB status has not been delineated. To address this issue, complex O-glycans were isolated from two distinct sources of human ovarian cyst glyco- proteins (HOC 89 and Cyst 19) and profiled by advanced mass spectrometry analyses, in conjunction with defining their binding characteristics against a panel of lectins and monoclonal antibod- ies. The major O-glycans of HOC 89 were found to correspond to sialyl Tn, mono- and disialyl Tstructures, whereas those of Cyst 19 were apparently much less sialylated, extended to larger sizes, and overall more heterogeneous. A minimal structure that carries both A and B determinants on the same molecule was identified, in which the A epitope is attached directly to the core GalNAc, whereas the B epitope is preferentially located on the six arm of a core two structure. Both arms can be further extended with internal fucosylation that appears to be restricted to those non-sialylated chains already carrying the terminal ABH determinants, thus giving rise to rather prominent A/B-Leb/y gly- cotopes on larger O-glycans. Diabetic cardiomyopathy is related directly to hyperglycemia that increases oxidative stress inducing cardiac damage in animals and humans with diabetes. Sirtuin 1 (SIRT1) has been implicated in modulating transcriptional silencing and cell survival. Resveratrol (RSV), exhibits potent antioxidative and anti-inflammatory effects, has potent pharmacological agonist of SIRT1 activity. The reduction of SIRT1 activity because of high glucose is corre-

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In this

P1–103 Using RNAi-based high-throughput screen to identify novel signaling pathways regulating oral cancer cells proliferation M. H. Yeh1, P. Y. Lee2, Y. L. Chen3, W. L. Tsai3, T. C. Tsai4, M. H. Tsai5 and M. C. Kao6 1National Defense Medical Center, Graduate Institute of life sciences, Taipei, TAIWAN, 2Graduate Institute of Basic Medical Science, China Medical University, Taichung, TAIWAN, 3Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung, TAIWAN, 4Department of Microbiology and Immunology, National Chiayi University, Chiayi, TAIWAN, 5Department of Otolaryngology, China Medical University Hospital, Taichung, TAIWAN, 6Department of Biological Science and Technology, China Medical University, Taichung, TAIWAN

lated to activation of Matrix metalloproteinase-9 (MMP-9) tran- scription. MMP-9, belong to a large MMP family capable of degrading the constituents of extracellular matrix, is decreased by hyperglycemia. The present study was designed to clarify the effect of RSV treatment on diabetic cardiomyopathy and to determine the MMP-9 and SIRT1 mRNA expression and ferrous oxidation xylenol orange (FOX), malondialdehite (MDA) levels and superoxide dismutase (SOD) activity in diabetic rat hearts. Three months-old, 44 Wistar albino male rats were used for the study. Rats were divided into six groups. After the induction of Streptozotocin (55 mg/kg), every day 10 mg/kg RSV was admin- istered intraperitoneally for 4 weeks. study, SIRT1 mRNA levels decreased in diabetic group (P < 0.05). SIRT1 mRNA levels increased in RSV treated diabetic group when com- pared to the diabetic group. (P < 0.05). On the other hand, RSV was no significant effects on MMP-9 mRNA expression among groups (P > 0.05). SOD activity decreased in RSV trea- ted sodium sitrat buffer (sham) control and RSV treated diabetic group (P < 0.05). In conclusion, higher doses or different peri- ods of RSV treatment may alter antioxidant capacity. Activation of SIRT1 by RSV may prevent proinflammatory phenotypic alterations in heart tissues.

P1–102 Molecular cloning of a wheat a-amylase inhibitor and its functional studies S. Yasothornsrikul1 and G. R. Reeck2 1Biochemistry, Naresuan University, Phitsanulok, THAILAND, 2Biochemistry, Kansas State University, Manhattan, NY, USA

Oral carcinoma is a worldwide healthy issue and the first leading cause of head and neck cancer death in Taiwan. Moreover, oral cancer patients often present symptoms at a late stage and show a high recurrence rate after treatment. Therefore, to identify novel biomarkers for early diagnosis or clinical oral caner ther- apy is an urgent topic. In this research we employed the ‘anti-ki- nome’ and ‘anti-phosphatome’ RNAi subsets (including 737 kinases, 209 phosphatases and 30 genes with dual function) from the RNAi consortium (TRC), to high-throughput screen and sys- temically identify the novel regulators of oral cancer cells. The screen results showed two groups of genes: growth inhibition group (GIG) contained 51 genes revealing more than 90% growth inhibition; and growth promotion group (GPG) con- tained 42 genes revealing more than one fold of growth promo- tion from human oral cancer HSC-3 cells. These two groups of genes were submitted to MetaCore(cid:4) analysis, resulting in four and three major putative molecular pathways in GIG & GPG respectively. They are TGF-beta receptor type II, FLT3, IPP-1, and EGFR pathways in GIG, and ErbB2/ErbB4-IGF-1 receptor, Insulin receptor and p57 pathways in GPG. These novel regula- tors may have potential as biomarkers for early diagnosis of the oral cancer. Moreover, these regulators will also be used as potential targets for anti-cancer drug design and help to develop a novel method to combat with oral cancer.

P1–104 Identification of GPI-anchored proteins using position specific score matrix Y. Ikeda1, O. Oura1, T. Sasaki1, Y. Yabuki2 and M. Ikeda3 1Department of Electronics and Bioinformatics, Meiji University, Kawasaki, JAPAN, 2Information and Mathematical Science Laboratory Inc., Life Science Group, Tokyo, JAPAN, 3Consoli- dated Research Institute for Advanced Science and Medical Care, Waseda University, Tokyo, JAPAN

and TMA at 80 ± 10%,

is

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Glycosylphosphatidylinositol (GPI) is one of the most important post-translational modifications, which can bind soluble polypep- tides to plasma membrane. In this regard, the development of computational methodology to predict GPI modification from protein sequences is highly awaited. GPI-anchored protein has signal peptides in the N-terminal region because the GPI anchor is modified in the endoplasmic reticulum. The propeptide of GPI- anchored protein, which is a polypeptide positioned behind the removed. The GPI- GPI-anchored position (omega-site), anchored protein has the propeptide consists of hydrophobic resi- dues and small amino acid residues exist near the omega-site. A prediction method based on the characteristics of C-terminal amino acids has been reported by Eisenhaber et al. (JMB, 1999). The kernels of wheat contain a number of proteins which can inhibit a-amylases of various origins. These protein inhibitors can be classified into subfamilies and families on the basis of their amino acid sequence similarity. The wheat 0.28 a-amylase inhibitor can inhibit Tenebrio molitor a-amylase but has very little effect on human salivary a-amylase. We have isolated a cDNA clone, W22.1, encoding a wheat a-amylase inhibitor and investigated its inhibitory function towards a-amylases from various sources. The cDNA clone consists of 435 nucleotides flanked by a 33 nucleotide 5¢-untranslated sequence and a 3¢- non coding sequence of 126 nucleotides. One putative polyaden- ylation signal is located 47 base pairs upstream from the poly- adenylation site. The longest open reading frame translates into 145 amino acid residues, with 30 residues of a signal peptide. The deduced amino acid sequence shows that it belongs to the wheat 0.28 a-amylase inhibitor family. We expressed and puri- fied the mature protein from clone W22.1 in E. coli, and its inhibitory activity against a-amylases from Sitophilus oryzae, Tribolium casteneum, and Tenebrio molitor was analyzed. Results reveal that the E. coli-expressed protein from the W22.1 clone can inhibit a-amylase activities of SOA1, SOA2, TCA1, TCA2, 37 ± 9%, 88 ± 8%, 20 ± 7%, and 73 ± 5%, respectively. These data suggest speci- ficity and selectivity of this a-amylase inhibitor towards a-amy- lases this from Sitophilus oryzae. Such characteristic of inhibitor could be further used as a model to study structure/ function relationships toward specificity and selectivity of other a-amylase inhibitors of the same family.

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FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Here we describe a new method for the discrimination of GPI- anchored proteins, which uses the position specific score matrix the (PSSM) combined with the hydropathy profile. First, sequences of mammalian GPI-anchored proteins (411 sequences from SWISS-PROT ver. 54.0) were scanned in terms of average hydrophobicity, and non-GPI-anchored proteins having similar hydropathy profiles to GPI-anchored proteins were used as nega- tive control (368 sequences). Sequences were aligned according to residue size in the C-terminal region, and position-specific amino acid propensity depending on alignment position was analyzed for both GPI-anchored proteins and negative controls. The PSSM was devised using each amino acid propensity and the matching score was estimated for each dataset. The threshold was set from the score distribution. The discrimination accuracy between GPI-anchored proteins and negative controls was higher than 90%, evaluated on the basis of the cross-validation test. Reference: 1. Eisenhaber B, Bork P & Eisenhaber F. Prediction of potential GPI-modification sites in proprotein sequences J. Mol. Biol 1999; 292: 741–758.

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Poster Presentations

P2 Protein Structure and Interactions

P2–1 Investigation of blip – beta-lactamase binding in-vivo A. E. Akkaya1, A. Hortacsu1, E. O. Olmez1 and B. S. Akbulut2 1Department of Chemical Engineering, Bogazici University, Istan- bul, TURKEY, 2Department of Bioengineering, Marmara University, Istanbul, TURKEY

FOR EARLY STAGE RESEARCH TRAINING (1/4/04–31/5/ 08) n MEST-CT-2004-504391, ‘NMR in Inorganic Structural Biology’; Financial support by the Access to Research Infrastruc- tures activity in the 6th Framework Program of the EC (Contract #RII3-026145, EU-NMR) for conducting the research at CERM is gratefully acknowledged. References: 1. Pereira AS, Tavares P, Folgosa F, Almeida RM, Moura I & Moura JJG. Eur. J. Inorg. Chem. 2007; 18: 2569–2581. 2. Auche´ re F, Pauleta SR, Tavares P, Moura I & Moura JJG. J. Biol. Inorg. Chem. 2006; 11(4): 433–444.

P2–3 A comparison of Polyphenoloxidases from different plants G. Arabaci and B. Duzcan Sakarya University, Chemistry, Sakarya, TURKEY

The widespread use of beta-lactam antibiotics has caused bacteria to acquire resistance to these antibiotics which is a major health problem. The use of beta-lactamase inhibitors is one of the strat- egies to activate the therapeutic value of beta-lactam antibiot- ics.Recent studies show that beta-lactamase inhibitor protein (BLIP) from Streptomyces clavuligerus has inhibitory properties against class A beta-lactamases such as TEM-1. In this work, experimental studies were carried out to investigate in-vivo beta- lactamase inhibition by BLIP. For this purpose, BLIP was cloned into expression vector pET-26b(+) and expressed as a his-tagged periplasmic proteinin E. coli BL21 (DE3) cells for binding and inhibition studies. In order to investigate in-vivo beta-lactamase inhibition by BLIP in the periplasm, pUC18 plasmid, carrying the gene for R-TEM-1 beta-lactamase was transformed into the E. coli BL21 (DE3) cells harboring recombinant pET-26b(+) vector. BLIP expression was under the control of IPTG induc- tion whereas beta-lactamase expression was constitutive. Beta-lac- tamase inhibition was monitored by analyzing the growth profile of the recombinant cells. Furthermore osmotic shock fluid from these cells was analyzed on SDS-PAGE for the complex forma- tion between BLIP and beta-lactamase.

P2–2 The electron transfer complex between D. gigas Superoxide Reductase and Rubredoxin R. Almeida, S. Pauleta, I. Moura and J. J. G. Moura REQUIMTE, Quimica, Caparica, PORTUGAL

Polyphenoloxidase (PPO) is widely distributed in nature and it is a copper-containing enzyme which catalyzes the hydroxylation of monofenols to o-diphenols and the oxidation of o-diphenols to o-quinones by using molecular oxygen. It is responsible for enzy- matic browning reaction occurring during the handling, storage and processing of fruits and vegetables. The present work sought to carry out a screening of PPO activity in four different Turkey (northwestern Turkey) plants, goosefoot plant (Chenopodium ant- helminthicum), common mallow (Malva sylvestris), dandelion (Ta- raxacum officinale) and cress (Lepidium sativum) consumed as food and also used for medicinal purposes by local populations. The all plants were homogenized in 0.1 M sodium phosphate buffer pH 7.0 with 0.5% PVP, 2% ascorbic acid and 5% Triton- X100 at 4(cid:2)C. The extract was filtered through 4 layers of cheese cloth and centrifuged at 10 000 g for 15 min at 4(cid:2)C. PPO activity was determined with catechol as a substrate by measuring the increase in absorbance at 420 nm. The enzyme from each plant was characterized for thermal stability, pH and kinetic with dif- ferent catechol concentrations at proper pH’s. The results showed that dandelion and goosefoot plant PPO have 11.74 and 11.41 mM Km values for catechol at pH 6.5 and 7.5. However, common mallow and cress have 23.5 and 20.99 mM Km values for catechol at pH 7.0 and 5.0 have 23.5 and 20.99 mM Km val- ues for catechol at pH 7.0 and 5.0. Dandelion and goosefoot plant PPO are more active and efficient to catechol substrate at different pH’s and 35(cid:2)C. Thus, different plants have different enzyme activities at the same conditions.

P2–4 Lipocalin-type prostaglandin D synthase: a transporter of prostaglandin D2 and scavenger of prostaglandin D2-degraded products K. Aritake1, M. Sakata1, S. Shimamoto2, T. Tsurumura1 and Y. Urade1 1Osaka Bioscience Institute, Molecular Behavioral Biology, Suita, JAPAN, 2Pharmaceutical Science, Osaka University, Suita, JAPAN

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is responsible for the production of PGD2, a potent endogenous sleep substance, and is secreted into the cerebrospinal fluid (CSF) as beta-trace, a major protein of human CSF. L-PGDS is a member of the lipocalin gene family, which consists of small secretory transporter proteins of various lipophilic ligands such Superoxide reductase (SOR) is a 29 kDa homodimeric metallopro- tein present in the cytoplasm of Desulfovibrio (D.) gigas cells. This protein allows this anaerobic organism to survive under mild expo- sure to aerobic atmospheres, by converting the radical anion superoxide to hydrogen peroxide without formation of molecular dioxygen. The D. gigas protein is a class II SOR, thus it contains only one iron center per monomer: a Fe atom bound to four histid- inyl residues’ side chains in the equatorial plane, with a fifth ligand provided by a cysteinyl sulfur in the axial plane (1). Several attempts have been made to identify and characterize the electron- transfer (ET) partners of SOR (2). Rubredoxin (Rd) is proposed to be one such partner. This small (6 kDa) monomeric metalloprotein is capable, among other things, of accepting electrons from NADPH:Rubredoxin Oxidoreductase and transferring them to the terminal oxidase of the oxygen detoxification pathway, Rubredox- in:Oxygen Oxidoreductase. Its metal center is very similar to center I of class I SORs.ET has been demonstrated to occur between SOR and Rd in vitro (2). In this work, we provide evidence for the formation of the ET complex via 2D NMR titration. This experi- ment showed that the complex is on fast exchange in the NMR time scale. We were also able to determine the Rd aminoacid resi- dues affected by the formation of the complex. Furthermore, we estimate a value for the dissociation constant (Kd) consistent with the formation of a low-affinity, high-turnover complex. Acknowledgements: Fundacio para a Ciencia e Tecnologia – MCTES (PhD grant SFRH/BD/25342/2005 and project POCI/ QUI/57741/2004); MARIE CURIE HOST FELLOWSHIPS

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molecules and/or membranes in the regulation of lipid trafficking. As an application of the acquired knowledge, we show the char- acterization of the interaction of FABPs with lipid-functionalized gadolinium chelates to be used as potential hepatospecific con- trast agents for MRI (4). References: 1. Furuhashi M & Hotamisligil GS. Fatty acid-binding proteins: role in metabolic diseases and potential as drug targets. Nat Rev Drug Discov. 2008;7(6): 489–503. Review.

2. Eliseo T, Ragona L, Catalano M, Assfalg M, Paci M, Zetta L, Molinari H & Cicero DO. Structural and dynamic determi- nants of ligand binding in the ternary complex of chicken liver bile acid binding protein with two bile salts revealed by NMR. Biochemistry 2007;46(44):12557–12567.

3. Pedo` M, D’Onofrio M, Ferranti P, Molinari H & Assfalg M. Towards the elucidation of molecular determinants of coop- erativity in the liver bile acid binding protein (Submitted).

4. Tomaselli S, Zanzoni S, Ragona L, Gianolio E, Aime S, Assfalg M & Molinari H. Solution structure of the supramo- lecular adduct between a liver cytosolic bile acid binding protein and a bile acid-based gadolinium(III)-chelate, a poten- tial hepatospecific magnetic resonance imaging contrast agent. J Med Chem. 2008;51(21):6782–6792. as retinoic acid, retinal, biliverdin, bilirubin, gangliosides with high affinities. PGD2 is chemically unstable in aqueous solution and non-enzymatically dehydrated to produce the J series of PGs, such as PGJ2, D12-PGJ2, and 15-deoxy-D12,14-PGJ2 in vitro. However, the stability and metabolism of PGD2 in vivo remain to be clarified. In this study, we characterized the binding affinity of L-PGDS to PGD2 and its degraded products. In the presence of L-PGDS but not albumin, we could detect only PGD2 in aqueous solution without any detection of neither metabolites nor catabolites over 6 h. Surface plasmon resonance analysis revealed that PGD2 bound to L-PGDS in a concentration-depen- dent manner with high affinities of Kd = 20 nM, and its binding was reversibly in the short duration. On the other hand, the degraded products, 15-deoxy-D12,14-PGJ2-binding to L-PGDS was irreversible. Using MALDI-TOF mass spectroscopy, we showed that the binding of PGD2 to L-PGDS was covalent and irreversible. 15-deoxy-D12,14-PGJ2 dose-dependently inactivated L-PGDS enzyme activities. We identified that Cys65 is the resi- dues for the PGD2-binding, proven by peptidase-digestion and site-directed mutagenesis. We therefore propose that PGD2 is sta- bilized via binding to L-PGDS providing a stable transport towards its receptors. Moreover, L-PGDS consecutively acts as scavenger for J-type PGs through covalent binding providing a balance in the biological PGD2 system.

P2–6 Dysferlin interacts with alpha-tubulin in skeletal muscle B. A. Azakir, C. Therrien, S. Di Fulvio and M. Sinnreich Montreal Neurological institute, Neurology, Montre´al, QC, CANADA

P2–5 Binding cooperativity and allosterism make liver fatty acid binding proteins ideal chaperones of lipids and lipid-functionalized drugs M. Assfalg, M. D’Onofrio, M. Pedo’, S. Zanzoni, C. Cogliati, M. Guariento and H. Molinari Dipartimento di Biotecnologie, Universita’ degli Studi di Verona, Verona, ITALY

The pathogenic basis for many muscular dystrophies is the inability of the muscle cell to maintain its sarcolemmal integrity, due to defective or deficient membrane associated structural pro- teins or components of the membrane repair machinery. An important protein implicated in surface membrane repair in mus- cle is dysferlin. Mutations in dysferlin are a frequent cause for recessively inherited limb girdle muscular dystrophies the (LGMD), defining the common subtype of LGMD2B. In addi- tion, dysferlin mutations also cause Miyoshi Myopathy (MM) and distal anterior compartment myopathy, both distal forms of muscular dystrophy. To better understand the cellular function of normal dysferlin, we set out to identify novel protein binding partners. Using affinity purification followed by liquid chroma- tography/mass spectrometry, we have identified a number of pro- teins in skeletal muscle, which showed association with dysferlin. Importantly, we were able to identify the few proteins that were previously shown to interact with dysferlin by co-immunoprecipi- tation. The newly identified proteins fall into categories of surface membrane proteins, proteins involved in cellular trafficking and in protein degradation. We have identified alpha-tubulin as a dysferlin binding partner and confirmed this interaction through co-immunoprecipitation assays and co-localization studies. We found the interaction to be calcium dependant and propose a role for alpha-tubulin in dysferlin trafficking after sarcolemmal injury.

P2–7 Structural studies on the catalytic subunit of the oxaloacetate decarboxylase Na+ pump M. Balsera, R. M. Buey, F. W. Winkler and X. D. Li Paul Scherrer Institute, Structural Biology, Villigen, SWITZERLAND

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Lipids are vital components of many biological processes and crucial in the pathogenesis of numerous common diseases, but the specific mechanisms coupling intracellular lipids to biological targets and signalling pathways are not well understood (1). Intracellular lipid chaperones known as fatty acid binding pro- teins (FABPs) coordinate lipid responses in cells and are strongly linked to metabolic and inflammatory pathways. FABPs display a wide range of sequence diversity, but share a common struc- tural fold. Among these proteins, human liver FABP is increas- ingly attracting the interest due to its capability of regulating not only fatty acids but also bile acids pathways. This protein, which is the most abundant cytosolic protein in hepatocytes, is proba- bly the most versatile chaperone in terms of its ligand repertoire. As a consequence of its abundance, it was suggested that L-FAB- Ps may be involved not only in the transport of endogenous lip- ids, but also of exogenous lipophilic drugs. Our group is focussing its research on the elucidation of the complex binding mechanisms of FABPs (2). Here we present recent results on the structural and the dynamic characterization of protein-ligand ad- ducts, obtained primarily by Nuclear Magnetic Resonance (NMR) spectroscopy. We describe measurements of translational diffusion and site-specific protein mobility that have highlighted functionally relevant protein regions. These data, performed both on L-FABP and on model proteins, have been combined with calorimetry and mass spectrometry data: the emerging picture is consistent with the occurrence of strong cooperative binding to multiple sites, originated by protein allosterism (3). Furthermore, we show very recent NMR data referred to experiments per- formed on FABPs in solutions containing phospholipids vesicles and in living cells. The obtained results suggest that these pro- teins can act as molecular switches allosterically activated by lipid The oxaloacetate decarboxylase Na+ pump (OAD) is a multi- functional enzyme that converts the free energy of a specific deca-

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P2–9 Inhibition of A277V mutant of human butyrylcholinesterase by malachite green K. Biberoglu1, O. Tacal1 and H. Akbulut2 1Department of Biochemistry, Hacettepe University School of Pharmacy, Ankara, TURKEY, 2Department of Medical Oncology, Ankara University School of Medicine, Ankara, TURKEY

rboxlylation reaction into an electrochemical gradient of Na+ ions across the membrane. This membrane protein complex is in the anaerobic citrate fermentation functional and essential pathway of some bacteria. It is composed of three subunits: a bi- modular soluble alpha subunit that is posttranslationally biotiny- lated and contains a metal ion in its catalytic center where the decarboxylation of oxaloacetate to pyruvate occurs, a bitopic gamma subunit that binds tightly to the alpha enzyme and local- izes it to the membrane, and a multispan transmembrane beta subunit that constitutes the Na+ channel and whose function is fully dependent on the transfer of the carboxyl group from the decarboxylation reaction on the alpha subunit. Large structural arrangements have been proposed for the alpha subunit in order to promote the carboxy-transfer reaction from its active site to the beta subunit niche through biotin. With the aim of under- standing the molecular mechanisms underlying OAD, we have carried out solution structural analyses by small-angle X-ray scat- tering in combination with homology modelling on the enzymatic subunit. Our results let us hypothesize about the conformational rearrangements associated with the carboxy-biotin transfer mech- anism within the complex.

Human butyrylcholinesterase (BChE) is a serine hydrolase which is toxicologically and pharmacologically important in scavenging and detoxifying cocain, organophosphorus pesticides, carbamate pesticides and chemical warfare agents. In this study, we tested the role of Ala277 in inhibition of BChE by malachite green. The mutation A277V was introduced into human BChE by PCR- mediated site-directed mutagenesis. The presence of A277V muta- tion was verified by automated sequencing. Recombinant wild- type and A277V mutant of human BChE were transiently expressed in human embryonic kidney cells and partially purified by procainamide-Sepharose affinity chromatography. For kinetic studies, BChE activity was assayed spectrophotometrically at 25(cid:2)C, in 50 mM MOPS buffer (pH 8), containing 0.05–0.4 mM butyrylthiocholine as substrate, and 0–3 lM dye. Malachite green acted as linear mixed inhibitiors of mutant enzyme and wild type BChE. Based on the rapid equilibrium inhibitory model, Ki val- ues were 0.166 mM for A277V mutant enzyme and 0.044 mM for recombinant wild type BChE. In conclusion, malachite green was found four-fold less effective as an inhibitor of A277V mutant, pointing Ala 277 may be important in the binding of malachite green to BChE. Acknowledgements: This study was supported by a grant (SBAG-3677) from Scientific and Technical Research Council of Turkey.

P2–8 Saturation transfer difference NMR study on the specific binding of sinomenine by human serum albumin G. Bazylak1, C. Ludwig2 and U. L. Gunther2 1Department of Pharmaco-Bromatology & Molecular Nutrition, Faculty of Pharmacy Collegium Medicum Nicolaus Copernicus University, Bydgoszcz, POLAND, 2HWB NMR, CR UK Institute for Cancer Studies University of Birmingham, Edbagston Birmingham, UK

P2–10 The study of multimerization leading to the assembly of immature particles of Mason– Pfizer monkey virus and Human immunodeficiency virus K. Bohmova, R. Hadravova, J. Stokrova, I. Pichova and M. Rumlova Gilead Sciences & IOCB Research Centre Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic, Prague, Czech Republic, Viral and microbial proteins, Prague, CZECH REPUBLIC

Sinomenine (SN), a natural dextrorotatory morphinan analog, is alkaloid isolated from the Chinese medicinal plant Sinomenium acutum. It has immunomodulating and anti-inflammatory acitivi- ties and is used in traditional Chinese anti-arthritic and anti- rheumatic therapies. The in vitro human serum albumin (HSA) binding rate of SN was estimated as 63% on the introductory results of equilibrium dialysis assay. Thus interaction process occurring at the protein-solvent interface for SN-HSA system was investigated by NMR using the saturation transfer difference (STD) technique which is valid in the lM–mM range of the ligand-protein binding constants. Similar procedure was used for the common anti-tussive alkaloid codeine and non-steroidal anti- inflammatory drug naproxen, thus the comparison of the binding ability of SN and codeine towards two various forms of HSA have been made. In contrast to naproxen, exhibiting high binding affinity to the both fatty-acid-loaded-HSA and fatty-acid-free- HSA, we observed that SN binds exclusively to the second men- tioned form of HSA, what is contrary to codeine which indicated no binding affinity to these both forms of HSA. The location of specific SN binding site(s) of fatty-acid-free-HSA have been pro- posed to explain these STD-NMR results, thus enabling in vitro prediction of HSA binding ability and evaluate pharmacokinetic properties of SN and co-administered NSAIDs during pharmaco- logical treatment of rheumatoid arthritic subjects. Acknowledgement: Supported by the grant from EU NMR project – Contract No. RII3-026145.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

The basic interactions leading to the assembly of immature retro- viral particles are mediated by protein-protein and protein-nucleic acids contacts. Protein-protein interactions are mediated mainly by capsid protein (CA) molecules whereas nucleocapsid protein (NC) is responsible for the binding of RNA. During the assem- bly, two Cys-His boxes of NC that are highly conserved among retroviruses specifically incorporate retroviral genomic RNA. However, the role of NC in the process of immature capsid assembly remains unclear. Studies from several laboratories sug- gest that the NC might function as a dimerization domain, as a replacement of NC protein by heterologous dimerization domain allows the polymerization of Gag and virus-like particles assem- bly. To study the multimerization effect on the assembly of immature particles, the nucleocapsid proteins from both, Mason- Pfizer monkey virus (M-PMV) and Human immunodeficiency virus (HIV-1), were replaced by heterologous dimerization and trimerization leucine zipper domains from GCN4 and the dimer- ization domain from CREB. Using a bacterial expression/assem- bly system, in vitro and in vivo assembly assays was found that

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the enzyme is present

replacement of entire M-PMV NC and HIV-1 NC domains with both, GCN4 and CREB multimerization domains, completely abrogated the assembly. The efficient assembly of M-PMV and HIV-1 particles was observed only in the presence of basic resi- dues of either N-terminal part of NC or CREB, suggesting the importance of these residues for an initiation of virus-like particle assembly. The impact of these replacements on the immature par- ticles assembly will be also studied in vivo in 293 cells.

to raise polyclonal antibodies in rabbits. The antiserum recog- nizes the major b-1,3-glucanase from midgut and by immunocy- in midgut tolocalization showed that columnar cells, associated to the cell glycocalyx and in large secretory vesicles from posterior midgut. This distribution agrees with that of tissue b-1,3-glucanase activity. SLAM produced in Pichia pastoris GS115 cells using a pPIC9K expression system has a Km of 0.13 ± 0.08% (w/v) and optimum pH of 9.0 with laminarin as substrate, similar to the parameters previously obtained for purified enzyme from the midgut. The action of SLAM against several substrates showed that laminarin is the best substrate. The straight substrate specificity of the enzyme may be due to an adaptation to hydrolyse fungal polysaccharides and the enzyme may help in preventing midgut infections by fungi.

P2–11 Role of the protein S1 in translation initiation and ribonuclease RegB activation; an NMR and SAXS analysis F. Bontems1, C. Sizun1, J. B. Cre´ chet2, J. Perez3, M. Uzan4, P. Aliprandi1, P. Giraud1, F. Mareuil1 and S. Caputo1 1Institut de Chimie des Substances Naturelles, RMN structurale, Gif-sur-Yvette, FRANCE, 2Ecole Polytechnique, RMN structurale, Palaiseau, FRANCE, 3Synchrotron Soleil, SWING beam line, Gif-sur-Yvette, FRANCE, 4Institut Jacques Monod, dynamique du ge´nome, Paris, FRANCE

P2–13 Kv1.2-based model of the calcium channel is consistent with published data obtained by substituted cysteine accessibility method I. Bruhova and B. Zhorov Biochemistry and Biomedical Sciences, McMaster University, Hamilton ON, CANADA

S1 is the largest protein of the Escherichia coli ribosome. It is strictly required to the correct recognition of the translation initi- ation codon of most, if not all, messenger RNAs by the ribo- somal 30S subunit. It is also used by several bacteriophages at their own benefit. It is one of the subunits of the phage Qb repli- case. It also enhances the reaction rate of the phage T4 endoribo- nuclease RegB, which inactivates the phage early mRNAs when their translation is no more required by cleaving them in the mid- dle of their Shine-Dalgarno sequence. We are interested in ana- lyzing and comparing S1 mode of action in these different functions. By combining biochemical, NMR and SAXS tech- niques, we identified a functional fragment of the protein formed of three domains and analyzed its structure and its interactions with different RNAs corresponding to different functions. We also undertook to compare the interactions of S1 with the ribo- somal 30S subunit and with the Qb replicase. All our results sug- gest that the different S1 functions are associated to an unique mechanism We also undertook to revisit the relationships between the forms of the S1 protein found in the gram-negative and the gram-positive bacteria.

Calcium channels are important drug targets, but their high-reso- lution structures are unavailable. Zhen et al. (2005) used the substituted cysteine accessibility method to identify pore-lining residues in CaV2.1 and concluded that their results are inconsis- tent with published sequence alignments between Ca2+ and K+ channels. This conclusion casts doubts on homology models of Ca2+ channels. Here we modeled MTSET-substituted Cys mutants (MTSETC) of CaV2.1 using the alignment reported for the Kv1.2-based model of CaV1.2 (Tikhonov & Zhorov, 2008). Energetically favorable orientations of MTSETC were predicted by Monte-Carlo minimizations. In our models, the inner-helix resi- dues in positions i15, i18, i19, and i22 face the pore. In agree- ment with this, MTSET blocks CaV2.1 with Cys substitutions in positions i15 and i19 of all four domains. In our models, Ci22 of any domain is surrounded by large hydrophobic residues, which would prevent reaction with MTSET. This explains why MTSET does not block channels with Ci22 in any domain. According to calculations, MTSETC2i18 and MTSETC4i18 occlude the inner pore, whereas MTSETC1i18 and MTSETC3i18 protrude into domain inter- faces. This explains why MTSET blocks C2i18 and C4i18, but not C1i18 or C3i18. the outer-helix Intriguingly, MTSET blocks mutants C2o10 and C4o10, but not C3o10. These observations also in whichMTSETC2o10 and MTSETC4o10 agree with our models, extend toward the inner pore, whereas access of MTSETC3o10 to the inner pore is prevented by F3i18. Our calculations validate the reported alignment between Ca2+ and K+ channels and suggest similar dispositions of transmembrane helices in these channels. Supported by CIHR.

P2–12 Partial purification, characterization, molecular cloning, recombinant expression and immunocytochemical localization of a beta-1,3-glucanase from the midgut of Spodoptera frugiperda larvae I. Bragatto1, F. Ariel Genta2, W. Ribeiro Terra1 and C. Ferreira1 1University of Sao Paulo Institute of Chemistry, Biochemistry, Sao Paulo, BRAZIL, 2Instituto Oswaldo Cruz FIOCRUZ Brazil, Dbbm, Sao Paulo, BRAZIL

laminarin, enzyme has the

P2–14 CV-IIL lectin: an ideal helper for understanding mutation-caused binding preference changes in the PA-IIL lectin family L. Brunova1, J. Adam1, M. Pokorna1 and M. Wimmerova2 1National Centre for Biomolecular Research, Masaryk University, Brno, CZECH REPUBLIC, 2National Centre for Biomolecular Research and Department of Biochemistry, Masaryk University, Brno, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Lectins are sugar-binding proteins that play important role in rec- ognition processes. Bacterial lectins could be crucial agents in the b-1,3-glucanases hydrolyze b-1,3-glucans from fungal or plant cell walls and are little known in insects. b-1,3-glucanase activity is similar along the midgut of S. frugiperda. The purified enzyme (SLAM) has a molecular mass of 37 kDa and optimum pH of kcat = 8.72 s-1, 9.0. Using Km = 0.14 ± 0.004% (w/v). SLAM does not signicantly form small products, hence being an endo-b-1,3-glucanase, with multi- ple attack degree of 0.4. A cDNA that codes for a protein similar to those in family 16 glucanase was cloned from a midgut cDNA library. The protein produced in Escherichia coli BL21-DE3 cells using a pET28-A expression system, although inactive, was used

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Results and Conclusions: The change in the fluorescence prop- erties of FA bound to the hidrofobic moiety of BSA upon the binding of PAA let us think about the change in conformation of BSA. Experiment will continue to examine this effect at differ- ent pH values.

P2–16 Investigation of in-vivo beta-lactamase inhibition by beta-lactamase-inhibitor-protein based peptides N. Budeyri1, E. O. Olmez2 and B. S. Akbulut3 1Bioengineering, Marmara University, Istanbul, TURKEY, 2Chemical Engineering, Bogazici University, Istanbul, TURKEY, 3Biongineering, Marmara University, Istanbul, TURKEY

process of colonization and in the bacterial virulence by binding to specific saccharide moieties at host cells´ surface. Understanding the nature of the interaction can be helpful in designing anti-adhe- sion therapy. CV-IIL is a lectin from human opportunistic patho- gen Chromobacterium violaceum and belongs to PA-IIL lectin family, which includes structurally similar lectins from several other pathogenic bacteria. The interaction between CV-IIL and the saccharide is mediated by two calcium ions in the binding site. The specificity of the interaction is directed by the composition of the binding site, namely a triad of amino acids called ‘specificity- binding loop’ in the positions 22-23-24. Opposed to other members of the PA-IIL family that prefer either mannose or fucose as a binding partner, CV-IIL lectin could recognize both sugars with high affinity and specificity (1). This feature makes CV-IIL an ideal candidate for investigating the change of sugar preference in either direction upon amino acid mutations. Specific mutations in bind- ing site were prepared by in vitro mutagenesis method. Surface plasmon resonance was used to characterize their binding prop- erty. The in silico mutagenesis method (complemented with molec- ular docking) offers additional insight into the structural reasoning behind the sugar preference. Acknowledgements: This work is supported by Ministry of Education of the Czech Republic (MSM0021622413, LC06030). Reference: 1. Pokorna´ , M. et al., Unusual Entropy-Driven Affinity of Chro- mobacterium violaceum lectin CV-IIL toward Fucose and Man- nose. Biochemistry 2006; 45(24): 7501–7510.

P2–15 Interaction of bovine serum albumin- polyelectrolyte followed by a novel polarity sensor Y. Budama Battal1, Z. Mustafaeva1, S. Ercelen2 and A. P. Demchenko3 1Bioengineering Department, Yildiz Technical University, Istanbul, TURKEY, 2Tubitak Marmara Research Center, Genetic Engineer- ing and Biotechnology Institute, Kocaeli, TURKEY, 3A.V. Palladin Institute of Biochemistry, Kiev, UKRAINE

Beta-Lactamase-Inhibitor-Protein (BLIP) is an effective inhibitor of class A beta-lactamases such as TEM-1, SHV-1 and SME-1 that render bacteria resistant to beta-lactam antibiotics. Due to the resistance of beta-lactamase developed against small organic inhibitors, beta-lactamase inhibition by BLIP is an interesting research field for peptide based inhibitor development. In-vitro studies have shown that small peptides derived from BLIP have the potential to be used as inhibitors (1,2). However there has yet been no report on the in-vivo behavior of these possible therapeu- tic agents against beta-lactamase inhibition. It is well documented that large biotinylated peptides can readily be transported into gram (-) bacteria such as E. coli (3). In this study, this tool has been used to investigate the in-vivo inhibition potential of two N- terminal biotinylated BLIP based peptides of different lengths [Biotin-NH2-AAGDYY-COOH (residues 46-51BLIP) and Biotin- NH2-CETGGSFGDSIHCRGHAAGD-COOH (residues 30- 49BLIP)]. Escherichia coli K12 strain harboring the pUC18 plas- mid that carries the gene of RTEM-1 beta-lactamase, was used for periplasmic beta-lactamase production. Following a predeter- mined time of incubation of the cells in the presence and absence of the biotinylated peptides, samples were taken to count colony forming units. In addition to this, osmotic shock fluids from both cultures were assayed for beta-lactamase activity using CENTA as the substrate. The results obtained were used to evaluate effec- tiveness of this method for peptide transport and potential of the two peptides for in-vivo beta-lactamase inhibition. References: 1. Rudgers et al., Protein Eng. 2001; 14(7) 487–492. 2. Rudgers et al., Antimicrob. Agents Chemother. 2001; 45(12) 3279–3286. 3. Walker et al., Appl. Environ. Microbiol. 2005; 71(4) 1850–1855.

P2–17 Allozymic variations in the genus dryomys (rodentia: gliridae) distributed in Turkey S. Bulut1, N. Yigit2, E. Colak2, R. Colak2, P. Cam3 and F. Saygili2 1Faculty of Science-Art, Biology, Corum, TURKEY, 2Faculty of Science, Biology, Ankara, TURKEY, 3Faculty of Science-Art, Biology, Sinop, TURKEY

3-hydroxychromone derivative

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Allozymic variations were analyzed in two species representing three populations of Dryomys nitedula from Central Anatolia, Turkish Thrace and the Black Sea region of Turkey and one population of Dryomys laniger, is endemic mammal species for Turkey, from the southern part of Turkey. The genetic analysis was carried out on 36 specimens on 17 enzyme systems. Nine of twenty loci (a-Gpdh, Me, Idh-1, G6pdh, Sod, Ca-2, Acon, Mpi, Pgm) were found to be polymorphic in D. nitedula populations Introduction: Serum albumin is the most abundant of all pro- teins in blood plasma of many species, where it reaches a concen- tration of about 40 mg/ml. Biological and pharmacokinetic functions of albumin are very important. It is the major trans- porter in the blood of non-esterified fatty acids as well as of many other low-polar metabolites and drugs. In our previous studies, the complex formation of Bovine Serum Albumin (BSA) with anionic polyelectrolyte (polyacrylic acid) in aqueous solution was examined using the turbidimetric titration, HPLC and fluo- rescence methods. It is revealed that the character of the interac- tions and solubility of the polycomplex particles depends on the protein/polyelectrolyte ratios and the pH of solution. Methods: The polycomplexes have been examined using UV- VIS Spectrophotometer, Steady-State Flourescence Spectrometer. Two seperate methods were used to determine the conformation of BSA in BSA-PAA complexes by adding prob, which is the new ratiometric 2-(6-diet- hylaminobenzo[b]furan-2-yl)-3-hydroxychromone (FA) displays a dramatic solvent-dependent transformation of fluorescence spec- tra in the range of low-polar envoriment. In the first method, BSA-PAA complexes were formed and examined under different ratios and pH values by adding equal amount of FA probe. In the second method, different ratios of polyelectrolyte solutions were added to BSA-FA complexes and examined under different pH values.

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and five polymorphic loci (Idh-1, Sod, Fum, Pgm ve Mpi) in D. laniger. Ldh and Gpi loci were fixed in the different alleles between D. nitedula and D. laniger. The percentage of polymor- phic loci varies from 25 to 40 in D. nitedula populations and is 25 in D. laniger population. The mean value of the fixation index was FST = 0.13 indicating moderate/high genetic differences between the subpopulations of D. nitedula. Nei’s measure of genetic distance varied from D = 0.006 to D = 0.030 between the subpopulations of D. nitedula and the highest D value appeared between the Black Sea subpopulation of D. nitedula and D. laniger (D = 0.187).

starting from monomers until

P2–18 Tracking of single A-beta oligomers on the plasmamembrane reveals an heterogeneous dynamic behaviour M. Calamai1, M. Zanni2, F. Chiti3 and F. Pavone1 1Lens – European Laboratory for Non-linear Spectroscopy, University of Florence, Sesto Fiorentino (Florence), ITALY, 2Department of Chemistry, University of Wisconsin, Madison, WI, USA, 3Department of Biochemistry, University of Florence, Florence, ITALY

located at microtubule-organizing centers. The majority of cen- trin in the cell is non-centrosomal, whose function is not yet clear. Two centrins, BeCen1 and BeCen3, from the aquatic fungi Blastocladiella emersonii were overexpressed, purified and charac- terized by CD, AFM and SAXS. Both proteins showed CD spectra compatible with alpha helix content and exhibited a self- assembly capacity depending on Calcium presence and tempera- ture. The transitional temperature (Tm) of 42(cid:2)C for BeCen1 and 49(cid:2)C for BeCen3 had an increase of 5(cid:2)C in Calcium presence. After denaturation process (90(cid:2)C), only BeCen1 showed a refold capacity (submitted manuscript). Recent results showed that both centrins are in soluble form below 35(cid:2)C (with or without Cal- cium) and above this temperature, the formation of insoluble organized filaments happens in calcium presence. The drastic conformational changes as a function of temperature in the Cal- cium presence (from monomeric, oligomeric and filaments forms) were identified by AFM and SAXS. AFM images of BeCen1 and BeCen3, deposited on mica slice, showed organized filaments at different temperatures. Measurements of SAXS showed the oligo- merization process, trimmers between 20 and 35(cid:2)C, above these temperatures the oligomers are formed, in the absence and presence of Calcium ions. Acknowledgement: Supported by Brazilian agencies: FAPESP, CNPq, CAPES.

laterally diffuse on the membrane

P2–20 The allosteric modulation of heme-serum albumin M. Coletta1,2, A. Bocedi3, C. Ciaccio1, A. Di Masi4, F. P. Nicoletti5, G. Fanali6, G. De Sanctis7, G. Smulevich5, M. Fasano6 and P. Ascenzi4 1Department of Experimental Medicine and Biochemical Sciences, University of Roma Tor Vergata, Roma, ITALY, 2Interuniversity Consortium for the Research on Chemistry of Metals in Biological Systems, Bari, ITALY, 3Department of Molecular Genetics and Microbiology, Duke University, Durham, NC, USA, 4Department of Biology, University of Roma Tre and National Institute for Infectious Diseases I.R.C.C.S. ‘Lazzaro Spallanzani’, Roma, ITALY, 5Department of Chemistry and Interuniversity Consortium for Science and Technology of Materials, University of Firenze, Sesto Fiorentino (FI), ITALY, 6Department of Structural and Functional Biology and Center of Neuroscience, University of Insubria, Busto Arsizio (VA), ITALY, 7Department of Molecular, Cellular and Animal Biology, University of Camerino, Camerino (MC), ITALY

Alzheimer’s disease (AD) is a neurodegenerative pathology char- acterized by the extracellular deposition in the brain of fibrillar aggregates consisting predominantly of A-beta peptide. Recent evidence points at early prefibrillar A-beta oligomers, rather than mature amyloid fibrils, as main cause for neurotoxicity. A-beta oligomers interact with the cellular membrane, but the specific manner through which they bind and cause neuronal dysfunction is still a matter of debate. Distinct results indicate that oligomers may insert non-specifically into the lipid bilayer, or bind to spe- cific targets, such as excitatory post-synaptic locations and gan- gliosides characteristic of lipid rafts. In general, most of these studies have been focused on the averaged features of an ensem- ble of molecules. Here, we have been able to successfully monitor the mobility of single A-beta oligomers on the plasmamembrane of living NRK fibroblasts. Preformed oligomers were incubated with cells and subsequently labelled with monoclonal primary antibodies and secondary Fab fragments coupled to small (10– 30 nm) and extremely photostable fluorescent probes called ‘quantum dots’ (QDs). Single QDs bound to the oligomers were then tracked. The analysis of the trajectories shows that the olig- omers display an heterogeneous dynamic behaviour. Some oligo- mers following a free Brownian motion, while others show a highly confined mobility. The latter result, although preliminary, suggests a potential inter- action of the oligomers with molecules linked to the cytoskeleton. The full characterization of the membrane dynamics of A-beta oligomers will enable a better understanding of AD and thus contribute to the development of new therapies.

P2–19 AFM and SAXS Structural Analises of two Centrins from Fungi Blastocladiella emersonii A. I. Camargo1, P. C. Camargo2, L. Barbosa3, R. Itri3 and L. Beltramini1 1Instituto de Fisica de Sao Carlos, Biophysics, Universidade de Sao Paulo, Sao Carlos, BRAZIL, 2Physics, Universidade Federal do Parana, Curitiba, BRAZIL, 3Biophysics, Universidade de Sao Paulo, Sao Paulo, BRAZIL

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Centrins are EF-Hand calcium binding protein required for duplication of centrioles. It may also play a role in severing of microtubules by causing calcium-mediated contraction, usually Human serum albumin (HSA) is the most abundant protein pres- ent in the bloodstream (reaching a plasmatic concentration of 0.7 mM) and it is characterized by an extraordinary ligand bind- ing capacity for several natural compounds, such as metabolites, drugs and hormones. It displays seven binding sites for fatty acids, one of which shows a very high tendency to bind heme. Heme-HSA, is able to bind gaseous ligands, such as O2 and CO, in the Fe (II) form and oxygen and nitrogen radicals, such as peroxynitrite, in the Fe (III) form. Furthermore, this binding pro- cess can be modulated by the allosteric interaction between the heme site and the six additional binding sites. Therefore, the occupancy of one of these sites can alter the reactivity of the heme. We have investigated in details the allosteric effect induced by two drugs (namely warfarin and ibuprofen) on the reactive properties of heme-HSA toward CO in the Fe (II) form (by war- farin) and toward peroxynitrite in the Fe (III) form (by ibupro- fen). This study allowed us to characterize most of the kinetic parameters for the binding of both heme ligands to heme-HSA and of drugs to their relative sites. These measurements have

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been carried out as a function of drugs concentration, rendering possible a quantitative description of the energetics associated to the allosteric interactions. Parameters obtained from this study turn out to be important for the knowledge of reciprocal interac- tions between different molecules bound to HSA, unraveling the possibility of interference between different ligands present in the bloodstream.

P2–21 Human scFv derived from a naive library that target gp41 are strong candidates to inhibit HIV-1 fusion process A. Couto and J. Goncalves Instituto de Medicina Molecular, URIA-CPM, Lisboa, PORTUGAL

addition of another methyl group in this highly evolutionary con- served region in the ribosome could have its biological cost. We investigated how the presence of the Sgm enzyme affects the exponential growth of E. coli cells in various media and tempera- ture conditions and the ability of bacteria to compete with the cells not expressing the enzyme. To see if m7G1405 methylation influences the ribosome assembly, we compared ribosomal pro- files of cells carrying Sgm methyltransferase with the wild type cells, both in the presence and the absence of kanamycin or gen- tamicin. Our results revealed that E. coli cells expressing Sgm enzyme grow slightly slower compared with the non expressing cells. Moreover, cells without Sgm outcompeted Sgm expressing cells when grown together, suggesting that the presence of the Sgm enzyme is advantageous only in the presence of antibiotics. Since the ribosome assembly was not influenced by the presence of Sgm enzyme, we propose that the biological cost of m7G1405 methylation in the ribosomal A-site could be caused by the hin- drance of the protein synthesis process.

P2–23 Structural and functional features of post- translationally modified cytochrome c, a bifunctional protein I. Diaz-Moreno1, J. M. Garcia-Heredia1, A. Diaz-Quintana1, P. Nieto2, M. Orzaez3, E. Perez-Paya3 and M. A. De la Rosa1 1Institute of Plant Biochemistry and Photosynthesis, Sevilla, SPAIN, 2Institute of Chemical Research, Sevilla, SPAIN, 3Centro de Investigacio´n Prı´ncipe Felipe, Valencia, SPAIN

The fusion process of the Human Immunodeficiency Virus type 1 (HIV-1), is mediated by the gp120 surface protein and the gp41 transmembrane protein. The gp120-CD4 interaction initiates this process, and induces conformational changes in gp41 that result in an active conformation of the fusion peptide (fusogenic confor- mation) promoting the fusion of the cellular and viral membranes. Since the fusion process depends on the conformational changes of the gp41 ectodomain, by interfering with this process, it is pos- sible to block viral entry into the cell. Thus the aim of this study is to select single chain antibodies (scFv) against epitopes present in the gp41 ectodomain critical to gp41 conformational changes, with the purpose of blocking HIV-1 fusion, and consequently inhibiting HIV-1 infectivity. In this work we have selected by phage display technology, single chain antibodies (scFv) against the gp41 ectodomain. These scFv were selected from a human scFv phage library obtained from HIV non-immunized donors. The inhibition of HIV-1infectivity is being tested in vitro using scFv that presented good expression levels and a strong binding to gp41. The scFv molecules that present higher inhibition of HIV-1 infectivity, revealed a high prevalence of Ser and Tyr in the CDR1 and CDR3 particularly in the light chain. These scFv were subjected to affinity maturation strategies restricted to ran- domization of only four amino acids, Ser, Tyr, Ala and Asp. These results suggest that this scFv anti-gp41 represent a promis- ing alternative in the development of new fusion inhibitors.

P2–22 Biological cost of the 16S rRNA modification conferred by aminoglycoside resistance methyltransferase Sgm S. Cubrilo, F. Babic and G. M. Vlahovicek Faculty of Pharmacy and Biochemistry, Department of Biochemis- try and Molecular Biology, University of Zagreb, Zagreb, CROATIA

the A-site of Cytochrome c (Cc) is a small soluble hemeprotein located in the mitochondrial inter-membrane space, where it serves as a mobile carrier in the respiratory chain shuttling electrons between the membrane-embedded complexes cytochrome bc1 and cytochrome c oxidase (1). In addition to its well-established role in energy metabolism, regulation of programmed cell death (PCD) has emerged as a second major function of Cc. Early events in PCD involve the release of Cc from mitochondria to the cytoplasm so as to trigger the assembly of the apoptosome by interacting with the Apoptotic protein-activating factor (Apaf-1) and to activate the cysteine-aspartic acid proteases (caspases) that finally leads to cell death (2). The function of Cc in cell signalling seems to be regulated by post-translational modification of the hemeprotein. Actually, one of the most common effects of the so-called RNOS (Reactive Nitrogen and Oxygen Species), which are being pro- duced in mitochondria, is protein nitration (3), with respiratory Cc – in particular, its five tyrosine residues – being an outstand- ing target for the RNOS (4). To determine the specific effect of tyrosine nitration on the structure-function relations of human Cc, we have produced a set of monotyrosine Cc mutants – in which all the tyrosine residues but one are replaced by phenylala- nines (5). Using both the nitrated and non-nitrated species of WT and mutant Cc, a biophysical approach has been performed by combining NMR and CD techniques with theoretical MD cal- culations. In addition, the impairment of procaspase-9 activation depending on Cc and Apaf-1 interaction has been investigated as a function of specific nitration of Cc tyrosines. References: 1. Maneg et al., Biochim. Biophys. Acta 2004; 1655: 274–281. 2. Kim et al., Proc. Natl Acad. Sci. USA 2005; 102: 16545–17550. 3. Aulak et al., Am. J. Physiol. Heart Circ. Physiol. 2004; 286: H30–H38.

4. Batthya´ ny et al., Biochemistry 2005; 44: 8038–8046. 5. Rodrı´ guez-Rolda´ n et al., Biochemistry 2008; 47: 12371–12379.

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Sgm methyltransferase from actinomycete Micromonospora zion- ensis is a member of the Arm family of enzymes that confer high level resistance to aminoglycoside antibiotics. Their mode of action is modification of the A-site of the ribosome by methyla- tion of the nucleotide G1405 at the position N-7. Structure and function of the ribosome is highly conserved throughout all three kingdoms of life and so are the nucleotide sequence and posttranscriptional modification content of the 16S rRNA contained within. Nucleotide G1405 is located in the upper part of helix 44 in 16S rRNA, in the decoding center of the ribosome. In the vicinity of G1405, there are several nucleo- tides modified by housekeeping methyltransferases, such as m4Cm1402, m5C1407 and m3U1498. Here we propose that the

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PON1 enzyme was purified to homogeneity using a new purifi- cation approach different from the literature, and for the first time the kinetic properties of the purified enzyme were investi- gated using phenylacetate and homocysteine thiolactone as substrates.

P2–24 Purification of Human Serum Paraoxonase 1 and Investigation of Its Kinetic Properties A. Bayrak, T. Bayrak, E. Demirpence and K. Kilinc Department of Biochemistry, Hacettepe University Faculty of Medicine, Ankara, TURKEY

P2–26 Spectroscopic investigation of protein-anionic polyelectrolyte complexes and conjugates S. Derman, K. Kizilbey, B. Mansuroglu and Z. Akdeste Bioengineering, Yildiz Technical University, Istanbul, TURKEY

Human serum paraoxonase 1 (PON1) is a high-density lipopro- tein (HDL)-associated, calcium-dependent enzyme. In addition to its arylesterase, lactonase and organophosphate hydrolyzing activities, PON1 is capable of hydrolyzing oxidized lipids. Many attempts have been made to purify serum PON1, however, purifi- cation to homogeneity has proven difficult. In this study we aimed to purify the human serum PON1, and to study its kinetic properties. Human serum PON1 was purified by ammonium sul- phate precipitation, Sephacryl S300HR gel filtration chromatog- raphy, DEAE ion-exchange chromatography, Cibacron Blue 3GA nonspecific affinity chromatography and a second DEAE ion-exchange chromatography, consecutively. The purity was controlled by using SDS-PAGE and the specificity of the PON1 protein band was shown by Western blotting. The activity of PON1 was measured spectrophotometrically using both paraoxon (paraoxonase activity) and phenylacetate (arylesterase activity) as substrates. PON1 was purified to homogeneity and obtained as a single band at SDS-PAGE. Purified PON1 had a specific activity of 1168 U/mg protein when paraoxon was used as a substrate with a purification fold of 590. When phenylacetate was used as a substrate, purified PON1 had a specific activity of 970 U/mg protein with a purification fold of 795. The purified enzyme had Km values of 0.8±0.06 and 1.025 ± 0.078 mM, and Vm values of 1380 ± 75 micromol/min/mg protein and 1256 ± 68 micro- mol/min/mg protein for the substrates paraoxon and phenylace- tate, respectively.

Conjugates of natural and synthetic macromolecules seem to be of great importance for medicine and biotechnology in particular with respect to drug delivery, to immobilize enzymes. Synthetic polyelectrolytes (PE) have been widely used to modify proteins via covalent attachment, increasing (or reducing) the immunore- activity and/or immunogenecity of originally antigenic proteins, and improving their in vivo stability with prolonged clearance times (1). Water-soluble and insoluble protein-polyelectrolyte complexes as functional biopolymer systems represent a specific class of polymer-protein compounds that have important applica- tions in various areas (2). The water-insoluble Protein-PE poly- complexes exhibited pH-dependent solubility and could be transformed to soluble state and form water-soluble complexes at a pH close to isoelectric point (pI) of the proteins (pIBSA = 4.9) (3). In this study, we investigated the interactions of bovine serum albumin (BSA) with different polymers in component’s dif- ferent molar ratios and pH by turbidimetric methods. From the analysis of the water-soluble and insoluble Protein-PE complexes, it was possible to analyze the mechanism of the polycomplex for- mation and structure of forming particles. In the present study we prepared Protein-PE compelxes and conjugates at different molar ratio (nProtein/nPE = 1,3,5,7,9,11 etc) and different pH (4,5,6,7). This complexes and conjugates characterized and inves- tigated by using Ultraviolet Spectrophotometer (at 280 and 500 nm). References: 1. Dilgimen AS, Mustafaeva Z, Demchenko M, Kaneko T, Os- ada Y & Mustafaev M. Biomaterials 2001; 22: 2383.

P2–25 Purification of Rabbit liver paraoxonase 1 and investigation of its kinetic properties T. Bayrak, A. Bayrak, E. Demirpence and K. Kilinc Department of Biochemistry, Hacettepe University Faculty of Medicine, Ankara, TURKEY

2. Akkilic N, Mustafaeva Z & Mustafaev M. High Performance Liquid Chromatography Study of Water-Soluble Complexes and Covalent Conjugates of Polyacrylic Acid with Bovine Serum Albumin. J. Appl. Polym. Sci. 2007; 105: 3108–3120. 3. Topuzogullari M, Cimen NS, Mustafaeva Z & Mustafaev M. Molecular-weight distribution and structural transformation in water-soluble complexes of poly(acrylic acid) and bovine serum albumin. Eur. Polym. J. 43, 2007; 43: 2935–2946.

and homocysteine thiolactone as

electrophoresis poliacrylamide gel

P2–27 Immunogenic Properties of VP1 Protein’s Peptide Epitops of of Foot-and-Mouth Disease Virus (FMDV) Covalent Conjugates With Anionic N-Vinylpyrolidone Copolymers B. Mansuroglu, S. Derman and Z. M. Akdeste Bioengineering, Yildiz Technical University, Istanbul, TURKEY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Synthetic peptides are small molecules thus they are not strong immunogens, because of this they coupled with a high molecular carriers such as BSA, KLH or different polymers. Proteins and synthetic polyelectrolytes (PE) and have been used to modify syn- thetic peptides via covalent attachment, increasing the immunore- activity and/or immunogenecity of originally antigenic proteins and improving their in-vivo stability with prolonged clearance Human paraoxonase 1 (PON1) is synthesized in the liver and secreted into theblood, where it is associated exclusively with HDL. Unlike serum PON1, the structural and kinetic proper- ties of the liver enzyme are not well defined. In this study we aimed to purify the rabbit liver paraoxonase, and to study its kinetic properties. Rabbit liver PON1 was purified through the preparation of liver microsomal fraction, Sephacryl S300 HR gel filtration chromatography, DEAE Trisacryl M ion exchange chromatography, hydroxyapatite chromatography and a second chromatography steps. DEAE Trisacryl M ion exchange the puried enzyme were studied using Kinetic properties of phenylacetate substrates. Using this method, rabbit liver PON1 was purified 668 times with a specific activity of 3160 U/mg protein. Sodium dodecyl (SDS-PAGE) sulphate revealed the purified enzyme as a single protein band close to 40 kDa. The specificity of the PON1 protein band was shown by Western blotting. The Km of the purified enzyme was found as 0.55 ± 0.024 mM for phenylacetate at 25(cid:2)C, in 50 mM Tris–HCl buffer (pH: 8) containing 1 mM Ca+2. For homocy- steine thiolactone substrate, the Km of the purified enzyme was found as 17.31 ± 1.2 mM at 37(cid:2)C, in 50 mM Hepes buffer (pH: 7.4) containing 1 mM Ca+2. In this study, rabbit liver

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2. Akkilic N, Mustafaeva Z & Mustafaev M. High Performance Liquid Chromatography Study of Water-Soluble Complexes and Covalent Conjugates of Polyacrylic Acid with Bovine Serum Albumin. J. Appl. Polym. Sci. 2007; 105: 3108–3120. 3. Topuzogullari M, Cimen NS, Mustafaeva Z & Mustafaev M. Molecular-weight distribution and structural transformation in water-soluble complexes of poly(acrylic acid) and bovine serum albumin. Eur. Polym. J. 2007; 43: 2935–2946.

P2–29 Phosphorylation-mediated unfolding of a KH domain regulates KSRP localization via 14-3-3 binding I. Dı´ az-Moreno1, D. Hollingworth2, T. A. Frenkiel2, G. Kelly2, S. Martin2, S. Howell2, M. F. Garcı´ a-Mayoral2, R. Gherzi3, P. Briata3 and A. Ramos2 1Institute of Plant Biochemistry and Photosynthesis, University of Seville-CSIC, Sevilla, SPAIN, 2National Institute for Medical Research, Medical Research Council, London, UK, 3Istituto Nazio- nale per la Ricerca sul Cancro, IST, Genova, ITALY

times. Such conjugates seem to be great importance for medicine and immunobiotechnology in particular with respect to drug delivery and vaccine innovation. Synthetic peptides are promising candidate vaccines for the control of viral diseases. Previous stud- ies with FMD virus have identified fragments of isolated VP1 protein which stimulate antibody production, albeit of poor neu- tralizing activity, or are recognized by anti-virus antibodies. Foot-and-mouth disease virus (FMDV) is an attractive model with which to study the potential of peptide-based synthetic vac- cines. In this study, VP1 protein (135–160 residues) were used as polypeptide antigens and we synthesized covalent conjugates of this synthetic peptide with synthetic copolymer of vinyl pyrroli- done-acrylic acide. We evaluate the immunogenicity of a candi- date containing 135–161 peptide epitops of VP1 protein of FMDV synthetic peptide. The immunogenic properties of the conjugates were also investigated and the relationship between immunogeneticity and structure formation in the solutions is ana- lyzed. A new high immunogenic protein-polymer complex and conjugates with antibody production, processing relatively pro- longed times was obtained. It was obtained that a single immuni- zation of mice with PE-peptide conjugates without classical adjuvant increased the primary and secondary peptide-specific immune response to FMDV.

P2–28 HPLC and UV study of water soluble and insoluble complexes of human serum albumin with copolymer of VP/AA S. Derman, K. Kizilbey and Z. Akdeste Bioengineering, Yildiz Technical University, Istanbul, TURKEY

The AU-rich element (ARE)-mediated mRNA-degradation activ- ity of the RNA binding K-homology splicing regulator protein (KSRP) is regulated by phosphorylation of a serine within its N- terminal KH domain (KH1). In the cell, phosphorylation pro- motes the interaction of KSRP and 14-3-3 protein and impairs the ability of KSRP to promote the degradation of its RNA tar- gets. Here we examine the molecular details of this mechanism. We report that phosphorylation leads to the unfolding of the structurally atypical and unstable KH1, creating a site for 14-3-3 binding. Using this site, 14-3-3 discriminates between phosphory- lated and unphosphorylated KH1, driving the nuclear localiza- tion of KSRP. 14-3-3/KH1 interaction regulates the mRNA- decay activity of KSRP by sequestering the protein in a separate functional pool. This study demonstrates how an mRNA-degra- dation pathway is connected to extracellular signaling networks through the reversible unfolding of a protein domain. Further reading: Dı´ az-Moreno I, Hollingworth D, Frenkiel TA, Kelly G, Martin S, Howell S, Garcı´ a-Mayoral M, Gherzi R, Briata P & Ramos A. Phosphorylation-mediated unfolding of a KH domain regu- lates KSRP localization via 14-3-3 binding. Nat Struct Mol Biol 2009;16: 238–246.

P2–30 Lipase from Bombus terrestris, their substrate specificity and effect of the detergents on their activity J. Dolejsova1, J. Kindl1, M. Mackova2 and M. Zarevucka1 1Institute of Organic Chemistry and Biochemistry, Academy of Sci- ences of the Czech Republic, Infochemicals, Prague 6, CZECH REPUBLIC, 2Department of Biochemistry and Microbiology, Institute of Chemical Technology, Prague 6, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Intermolecular complexes and, in particular, protein containing polycomplexes (PECs) can be considered as a special class of polymeric compounds. Studies of mechanisms of cooperative binding of protein by synthetic polyelectrolytes is of interest for the construction of artificial immunomodulators and immuno- gens, for immobilization of enzymes, elucidation of the mecha- nism of polyelectrolytes physiological activity (1). The formation of some water soluble and insoluble complexes of poly acrylic acid (PAA) with bovine serum albumin was investigated by sedi- mentation analysis, turbidimetric titration, fluorescence, GPC and UV spectroscopy in neutral aqueous media (2–3). In this study we used high pressure liquid chromatography (HPLC) to investigate the complex formation when N-vinyl-2-pyrrolidone- co-acrylic acid (VP/AA) reacts with Human serum albumin (HSA) in water media at different pH value and we also used UV Spectrometer to for Optic density analysis. It also illustrates the use of HPLC and UV Spectrophotometer to facilitate under- standing the formation mechanism of polymer-protein complexes (1). The water-insoluble HSA-VP/AA complexes exhibited pH- dependent solubility and could be transformed to soluble state and form water-soluble complexes at a pH close to isoelectric point (pI) of the proteins (3). In the present study we prepared HSA-VP/AA complexes at different molar ratio (nHSA/nPE) of components and at different pH value. This complexes character- ized by using Ultraviolet Spectrophotometer, High Pressure Liquid Chromatography (HPLC) and from the analysis results it was possible to analyze the formation mechanism and structure of complexes. References: 1. Tasdelen BD & Bayulken S. HPLC investigation of water sol- uble ternary polyacrylic acid-Cu2+-protein complexes. Polym. Adv. Technol. 2004; 15: 716–719. Lipases are ubiquitously produced by animals, plants, and micro- organisms. Recently, microbial lipases have been widely studied and used as a catalyst for the biosynthesis and in different bio- technological applications. In contrast to the microbial lipases and the lipases from insects, their properties and importance are poorly investigated area. The lipases from the labial gland, the midgut and the fat body of Bombus terrestris were isolated and their catalytic properties studied. The enzyme activity was moni- tored spectrophotometrically by measuring released p-nitrophenol from p-nitrophenol palmitate. The maximum activity was

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(MCOs)

observed in the case of labial gland lipase of 2 and 12 day old individuals. The lipase activity of the midgut and the fat body was on the same level in the whole tested period (from 1 to 15- day-old individuals). The specificities of the lipases were tested by hydrolysis p-nitrophenyl esters with different chain lengths (C8– C18). The enzymes showed the strongest specificities to short- chain fatty acids esters (C8 and C10). The lipases have a strong tendency towards aggregation. The effect of different concentra- tions of detergents Tween 20, Tween 80 and Triton X-100 on the enzyme stability and activity was determined. The highest activity of lipases was observed at the lower concentration of the used detergents (0.05% v/v). Triton X-100 was strong inhibitor of the lipase from the midgut in both used concentrations. On the other hand, Triton X-100 showed an activating effect on the lipase from labial gland. The effects of the detergents showed different rates of lipase inhibition at the same conditions probably because of different type of the lipases. Acknowledgement: The study was supported by the Czech Sci- ence Foundation GA CR 203/09/1446.

gen to water: the copper- and heme-containing terminal oxidases (EC 1.9.3.X) and the multicopper oxidases (EC 1.10.3.X). Laccases are the simplest members of MCO that are characterized by having three distinct types of copper sites, namely type 1 (T1), type 2 (T2) and type 3 (T3). The function of the T1 Cu site is to shuttle electrons from substrates to the T2/ T3 trinuclear centre where molecular oxygen is reduced to water. Mutations at the E498 residue, located within the entrance chan- nel to the trinuclear copper center of the CotA laccase, have been studied by spectroscopic and kinetic analysis to evaluate its role in the mechanism of dioxygen reduction. Replacement of gluta- mate by aspartate allows the enzyme to retain its catalytic activ- ity at levels close to those exhibited by wild type, while the absence of an acidic group at that position as in E498L and E498T mutants result in enzymes with severe catalytic impair- ment. Furthermore, these latter mutants present decreased values of affinity for dioxygen, in contrast with mutant E498D that pre- sents values similar to the wild-type enzyme. Overall, our results strongly suggest that E498 plays a critical role during the cata- lytic mechanism of CotA-laccase by promoting the binding of di- oxygen to the trinuclear cluster and, importantly, by supplying protons to the initial reaction intermediates of dioxygen reduc- tion to water.

P2–31 Effect of ADP-actin filaments on the ATPase activity of two myosin S1 isoforms R. Dudas, T. Kupi, J. Orban, M. Nyitrai and G. Hild Department of Biophysics, University of Pecs, Pecs, HUNGARY

P2–33 L-type pyruvate kinase is non-allosteric enzyme, which is converted into allosteric enzyme through phosphorylation I. Faustova and J. Ja¨ rv Institute of Chemistry, University of Tartu, Tartu, ESTONIA

The basis of muscle contraction is the sliding of the thick fila- ments on thin filaments, which is achieved by the action of the actomyosin complexes. Thin filaments are actin filaments with bound actin-binding proteins. Actin filaments are built up from ATP-actin monomers. Under ATP-deficient conditions (fatigue, ischemia) filaments are formed from ADP-actin monomers. The structure of the actin monomers is nucleotide dependent, and structural differences were also observed between the filaments formed by ADP-binding actin monomers and those constructed from ATP-binding monomers. Myosin is one of the most impor- tant actin-binding proteins. The number of cleaved ATP mole- cules in unit time (ATPase activity of myosin) describes the effectivity of the actomyosin complex. It was shown previously that the cardiac isoform of myosin subfragment-1 (S1) has a lower ATPase activity compared to the skeletal isoform. The AT- Pase activity of S1 can be increased by actin filaments. This acti- vation may depend on the actual conformation of the filaments. We investigated the effect of filaments polymerized from ADP- actin monomers on the ATPase activity of both skeletal and car- diac muscle myosin subfragment-1. Our results confirmed that the basal ATPase activity of the cardiac S1 was lower than that of the skeletal S1. Actin increases the ATPase activity of cardiac S1 to a lower extent than in the case of skeletal S1. The activa- tion of the S1 ATPase activity by the filaments from ADP-actin and ATP-actin monomers was similar. No difference was found between the activation by skeletal and cardiac actin isoforms.

P2–32 Evidences for the role of glutamate 498 as a proton donor in the reduction of dioxygen by CotA-laccase P. Durao, Z. Chen, M. M. Pereira, S. Todorovic, I. Bento, P. F. Lindley and L. O. Martins ITQB, MET group, Lisboa, PORTUGAL

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Four pyruvate kinase isoenzymes are found in mammals: M1, M2, R and L. If these isoenzymes are classified according with their allosteric properties, only the M1 type is considered as non- allosteric enzyme, as it shows hyperbolic kinetics toward PEP and is not subject to allosteric control by FBP. However we have revealed that allosteric properties are also missing in non-phos- phorylated L-type pyruvate kinase, which shows hyperbolic dependence of reaction rate upon PEP concentration. The non- phosphorylated enzyme was obtained by overexpression of cDNA of rat L-type pyruvate kinase in E. coli. As these bacterial cells do not have mammalian protein kinases, which phosphory- late this enzyme in liver tissue, the non-phosphorylated protein can be isolated from this expression system. Thus the list of non- allosteric pyruvate kinases should be supplemented with the non- phosphorylated form of L-type pyruvate kinase. The non-phos- phorylated enzyme can be turned into allosteric enzyme through phosphorylation of its subunits at Ser12 residue of their N-termi- nal domain. The catalytic properties of this phosphorylated enzyme matched well with properties of this enzyme extracted from liver tissue (1). However, more importantly, the possibility of switching between non-allosteric and allosteric forms of this enzyme by phosphorylation provides new possibilities for investi- gation into the mechanism of allostery and more specifically, its regulation by protein phosphorylation. For example, phosphory- lation of the first subunit in the tetrameric molecule of L-type pyruvate kinase seems to be sufficient for engaging the allosteric mechanisms in this multimeric protein, while further phosphory- lation of subunits only modulates this effect. In the same way the enzyme affinity for substrate (PEP) seems to be controlled. These mechanisms may have some importance for understanding of the regulatory phoaphorylation phenomena in general. Reference: 1. Faustova I & Ja¨ rv J. Kinetic analysis of cooperativity of phos- phorylated l-type pyruvate kinase. Proc Estonian Acad Sci Chem 2006;55: 179–189. The reduction of dioxygen is a key step in biological processes and yet the catalytic mechanisms employed are not fully under- stood at the current time. There are only two enzyme classes that include members catalyzing the four-electron reduction of dioxy-

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P2–34 Molecular and catalytic properties of novel nitrate reductase from nitrate-reducing haloalkaliphilic bacterium Thioalkalivibrio nitratireducens A. Filimonenkov1, T. Tikhonova2, R. Zvyagilskaya3 and V. Popov2 1Biological Oxidation Laboratory, A.N. Bach Institute of Biochem- istry RAS, Moscow, RUSSIA, 2Enzyme Engineering Laboratory, A.N. Bach Institute of Biochemistry RAS, Moscow, RUSSIA, 3Biological Oxydation Laboratory, A.N. Bach Institute of Biochemistry RAS, Moscow, RUSSIA

obligately nitrate-reducing chemolithoautotrophic

juice. The serum level of these enzymes as well as their zymogens reflects the physiological state of gastric mucosa and can serve as a marker for gastric diseases. The affinity-based separation of pepsins can be achieved using either substrate-derived analogs or the enzyme inhibitors. For the separation of pepsin A and pepsin C we have chosen synthetic heptapeptide containing D-amino acid residues (Val-D-Leu-Pro-Phe-Phe-Val-D-Leu) that differ in its affinity to both enzymes and its analog differing in the substi- tution of one L-phenylalanine residue with L-tyrosine one (Val- D-Leu-Pro-Tyr-Phe-Val-D-Leu). Both heptapeptide inhibitors coupled to Glyoxal agarose magnetic beads (BioScience, USA) were used for the separation of pepsin A and pepsin C. The affinity of porcine pepsin A, rat pepsin C and human samples containing both pepsins to magnetic inhibitor modified beads was investigated. While pepsin A interacted with both inhibitors, pepsin C exhibited an affinity to the analog containing L-tyrosine residue. Acknowledgements: This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (grant MSM 0021620806 and project CEH LC 06044) and by the Czech Science Foundation (grant 203/09/0857).

P2–36 Bordetella adenylate cyclase: translocation into lipid rafts directs toxin endocytosis R. Fiser1, J. Masin2, L. Bumba2, C. Fayolle3, M. Basler2, L. Sadilkova2, J. Cerny4, I. Konopasek1, R. Osicka2, C. Leclerc3 and P. Sebo2 1Faculty of Science Charles University in Prague, Department of Genetics and Microbiology, Prague, CZECH REPUBLIC, 2Institute of Microbiology of the Academy of Sciences, Laboratory of Molecular Biology of Bacterial Pathogens, Prague, CZECH REPUBLIC, 3Institut Pasteur, Unite´ de Re´gulation Immunitaire et Vaccinologie, Paris, FRANCE, 4Faculty of Science Charles University in Prague, Department of Cell Biology, Prague, CZECH REPUBLIC

The and haloalkaliphilic sulfur-oxidizing bacteria Thioalkalivibrio nitratire- ducens isolated from the Egyptian soda lake can grow aerobically with reduced sulfur compounds and anaerobically with nitrate as an electron acceptor. Multiheme cytochrome c nitrite reductase isolated earlier from this microorganism has a number of specific structural features and properties that distinquish it from other known enzymes of this group. Presumably, the unique properties of this enzyme are associated with the extreme living conditions of the bacterium. In the present study nitrate reductase (TvNaR) was isolated from a soluble fraction of the cell extract of the bac- terium Thioalkalivibrio nitratireducens and characterized. The properties and structure of TvNaR were studied by kinetic and spectroscopic (UV and EPR spectroscopy and mass spectrome- try) methods. TvNaR exists in solution as a monomer with a molecular weight of 140 kDa and a homotetramer with a molec- ular weight of about 600 kDa. The monomer contains the mo- lybdopterine cofactor and the [4Fe–4S] cluster, exhibits nitrate and chlorate reductase activity, and is inhibited by azide and cya- nide ions which indicates that this enzyme belongs to the mem- brane respiratory nitrate reductase family (Nar). According to mass-spectrometric data, TvNaR is homologous with the NarG subunit from Halomonas halodenitrificans. However, unlike the classical Nar enzymes, which are localized in the membrane frac- tion and synthesized in anaerobic conditions in the presence of nitrate as the electron acceptor, TvNaR was isolated from a solu- ble fraction and it synthesis was not controlled by oxygen, nitrate, and ammonium, which indicates that TvNaR belongs to dissimilatory periplasmatic nitrate reductases (Nap). Acknowledgement: The work was supported by the grants of RFBR (07-04-01559-a).

P2–35 Immobilization of heptapeptide inhibitors to magnetic particles and their use for the separation of different forms of aspartic proteinases M. Filuszova1, P. Prikryl1, M. Ticha1, M. Ticha2 and Z. Kucerova1 11st Faculty of Medicine Charles University in Prague, Institute of Pathophysiology and CEH, Praha 2, CZECH REPUBLIC, 2Faculty of Science Charles University in Prague, Department of Biochemistry, Praha 2, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Affinity based separation techniques represent and important and useful tool not only for isolation of specific proteins, but also for studying their binding properties and for the resolution of indi- vidual forms of one protein that differ only slightly in binding properties. Combination of these techniques with magnetic-based separations is an advantageous instrument for structural studies and for analytical purposes. Two major groups of aspartate pro- teinases (pepsin A and pepsin C) are present in human gastric The Bordetella adenylate cyclase toxin (CyaA) targets phagocytes expressing the CD11b/CD18 integrin, permeabilizes cell mem- branes via formation of cation-selective pores and delivers into cells adenylate cyclase (AC) enzyme that converts cytosolic ATP into cAMP. The genetically detoxified CyaA is used for highly efficient in vitro and in vivo delivery of foreign CD8+ and CD4+ T-cell epitopes into the MHC class I and class II-dependent anti- gen presentation pathways. Recently, we described a third activ- ity of CyaA that consists in elevating [Ca2+]i in monocytic cells. This requires structural integrity and membrane translocation of the AC domain. We also showed that AC domain translocation- mediated entry of Ca2+ into cells was found to be required for relocalization of the CD11b/CD18 receptor from the bulk of the membrane into cholesterol- and sphingolipid-rich microdomains. In thisour present study, we demonstrate that the structural integrity of hydrophobic pore-forming domain of the CyaA plays a key role in delivery of a CD4+ T-cell MalE epitope into MHC class II antigen presentation pathway. We show that amino acid substitutions in the hydrophobic domain of CyaA, which abolish relocalization of mutated CyaA into lipid microdomains, direct the toxoid into a rapid, clathrin- independent pathway of cell entry. In contrast, the intact CyaA toxoid reaches after transloca- tion to the lipid rafts reaches with some delay an intracellular compartment containing also the clathrin-dependent endocytosis marker transferrin. Despite of completely different pathways and kinetics of internalization, the mutated and intact CyaA toxoids, however, end up in the late endosomal/ lysosomal compartment.

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diminished amount of COX holoenzyme with an altered assembly pattern. Our results argue for importance of Cox5a, Cox4 and Cox6a subunits to COX biogenesis. Acknowledgement: Supported by GACR 303/07/0781 and GAUK 1/2006/R.

P2–37 Cloning and characterization of a La protein that inhibits Glut1 expression J. Fong, Y. H. Cheng and Y. s. Huang Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, TAIWAN

expression of LARP4 gene

P2–39 Hydrolytic cleavage of cytokinin and its derivatives by eukaryotic adenine and adenosine deaminases I. Fre´ bort1, H. Pospı´ silova´ 1, O. Nova´ k2 and N. Kostla´ nova´ 3 1Department of Biochemistry, Palacky University, Olomouc, CZECH REPUBLIC, 2Laboratory of Growth Regulators, Palacky University/Institute of Experimental Botany AS CR, Olomouc, CZECH REPUBLIC, 3National Centre for Biomolecular Research, Masaryk University, Brno, CZECH REPUBLIC

Immunofluorescence analysis

By means of suppressive subtractive hybridization, we found that the (NCBI accession number XM_128090; a La ribonucleoprotein domain-containing protein) was increased in 3T3-L1 adipocytes treated with b-adrenergic agonist isoproterenol for 24 h. The result was confirmed by RT- PCR analysis. LARP4 has a higher expression in preadipocytes than adipocytes and is ubiquitously expressed in various mouse tissues. LARP4 seems to be a highly conserved protein, as com- parison of the protein sequences of mouse LARP4 (NCBI acces- sion number XP_128090) and human LARP4 (NCBI accession number NP_954658) shows a high degree of homology (83% identity). LARP4 gene is expressed in human MCF-7, Ovcar-3 and H1299 cancer cells and MCF-7 cells have a lower expression level than the other two. Similar to 3T3-L1 adipocytes, expres- sion of LARP4 gene in MCF-7 cells can be induced by isoprote- renol. To further characterize LARP4 protein, we cloned LARP4 gene from 3T3-L1 adipocytes and constructed expression vector for FLAG-tagged LARP4 (pFLAG-LARP4) to examine LARP4 distribution in 3T3-L1 adipocytes and MCF-7 cells transfected with this vector. showed that LARP4 was found predominantly in the cytoplasm. In addition, cotransfection of 3T3-L1 or MCF-7 cells with pFLAG-LARP4 and pLuc-GT1/E1/E2, a luciferase reporter under the control of glucose transporter 1 (Glut1) promoter, showed that LARP4 was able to inhibit Glut1 promoter activity induced by various stim- uli. Thus we demonstrate for the first time that LARP4 is an iso- proterenol-inducible La protein that suppresses Glut1 expression.

P2–38 Knock-down of cytochrome c oxidase structural subunits in HEK293 cells D. Fornuskova, L. Stiburek, K. Vinsova and J. Zeman First Faculty of Medicine, Department of Pediatrics, Charles University, Prague 2, CZECH REPUBLIC

obtaining macrocrystals for of

Homogeneous adenine deaminases (EC 3.5.4.2) from the yeast Saccharomyces cerevisiae (AAH1) and Schizosaccharomyces pom- be (SPBC1198.02) and a putative adenosine deaminase (EC 3.5.4.4) fromArabidopsis thaliana (At4g04880) were obtained for the first time as purified recombinant proteins by molecular clon- ing of the corresponding genes and their overexpression in E. coli. The enzymes showed comparable molecular properties to well known mammalian adenosine deaminases, but exhibited lower kcat values. Adenine was the most favored substrate for the yeast enzymes, whereas the plant enzyme showed only very low activities with either adenine, adenosine, AMP or ATP. Interest- ingly, the yeast enzymes also hydrolyzed N6-substituted adenines from the group of plant hormones cytokinins, cleaving them to inosine and the corresponding side-chain amine. The hydrolytic cleavage of synthetic cytokinin 2,6-disubstituted analogues that are used in cancer therapy, such as olomoucine, roscovitine and bohemine, was subsequently found also for a reference sample of human adenosine deaminase (ADA1). ADA1, however, showed a different reaction mechanism than the yeast enzymes, hydrolyzing the compounds to an adenine derivative and a side chain alcohol. The reaction products were identified using reference compounds on HPLC coupled to UV and Q-TOF detectors. The ADA1 activity may constitute the debenzylation metabolic route already described for bohemine and as a consequence, it may compro- mise physiological or therapeutic effect of exogenously applied cytokinin derivatives. All three recombinant proteins were sub- jected to sparse matrix crystallization screenings that revealed optimum conditions the At4g04880 protein. Further screenings and analyses are in progress.

P2–40 Structural and dynamic characterization of hSOUL, a heme-binding protein F. Freire1, A. L. Macedo2, S. S. Aveiro3, M. J. Romao2, A. L. Carvalho2 and B. J. Goodfellow3 1Department of Quı´mica, REQUIMTE, Caparica, PORTUGAL, 2Department of Quı´mica FCT-UNL, REQUIMTE, Caparica, PORTUGAL, 3Department of Quı´mica, CICECO, University Aveiro, Aveiro, PORTUGAL

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

The SOUL protein, a 23 kDa heme-binding protein, was initially identified in the retina and pineal gland of chicken. SOUL is pro- posed to be involved in photoreceptive functions, act as a circa- dian clock, or in the transport of heme to other heme proteins. Recent studies suggest that SOUL is also involved in necrotic cell death by inducing mitochondrial membrane permeability. A pre- Mammalian cytochrome c oxidase (COX), the terminal enzyme of respiratory chain, is a multiprotein complex of approx. 200 kDa composed of 13 subunits. COX assembly within the inner mitochondrial membrane is a sequential and relatively slow process. We used RNA interference in order to down-regulate steady-state levels of three different COX subunits and to analyze an impact of the subunit depletion on COX assembly. We chose Cox5a and Cox4 subunits, both of which enter the initial stage of the process and Cox6a subunit taking a part in a terminal step of COX holocomplex assembly. We constructed 11, 13 and 9 derivatives of pCMV-GIN-ZEO plasmid coding for hairpins tar- geted to different positions of COX5A, COX4I1 and COX6A1 mRNA, respectively. RNAi inducing transcript of pCMV-GIN- ZEO plasmid contains GFP and Neomycin Phosphotransferase coding sequences situated in tandem, upstream of the miR30-like hairpins. The recombinant plasmids were transfected to the HEK293 cells and the stable polyclonal cell populations were selected in a medium containing G418. The depletion of Cox5a, Cox4 and Cox6a was confirmed by SDS-PAGE immunoblot analysis. In selected cells with the lowest residual amounts of Cox4 and Cox6a subunits, transcripts of both tissue-specific iso- forms were quantified by qRT-PCR. In the HEK293 cells with depleted Cox5a, Cox4, and Cox6a subunits, BN-PAGE showed

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with HSA with more power than the same Zn2+ ions. To base on obtained results it can be form the conclusion that dendrimers could not be taken as chelators to remove the zinc ions and is not a good protection factor for human serum albumin before zinc ions.

P2–42 Stereospecificity of the suicide inactivation of tyrosinase. A kinetic study J. R. Acosta-Motos, J. L. Munoz-Munoz, F. Garcia-Molina, M. Garcia-Molina, J. Tudela, F. Garcia-Canovas and J. N. Rodriguez-Lopez Departamento Bioquı´mica y Biologia Molecular A, Universidad de Murcia, Murcia, SPAIN

D > Km

liminary model of the 3D structure of human SOUL (hSOUL) was obtained by molecular replacement, using the NMR struc- ture solution of murine p22HBP (2GOV) (1) as a search model. hSOUL structure superimposed with the solution structure of murine p22HBP shows a conserved overall fold (2). The protein is proposed to become hexameric upon heme binding with a His residue being involved as an axial ligand of the Fe (III) (3). 1H,15N-HSQC experiments were used to follow protein reso- nances shift due to structural alterations upon heme titration and relaxation data was collected to probe dynamic alterations induced by heme binding. Triple resonance techniques were applied to 13C/15N and 2H/ 13C/15N labelled SOUL samples in order to perform spectral assignment and determine the structure of the protein in solution. Dissociation constants for porphyrin binding were determined and found to be in the micromolar range. All these studies will allow us to elucidate the mechanism of heme binding. Acknowledgement: FCT-MCTES (SFRH/BD/30239/2006, PTDC/ QUI/64203/2006). References: 1. Dias J.S. et al., J. Biol. Chem. 2006; 281: 31553–31561. 2. Freire F. et al., Biochem. Biophys. Res. Commun. 2009; submitted. 3. Sato E. et al., Biochemistry 2004; 43: 14189–14198.

P2–41 Fluorescence study of interactions between dendrimer-protein, zinc-protein and dendrimer-zinc complex with protein T. Gabryelak and S. Sekowski Department of General Biophysics, University of Lodz, Lodz, POLAND

We study the suicide inactivation of tyrosinase acting on enantio- mers of o-diphenols. The electronic densities of the carbon atoms in the meta (C-3), d3, and the para (C-4), d4, positions of the benzene ring were determined by 13C-NMR assays. These values are related to the nucleophilic power of the oxygen atom belong- ing to the hydroxyl group and are equal for both enantiomers. So the kinetic properties of these stereoisomers that depend on this nucleophilicity, (catalytic constant) and (maximum inactiva- tion constant), are equal. However, the spatial orientation of the DL > substituents influence the values of Km, so that Km L. From these data, it can be deduced that mushroom tyrosi- Km nase shows stereospecificity in its affinity towards its substrates (Km), but not in the catalysis rate (kcat) or in the inactivation rate, ki. This implies that the partition ratio (r), or number of turnovers that one mol of enzyme must make before being inacti- vated, is the same for each pair of enantiomers. Acknowledgements: This work has been partially supported by grants from several Spanish organizations. Projects BIO2006- 15363 (MICINN, Madrid), BIO-BMC 06/01-0004 (BioCARM, Murcia) and 08856/PI/08 (Fundacion Seneca, CARM, Murcia). Predoctoral fellowships JLMM AP2005-4721 (FPU, MICINN, Madrid) and FGM (Fundacion CajaMurcia, Murcia).

P2–43 Tetrameric restriction enzyme Cfr42I belongs to the GIY-YIG nuclease family G. Gasiunas, G. Sasnaukas, G. Tamulaitis and V. Siksnys Laboratory of protein-DNA interaction, Institute of Biotechnology, Vilnius, LITHUANIA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Dendrimers are highly branched, three dimensional polymers, possess many interesting properties. Zinc ions are very important for correct function of human body but sometimes can be observed the poisoning by zinc. Many works describes applica- tions of dendrimers to remove different metal ions (also zinc). The main purpose of the research was study the interaction between G3.5 PAMAM dendrimer, zinc ions, dendrimer-zinc complex and human serum albumin (HSA). If the effects of den- drimer-zinc complex on the protein conformation will not be sig- nificant G3.5 PAMAM dendrimer could be used as potential chelators for removing zinc ions. The methods used in the experi- ments were the fluorescence measurements. The measured param- eter was quenching of HSA fluorescence descended from tryptophan amino acid and anisotropy of fluorescence. For that purpose we used G3.5 PAMAM dendrimers solution in PBS (5– 100 lM), water solution of Zn(SO4)2 · 7H2O (5–100 lM), PA- MAM G3.5-zinc complex (5–100 lM) and human serum albumin (5 lM). The effect of these compounds were determined on Per- kin-Elmer LS-50B spectrofluorimeter in 37(cid:2)C. The results show that all three ligands interact with human serum albumin. PA- MAM G3.5 dendrimer causing the highest decrease of HSA fluo- rescence. The least toxic are Zn ions even in the highest concentration of these compounds (100 lM). The fluorescence quenching is lower to compare with the same dendrimer or den- drimer-zinc complex. The Stern-Volmer constants obtained for all ligands are: 1.6–0.3 (mmol/l)-1, 0.24–0.001 (mmol/l)-1 and 1.0– 0.03 (mmol/l)-1 for G3.5 PAMAM dendrimer, zinc ions and den- drimer-zinc complex, respectively. The obtained results are good confirmed by anisotropy measurements. From preliminary studies we can conclude that G3.5 PAMAM dendrimers possess an abil- ity to interact with human serum albumin and with zinc ions. On the other hand dendrimer-zinc complex have an ability to interact The GIY-YIG nuclease domain was originally identified in hom- ing endonucleases and enzymes involved in DNA repair and recombination. Despite wide distribution of this domain, bio- chemical and structural studies of GIY-YIG proteins are often hampered by their multi-domain organization and complex func- tional requirements. Many of the GIY-YIG family enzymes are functional as monomers. We show that the Cfr42I restriction endonuclease which belongs to the GIY-YIG family and recog- nizes the symmetric sequence 5¢-CCGC/GG-3¢ (‘/’ indicates the cleavage site) is a tetramer in solution. Moreover, biochemical and kinetic studies demonstrate that the Cfr42I tetramer is cata- lytically active only upon simultaneous binding of two copies of its recognition sequence. In that respect Cfr42I resembles the ho- motetrameric Type IIF restriction enzymes that belong to the dis- tinct PD-(D/E)XK nuclease superfamily. Unlike the PD-(D/ E)XK enzymes, the GIY-YIG nuclease Cfr42I accommodates an extremely wide selection of metal ion cofactors, including Mg2+, Mn2+, Co2+, Zn2+, Ni2+, Cu2+ and Ca2+. To our knowledge, Cfr42I is the first tetrameric GIY-YIG family enzyme. Similar

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structural arrangement and phenotypes displayed by restriction enzymes belonging to the PD-(D/E)XK and GIY-YIG nuclease families point to the functional significance of tetramerization. In order to understand structure-function relationship within Cfr42I restriction enzyme we aim to determine its 3D structure by X-ray crystallography.

P2–44 Molecular basis of D-bifunctional protein deficiency T. Glumoff, M. Malin, L. Pietika¨ inen and K. Hiltunen Biochemistry, University of Oulu, Oulu, FINLAND

knock-down ticks totally lost its hemagglutination activity and the function of both genes is further studied by RNAi. Glycopro- teins and sialic acid (SA) in salivary glands (SG) of the tick Ix- odes ricinus: SG in ticks are of fundamental significance for the pathogen transmission to the host, amongst others. We mapped the glycoproteins of tick SG, potential receptors for the transmit- ted pathogens. We identified SA in glycoproteome of SG as well as a protein binding this carbohydrate. Based on the molecular features of the protein, it is similar to mouse sialoadhesin Siglec- 1. The origin of this protein and its function in SG are further studied. Acknowledgements: Supported by the LC06009 research cen- tre project of the Ministry of Education, Sport and Youth of the CR.

P2–46 PIP2 interacts with cytosolic C-terminal part of the vanilloid receptor TRPV1 L. Grycova, B. Holakovska and J. Teisinger Institute of Physiology AS CR v.v.i., Protein Structure, Prague 4, CZECH REPUBLIC

Structure-function studies and clinical data were used to establish a genotype-phenotype correlation for D-bifunctional protein defi- ciency, a metabolic syndrome resulting from nonfunctional or residually active multifunctional enzyme type 2 (MFE-2) of per- oxisomal fatty acyl beta-oxidation in humans (1). A milder form of the disease associated with expanded life time is apparent in patients carrying certain types of mutations. We are testing mutant MFE-2 variants for their stability and reversal of the sta- bility/folding defect. Methods include urea and guanidinium chlo- ride denaturation monitored with tryptophan fluorescence, thermal stability measured with CD spectroscopy, and chemical or pharmacological chaperone screening. Enzyme activities are determined, and results correlated with the known crystal struc- ture and properties of the wild-type protein. Mutant proteins are also subject to crystallization trials. Mutant proteins under study are T15A, N158D, E232K, R248C and W249G, which based on the available structural information are expected to be rather folding or stability defective than inactivated through substrate binding or catalytic site effects. All of these variants have been expressed as recombinant proteins and purified. Preliminary results on stability have been obtained. Latest results on stability and structural studies will be presented. Reference: 1. Ferdinandusse S, Ylianttila MS, Gloerich J, Koski MK, Oostheim W, Waterham HR, Hiltunen JK, Wanders RJA & Glumoff T. Mutational spectrum of D-Bifunctional protein deficiency and structure-based genotype-phenotype analysis. Am. J. Hum. Genet. 2006; 78: 112–124.

Transient receptor potential vanilloid 1 (TRPV1) plays a role in transducing painful stimuli. Physiological function of this channel is connected with general aspects of channel activity regulation and modulation. Recently has been suggested a role for the mem- brane phospholipid PIP2 as a modulator of transient receptor potential (TRP) channels. Similarly to other TRP channel, PIP2 modulates TRPV1 activity. Despite the PIP2 binding site has been suggested, its exact location and the way whereby PIP2 binding influences the channel activity remain controversial. The aim of our study was to characterize the TRPV1 CT/ PIP2 inter- action and to find the exact location of the putative PIP2 binding site. Recombinant fusion proteins as well as synthesized peptides were prepared and its binding abilities were verified by fluores- cence spectroscopy and the dissociation constants for PIP2 were estimated. We have provided the structural insight to the of C- terminal part of TRPV1 (TRPV1-CT) receptor/ PIP2 interaction using homology modeling and computer ligand docking. More- over our results confirm the ability of previously predicted region in the TRPV1-CT as a possible interaction site to bind PIP2. Acknowledgements: This work was supported by Grant GAAV IAA600110701, GACR 303/07/0915, project (No. H148), Centre of Neurosciences No. LC554 MSMT CR, Research pro- ject No. AV0Z.

P2–45 Glycobiochemistry of ticks, vectors of infectious diseases: carbohydrate-binding proteins and glycans L. Grubhoffer1, O. Hajdusek1, M. Vancova1, J. Sterba1 and N. Rudenko2 1Faculty of Science, University of South Bohemia, Ceske Budejo- vice, CZECH REPUBLIC, 2Biology Centre of ASCR, Institute of Parasitology, Ceske Budejovice, CZECH REPUBLIC

P2–47 Dimer organization and the fructose phosphate catalytic and allosteric sites of mammalian phosphofructokinase E. D. Herna´ ndez, C. Ferreras, J. J. Arago´ n and O. H. Martı´ nez-Costa Facultad de Medicina Universidad Auto´noma de Madrid, Departamento Bioquimica, Madrid, SPAIN

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Carbohydrate-binding molecules of the tick Ixodes ricinus: Ixod- erin A (IxoA) and chitinase 1 (Chix1). The lectin IxoA and enzyme chitinase 1 are potential factors of innate immunity in I. ricinus, especially active against bacterial and fungal infections. Sequence analysis of phage cDNA library revealed complete gene sequences of both IxoA and Chix1, and the expression of these genes in all developmental stages of the tick and in tissues of the adult tick was mapped. The IxoA molecule contains one fibrino- gen domain (FreD). Analysis of the gene for Chix1 confirmed, that the Chix1 molecule contains one domain Glyco-18, which is typical for chitinases, but it lacks the signal sequence and the chi- tin-binding domain. We succesfully knock-down the genes for both IxoA and Chix1 by RNA inhibition. Hemolymph of IxoA evolved after duplication, Mammalian phosphofructokinase fusion and divergence of an ancestral prokaryotic gene. Sequence conservation suggested that the duplicated fructose 6-phosphate catalytic site in the C-terminal half became an allosteric site for the activator fructose 2,6-bisphosphate, and that both sites are shared across the interface between subunits antiparallely ori- ented. The composition of these binding sites and the way in which subunits interact to form the dimer within the tetrameric enzyme have been examined by systematic point mutations to alanine of the amino acid residues of human muscle phospho-

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fructokinase which participation in either site depends on how subunits associate. We found that residues His-199, His-298, Arg-201, and Arg-292 contribute to the catalytic site and not to the allosteric site, as their mutation decreased the affinity for fructose 6-phosphate without affecting the activation by fructose 2,6-bisphosphate or its binding ability. In contrast, residues Arg- 566, Arg-655, and His-661 were identified as critical components of the fructose bisphosphate allosteric site, because of the strong dysfunction of this site elicited by their replacements, with no alteration of the active site. Our results suggest that mammalian PFK subunits associate by facing each terminal half the same domain of the neighbour subunit, so that the two catalytic sites per dimer are restricted to the N-terminus and the two allosteric sites are located in the repeated C-terminus. Additionally, resi- dues were identified as involved in the signal transduction route of the activator fructose 1,6-bisphosphate, and not in that of fructose 2,6-bisphosphate despite binding to the same site. MIC- INN (BFU2008-02364)

tus is the aim of most studies. It is a harmless saprophytic fungus for immunocompetent person, but once its conidia enter the body of immunodeficient human, they can cause severe allergic-like dis- eases. So called invasive aspergillosis harms mainly respiratory tract, but infection of heart, kidneys, brain and other organs has been described as well. Aspergillus leads the cause-of-death list in organ transplantation units and leukaemia treatment centres. The way Aspergillus can attach the epitelials is via sugar-binding pro- teins - lectins. In recently sequenced genome of Aspergillus fumiga- tus (strain Af293), we identified a few lectin-like protein sequences differing in length, putative domain composition and predicted subcellular localization. They are supposed to have low sequence homology and similarity, respectively, what makes them heteroge- nous group of fungi’s binding proteins. Some of them not studied so far are being investigated in our group now by molecular-bio- logical methods, revealing the variability that can hardly be esti- mated according to sequences published in protein databases. Acknowledgements: This project has been supported by Min- istry of Education (MSM0021622413) and Grant Agency (GA/ 303/09/1168) of the Czech Republic.

P2-48 Investigation of calmodulin binding site on C- tail of TRPV2 channel B. Holakovska, L. Grycova, H. Janouskova and J. Teisinger Department of Protein Structures, Institute of Physiology AS CR v.v.i., Prague 4, CZECH REPUBLIC

P2–50 Secreted aspartic proteinases of Candida parapsilosis: analysis of gene expression and catalytic properties O. Hruskova-Heidingsfeldova1, J. Dostal1, F. Majer2, J. Havlikova1, M. Hradilek1 and I. Pichova1 1Institute of Organic Chemistry and Biochemistry AS CR v.v.i., Biochemistry, Prague, CZECH REPUBLIC, 2Charles University in Prague 1st Faculty of Medicine, Institute of Inherited Metabolic Disorders, Prague, CZECH REPUBLIC

TRP (transient receptor potential) channels are calcium- perme- able cation channels with polymodal activation properties. By integrating multiple concomitant stimuli and coupling their activ- ity to downstream cellular signal amplification via calcium per- meation and membrane depolarization, TRP channels appear well adapted to function in cellular sensation, guidance and che- motaxis. Calmodulin (CaM) and other Ca2+ binding proteins are involved in the regulation. In our project we focused on TRPV2 channel. We predicted amino acids on intracellular C-terminus that could be involved in calmodulin binding. We performed pro- tein binding steady-state fluorescent measurement by titrating 30- amino acids peptide labeled with FITC on N-terminus with increasing amounts of CaM and calculated anisotropy and equi- librium dissociation constant of the complex. We mutated amino acids in the calmodulin binding motif of this peptide. We created two single mutants R26 and K28 and one double mutant K8K11 of the 30-amino acid peptide. We performed protein binding measurements and compared the results with the wild type. We proposed a mechanism of calmodulin binding to intracellular TRV2 C-tail by using comparative modeling and docking. Acknowledgements: This work was supported by Grant GAAV IAA600110701, GACR 303/07/0915, project (No. H148), Centre of Neurosciences No. LC554 MSMT CR, Research pro- ject No. AV0Z.

Candida parapsilosis is a human fungal pathogen, associated mainly with opportunistic bloodstream and skin infections. Can- dida pathogenicity is enhanced by several virulence determinants including secretion of aspartic proteinases (Sap). Three genes encoding Saps have been identified in C. parapsilosis: SAPP1, SAPP2 and SAPP3. We analyzed expression of these genes and production the respective proteinases in presence of different nitrogen sources. While the SAPP2 and SAPP3 transcripts were present under all of the conditions tested, expression of SAPP1 was induced only by the presence of exogenous protein as the sole nitrogen source. The concentration of Sapp1p in the medium upon induction was always at least one order of magnitude higher than the concentration of Sapp2p in any medium tested. Sapp3p did not act as a soluble secretory protein; it remained attached to the cell surface. Characterization of purified Sapp1p and Sapp2p showed that Sapp2p has more restricted substrate specificity and significantly lower catalytic activity than Sapp1p. Homology models of Sapp1p and Sapp2p revealed structural motifs that may be responsible for the differences between these two enzymes. Our results indicate that C. parapsilosis secretes a low level of Sapp2p proteinase with narrow substrate specificity and low proteolytic activity under most conditions, while expres- sion and secretion of an abundant amount of catalytically effi- cient Sapp1p is triggered by the presence of exogenous protein. Acknowledgements: This work was supported by the Czech Science Foundation (grant No. 310/09/1945) and by Ministry of Education of the Czech Republic (grant No. LC531).

P2–49 Aspergillus fumigatus host-interacting proteins – potential therapeutic targets J. Houser1, V. Dankova2 and M. Wimmerova1 1National Centre for Biomolecular Research, Masaryk University, Brno, CZECH REPUBLIC, 2Department of Biochemistry, Masaryk University, Brno, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Among the lungs-invading opportunistic eukaryotic pathogens, Aspergillus spp. belongs to the best studied genera in last years. From more than hundred species in this genus, Aspergillus fumiga-

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had been applied.We have used different strategies to confirm the presence of the DNA inside the crystal (3), but only in some cases the DNAs could be seen an electron density that corresponds to a little part of the DNA in the map. To complement and verify this crystallographic results the complex was studied in solution by SAXS and SANS. Different experiments with the truncated and the full length protein, alone and in complex with DNA have been performed at different concentrations, and in the case of SANS, also at different % of D2O (4). References: 1. Heras B & Martin JL. Acta. Cryst. 2005; D61: 1173–1180. 2. Kettenberger H & Cramer P. Acta. Cryst. 2006; D62: 146–150. 3. Strong M, Sawaya MR, Wang, S, Phillips M, Cascio D & Ei-

P2–51 Protein engineering of the horse cytochrome c: mutant proteins as an effective detector for quantitative measurement of superoxide radical generation R. Chertkova1, T. Pepelina1, T. Ostroverkhova2, V. Grivennikova3, A. Vinogradov3, D. Dolgikh1 and M. Kirpichnikov2 1Protein engineering Department, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, RUSSIA, 2Bioengineering Department Biological Faculty, Lomonosov Moscow State Univer- sity, Moscow, RUSSIA, 3Lomonosov Moscow State University, Biochemistry Department Biological Faculty, Moscow, RUSSIA

senberg D. PNAS. 2006; 103: 21: 8060–8065.

P2–53 Structural bases for antagonistic repressor- operator and repressor-antirepressor interactions in a photo-inducible switch in Myxococcus xanthus E. Leon1, G. Navarro-Aviles2, C. M. Santiveri1, C. Gonzalez1, M. Rico1, F. J. Murillo2, M. Elias-Arnanz2, S. Padmanabhan1 and M. A. Jimenez1 1Instituto de Quimica-Fisica Rocasolano, CSIC, Madrid, SPAIN, 2Departamento de Genetica y Microbiologia, Facultad de Biologı´a, Universidad de Murcia, Murcia, SPAIN

ligand for a transcription factor,

reveal that CarA(Nter) adopts

Cytochrome c is one of the key proteins involved in electron transfer and programmed cell death. Chemically modified (partly acetylated) cytochrome c is traditional analytical reagent for the detection of superoxide radical generation. We have constructed a number of horse heart cytochrome c vari- ants with directed substitutions of surface charged amino acid residues both surrounding the heme crevice and located on the opposite side of the molecule. These residues are involved in spe- cific interactions of the cytochrome c with its respiratory chain partners – ubiquinol : cytochrome c reductase (complex III) and cytochrome c oxidase (complex IV). The reactivity of all cyto- chrome c mutants with complex III and IV in rat liver mitoplasts depleted in cytochrome c was investigated. Three mutant proteins with six or eight substitutions possessed essential lower compared with acetylated cytochrome c reactivity in interactions with com- plex III whereas in the reaction with complex IV these proteins were inactive. Their reactions with superoxide radical produced by NADH:ubiquinone reductase (complex I) were studied. The rate of superoxide generation by tightly coupled bovine heart submitochondrial particles was measured as the superoxide dismutase(SOD)-sensitive reduction of cytochrome c. In case of mutant cytochromes with six or eight substitutions the contribu- tion of SOD-sensitive reactions reached 80–90% whereas the reduction of the acetylated cytochrome c under the same condi- tions was significantly lower (about 40%). Three of the engi- neered cytochrome c mutants are most effective detectors forquantitative measurement of superoxide generation.

P2–52 Biochemical, crystallographic and in solution studies of a protein-DNA complex N. Jime´ nez1, A. Rubio1, P. Timmins2, I. Uson1 and M. Sola1 1Instituto de Biologı´a Molecular de Barcelona (IBMB-CSIC), Structural Biology, Barcelona, SPAIN, 2Institut Laue-Langevin, Grenoble, FRANCE

The CarA repressor and its antirepressor CarS regulate expres- sion from PB, a photoinducible promoter that groups all but one of the genes for carotenogenesis in M. xanthus. CarA binding to its operator represses PB in the dark, while light induces produc- tion of CarS that interacts with CarA to derepress PB. CarA con- tains two structural and functional domains. Its C-terminal domain directs dimerization, and though it can bind vitamin B12, a novel its activity is not B12-dependent. The autonomously stable N-terminal domain, CarA(Nter), interacts with both operator and CarS. NMR and site-directed mutagenesis the ‘winged-helix’ fold and conserves the DNA-binding determinants of bacterial MerR-type transcriptional factors. The DNA recog- nition helix is also crucial in CarS-binding, indicating that com- petition between operator DNA and CarS for binding to the same region in CarA underlies regulation at PB. CarS, a highly acidic protein, has no known sequence homologues. The NMR structure of CarS1, a truncated yet functional form of CarS lack- ing the latter´ s 25 C-terminal residues, consists of a five-stranded antiparallel ß-sheet in a ß5-ß1-ß2-ß3-ß4 topology similar to SH3 domains, protein–protein interaction modules in diverse eukary- otic cellular signaling pathways, but less common in prokaryotes. We provide a model for the CarA–CarS1 complex based on anal- yses using NMR, site-directed mutagenesis and peptides encom- passing the CarA recognition helix that reveals the structural basis for antirepression involves occlusion of the DNA recogni- tion element of a repressor through its physical interaction with a DNA-like surface on the antirepressor.

P2–54 The role of conserved arginine of PDI in catalysis A. Karala, A. K. Lappi and L. Ruddock Biochemistry, University of Oulu, Oulu, FINLAND

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

A conserved arginine has been shown to be important for the catalytic function of protein disulfide isomerases (PDIs), specifi- Our project deals with a transcription factor (39 kD) in complex with the DNA that harbours the specific binding sequence. Crys- tallization experiments with a shorter proteolysis-based stable frag- ment yielded small crystals that could be optimised (1). These crystals belong to the trigonal space group P3(2) (cell parameters a = b = 101 A˚ , c = 35.5 A˚ , a = g = 90(cid:2), b = 120(cid:2)) although c axis could change according to the DNA length. They diffract any- sotropically to 2.7 A˚ resolution in a and b axes direction while 3.5 A˚ in the shortest c-axis. As no homology model was available for phase determination by molecular replacement, phases needed to be determined experimentally. Combination of data from differ- ent isomorphous SeMet-labeled crystals resulted in three Se posi- tions whose phases produced interpretable electron-density maps only if the strong anisotropy of the data was corrected (2), and sol- vent correction at low resolution and automatic main-chain tracing

143

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Poster Presentations

P2–56 The adrenodoxin redox chain protein complexes studied by paramagnetic NMR P. Keizers1, B. Mersinli2, W. Reinle2, Y. Hiruma1, F. Hannemann2, R. Bernhardt2, M. Overhand3 and M. Ubbink1 1Protein Chemistry, Leiden Institute of Chemistry, Leiden, THE NETHERLANDS, 2Biochemie, Universita¨t des Saarlandes, Sa- arbru¨cken, GERMANY, 3Bioorganic Synthesis, Leiden Institute of Chemistry, Leiden, THE NETHERLANDS

cally for re-oxidation. The movement of R120 into the catalytic site lowers the normally high pKa of the C-terminal cysteine resi- due and promotes the formation of thiolate at this position. The thiolate can then act as a nucleophile on any mixed disulfide formed by the N-terminal active site cysteine allowing re-oxida- tion and completion of the catalytic cycle. The same mechanism was postulated to play a role in the release of kinetically trapped mixed disulfides doing isomerisation reactions. To test the effect of R120/R461 on the protein oxidase and isomerase activity of PDI, the refolding of bovine pancreatic trypsin inhibitor (BPTI) was followed in the presence of PDI wild-type, PDI R120QR461Q and PDI R120DR461D. Results indicate that the initial rate of oxidation is slower, but the formation of the native conformation with three intramolecular disulfides is as fast or even faster for the R120/R461 mutants of PDI than for the wild- type. The effect of R120/R461 on protein reduction activity of PDI was also analyzed by following the precipitation of the insol- uble B-chain of bovine insulin. The results show that the R120/ R461 mutants of PDI are not as active in the insulin reduction as the wild-type enzyme. The R120/R461 mutants of PDI form also more stable mixed disulfides with BPTI than the wild-type, when the reactions of the reduced BPTI and oxidized PDIs are analyzed in the non-reducing SDS–PAGE.

Cytochrome P450 11A1 (CYP11A1) catalyzes the rate-limiting step in the biosynthesis of steroid hormones in mammals. To support oxygen cleavage and substrate hydroxylation, CYP11A1 requires electrons, which are transferred via a FAD-containing reductase, adrenodoxin reductase (AdR) and an iron–sulfur pro- tein, adrenodoxin (Adx). In this study, the structure of the com- plex between AdR and Adx is studied by paramagnetic NMR. Structures of proteins and their complexes can be refined using such long-range distance and angular restraints obtained from paramagnetic NMR experiments. The introduction of a lantha- nide ion to the protein is a good way to retrieve such paramag- netic restraints. The quality of the restraints obtained depends strongly on the rigidity of the lanthanide attachment. Recently we reported the design, synthesis and application of a novel lan- thanide chelator, CLaNP-5, which is attachable via two surface cysteine residues of a protein. Through its design this probe has conformational restrictions and is therefore well suited to deliver high quality restraints (1). Double cysteine mutants of AdR are labeled with CLaNP-5 ligated to Tm and titrated to 15N2H Adx while recording NMR spectra. From the spectral changes inter- protein distances were calculated and used to model the complex structure. Reference: 1. Keizers PH, Saragliadis A & Hiruma Y, Overhand M & Ubb- ink M. Design, synthesis, and evaluation of a lanthanide che- lating protein probe: CLaNP-5 yields predictable paramagnetic effects independent of environment. J. Am. Chem. Soc. 2008; 130: 14802–14812.

P2–55 Porphyromonas gingivalis adhesins involved in the binding of human plasma kinin-generating proteins J. Karkowska-Kuleta1, A. Sroka2, J. Potempa2, A. Kozik1, M. Rapala-Kozik1, G. Cholewa1 and B. Chruscicka1 1Department of Analytical Biochemistry, Jagiellonian University Faculty of Biochemistry Biophysics and Biotechnology, Krako´w, POLAND, 2Department of Microbiology, Jagiellonian University Faculty of Biochemistry Biophysics and Biotechnology, Krako´w, POLAND

P2–57 Does enolase from rabbit muscle interact with phospholipids bilayer? G. Kijanka, M. Stasiuk and A. Kozubek Department of Lipids and Liposomes, University of Wroclaw Faculty of Biotechnology, Wroclaw, POLAND

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Enolase (EC 4.2.1.11 phosphopyruvate hydrolase) is a glycolityc enzyme which is responsible for the catalysis of 2-phosphoglycer- ate to phosphoenolopyruvate. Mg2+ ions are necessary for eno- lase catalytic activity and stabilization of its dimeric structure. Enolase is a homologue of cristalline (1), which is encoded by the same gene, and plasminogen receptor (1,2). Some recent studies show its interactions with sucelular organelles (3). To determine of enolase interactions with cell membrane, we investigated quenching of tryptophan residues by 2,2,2-Trichloroethanol, Acrylamide and Potassium Iodide in the presence of liposomes composed of phospholipids presented in the cell membrane. Acrylamide and Potassium Iodide are hydrophilic quenchers while Trichloroethanol is a hydrophobic one. We use LUV‘s liposomes composed of egg phosphatidylcholine, dioleoylphosphatidylcho- line, dipalmitoylphosphatidylcholine, and phosphatidylcholine/ phosphatidylserine, phosphatidylcholine/phosphatidylinositol, phosphatidylcholine/phosphatidylethanolamine, phosphatidylcho- line/ phosphatidic acid in molar ratio 4 : 96 and 16 : 84, and Porphyromonas gingivalis is one of the principal microorganisms involved in the initiation and progression of periodontal disease. It is well documented that its major virulence factors, cysteine proteinases (gingipains), cause an overproduction of vascular per- meability-enhancing bradykinin-related peptides, kinins, either through a direct degradation of high molecular mass kininogen (HK) or indirectly, by an activation of plasma prekallikrein (PK). In this work, we investigate an alternative mechanism of kinin-production by this pathogen, triggered by an adsorption of the components of plasma kinin-forming cascade (‘the contact system’), including the coagulation factor XII, PK and HK, on the bacterial cell surface. The most virulent W83 strain of P. gin- givalis bound the contact system proteins with the strength decreasing in the order: FXII >HK>pHPK (Kd = 28, 5 ± 5, 9 nM; 39, 3 ± 12, 7 nM; 69, 6 ± 19, 2 nM, respectively). A com- parison of protein binding to the surface of wild strain and mutants deficient in the major surface adhesins, fimbriae and gin- gipains, indicated that the binding occurred predominantly via the hemagglutinin domains of RgpA (Arg-gingipain) and Kgp (Lys-gingipain) and not through their catalytic domains. On the other hand, the gingipain catalytic activities caused degradations and/or detachments of the adsorbed contact system proteins. The fimbriae were not involved in the binding of factor XII or HK, and only partially contributed to the pHPK adsorption. The abil- ity of bacterial surface proteins to bind and assemble the contact system elements for efficient kinin generation may help the path- ogen to colonize the gingival tissue.

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Abstracts

dioleoylethanolamine/dipalmitoylphosphatidylcholine in molar ratio 70 : 30. The molar ratio lipids/protein used in the experi- ments were 20, 50, 80, 110 and 140. The final range of quencher‘s concentration was from 50 mM to 500 mM for Potassium Iodide and acrylamide and from 5 mM to 50 mM for Trichloroethanol. For each quencher Stern-Volmer plots were prepared. The results clearly show that enolase doesn’t interact with liposomal bilayer in experimental conditions. References: 1. Pancholi V. Mol. Life. Sci. 2001; 58: 902–920. 2. Nakajima K, Hamanoue M, Takemoto N, Hattori T, Kato K & Kohsaka SJ. Neurochem. 1994; 63: 2948–2057.

3. Keller A, Demerieu J, Merkulova T, Geraud G, Cywiner-Golen- zer C, Lucas M & Chatelet FP. Biol. Cell. 2000; 92: 527–535.

fibrils with infectious properties (PrPsc). Several authors proposed the molecular chaperones to be involved into this process. Differ- ent chaperones were shown to promote the formation of PrP aggregates which share some certain characteristics of PrPsc. In current work we demonstrated the de novo formation of beta- structured aggregates of two ovine prion protein allelic variants PrP-VRQ and PrP-ARR in the presence of bacterial chaperonin GroEL without using any denaturants. GroEL was shown to enhance the aggregation of both variants having the strong influ- ence on the aggregate size, but this effect was much higher in the case of VRQ than that observed with ARR variant. The signifi- cant increase of beta-sheet content, observed (by thioflavin T flu- orescence assay) only after the GroEL dependent aggregation in the presence of MgATP, was significantly higher in the case of VRQ. These results might explain the large difference between these allelic variants in their susceptibility to scrapie. Acknowledgements: The work was supported by CNTP (Rus- sia, 02.512.11.2249), RFBR (08-04-00231, 08-08-00540), grant of the President of RF (MC-467.2008.4), NATO (PDD(CP)- (CBP.NR.RIG 982779)).

P2–58 Identification of the strong Ca2+-binding site of human transglutaminase 2 R. Kiraly1, E. Csosz1, T. Kurtan2, K. Szigeti3 and L. Fesus1 1Department of Biochemistry and Molecular Biology, University of Debrecen Medical and Health Science Center, Debrecen, HUNGARY, 2Department of Organic Chemistry, University of Debrecen, Debrecen, HUNGARY, 3Semmelweiss University, Research Group for Membran Biology Hungarian Academy of Sciences, Budapest, HUNGARY

P2–60 Can be matrix metalloproteinases activated by metallothioneins? O. Zitka1, D. Huska1, S. Krizkova1, V. Adam1, J. Kukacka2, R. Prusa2, V. Zizkova3 and R. Kizek1 1Department of Chemistry and Biochemistry, Mendel University of Agriculture and Forestry in Brno, Brno, CZECH REPUBLIC, 2Department of Clinical Biochemistry and Pathobiochemistry, Charles University, Prague, CZECH REPUBLIC, 3Vyzkumny ustav pletarsky, Department of Research and Development, Prague, CZECH REPUBLIC

Tissue transglutaminase (TG2) is a Ca2+-dependent acyltransfer- ase which plays important roles in several biological processes. It has complex structure and enzyme activities including hydrolyses of GTP. The Ca2+-binding sites of TG2 have not been fully char- acterized. Our aim was to study the Ca2+-binding of TG2 using site directed mutagenesis and isothermal titration calorimetry (ITC). Based on homology to Ca2+-binding sites of transglutamin- ase 3 (TG3) multiple mutation were generated at the potential strong Ca2+-binding site (S1). Recombinant TG2 was produced in Escherichia coli in (His)6-tagged form. The results show that TG2 has five weak and one strong Ca2+-binding site. This S1 site can permanently bind Ca2+ based on the comparison of the wild and S1 mutant enzyme using inductive coupled plasma – atomic emis- sion spectroscopy (ICP). The S1 mutant is deficient in trans- glutaminase activity even at high Ca2+ concentrations. 45Ca equilibrium dialysis and ITC have demonstrated decreased Ca2+- binding of the mutants. Circular dichroism spectroscopy, antibody binding test and GTPase activity measurements indicates that the amino acid substitutions do not cause major structural alterations. These results suggest that we identify the strong Ca2+-binding site of TG2 which site play important role in the Ca2+-binding/trans- glutaminase activity and regulation of TG2 function.

Matrix metalloproteinases (MMPs) represent a family of struc- turally related zinc-dependent metallopeptidases. MMPs are able to degrade both majority and minority components of extracellu- lar matrix include the native helix of collagen. These enzymes take part in normal biological processes but also in inflamma- tory, generative, and especially malignant processes. Zinc ion plays structural and regulatory role in MMPs but it is mainly involved directly in the catalytic hydrolysis of peptide or protein substrates. The aim of this work was to use two electrochemical (chronopotentiometric stripping analysis-CPSA and methods Brdicka reaction) for detection of MMP-9. In addition we aimed our attention to study various ways how to activate MMP-9 cleaving collagen as its substrate. Under the optimized conditions the detection limit for MMP-9 (3-signal/noise) was estimated as being 100 pM (CPSA) or 1 nM (Brdicka reaction). The tech- niques were used for study of interactions between MMP-9 and collagen. Due to cleavage of collagen by MMP-9 we were able to detect enhancing signal corresponding to collagen and thus evalu- ate the activity of MMP-9 electrochemically. The cleavage of col- lagen was confirmed by SDS–PAGE and gel electrophoresis on chip. Then, we aimed our attention on testing various ways how to activate MMP-9 including in vitro interaction with zinc-trans- porting-protein metallothionein. We found the zinc(II) ions as the best activator. Acknowledgements: This work was supported by GA AV IAA401990701, Liga proti rakovine (2009) and 2A-1591/122- MPO. References: 1. Kizek R. et al. Anal. Chem. 2001; 73(20): 4801–4807. 2. Huska D. et al.. Electroanalysis 2009; 21(3–5): 536–541.

P2–59 GroEL-mediated de novo formation of beta- structured aggregates of two ovine prion protein allelic variants PrP-VRQ and PrP-ARR G. Kisselev1, I. Naletova2, Y. Shchutskaya3, T. Haertle3 and V. Muronetz2 1School of Bioengineering and Bioinformatics, Moscow State Uni- versity, Moscow, RUSSIA, 2Laboratory of Animal Cell Biochemis- try, Belozersky Institite of Phisico-Chemical Biology of Moscow State University, Moscow, RUSSIA, 3Laboratoire d’Etude des Interactions des Molecules Alimentaires (LEIMA), Institute National de la Recherche Agronomique BIA-FIPL, Nantes, FRANCE

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

The development of prion diseases takes place due to the aggre- gation of prion protein PrP forming insoluble beta-structured

145

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Poster Presentations

P2–61 Adjuvant properties of polyelectrolytes B. Mansuroglu, S. Derman, K. Kizilbey and Z. Mustafaeva Akdeste Bioengineering Department, Yildiz Technical University, Istanbul, TURKEY

ity of anionic copolymers to form complexes with bovine serum albumin depend both on the composition of the copolymers and on the pH of the medium (2). Polymer-Protein complexes are be formed as a consequence of interaction of polyion chain and oppositely charged groups in proteins (3). Binding of polyelectro- lyte to protein is depent on the molar ratio of components, as a result of binding polycomplex (4–5), complex coaservats or amor- ph precipitate can be occours. In this study we aimed to prepare protein-polymer complexes and conjugates of Bovin Serum Albu- min and 1-Vinyl-2-pyrrolidone/acrylic acid (VP/AA) copolymers. We synthesized the conjugates at different molar ratio [for one protein molecule, different ratios of polymer (nprotein/npolymer)] with carbodiimide method and investigated the binding mecha- nism of BSA to polyelectrolyte matrix. Complexes and conju- gates are characterized and the mechanism of the binding process was investigated comparatively with BSA and Polymer by HPLC. Water soluble VP/AA-BSA conjugates occurrence and changes in their structure are determined according to components ratio and pH degree of media. References: 1. Topuzogullary´ M, Cimen NS, Mustafaeva Z & Mustafaev Mehmet. ‘Molecular-weight distribution and structural trans- formation in water-soluble complexes of poly(acrylic acid) and bovine serum albumin’. Euro. Poly. J. 2007; 43: 2935–2946. 2. Kabanov VA, Mustafaev MI, Belova VV & Evdakov VP. ‘2 types of soluble complexes of bovine serum-albumin with polyelectrolytes’. Biophysics 1978; 23: 789–795. 3. Morawetz H & Hughes WLJ. ‘The Interaction of Proteins I. Complexing of Bovine with Synthetic Polyelectrolytes. Serum Albumin’. J. Phys. Chem. 1952; 56: 64–69.

4. Berdick M & Morawetz H. J. Phys. Chem. 1953; 57: 959. 5. Mustafaev MI. Functionally Biopolymer Systems. J. Engg. Nat. Sci. 2004; 4: 1–201.

Synthetic polyelectrolytes (PE) have been widely used to modify proteins via covalent attachment, increasing (or reducing) the immunoreactivity and/or immunogenicity of originally antigenic proteins, and improving their in vivo stability with prolonged clearance times. Besides, the conjugates of PE with individual microbe antigens develop strong protective properties (1) and they can be considered as a new generation of vaccinating com- pounds (2–4). The purpose of this study is to examine the cova- lent binding mechanism of 1-Vinyl-2-pyrrolidone/acrylic acid (VP/AA) copolymer and Bovine Serum Albumin (BSA) mole- cules as carrier with 135–161 peptide sequences of VP1 capsid protein of Foot-and-Mouth Disease Virus (FMDV), causative agent of the economically most important animal viral disease world-wide. The immunogenic properties of water soluble BSA- Peptide and VP/AA-Peptide conjugates were investigated, and specificity of antibodies produced was analyzed. This epitope of Foot-and Mouth Disease virus VP1 protein (135–161 amino acid sequences) were used as a polypeptide antigen. Immunogenic activity of BSA-Peptide and VP/AA-Peptide conjugates were investigated depending on the peptide amount in conjugates per mouse and carrier (synthetic polyelectrolyte or protein molecule) structure. VP/AA-Peptide and BSA-Peptide conjugates revealed very high immunogenicity which are different in regards to the specificity of the antibody produced. The BSA-Peptides conju- gates were able to generate specific antibodies both for 135–161 peptide epitope and BSA. In contrast to VP/AA-Peptide conju- gates system were able to generate only 135–161 peptide epitope antibodies. References: 1. Petrov RV, Mustafaev MI & Norimov AS. Physico-chemical criteria for the construction of articial immunomodulators and immunogens on the bases of polyelectrolyte complexes. Sov. Med. Rev. D. Immunol. 1992; 2(4): 1113.

2. Salman Dilgimen A, Mustafaeva Z, Demchenko M, Kaneko T, Osada Y & Mustafaev M. Water-soluble covalent conjugates of bovine serum albumin with anionic poly(N-isopropyl- acrylamide) and their immunogenicity. Biomaterials 2001; 22: 2383–2392. 3. Kabanov VA. From Synthetic Polyelectrolytes to Polymersub- unit Vaccines. Pure Appl. Chem. 2004; 76(9): 1659–1677.

4. Mustafaev M 2004 Functionally Biopolymer Systems. Sigma, Journal of Engineering and Natural Sciences ISSN: 1304-7191, Yildiz Technical University, Zstanbul, 34349.

P2–63 Structural analysis of extrinsic PsbP protein of PSII from Spinacea oleracea and its interaction with the oxygen-evolving complex J. Kohoutova1, V. Kopecky Jr2, M. Lapkouski1, K. Hofbauerova3, Z. Sovova1, S. Gonza´ lez-Pe´ rezc4, I. Kuta Smatanova5, J. L. Revuelta6, J. B. Arellano4 and R. Ettrich1 1Institute of Systems Biology and Ecology Academy of Sciences of the Czech Republic, Department of Structure and Function of Pro- teins, Nove Hrady, CZECH REPUBLIC, 2Faculty of Mathemat- ics and Physics Charles University, Institute of Physics, Prague, CZECH REPUBLIC, 3Division of Immunology and Gnotobiology, Institute of Microbiology Academy of Sciences of the Czech Republic, Prague, CZECH REPUBLIC, 4Departamento de Estre´s Abio´tico, Instituto de Recursos Naturales y Agrobiologı´a de Salamanca (IRNASA-CSIC), Salamanca, SPAIN, 5Institute of Physical Biology, University of South Bohemia, Nove Hrady, CZECH REPUBLIC, 6Departamento de Microbiologı´a y Gene´tica, Universidad de Salamanca/CSIC, Salamanca, SPAIN

P2–62 HPLC investigations of bovine serum albumin- VP/AA copolymer complexes and conjugates K. Kizilbey, S. Derman, B. Mansuroglu and Z. Mustafaeva Akdeste Bioengineering Department, Yildiz Technical University, Istanbul, TURKEY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Water-soluble and insoluble protein-polyelectrolyte conjugates are important for applications in various areas. Covalent conju- gation of proteins to synthetic polymers improves proteins prop- erties. Polyacrylic acid (PAA) is a well known bioactive polymer membrane bags, ultrafine cellulose fiber surfaces grafted with PAA for enzyme immobilization, PAA-polyvinylpyrrolidone bio- polymeric systems for the treatment of the dry eye (1), etc. Abil- Photosynthesis is the process by which light energy is converted into chemical energy. It takes place in the thylakoid membrane of higher plants, algae and cyanobacteria, where membrane- embedded, pigment-protein PSII complex performs light-driven oxidation of water with concomitant reduction of the plastoqui- none pool. Water splitting is performed in a cluster of four Mn2+ ions located on the lumenal side of photosystem II (PSII) and Ca2+ and Cl- ions are required for optimal activity of this water-oxidase complex. In higher plants function of Ca2+ and Cl- is modulated by the presence of four extrinsic proteins, PsbP, PsbQ, PsbO, and PsbR, at the lumenal surface, the so called oxy-

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Abstracts

P2–65 Conformational dynamics of DNA glycosylases and AP endonucleases in the DNA repair V. Koval, L. Kanajevskaya, N. Kuznetsov, N. Timofeyeva and O. Fedorova Institute of Chemical Biology & Fundamental Medicine, Novosibirsk, RUSSIA

gen-evolving complex. To understand the molecular mechanisms of the oxygen-evolving reaction, an essential prerequisite is the structural knowledge of these proteins and their relative interac- tions. The structure of recombinant protein PsbP of the oxygen- evolving complex from Spinacia oleracea was determined at a 1.98 A resolution by X-ray crystallography and the unresolved parts further refined by Raman and FTIR spectroscopy. Raman spectroscopy gives a unique opportunity to study protein samples in different phases, we report the spectra of PsbP in crystal, solu- tion and as DCDR deposit. For protein preparation the overex- pression system Escherichia coli (BL21DE3) transformed by plasmid DNA with gene PsbP (recombinant HisPsbP) was used to produce protein stable in bisTRIS buffer (pH = 600) in a concentration of 15 mg/ml. A clearer picture about the formation of the oxygen-evolving complex and the location of the extrinsic protein in higher plants is emerging from interaction studies between PsbP and PsbQ both on a theoretical (Protein-protein docking, MD analysis) and experimental level (affinity chroma- topgraphy, AFM, Raman difference spectroscopy). Acknowledgements: Funding by MSMT No: LC 06010.

P2–64 Structure-function aspects of L-fucose binding lectin from Aspergillus fumigatus N. Kostlanova1, J. Komarek2, M. Delia1, J. Houser1 and M. Wimmerova3 1National Centre for Biomolecular Research, Masaryk University, Brno, CZECH REPUBLIC, 2Department of Biochemistry, Masaryk University, Brno, CZECH REPUBLIC, 3Department of Biochemistry and National Centre for Biomolecular Research, Masaryk University, Brno, CZECH REPUBLIC

conformational transitions to detect in the

Understanding the molecular mechanisms of protein-nucleic acids interactions is a primary goal of modern structural biology. Molecular basis of its specificity lies in conformational properties of a protein, its target DNA site, and the changes that ensue as a consequence of their interaction. Reactive oxygen species, includ- ing hydrogen peroxide, superoxide anion radical, hydroxyl radi- cal and singlet oxygen, are formed under conditions of oxidative stress, UV or ionizing irradiation, and during normal cell metab- olism. 7,8-Dihydro-8-oxoguanine (8-oxoG) and AP site are major DNA lesions formed by reactive oxygen species that can result in transversion mutations following replication if left unrepaired. In human cells, the effects of 8-oxoG are counteracted by OGG1, a DNA glycosylase that catalyzes excision of 8-oxoguanine base. These AP sites are then cleaved by the hydrolytic AP endonucle- ase Ape1, which are the most abundant type of activity in many cell types. The main research question in this work is the follow- ing: Is the high specificity of substrate recognition by DNA repair enzyme due to dynamic processes of enzyme and substrate conformational changes and formation of specific transient con- formations of the enzyme-DNA complexes during lesion search and recognition? In this study, we analyzed the substrate specific- ity and the catalytic mechanism of OGG1and Ape1 acting on various DNA substrates using stopped-flow kinetics with fluores- cence detection. Combining data on intrinsic tryptophan fluores- enzyme cence molecule, 2-aminopurine reporter fluorescence and changes in FRET-probe fluorescence to follow DNA dynamics, we defined all dynamic steps and assigned them to catalytic processes. The data obtained allow us to look at DNA repair processes in its dynamic flow and transformation. Acknowledgements: This work was supported by grants from the Russian Foundation of Basic Research (07-04-00191, 08-04- 12211), Russian Ministry of Education and Science (NSh- 652.2008.4.), and the Siberian Branch of the Russian Academy of Sciences (Nos.: 88, 21.22).

protein was expressed

P2–66 Isolation and characterization of plant antimicrobial peptides and small proteins M. Kralova1, T. Neubauerova1, T. Neubauerova2, T. Macek1, T. Macek2, M. Sanda3, M. Mackova1 and M. Mackova2 1Institute of Chemical Technology Prague, Biochemistry and Microbiology, Prague, CZECH REPUBLIC, 2Biochemistry and Microbiology, Institute of Organic Chemistry and Biochemistry AS CR, Prague, CZECH REPUBLIC, 3Mass Spectrometry, Institute of Organic Chemistry and Biochemistry AS CR, Prague, CZECH REPUBLIC

Aspergillus fumigatus is a fungus causes life-threatening infection in immunocompromised patients. The organism can cause rapidly progressive bronchopneumonia that is difficult to diagnostic and treat. The virulence factors of A. fumigatus have been identified but their role in pathogenicity of organism is not clear. The already identified 32 kDa lectin binds to L-fucose (1), a compo- nent of a major blood group H-antigen and thus the lectin may contribute to the pathogenicity and virulence of A. fumigatus. This contribution describes molecular cloning, expression, purifi- cation and preliminary structure-function studies of recombinant protein AFL1, which shows high sequence similarity to fucose- binding lectins AAL from orange peel mushroom Aleuria Auran- tia (2) and RSL from plant pathogen bacterium Ralstonia solana- cearum (3). Recombinant in BL21(DE3)Gold (Stratagene) bacterial host strain and purified on mannose-agarose column. AFL1 in concentration from 10 to 20 mg/ml was used for crystallization screening and some prom- ising conditions have been optimized in order to prepare suitable protein crystal for X-ray crystallography. The thermodynamics parameters of AFL1 binding have been determined by ITC mic- rocalorimetry and further function studies have been supple- mented by determination of sugar specificity by Surface Plasmon Resonance. The detailed description of AFL1 lectin can lead to number applications in diagnostic, therapy and prevention of the A. fumigatus infection. References: 1. Ishimaru, et al. Letter. Clin. Infect. Dis. 1996; 23: 898. 2. Wimmerova, et al. J. Biol. Chem. 2003; 278(29): 27059–27067. 3. Kostlanova, et al. J. Biol. Chem. 2005; 280(30): 27839–27849.

antimicrobial coneflower known for its is

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Decreasing efficiency of antibiotics developed during last century is responsible for efforts of the microbiological research to find new antibiotics and compounds with antimicrobial effect, for example peptide based antibiotics. Tissues of two plants – purple coneflower (Echinacea purpurea) and tomato (Solanum lycopersi- cum) – were chosen as sources for isolation of such peptides. Pur- ple and immunostimulating effects while tomato for comparison due to its wide availability. Extraction of peptides was performed using

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P2–68 Differences in conformational behavior of amyloid beta in solvent with different ionic strength Z. Kriz1, Z. Kristofikova2 and J. Koca1 1National Centre for Biomolecular Research, Masaryk University Fac Sci, Brno, CZECH REPUBLIC, 2Laboratory of biochemistry and brain pathophysiology, Prague Psychiatric Centre, Prague, CZECH REPUBLIC

extraction buffer containing protease inhibitors. Peptides were then precipitated with ammonium sulphate and fractionated using chromatographic methods (SPE column, RP-HPLC) and membrane filtration. Fractions were characterised by electropho- resis (PAGE) and by mass spectrometry MALDI-TOF. The anti- microbial activity of the fractions was screened by diffusion method. Several fractions exhibited inhibitory effects to model microorganisms – bacteria (Bacillus megatherium, Enterococcus faecalis, Escherichia coli, Micrococcus luteus, Pseudomonas aeru- ginosa, Staphylococcus aureus, Streptococcus equi) and fungi (Candida scotii, Fusariumsp., Mucor sp., Trichoderma virens). Crude coneflower extract exhibited antimicrobial activity, but it was lost due to unsuitable processing conditions. Hydrophilic peptide crude fraction isolated from tomato and purified by reverse phase chromatography was analysed by Tricine electro- phoresis. Obtained bands were identified by mass spectrometry MALDI-TOF. The analysis showed presence of glutamate dehy- drogenase (EC 1.4.1.3) with score 30.2 with molecular weight 44 813 Da, cystein proteinase 3 precursor (EC 3.4.2.-) with score 10.1 and 50.3 with molecular weight 38 945 Da, antifungal pro- tein NP24 precursor with score 10.1 with molecular weight 26 646 Da and osmotin-like protein TPM-1 precursor with score 10.1 with molecular weight 25 840 Da. Acknowledgement: Thanks to the grant MSM 6046137305 and grant GACR 522/09/1693.

Introduction: Amyloid b-peptide (Ab) is the major component of plaques found in the brains of Alzheimer’s patients. Among its two predominate forms – Ab(1–40) and Ab(1–42), the latter possesses stronger aggregation and deposition propensity than the former. To explore the conformational preference of Ab(1– 42) in solvent with different ionic strength, molecular dynamics (MD) simulations were performed. Methods: The human Ab(1–42) structure presented in PDB database (code 1Z0Q) was used as starting point for MD simula- tions. The rat structure was prepared from human by mutation of three residues using Modeller software. The input structures were solvated by program SOLVATE and sodium and chloride ions in different concentrations were added to the system. For each system, 50 ns long trajectory was run and stability of the secondary structures of the protein was investigated. Results: Although the differences between the human and rat peptides are only in three residues, they show different conforma- tional behavior in the solvent with different ionic strength. In all cases, the conformationally stable helix core (residues 11–20) was found. Conclusions: The used computational method is able to describe even small differences in behavior of similar peptides. The results are in agreement with experimental data. Acknowledgement: Supported by LC06030, MSM0021622413, GA305/09/0457.

P2–67 Complexes of amyloid beta peptides and of 17beta-hydroxysteroid dehydrogenase type 10 in cerebrospinal fluid Z. Kristofikova1, K. Hegnerova2, M. Bockova2, A. Bartos1, J. Ricny1, D. Ripova1 and J. Homola2 1Prague Psychiatric Centre, Alzheimer Disease Centre, Prague, CZECH REPUBLIC, 2Institute of Photonics and Electronics, Optical Sensors, Prague, CZECH REPUBLIC

P2–69 Investigation of the relationship between Flotillin-1 and APP processing/AD pathogenesis F. L. Kung, T. Y. Liu, L. Chiu and I. T. W. Wang School of Pharmacy, National Taiwan University, Taipei, TAIWAN

It

the Abeta peptide,

Data in literature suggest overexpression of mitochondrial enzyme 17beta-hydroxysteroid dehydrogenase type 10 in the brains of Alzheimer disease patients; however, the changes in the enzyme levels do not seem to be fully specific for this type of dementia. In our earlier experiments, we have evaluated levels of enzyme, free or bound to amyloid beta peptides, in cerebrospinal fluid of people with Alzheimer disease and multiple sclerosis. Total enzyme levels were elevated in Alzheimer disease/multiple sclerosis groups when compared to the controls. The levels of enzyme bound to amyloid beta peptides were small when com- pared to these of total; however, they were elevated only in patients with Alzheimer disease. On the other hand, these results indicated that the more sensitive methods are needed to quantita- tively detect In the study, the above-mentioned complexes. ELISA and optical biosensor based on surface plasmon reso- nance were combined with new antibodies. These methods were used to analyse new samples of people with Alzheimer disease and of the nondemented controls. A comparison of both the approaches was performed. We suggest that the complex of amy- loid beta peptides and of the above-mentioned enzyme in cere- brospinal fluid can be used as a biomarker of Alzheimer disease. Nevertheless, detailed analysis of particular amyloid beta peptide fragments needs to be performed from the point of view of their role in the binding. Acknowledgement: This work was supported by IGA MHCR (project NR/9322-3).

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Alzheimer’s disease (AD), especially the late-onset AD, is a pro- gressive neurodegenerative disorder often associated with elderli- ness. is characterized by insoluble extracellular amyloid plaques and intracellular neurofibrillary tangles (NFTs). The major constituent of amyloid plaque, is derived from the amyloid precursor protein (APP) by two sequential proteolytic cleavages, which also generate the APP intracellular domain (AICD). It has been suggested that AICD may interact with other proteins and the newly formed complexes would then translocate to other cellular locations, including the nucleus, and modulate some cellular processes. The precise cellu- lar function(s) of AICD, however, still remain obscure. We have previously reported the identification of a raft-associated mem- brane protein, flotillin-1, as an AICD-interacting partner. As the accumulation of flotillin-1 in human brain has been suggested to be associated with the progression of AD pathology, it was pro- posed that flotillin-1 may recruit APP to lipid rafts and therefore be involved in the localization and processing of APP. To further understand the roles lipid raft plays in APP processing, a series of experiments were performed. Our preliminary results revealed that phosphorylation at Thr668 of APP interfered with the inter- action between flotillin-1 and AICD, as well as the cellular distri-

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Abstracts

of Education Research Project grants 1M6837805002 and MSM 6046137305. bution of APP. These changes may eventually contribute to the observed shift of APP processing to a more pathogenic pathway.

P2–70 Better binding: stronger allostery in protein kinase A A. Kuznetsov and J. Ja¨ rv Institute of Chemistry, University of Tartu, Tartu, ESTONIA

P2–72 Interaction of DNA topoisomerase I with DNA intermediates and proteins of base excision repair N. Lebedeva, N. Rechkunova and O. Lavrik laboratory of bioorganic chemistry, Institute of chemical biology and fundamental medicine, Novosibirsk, RUSSIA

Protein kinase A (cAMP-dependent protein kinase catalytic sub- unit, EC 2.7.11.11) is a highly dynamic monomeric enzyme that interacts simultaneously with ATP and the phosphorylatable pep- tide or protein substrate. Our kinetic study has revealed that cat- alytic activity of this enzyme is governed by allosteric interaction between binding sites for these substrates, and the principle ‘bet- ter binding: stronger allostery’ was formulated (Kuznetsov & Ja¨ rv, Proc. Est. Acad. Sci. 2008; 57: 247–254). This principle can be expanded to most experimental data, which allow quantitative characterization of the allosteric effect in protein kinase A, and was formalized in terms of linear free-energy relationships. These relationships demonstrate that the allosteric interactions between the enzyme bound ligands: (i) have similar mechanism for differ- ent types of ligands and thus could be a characteristic feature of the protein molecule; (ii) are governed by ligand binding effec- tiveness and through this prameter by ligand structure; (iii) can be inverted from positive allostery in the case of efficiently bind- ing ligands into negative allostery in the case of badly binding compounds and (iv) may significantly contribute to substrate (ligand) binding specificity of the protein. In summary, the out- come of the allosteric regulation in this monomeric enzyme is the same as defined by classical theories for multimeric enzymes: making the enzyme response more efficient if proper ligand binds.

P2–71 HIV-1 integrase interacts with Ubc9, a component of the SUMOylation pathway M. Landova, Z. Knejzlik and T. Ruml Department of Biochemistry and Microbiology, Institute of Chemi- cal Technology Prague, Prague, CZECH REPUBLIC

DNA topoisomerases are involved in multiple cellular processes including replication, transcription and recombination. These enzymes are required for a solution of the topological problems arising in the processes of DNA metabolism and influence on their activity. Topoisomerase I (Top1) performs cleavage and re- ligation of DNA strand within specific cleavable site via the for- mation DNA-protein covalent intermediate between the catalytic tyrosine residue of the enzyme and 32-phosphate of the cleaved strand. Yeast Top1 from Saccharomyces cerevisiae was shown recently to interact with nicked and gapped DNA lacking Top1 specific site. Such structures can originate in cell during the pro- cesses inducing DNA damages or during DNA repair. In the present work the interactions of human and yeast Top1 with DNA structures imitating base excision repair intermediates were studied in details. DNA duplexes containing nick or short gap were designed. To increase selectivity of the protein modification in cellular and nuclear extracts, circular double stranded DNA containing uracyl residues distributed statistically within one strand was synthesized using M13 mp19 phage DNA. DNA structures with single strand breaks were produced by the treat- ment of dUMP containing DNA with purified uracyl DNA gly- cosylase and apurinic/apyrimidinic endonuclease or endogenous enzymes of cellular extracts. Circular DNA was shown to cross- link with cellular extract proteins more selectively than short DNA duplexes. Major modified product in bovine testis nuclear extract was identified as Top1 by immunoprecipitation with poly- clonal antibodies against Top1. The level of Top1 modification decreased when purified poly(ADP-ribose) polymerase (PARP1) was added in the extract. The data obtained suggest that PARP1, one of the main nick sensors in mammalian cells, can compete with Top1 for available single strand breaks in DNA, so inhibits the formation of Top1-DNA suicide adduct. Acknowledgements: This work was supported by a grant from the RFBR (07-04-00389, 09-04-91320), BRHE fellowship (2.2.2.3.10031) and HRJRG-102.

P2–73 A structural understanding of the substrate specificity and reaction mechanism of bacterial aminopeptidase PepS S. Lee1, K. K. Kim2, M. H. Ta2 and H. Park2 1Biological Science, Sungkyunkwan University, Suwon, SOUTH KOREA, 2Molecular Cell Biology School of Medicine, Sungkyunkwan University, Suwon, SOUTH KOREA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Human immunodeficiency virus type-1 (HIV-1) integrase cata- lyzes the integration of the retroviral cDNA into the host genome during early stage of infection. Retroviral cDNA integration results in stable infection and is necessary for efficient expression functions of many proteins of retroviral proteins. Biological including retroviral ones can be altered by their covalent modifi- cation by polypeptide modifiers such as ubiquitin or SUMO. It has been reported that integrase is ubiquitinylated and degraded by the N-end rule pathway by the 26S proteasome and that SU- MOylation of some other retroviral proteins has an important role in retroviral lifecycle. The aim of this study is to investigate the relationship between the HIV-1 integrase and SUMO conju- gation pathway. We have found three potential SUMOylation sites within HIV-1 integrase molecule corresponding to YKXE consensus sequence. The HIV-1 integrase was able to interact with SUMO-conjugating enzyme Ubc9 in GST pull-down assay and in yeast two-hybrid system. However, the SUMOylation of HIV-1 integrase has not been proved yet. Potential SUMOylation of HIV-1 integrase could play a regulatory role in HIV-1 infec- tion or it is possible that HIV-1 integrase is not SUMOylated and its interaction with Ubc9 plays different biological role. Acknowledgement: This work was supported by the Czech Science Foundation grant SCO/06/E001 and the Czech Ministry Aminopeptidases play important roles in diverse physiological pro- cesses such as the cleavage of N-terminal methionine and the cata- bolic turnover of peptides into amino acids. Their substrate specificities must be tightly regulated because unnecessary peptide cleavages may result in the loss of essential cellular functions and cell death. PepS, a key metallopeptidase in protein turnover, shows substrate specificity towards small peptides possessing arginine or aromatic amino acids at N-terminus, however, the mechanisms

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IBMP) and a small hydrophilic ligand. We further demonstrate that IBMP binds ASP2 with two stable alternative conforma- tions inside the ASP2 binding pocket. The 15N NMR relaxation study suggests that significant backbone mobility occurs at the ligand entry site at the millisecond rate, which likely plays a role in the recognition and the uptake/release mechanism of ligands by ASP2. We propose that the broad ligand specificity of GOBPs compared with PBPs is conferred by the cumulative effects of weak non-specific protein/ligand interactions and of enhanced protein internal dynamics at the ligand entry site.

underlying this specificity are not clearly understood. Using the high resolution crystal structures of PepS from Streptococcus pneu- moniae and its complexes with substrate or inhibitors, we were able to elucidate how PepS differentiates its substrates from other func- tional proteins and how it hydrolyzes them. PepS exists as an elon- gated dimer in which each monomer consists of N- and C-terminal domains. Depending on the relative orientation of the two domains, the conformation of PepS can be closed or open. The closed conformation of PepS prevents the substrate entry, but the open conformation accommodates the substrates into its binding pocket which is 20 A˚ deep and 10 A˚ wide. However, PepS cannot be fully activated in the open conformation since the catalytic resi- dues are rearranged into the active configuration only in the closed state. Taken these structural data together, we propose that PepS alternates between open and closed conformations by domain movements during each catalytic cycle; opening for swallowing in the substrate, closing for the hydrolysis of the bound substrate, and re-opening afor the release of the products.

P2–76 Role of the C-terminal region of a recombinant DnaK from Bacillus licheniformis as evaluated by a serial deletion L. L. Lin1, M. G. Lin2, W. C. Liang1 and M. C. Chi1 1Department of Applied chemistry, National Chiayi University, Chiayi, TAIWAN, 2Department of Biochemical Science and Tech- nology, National Chiayi University, Chiayi, TAIWAN

P2–74 Direct binding between D68 and PE in LacY M. Lensink, C. Govaerts and J. M. Ruysschaert 1Structure and Function of Biological Membranes, Universite Libre de Bruxelles, Brussels, BELGIUM

Proton motive force-dependent transport occurs in a number of proteins in the Major Facilitator Superfamily. The lactose trans- porter LacY of Lactococcus lactis uses a proton gradient to transport lactose in and out of the cell. A dual dependence on PE lipids has recently been suggested for this transporter family, when PE was not only found to ensure proper folding in the membrane, but a combination of Asp-68 and a resident hydrogen on the ethanolamine moiety of PE was found to be essential for activity in the multidrug transporter LmrP. Here we use LacY to show a direct interaction between PE and the conserved Asp-68 that requires a two-step process to be broken.

The full-length Bacillus licheniformis DnaK (BlDnaK) and its T86W and three C-terminally truncated mutants were con- structed to evaluate the role of up to C-terminal 255 amino acids of the protein. The specific ATPase activity for BlDnaK, T86W, T86W/?C120, T86W/DC249, and T86W/DC255 was 65.68, 53.21, 116.04, 321.38, and 90.59 nmol Pi/min/mg, respectively. In vivo, dnaK, dnaK/T86Wand dnaK/T86W/DC120 genes allowed an E. coli dnaK756-ts mutant to grow at 44 (cid:2)C. Except for T86W/ DC255, simultaneous addition of B. licheniformis DnaJ and GrpE, and NR-peptide (NRLLLTG) synergistically stimulated the ATPase activity of BlDnaK, T86W, T86W/DC120, and T86W/DC249 by 16.9-, 13.9-, 33.9-, 9.9-fold, respectively. Mea- surement of intrinsic tryptophan fluorescence revealed the signifi- cant alterations of the microenvironment of aromatic amino acids in the C-terminally truncated mutants. The temperature- dependent signal in the far-UV region for T86W was consistent with that ofBlDnaK, but the C-terminally truncated mutant pro- teins showed a higher sensitivity towards temperature-induced denaturation. These results suggest that C-terminal truncations alter the ATPase activity and thermal stability of BlDnaK and induce the conformation change of the ATPase domain.

P2–75 Structural basis of the broad specificity of a general odorant-binding protein from honeybee E. Lescop1, L. Briand2, J. C. Pernollet2 and E. Guittet1 1Institut de Chimie des Substances Naturelles (ICSN-CNRS), Laboratoire de Chimie et Biologie Structurales, Gif-sur-Yvette, FRANCE, 2Institut National de la Recherche Agronomique, Neu- robiologie de l’Olfaction et de la Prise Alimentaire Biochimie de l’Olfaction et de la Gustation UMR1197, Jouy-en-Josas, FRANCE

P2–77 Neisseria meningitidis FrpC protein and its autocatalytic protease activity: characterisation of putative calcium binding site by terbium phosphorescence P. Liskova, R. Fiser and I. Konopasek Department of Genetics and Microbiology, Faculty of Science Charles University in Prague, Prague, CZECH REPUBLIC

in the packing of

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

FrpC protein from RTX protein family is produced by all clinical isolates of Neisseria meningitidis, causative agent of bacterial men- ingitis. Despite its unknown biological function, FrpC was found be highly immunogenic and induces a novel type of calcium- dependent autocatalytic cleavage of the peptide bond between its residues Asp414 and Pro415. After cleavage, FrpC catalyzes a for- mation of isopeptide bond between Asp414 and epsilon-amino group of Lys of another FrpC molecule. The segment responsible for processing is localized between residues 400–657 and shares sequence homology with eukaryotic EF-hand calcium-binding motives (Osicka et al., 2004). In our work we studied the relation- ship between putative calcium binding site conformation and FrpC auto-cleavage activity. For this purpose we used FrpC vari- General odorant-binding proteins (GOBPs) are believed to transport a wide range of volatile hydrophobic molecules across the aqueous sensillum lymph towards olfactory receptors in insects. GOBPs are involved in the first step of odorant recogni- tion, which has a great impact in agriculture and in insect-medi- ated human disease control. We report here the first structural study of a GOBP, the honeybee ASP2, in complex with a small hydrophilic ligand. The overall fold of the NMR structure of ASP2 consists six a-helices creating an internal cavity and closely resembles that of the related phero- mone-binding proteins (PBPs). The predominantly hydrophobic internal cavity of ASP2 provides additional possible interactions (p-stacking, electrostatic contact) for ligand binding. We also show that the internal cavity of ASP2 has the ability to bind ligands of different structures and properties, including a hydrophobic (2-isobutyl-3-methoxypyrazine, component of the floral scent

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P2–79 Structure and binding properties of Burkholderia cenocepacia lectin with dual specificity L. Malinovska1, A. Varrot2, A. Imberty2 and M. Wimmerova3 1National Centre for Biomolecular, Research Masaryk University, Brno, CZECH REPUBLIC, 2Centre National de la Recherche Scientifique, Centre de Recherches sur les Macromolecules Vegetales, Grenoble, FRANCE, 3Department of Biochemistry and National Centre for Biomolecular Research, Masaryk University, Brno, CZECH REPUBLIC

ant without RTX domain (with only first 862 residues), with point substitution D521K in putative calcium binding site. Phosphores- cence of trivalent terbium Tb3+, a calcium binding analogue, was used for determination of calcium binding constants. Tb3+ was also shown to induce FrpC auto-cleavage activity in similar con- centration range as Ca2+. We showed that the binding constants for terbium were higher in case of FrpC variant D521K compared with FrpC without substitution and that the binding kinetics in higher Tb3+ concentrations of the latter was affected by auto- cleavage. The effect of D521K point substitution on the structure of putative calcium binding site was also modelled by ROSETTA 2.3.0 software that allowed Ab initio 3D prediction. Reference: 1. Osicka, R. et al. J. Biol. Chem. 2004; 279: 24944–24956.

P2–78 Phytoecdysteroids new role – regulation of plant enzyme activities T. Macek1, O. Uhlik1, M. Kamlar1, I. Chlubnova2, A. Pisvejcova2, R. Jezek3, J. Harmatha4 and L. Kohout5 1Joint Laboratory of ICT and IOCB, Institute of Organic Chemis- try and Biochemistry Czech Academy of Sciences, Prague, CZECH REPUBLIC, 2Department of Biochemistry and Microbi- ology, Institute of Chemical Technology Prague, Faculty of Food and Biochemical Technology Prague, CZECH REPUBLIC, 3Department of Peptides, Institute of Organic Chemistry and Bio- chemistry CAS, Prague, CZECH REPUBLIC, 4Department of Natural Products, Institute of Organic Chemistry and Biochemistry CAS, Prague, CZECH REPUBLIC, 5Department of Steroids, Institute of Organic Chemistry and Biochemistry CAS, Prague, CZECH REPUBLIC

Gram-negative bacterium Burkholderia cenocepacia is mainly a plant pathogen but it also infects immunocompromised humans, especially cystic fibrosis patients. Chronic pulmonary colonization by opportunistic bacteria is the leading cause of death among individuals suffering from this disease. Bacterial lectins can play a crucial role in the infection development. These carbohydrate- binding proteins can mediate adhesion to host cells and could be important virulence factors. This current work is focused on lec- tin from B. cenocepacia - Burkholderia cenocepacia lectin D (BclD). BclD consists of C-terminal domain with a high homol- ogy to lectin PA-IIL from Pseudomonas aeruginosa and N-termi- nal domain of unknown function. Binding specificity of BclD was determined using biosensor with surface plasmon resonance detection (SPR). Lectin recognises preferentially L-fucose but it also binds D-mannose with high affinity. C-terminal lectin domain (BclDLec) was separated on gene level and, surprisingly, single lectin domain is strictly D-mannose specific as was con- firmed by SPR and glycan array. BclDLec displays high struc- tural homology to another protein from B. cenocepacia, lectin BclA. BclA is also strictly mannose specific and lacking any addi- tional N-terminal domain (1). BclD probably recognizes L-fucose via its N-terminal domain or this domain participates in carbohy- drate-binding. Hypothesis of this dual specificity will be con- firmed after separate expression of N-terminal domain (which appeared to be difficult task) and determination of its carbohy- drate-binding properties. Acknowledgements: This work is supported by Ministry of Education (MSM0021622413) and Grant Agency of Czech Republic (303/09/1168/). Reference: 1. Lameignere E, Malinovska L, et al. Biochem. J. 2008; 411: 307–318.

P2–80 Structure of the Schistosoma mansoni guanidino kinase O. Marcillat1, S. Laurent1, A. Awama1, P. Paracuellos2 and P. Gouet2 1ICBMS-UMR CNRS 5246, Universite´ Lyon 1, Villeurbanne, FRANCE, 2IBCP-UMR CNRS 5086, Universite´ Lyon 1, Villeurbanne, FRANCE

Z40550506 projects to

Bioaffinity chromatography on immobilized steroid ligands revealed that a number of plant proteins exhibit high affinity to some oxysterols (ecdysteroids, brassinosteroids), and that these low molecular weight organic compounds influence biological activity of some proteins. As example can serve the recently described activity increase of the plant ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) by effect of ecdysteroids. We found insect hormones ecdysteroids, which are synthesized in many plant species and their function has not yet been fully understood, to be able to increase the carboxylase activity of iso- lated RuBisCO by more than 10%. This results in the photosyn- thetic yield increase. We tested 20-hydroxyecdysone, ajugasterone C, polypodine B and a mixed ecdysteroid fraction isolated from the roots of Leuzea carthamoides, all with similar impact on the carboxylase activity of RuBisCO (Uhlı´ k et al., 2008). This protein is considered to be the most abundant protein in the world – it makes up 30–50% of the soluble proteins in leaves, for example in spinach leaves, RuBisCO content is about 4 grams per square meter. These large amounts of RuBisCO are necessary since, unlike other enzymes, its catalytic rate is very slow, there- fore, the reaction catalyzed by RuBisCO is accepted as the limit- ing step in photosynthesis. Thus increase of its activity is very important. Acknowledgements: The support of the Czech Ministry of Education and 1M06030, MSM6046137305 is highly acknowledged. Reference: 1. Uhlı´ k, et al. Steroids 2008; 73: 1433–1440.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Guanidino kinases (GK) catalyze the reversible transfer of a phosphoryl group between ATP and a guanidino compound. These enzymes, named according to their specificity towards their guanidino substrate -e.g. creatine kinase-, play a prominent role in energy management in a wide range of eukaryotic cells. Schis- tosoma mansoni (Sm) is the etiological agent of schistosomiasis, a disease that affects about 200 million people worldwide. Sm cercariae express a duplicated GK in which the two domains (1– 363 and 364–716) are very similar (53% identity) and present the characteristic features of the GK family. Crystals of the 80 kDa

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recombinant SmGK (monoclinic space group P21, unit-cell parameters: a = 52.7, b = 122.1, c = 63.2 A˚ and b = 108.5) have been used to collect synchrotron data with a resolution of 2.8 A˚ . SmGK structure has been solved by molecular-replace- ment, using the 357-amino-acid Trypanosoma cruzi arginine kinase as a search model. SmGK, which show taurocyamine kinase activity (120 U/mg), has an original U-shaped structure, in which the C-terminal side of the first domain interacts with the N-terminal side of the second domain. This arrangement dif- fers from the classical banana-shape observed in dimeric and oc- tameric GKs, in which pairs of monomers show a twofold rotation axis. In spite of this difference, each domain shows the characteristic GK fold with a first a-helical sub-domain (about 100 amino acids) and a second larger one (260 amino acids) with an anti-parallel b-sheet and long a-helices

P2–81 Myotropic peptides are predominant in beetles ventral nerve cord – mass spectrometry morphology P. Marciniak1, N. Audsley2 and G. Rosinski1 1Department of Animal Physiology and Development, Adam Mickiewicz University, Poznan, POLAND, 2Enviromental Biology Group, Central Science Laboratory, Sand Hutton York, UK

supported by project catalyze hydroperoxidation of fatty acids by introduction of molecular oxygen into the cis,cis-1,4-pentadiene structure of its substrate. In plants, the hydroperoxide fatty acids are further metabolized into physiologically active lipid-breakdown products. In different plant LOX expression and/or activity are modulated by a variety of stimuli. The objective of this work is to character- ize LOX activity in new varieties of spring malting barley. The goal of breeding was to incorporate of low termostable LOX-1 gene into spring barley genotypes. LOX type and its activity in barley can influence final flavour stability and sensory quality of beer. Twenty different new varieties of spring barley and appro- priate malts were enrolled into this study. Malt samples were pre- pared by standard microprocedure at Malting Institute in Brno. Barley varieties were breeded to at Agrotest fyto Ltd. LOX-1 gene polymorphisms were analyzed by PCR using CAPS markers at LoxA locus 4 h chromosome. LOX activity was measured spectrophotometrically at 234 nm using linoleic acid as substrate. Fatty acids and their derivatives in barley grains and malt were analyzed by GC/MS. Volatile trans-2-nonenal was extracted by HS-SPME. Total antioxidant status of barley and malt was mea- sured by ABTS method. In parent varieties no polymorphisms in LoxA locus were observed, while analysis of offsprings exhibited changes in four varieties. In barley grains one isoform of LOX was found, however, in all malt samples two isoforms (LOX-1 and LOX-2) were present. Analyzed barley varieties differed in LOX-1 activites in range of 17.4 and 33.4 U/mg. Total LOX activity in all malt samples was substantially higher according to barley variety. Substrate specificity of new varietes was similar; li- noleic acid was preferred in all samples. Inhibitory activity of barley and malt extracts to soybean LOX-1 exhibited slight dif- ferences between individual varieties. LOX activtiy was connected with trans-2-nonenal level as well as with total antioxidant status. (including four varieties exhibiting In several new varieties LoxApolymorphism) lower temperature optimum was observed. Results obtained in this study will be used for choice of parent varieties in new series of breeding experiments. Acknowledgements: This work was QH81056 OF the Ministry of Agriculture.

P2–83 Solution structure of EB proteins determined by SAXS R. M. Buey and M. O. Steinmetz Structural Biology, Paul Scherrer Institute, Villigen, SWITZERLAND

In insects neuropeptides are important messenger molecules that regulate developmental, reproductive and behavioural processes. In the past few years large numbers of new neuropeptides has been identified from insects. They can act as transmitters, modu- lators or classical hormones and often exhibit plejotropic func- tions, including myotropic activity. The ventral nerve cord (VNC) together with the brain and the corpus cardiacum/corpus allatum complex comprise the central nervous system in insects. The aim of this study was to isolate and identify neuropeptides from methanolic extracts of the ventral nerve cord of two beetle species Tenebrio molitor and Zophobas atratus using a combina- tion of high performance liquid chromatography (HPLC), matrix assisted time of flight mass spectrometry (MALD-TOF MS) and tandem mass spectrometry (MS/MS). In both beetles at least 10 abundant ion signals were detected, the most predominant mono- isotropic masses (649.5 and 1257.9) correspond to two previously known in insects myotropic peptides proctolin (RYLTP) and leu- comyosuppressin (pEDVDHVFLRFa). Furthermore in Z. atra- tus VNC extract, NVPL-4 (GRWGGFA) was identified, a peptide previously found in Tenebrio castaneum. Direct peptide profiling from dissected single ganglia using MALDI-TOF MS resulted in assigning identified peptides to certain ganglia. The HPLC fractions were also analyzed by a homological semi-iso- lated heart bioassay to screen for potent myotropic activity. The HPLC fraction containing proctolin caused a positive chrono- tropic effect on the heart whereas the fraction corresponding to leucomyosuppressin caused a negative effect. These are the first neuropeptides identified from the ventral nerve cord of beetles.

P2–82 Regulation of lipoxygenase activity in barley and malt I. Marova1, A. Halienova1, R. Mikulikova2 and Z. Svoboda2 1Faculty of Chemistry, Brno University of Technology, Brno, CZECH REPUBLIC, 2Institute of Brewing and Malting, Malting Institute, Brno, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

End binding (EB) proteins are highly conserved core components of microtubule (MT) plus-end tracking protein (+ TIP) net- works. EBs are 60 kDa dimeric proteins composed of mainly two distinct and highly conserved functional modules connected by a long poorly conserved linker: a calponin homology (CH) domain, responsible for MT binding, and a coiled-coil (CC) structural motif, responsible for dimerization and partner binding. Despite the fact that a number of high-resolution structures of the CH and CC domains have been solved, no structural data at all is available for a functional full-length dimeric EB protein. In the present work, we have used Small Angle X-ray Solution Scatter- ing (SAXS) to determine the low-resolution structure of full- length EB proteins. To our best knowledge, our SAXS-derived models represent the first structural information of a full-length functional EB protein, providing insights into the mechanisms of action of these master regulators of + TIP networks. linoleate:oxygen (LOX, Lipoxygenases oxidoreductase, EC1.13.11.1 2) are non-heme iron-containing dioxygenases that

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P2–84 Allosteric inhibition of Saccharomyces cerevisiae aspartate kinase by threonine: mutational analysis and structural organization O. H. Martı´ nez-Costa, V. Sa´ nchez, J. Delgado, A. Gala´ n, C. Hermida and J. J. Arago´ n Facultad de Medicina Universidad Auto´noma de Madrid, Bioquı´mi- ca, Madrid, SPAIN

macrophage single cell patch-clamp we show that a synergic com- bination of substitutions within the pore-forming (E570Q) and acylation-bearing (K860R) domains of CyaA ablates selectively its cell-permeabilizing activity, thus eliminating the residual cyto- lytic activity of the CyaA/AC- toxoids on CD11b+ cells. At the same time, the E570Q + K860R construct retains a full capacity to translocate the AC domain into cell cytosol and its toxoid is fully capable to deliver the reporter OVA CD8+ T cell epitope to the cytosolic pathway of dendritic cells for MHC class I- restricted presentation and induction of specific cytotoxic T cell responses in vivo. This shows that CyaA employs an unprece- dented mechanism of translocation of the AC enzyme domain across cellular membrane that does not involve passage across the cytolytic pore.

P2–86 Characterisation of the D60A mutant of the elongation factor 1a from the archaeon Sulfolobus solfataricus M. Masullo1, I. Ruggiero1, C. Piergiuseppe2, A. Lamberti2, A. Sorrentino2, N. M. Martucci1, A. Ruggiero3, R. Arcone1, L. Vitagliano3 and P. Arcari2 1Dipartimento di Scienze Farmacobiologiche, Universita’ degli Studi ‘Magna Graecia’ di Catanzaro, Roccelletta di Borgia (CZ), ITALY, 2Dipartimento di Biochimica e Biotecnologie Mediche, Universita’ degli Studi di Napoli Federico II, Napoli, ITALY, 3Istituto di Biostrutture e Bioimmagini, Consiglio Nazionale delle Ricerche, Napoli, ITALY

Aspartate kinase (AK) constitutes the first step in the biosynthe- sis of lysine, threonine and methionine. The AK from Saccharo- myces cerevisiae (ScAK) is a monofunctional enzyme and it is regulated through feedback inhibition by the end product threo- nine. ScAK catalytic domain is located at its N-terminus, while the regulatory region containing two ACT subdomains (ACT1 and ACT2) is confined within the C-terminus of the protein. The native enzyme is a homohexamer and threonine promotes an important conformational change on the protein, decreasing its molecular mass from 346 to 280 kDa (1). We have found that the hexameric form of ScAK dissociates into inactive trimers and monomers in the absence of phosphate, being the hexamer stabi- lized by the addition of the substrates, aspartate or ATP. The structural motifs involved in the inhibitory effect and binding of threonine have been investigated by deletions and point muta- tions of the ACT subdomains. Threonine inhibition was abol- ished when either ACT2 or both ACTs were eliminated, and mutation to alanine of residues S390 and S472 (pertaining to ACT1 and ACT2 subdomains, respectively) markedly reduced the action and binding of the inhibitor. These results indicate that the hexamer is the minimal active form of ScAK, which may be organized as a dimer of trimers, and that both ACT1 and ACT2 subdomains are involved in the threonine allosteric inhibi- tion of the enzyme. MEC (BFU2005-09071) and MICINN (BFU2008-02364). Reference: 1. Marina, P. et al. Biochem. Biophys. Res. Commun. 2004; 321: 584–591.

P2–85 Adenylate Cyclase toxin translocates across target cell membrane without forming a pore J. Masin1, A. Osickova1, C. Fayolle2, J. Krusek3, M. Basler1, C. Leclerc2, R. Osicka1 and P. Sebo1 1Institute of Microbiology, Cell and Molecular Microbiology, Prague, CZECH REPUBLIC, 2Institut Pasteur and INSERM, Unite´ de Re´gulation Immunitaire et Vaccinologie, Paris, FRANCE, 3Institute of Physiology, Cellular Neurophysiology, Prague, CZECH REPUBLIC

The elongation factor 1a from the hyperthermophilic archaeon Sulfolobus solfataricus (SsEF-1a) belongs to the family of GTP- binding proteins (GNBP). In the fulfilment of its biological func- tion SsEF-1a switches between an active GTP-bound and an inactive GDP-bound form and elicits an intrinsic GTPase activ- ity. Surprisingly, the 3D structure of SsEF-1A in complex with GDP showed that, in contrast with most of the other GNBP, no magnesium ions have been found in the nucleotide binding site, despite the presence of the cation in the crystallization medium. Furthermore, a conserved aspartic acid (D60 in SsEF-1a), although present in the substrate binding site of the factor, is located more far away with respect to its position occupied in all the other GNBPs with known structure. To study the involve- ment of this amino acid position on the functional properties of SsEF-1a, a mutagenic analysis on this position has been started. In this communication, the results on the biochemical character- ization of a first mutated form of SsEF-1a is reported.The D60A mutant of SsEF-1a, was obtained as heterologous expressed puri- fied protein and characterised. The expression system used pro- duced a folded protein able to catalyse, although to a slightly lower extent with respect to the wild type enzyme, protein synthe- sis in vitro and NaCl-dependent intrinsic GTPase activity. The affinity for guanosine nucleotides and heterologous aa-tRNA was almost identical to that exhibited by wild type SsEF-1a; vice versa, the GDP exchange rate was one order of magnitude faster on the mutated elongation factor, a property partially restored when the exchange reaction was analysed in the presence of the magnesium ions chelating agent EDTA. Finally, the D60A sub- stitution only a little affected the high thermal stability of the elongation factor. From a structural point of view, the analysis of the data reported confirmed that this conserved carboxyl group belongs to a protein region differentiating the GDP bind- ing mode among elongation factors from different organisms.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

The RTX Bordetella adenylate cyclase toxin-hemolysin (CyaA) exhibits two distinct and synergic biological activities that oper- ate simultaneously on CD11b-expressing myeloid phagocytic tar- get cells. CyaA forms cation-selective pores, permeabilizing cell membrane and eventually provoking colloid-osmotic cell lysis. In parallel, CyaA reaches the cytosolic compartment and subverts phagocyte functions by catalyzing unregulated conversion of cytosolic ATP into cAMP. Unlike other enzymatically active tox- ins, CyaA does not depend on receptor-mediated endocytosis and translocates its adenylate cyclase enzyme (AC) directly across the cytoplasmic membrane of target cells. CyaA with ablated AC enzyme activity can be exploited for delivery of inserted passen- ger antigens into the cytosolic MHC Class I pathway of process- ing and presentation on dendritic cells, to trigger vaccinal or immunotherapeutic cytotoxic CD8+ T cell responses. Using

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P2–87 Restoration of glycosylation of alpha- dystroglycan by a glycosyltransferase LARGE in cultured cells K. Matusmura, F. Saito and T. Shimizu Teikyo University, Neurology, Tokyo, JAPAN

ity and influence(s) of particular residue on kinetics and structure of the enzyme (3). That approach, however, has several limita- tions. Therefore we have adopted a strategy of random site direc- ted mutagenesis followed by directed evolution to investigate the functional relationships between amino acid residues. The corner- stone of this procedure is a screening method for glucosidase activity we have just developed. Generally, this work will shed more light on the complex evolution of enzyme substrate interac- tion at the active site. Simultaneously, the ability to modulate specificity of b-glucosidases holds substantial promise in terms of biotechnological applications. Acknowledgments: This project was supported by grants GACR203/02/0865 from the Grant Agency of the Czech Repub- lic, LC06034, LC06010, MSM0021622415 and MSM0021622412 from the Ministry of Education, Youth and Sports of the Czech Republic, and AVOZ50040507 from the Academy of Sciences of the Czech Republic. References: 1. Brzobohaty´ , et al. Release of active cytokinin by a ß-glucosi- dase localized to the maize root meristem. Science 1993; 262: 1051–1054.

2. Kiran, et al. Ectopic over-expression of the maize b-glucosi- dase Zm-p60.1 perturbs cytokinin homeostasis in transgenic tobacco. J. Exp. Bot. 2006; 57(4): 985–96.

3. Dopitova´ , et al. Functional analysis of the aglycone-binding site of the maize b-glucosidase Zm-p60.1. FEBS J. 2008; 275: 6123–6135.

glycosyltransferases including other

P2–89 Electrochemically-driven turnover of tamoxifen by immobilized human flavin-containing monooxygenase R. Meirinhos, S. Sadeghi and G. Gilardi Department of Human and Animal Biology, University of Turin, Turin, ITALY

Dystroglycan (DG) links the extracellular matrix and cytoskele- ton. Alpha-Dystroglycanopathy is a disorder characterized by muscular dystrophy, brain anomaly and eye abnormalities. Muta- tions of known or putative glycosyltransferases, POMGnT1, POMT1, POMT2, fukutin, FKRP and LARGE, were identified and defective glycosylation of alpha- DG has been implicated in the pathogenesis of this disorder. Recently, it was reported that overexpression of Large recover a function of alpha-dystroglycan and bypasses the defective glycosylation of alpha-DG in cultured cells deficient in fukutin, POMGnT1 or POM1. In this study, we characterized glycosylation status of alpha-DG in several cul- tured cell lines and examined the restorative effect of glyco- syltransferases on the defective glycosylation of alpha-DG. We demonstrated that benign tumor or embryonic cell lines such as RT4, HEK293 or C2C12 express alpha-DG detectable by an antibody against sugar chain of alpha-DG, whereas malignant carcinoma cell lines such as HeLa or MCF7 did not. Using an antibody against core protein, alpha-DG with molecular mass of 75 kD was detected in these carcinoma cells, suggesting that functional alpha-DG is lost in carcinoma cells. Next, we exam- ined if the function of alpha-DG is restored in these cells by gene transfer of glycosyltransferases, mutation of which leads to alpha-dystroglycanopathy. Transfection of the cells with LARGE greatly restored the laminin binding activity of alpha-DG, whereas POMGnT1, POMT1, POMT2, fukutin or FKRP did not. These results dem- onstrate that the restoration of functional alpha-DG by LARGE is a possible molecular target when developing therapeutic strate- gies for alpha-dystroglycanopathy.

P2–88 Protein design and directed evolution in understanding maize b-glucosidase Zm.p60.1 structure-function relationships P. Mazura1, R. Dopitova2, J. Damborsky3, T. Filipi1, N. S. Kiran1 and B. Brzobohaty1 1Department of Molecular Biology and Radiobiology, Mendel University of Agriculture and Forestry in Brno, Brno, CZECH REPUBLIC, 2Department of Functional Genomics and Proteomics, Masaryk University, Brno, CZECH REPUBLIC, 3Loschmidt Laboratories, Masaryk University, Brno, CZECH REPUBLIC

Flavin-containing monooxygenases (FMOs) are the second most important family of drug-metabolising enzymes in humans and catalyze the oxygenation of a wide variety of drugs, pesticides and other xenobiotics. They use FAD, NADPH and molecular oxygen for their detoxification reaction. FMO3 appears to be the predomi- nant member in the adult human liver and is associated with the majority of FMO-mediated hepatic metabolism, playing a promi- nent role in the metabolism of clinically important drugs such as the anticancer tamoxifen. The presence of the cofactor FAD in the active site of the FMO3 makes this protein amenable to electro- chemical measurements. The reducing equivalents of NADPH co- factor, necessary for the monooxygenation reaction of hFMO3 in vivo, can be substituted by use of electrodes and application of a controlled potential. The achievement of a direct electron transfer between solid electrodes and the protein on the surface provides a simple and efficient way of coupling enzymatic turnover events with signal transduction. Herein we present two methods for elec- trochemical characterization of human FMO3: non-covalent immobilization on glassy carbon (GC) together with covalent immobilization on gold electrodes. The ability of the immobiliz- edhFMO3 to catalyse the turnover of tamoxifen was investigated by chronoamperometry methods in fully oxygenated buffer where a potential bias of -400 mV (vs. NHE) was applied to the electro- chemical cell with constant stirring.The catalysis products were separated by HPLC and the presence of tamoxifen N-oxide con- firmed the electrocatalytic behaviour of the immobilized enzyme. Finally, the direct correlation between the catalytic current and drug turnover allows us to use these electrodes in drug sensing.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Our study focuses on attempting to modulate enzyme specificity and understanding the functional relationships between key amino acid residues that form the entrance part of the active site. Our model enzyme, the maize b-glucosidase Zm-p60.1, is impor- tant for the regulation of plant development due to its role in the targeted release of active phytohormons (cytokinins) from their inactive storage forms, cytokinin-O-glucosides (1,2). Among the glucosidases involved in plant hormonal metabolism, Zm-p60.1 is the best described. b-glucosidases comprise a highly diverse group of proteins in terms of homologous enzymes, which enables bio- informatics as well as the elucidation of their biological signifi- cance. Methods of rational design in protein engineering are the foundation of our work. By changing amino acid residues at the important spots we study the determination of substrate specific-

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counterparts. In addition, they are less resistant to urea-induced denaturation and more susceptible to proteolytic (subtilisin, V8 protease, trypsin) digestion. The structural basis of the relative instability and the phylogenetic distribution of actin unique to slow skeletal muscle will be discussed.

P2–90 Thioflavin T may influence fibrillation of proteins of different structural properties A. A. Meratan1, A. Es-haghi1, D. Morshedi2, A. Ebrahim-Habibi3 and M. Nemat-Gorgani4 1Institute of Biochemistry and Biophysics, University of Tehran, Tehran, IRAN, 2National Institute of Genetic Engineering and Bio- technology (NIGEB), Industerial and Envionmental Biotechnology, Tehran, IRAN, 3Endocrinology and Metabolism research center, Tehran University of Medical Sciences, Tehran, IRAN, 4Stanford Genome Technology Center, Stanford University, Palo Alto, USA

P2–92 Towards the design of RNases with improved antitumor activity A. Merlino, I. Russo Krauss, C. Ercole, E. Pizzo, A. Vergara, L. Mazzarella and F. Sica Universita di Napoli Federico II, Chemistry, Naples, ITALY

Amyloid fibrils are polymeric structures, containing a cross-b spine, with b-strands perpendicular to the long axis of the fibril. The ability to form amyloid fibrils is a common property of poly- peptides with structural diversity and no sequence similarity, rang- ing from small peptides (amyloid b-peptide, amylin, insulin), through natively unfolded proteins (a-synuclein) to natively folded monomeric proteins (lysozyme) or even protein assemblies (trans- thyretin). In vitro detection of these fibrils is often performed using thioflavin T, a cationic benzothiazole dye, which binds in a specific and regular fashion to amyloid fibrils such that their long axes are parallel, leading to enhancement of fluorescence. In the present study, the effect of different concentrations of thioflavin T (0, 2, 5, 10 and 20 lM) on fibrillation of three model proteins, a-synuclein (natively unfolded), lysozyme (about 15% b-sheet) and insulin (all a-helix) were investigated. Thioflavin T fluorescence and Congo Red absorbance assays were employed to detect amyloid fibril for- mation and secondary structural determination was carried out using circular dichroism spectroscopy. The presence of thioflavin T was found to accelerate the rate of fibrillation of a-synuclein, but reduced fibrillation of lysozyme, in a concentration-dependent manner. However, fibrillation of insulin was not affected by this probe, even at high concentrations. The observed different effects of Thioflavin T on fibrillation of these proteins appears to be due to specific interactions occurring at varying efficiencies and further confirms previous reports suggesting that the morphology of amy- loid fibrils may determine fluorescence of this small molecule in a specific manner.

P2–91 Distribution and some properties of an actin isoform unique to slow skeletal muscle R. C. C. Mercer, W. A. K. A. Mudalige and D. H. Heeley Biochemistry, Memorial Univeristy, St John’s, CANADA

The ribonucleolytic activity of ribonucleases (RNases) provides the potential to use these enzymes as therapeutics for tumor treatment. To unfold their cytotoxicity, RNases have to reach the cytosol of target cells and have to avoid inhibition by the ribonu- clease inhibitor (RI). The antitumor activity of bovine seminal ribonuclease (BS-RNase), which exists in the native state as an equilibrium mixture of a swapped and an unswapped dimer, is a peculiar feature of the swapped isomer and in particular of its non-covalent form (NCD-BS), the species produced by reduction of inter-chain disulfides (1–2). NCD-BS escapes inhibition by RI because it adopts a compact quaternary structure that interferes with binding and, moreover, maintains the proximity of the reduced cysteines that facilitates the reformation of disulfide bonds. The stability of this quaternary shape was attributed to the combined presence of Pro19 and Leu28 (1). Using the homol- ogy mutation approach, few structural features of BS-RNase has been introduced in bovine pancreatic ribonuclease (RNaseA), which does not show antitumor action, with the long term aim of designing a RNase with improved antitumor activity and lower immunogenicity. The introduction of Leu28, Cys31 and Cys32 and, in addition, of Pro19 or the introduction of the hinge pep- tide sequence (residues 16–22) of BS-RNase and the two cysteines in the sequence of RNaseA produced dimeric variants which do exhibit a cytotoxic activity, though at a much lower level than BS-RNase (3–4). The crystal structure analysis of the NCD form of these mutants has revealed that these dimers adopt an opened quaternary structure, that does not hinder the binding of RI. The comparison of these structures with those previously reported for other ribonuclease swapped dimers strongly suggests that the presence of the three residues Gly16, Pro19 and Leu28 may rep- resent a minimal requirement to convert RNaseA in a dimer with the compact quaternary organization observed for NCD-BS (3).Work is now in progress to test this hypothesis. Acknowledgement: Grant Sponsors: FIRB RBNE03B8KK and PRIN2007. References: 1. Sica F, Di Fiore A, Merlino A & Mazzarella L. J. Biol. Chem. 2004; 279: 36753–36760. 2. Merlino A, Ercole C, Picone D, Pizzo E, Mazzarella L & Sica F. J. Mol. Biol. 2008; 376: 427–437.

3. Merlino A, Russo Krauss I, Perillo M, Mattia CA, Ercole C, [Epub Picone D, Vergara A & Sica F. Biopolymers 2009; ahead of print]. DOI:10.1002/bip.21183.

4. Ercole C, Colamarino R, Pizzo E, Fogolari F, Spadaccini R & 2009; DOI: [Epub ahead of print]. Picone D. Biopolymers 10.1002/bip.21176.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Actin is the most conserved of the sarcomeric proteins. It was believed that all skeletal muscles utilise a single actin gene. How- ever, the present work demonstrates the existence of two separate genes coding for skeletal muscle actin in certain fishes. The actin in fast muscle contains three replacements when compared with rab- bit skeletal actin. Conversely, slow muscle actin contains at least ten substitutions when aligned with either of the above, which is a record for a vertebrate muscle actin. The heterogeneity, restricted to sub-domains one and three, includes a number of non-conserva- tive substitutions, most notably at residue 360. Regarding the slow isoform, this residue confers an extra negative charge at neutrality. Electrophoresis has been used to track the distribution of this charge variant in various vertebrates. Absent in some classes (Aves and Amphibia) it has been detected in separate orders in the class Actinopterygii or ray finned fishes, for example Brown trout (Salmo trutta) and Atlantic herring (Clupea harengus). With a sin- gle substitution between them, the sequences of these slow muscles actins are virtually identical. The melting temperature of G-actins (CaATP), as observed by circular dichroism, is 5–10 (cid:2)C lower in the case of the slow muscle actins compared to their fast muscle

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with human proteins looking for a possible use of trout as a suit- able model for human cholesterol transport.

P2–93 The unique primary structure of plant heme oxygenases makes the enzyme function by use of unique key residues C. Migita Biological Chemistry Faculty of Agriculture, Yamaguchi University, Yamaguchi, JAPAN

P2–95 Exploring beta-1,3-Exoglucanase- polysaccharide interactions: molecular dynamics of the enzyme-substrate complex with or without a polysaccharide ligand bound in the flexible enzyme loops S. A. Moura-Tamames, M. J. Ramos and P. Alexandrino Fernades Faculty of Sciences of the University of Porto, Chemistry, Porto, PORTUGAL

Heme oxygenase (HO) converts hemin into biliverdin, CO, and free iron. During successive reactions, two intermediates, alpha-hy- droxyhemin and verdoheme, are generated. The heme degradation by plant HOs has been considered to follow similar mechanism to that of mammalian HOs, though it shows some apparent differ- ences. In particular, the oxidation state of the verdoheme-HO com- plexes is different. To address this problem, we applied EPR on the reaction mixtures of the heme degradation by HOs. In soybean heme oxygenase isoform-1 (GmHO1) reactions carried out in the presence of NADPH/FNR/Fd, a low-spin ferric intermediate was detected, which was undetectable in ratHO1 reactions in the pres- ence of NADPH/CPR, suggesting the verdoheme in the six-coordi- nate ferric state in GmHO1. Optical absorption spectra revealed that the heme degradation by GmHO1 was extremely retarded at verdoheme though this reaction was not inhibited under CO, unlike the ratHO1 reaction. On the contrary, the heme degrada- tion by GmHO1 and ratHO1 with H2O2, in place of O2/electrons, both provided ferric low-spin intermediates though their yields were different. Optical absorption spectra suggested that the ferric and ferrous verdoheme coexisted in the reaction mixtures by H2O2. In physiological HO reactions, the verdoheme was found to be stabilized in the ferric state in GmHO1, probably guided by protein distal residues, and in the ferrous state in ratHO1. In the H2O2-supported HO reactions, hydroperoxide/hydroxide coordi- nation might stabilize the ferric state of verdoheme in both HOs. Site-specific mutagenesis on putative distal helix part of GmHO1 revealed that H150 was a center player of the regulation of verdo- heme oxidation state in soybean HO1.

P2–94 Fish oil diets fed to rainbow trout (Oncorhynchus mykiss): Effect on cholesterol lipoprotein transport and muscle deposition. A preliminary study J. C. G. Milicua1, A. M. Salvador1, J. A. Fernandez-Higuero1, G. Choubert2 and J. R. Arrondo3 1Faculty of Science and Technology, Biochemistry and Molecular Biology, Bilbao, SPAIN, 2INRA UMR1067, Nutrition Aquaculture an Genomic Pole d’hydrobiologie, Saint Pee-sur-Nivelle, FRANCE, 3Unidad de Biofisica, CSIC-UPV, Bilbao, SPAIN

Glycoside hydrolases are a class of enzymes that break/form the glycosidic bond existing in oligo- or polysaccharide molecules. Candida albicans’s b-1,3- Exoglucanase (Exg), a family 5 glycosi- dase, belongs to this class of enzymes. In a previous work (accepted for publication), we created several models for enzyme- small sub- strate complexes starting from a crystallographic glucanase-inhibi- tor structure. With this lead, we now model another enzyme- substrate complex using a very long substrate (with 18 glucose units – Exg-18Glc). Additionally we also created an enzyme-ligand complex (Exg + 18Glc), where we bind an 18Glc ligand between the flexible enzyme loops we noticed in our previous work (corre- sponding to aminoacids 36–47 and 101–106). The last complex we designed is a complex with both the substrate and the ligand bound to the enzyme. We used Molecular dynamics to study the flexibility of the enzyme-substrate/ligand interactions for each complex. The results are in agreement with the previous work we conducted, indicating that the enzyme overall conformation is quite rigid, with only the two flexible loops showing greater variation in their posi- tion. When we bound the polysaccharide ligand between the loops, we found that the structure of loop 36–47 became stabilised during the dynamics run, with no alteration of the overall enzyme confor- mation. The enzyme-ligand complexes proved to be very stable, and the enzyme-ligand interactions formed were maintained with- out any distortion of the intervening molecules. This supports the hypothesis we proposed earlier that these flexible loops allow the enzyme to be bound on its surface to the fungus cell wall, where it acts to shape glucan structure. The dynamic behaviour of the two substrate complexes, with or without the ligand, is very similar; however we notice some differences. For the Exg-18Glc complex, 6 ns into the dynamics run the substrate shifts its position in the active site and adopts a position unlikely to favour catalysis. For the Exg-18Glc + 18Glc the substrate maintained an apparently favourable positioning during the entire 10 ns of the dynamics run. It seems that the presence of the ligand stabilised the conformation of Tyr29 and its interaction with the substrate. This interaction was lost in the Exg-18Glc complex, and hence the substrate was allowed to change its position in the active site. These results show that the ligand binding could potentially have a role in enzyme structure stabilization during the reaction.

P2–96 Modelling beta-1,3-Exoglucanase- saccharide interactions: structure of the enzyme-substrate complex and enzyme binding to the cell wall S. Moura-Tamames, M. J. Ramos and P. Alexandrino Fernades Chemistry, Faculty of Sciences of the University of Porto, Porto, PORTUGAL

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

In this work, we have examined the effects of high cholesterol diet on cholesterol lipoprotein transport and its muscle deposi- tion in rainbow trout. Two sets of twelve immature rainbow trout (mean weight 300 g) were fed ad libitum during 8 days with diets containing 7% (1.5% fish oil) or 24% (19% fish oil) lipid, which correspond to 1.9 and 6.4 g of cholesterol per kg of feed, respectively. Trout fed the high cholesterol level diet, contained a highest cholesterol amount in serum and in muscle, with an increase in cholesterol of 16% in serum and 20% in trout muscle than trout fed the control diet with a lower cholesterol level. This is of particular interest for consumers who demand products with low cholesterol contents due to the relationship of this compound with atherosclerosis. On the other hand, trout have serum lipo- proteins similar as humans: HDLs, LDLs, and VLDLs, with sim- ilar densities and lipid composition. FTIR of trout lipoproteins has been performed for comparison of trout lipoprotein structure Glycoside hydrolases are a class of enzymes that break/form the bond between sugar monomers (monosaccharides). Candida albi-

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several starting enzyme-substrate models glucanase-inhibitor

P2–98 Hydrolysis some esters by BCA and stomach CA and again explain of CA enzymes’ active site and catalysis mechanism H. Nadaroglu1, N. Demir2 and Y. Demir2 1Oltu Vocational Trainig School, University of Ataturk, Erzurum, TURKEY, 2Science Faculty, University of Ataturk, Erzurum, TURKEY

cans’s b-1,3- Exoglucanase (Exg), a family 5 glycosidase, belongs to this class of enzymes. This small protein is an ideal computa- tional model for its family of enzymes and was used here to cre- ate from a crystallographic structure. A series of enzyme-substrate complexes where generated using molecular docking, ranging from Exg-glucose (Exg-1Glc) to Exg-laminari- hexaose (Exg-6Glc). Structure optimizations followed by Molecu- lar Dynamics provided a picture of the way the enzyme and substrates interact. Molecular dynamics was conducted for each complex to assess the flexibility of the substrate, of the enzyme as a whole, and of enzyme-substrate interactions. The enzyme over- all conformation was found to be quite rigid, although most enzyme residues increase mobility upon substrate binding. How- ever, two surface loops stand out by having large fluctuations and becoming less flexible when the substrates were bound. These data point to a possible biological role for the mentioned loops, corresponding to amino acids 36–47 and 101–106. We propose that these loops could bind the enzyme to a glucan chain in the cell wall. The polysaccharide and enzyme structures have very complementary shapes and form numerous interactions; so it appears likely that the flexible loops connect the enzyme to the cell wall and allow it to navigate the wall to shape glucan struc- ture.

P2–97 Chaperone-like activities and structural properties of different molecular forms of ß-casein N. Mukhametshina1, N. Zaharchenko2, T. Konnova2, J. C. Gaudin3, Y. Gogolev1, Y. Zuev2 and T. Haertle´ 3 1The Group of Molecular Biology, Kazan Institute of Biochemistry and Biophysics, Kazan, RUSSIA, 2Laboratory of Biophysical Chemistry of Nanosystems, Kazan Institute of Biochemistry and Biophysics, The Kazan, RUSSIA, 3Biopolymeres Interactions Assemblages – INRA, Equipe Fonctions et Interactions des Proteines Laitieres, Nantes, FRANCE

Carbonic anhydrase (CA: carbonate hydrolase; 4.2.1.1) is a zinc- containing metallo enzyme, and was first isolated from mamma- lian blood cells. CA catalyzes the hydration of CO2. The hydro- lysis of carboxylic acid esters is one of the most studied chemical reactions because of its importance both in chemistry and bio- chemistry (1). The base-catalyzed hydrolysis of the majority of common esters occurs through the nucleophilic attact of the hydroxide ion at the carbonyl carbon. As a matter of fact, a de- protonated zinc-bound water molecule is involved in many bio- logical proses, such as those catalyzed carbonic anhydrase (2–3) where the cataltic role of zinc is generally ascribed to two main functions: (i) binding and/or activation of substrates and (ii) de- protonation of Zn(II)-coordinated water molecules to give Zn- OH functions, which can act as nucleophilic in the hydroplytic mechanism. Carbonic anhydrases were separately purified and characterisated from bovine stomach and bovine erythrocytes using sepharose-4B-L-tyrosine-sulphanylamide affinity chroma- tography. Catalytic parameters of bovine erythrocyte and stom- ach carbonic anhydrases for hydrolysis of homologous pairs of nitrophenyl esters and non-nitrophenyl esters were compared. 4- Nitrophenyl-2,2,2-trifloroacetate, 4-nitrophenyl butyrate, phenyl benzoate, methyl-2-oxo-2-phenylacetate, ethyl-2- phenylacetate, phenylethyl acetate and ethyl benzoate were hydrolysed in the catalysis of purified stomach’s CA and BCA. Esters hydrolysis was carried out during 10 h. To be able to determine wheather reactions occur, defined volumes of samples were taken from reaction mixtures at interval times and were extracted by an organic solvent than analyzing on TLC, UV and NMR spectros- copies. It was observed that bovine stomach CA and BCA have a different effects on esters. From this results some suggestion can be made about the structure of active domain of CA enzyme. References: 1. Supuran CT & Scozzafava A. Eur. J. Med. Chem. 2001; 35: 867–874. 2. Demir N, Nadaroglu H & Demir Y. Int. J. Chem. 2004; 14: 41– 46. 3. Demir N, Nadaroglu H & Demir Y. Int. J. Chem. 2004; 14: 205–209.

P2–99 The role of galectin-3 in immune cells activation R. Novak, A. Lepur, S. Dabelic and J. Dumic Biochemistry and Molecular Biology, University of Zagreb – Faculty of Pharmacy and Biochemistry, Zagreb, CROATIA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Recently diagnostic aids and treatments of illnesses connected with structural diseases are actively developed. A novel function for caseins has been proposed as ‘molecular chaperones’ protect- ing many proteins including whey proteins against heat, chemical and UV light-induced aggregations. ß-casein (ß-CN) contains rel- atively high amount of prolyl residues, adopts non-compact and flexible structure and exhibits chaperone-like activity in vitro. Like many chaperones, native ß-CN does not contain cysteinyl residues and exhibits strong tendencies for self-association. The chaperone-like activities of three recombinant ß-CNs wild type (WT) ß-CN, C 31 ß-CN (with cysteinyl residue in position 31) and C3–210 ß-CN (with cysteinyl residue in position 3 and 208), expressed and purified from Escherichia coli were examined and compared using alcohol dehydrogenase (ADH) and lysozyme as target/substrate proteins. We studied the structural proprieties of dimeric and monomeric forms (ß-CND) of C 31-ß-CN and C3- 210 ß-CN and their chaperone-like activities. Dimerisation of C3-210 ß-CN in different buffers exhibited formation of various forms. Dimers of C3-210 ß-CN exhibited higher chaperone-like activities, compared with dimers of C 31-ß-CN and WT. Mono- meric forms C3-210 ß-CN, C31 ß-CN exhibited lower chaper- one-like activities compared with WT. Micelle formation of dimeric forms of C3-210 ß-CN, C31 ß-CN occured at lower tem- perature in comparison with monomeric form and WT. The obtained results demonstrate the significant role played dimerisa- tion and micelle formation proprieties of ß-caseins in modulation the chaperone-like activity. Galectin-3, a b-galactoside binding lectin exerts important roles in many (patho) physiological processes (adhesion, proliferation, differentiation, apoptosis, inflammation, neoplastic transforma- tion, spreading metastases). Being one of the key lectins of innate and acquired immunity, galectin-3 is considered a powerful pro- inflammatory signal. It triggers/promotes respiratory burst in monocytes, acts as a monocyte/macrophage chemoattractant and promotes survival of inflammatory cells through its anti-apopto- tic activity. The aim of this study was to ascertain the level of galectin-3 expression and explore it’s role in (patho)physiological processes of monocytes and lymphocytes. Flow cytometry was

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responsible for

surface of microtubule, however

used to determine the level of membrane and intracellular galec- tin-3 expression on a model THP-1 monocytic cell-line. We used lipopolysaccharide (LPS) activated and phorbol 12-myristate-13- acetate (PMA) differentiated, THP-1 cells cultured in RPMI 1640, supplemented with FCS and antibiotics. Galectin-3 was detected using anti-human galectin-3 primary, and labeled sec- ondary antibodies. Dead cells were excluded by 7AAD. Macro- phage-like morphology was confirmed by an elevated CD14 stain and surface adhesion. LPS-activated THP-1 cells have markedly up-regulated expression of intracellular galectin-3, while the sur- face level remains largely unchanged. Differentiation of mono- cytes to macrophages is associated with an increase of surface galectin-3 in respect to control cells. Using cytokine capture beads, we also studied the effects of recombinant galectin-3 on inflammatory cytokine secretion by human PBMC’s from healthy volunteers. It was shown that exogenous galectin-3 influences cytokine secretion in human monocyte derived macrophages and lymphocytes. Given the importance of galectin-3 in monocyte and lymphocyte physiology, further experiments are necessary to elucidate it’s roles in immune cells.

of microtubules to switch between phases of assembly and dis- assembly is crucial to fulfil their functions in the cell. In previ- ous studies we have demonstrated that prion protein (PrP), a key molecule implicated in pathogenesis of prion diseases, binds directly to tubulin and this interaction inhibits microtu- bule formation by inducement of tubulin oligomerization. In this report we focused on (i) mapping the regions of PrP and tubulin involved in the interaction and (ii) identifying of PrP domains tubulin oligomerization. Employing proteolytic fragments of a- and b-tubulin we have found that docking sites for PrP are localized to the C-terminal domains constituting the outer the extreme C-termini of either isoform are not involved in the interaction. Using a panel of PrP deletion mutants as well as proteolytic fragments of prion protein we identified two micro- tubule binding regions: the major binding site spanning resi- dues 23–32 and the weak binding site comprising residues 101– the latter structurally supported by the region linking 110, both sites. Light scattering measurements have revealed that deletion of the sequence 23–32 markedly lowers the capability of PrP to induce tubulin oligomerization. A synthetic peptide corresponding to this sequence, but not that identical with the the full-length PrP on fragment 101–110, mimics effects of tubulin oligomerization and microtubule assembly. Further- more, at the cellular level, peptide composed of the PrP motif 23–30 and cell penetrating sequence disrupts microtubular cyto- skeleton.

P2–100 Conformational behavior of C-terminal tails of yeast 14-3-3 isoforms D. Urychova1, P. Herman2, J. Vecer2, T. Obsil3 and V. Obsilova1 1Protein structure, Institute of Physiology AS CR v.v.i., Prague, CZECH REPUBLIC, 2Faculty of Mathematics and Physics Charles University, Institute of Physics, Prague, CZECH REPUBLIC, 3Faculty of Science Charles University, Physical and Macromolecular Chemistry, Prague, CZECH REPUBLIC

P2–102 The cathepsin D-like proteinases from the midgut of Musca domestica larvae: sequence, properties, localization and function M. H. P. Padilha, A. C. Pimentel and W. R. Terra Biochemistry, University of Sao Paulo Institute of Chemistry, Sao Paulo, BRAZIL

for three coding cDNAs

funded

14-3-3 proteins are abundant binding proteins involved in many biologically important processes. They bind to other proteins in a phosphorylation-dependent manner and function as scaffold mol- ecules. Many organisms express multiple 14-3-3 isoforms and maximal sequence variability occurs within the C-terminal stretch, a region that is believed to be flexible. The structure of this part of 14-3-3 molecule is unknown. It has been shown that 14-3-3 C-terminal stretch is involved in the regulation of ligand binding. Yeast 14-3-3 isoforms (BMH1 and BMH2) posses unusually longer C-terminal tails that contain polyglutamine (polyQ) sequences with unknown function. The polyQ sequences of certain proteins are known to induce protein aggregation. We used methods of time-resolved fluorescence spectroscopy and studied conformational properties of these segments. Fluores- cence measurements suggest that the C-terminal tails of BMH proteins do not function as autoinhibitors which are ejected from the ligand binding groove during the ligand binding. In addition, fluorescence measurements revealed that BMH proteins form oligomers bigger than expected dimmers (likely tetramers). Acknowledgements: This work was by Grant IAA501110801 of the Grant Agency of the Academy of Sciences of the Czech Republic, by Research Project MSM0021620857 and Centre of Neurosciences LC554 of the Ministry of Education, Youth, and Sports of the Czech Republic, and by Research Project AV0Z50110509 of the Academy of Sciences of the Czech Republic.

Aspartic proteinases are key digestive enzymes in larval flies and some beetles. Knowledge on these enzymes is scarce and for this a study was undertaken with our fly model, Musca domestica. preprocathepsin-D (ppAspMD01, 02 and 03) were cloned and sequenced from a cDNA library prepared from Musca domestica larval midguts and their sequences are homologous to cathepsin-D enzymes. pAspMD01 was expressed in bacterial systems. The recombi- nant pAspMD01 was purified and once activated was able to hydrolyse hemoglobin under acidic conditions. Sequence trees of cathepsin-D sequences from insects and other organisms was prepared. ppAspMD03 was most closely related to lysosomal enzymes of insects. ppAspMD01 and ppAspMD02 sequences branch alone and are more derived than the other sequences. RT-PCR analysis showed that pAspMD03 mRNA transcripts were expressed in all major tissue homogenates. Differently, pAspMD01 and pAspMD02 mRNA transcripts were expressed only in anterior and middle midgut from M. domestica larvae. Western blot detection recognized pAspMD01 translation in anterior and middle midgut lumen, where cathepsin D-like activity is found. The data suggested that Diptera cyclorrhapha have a digestive cathepsin-D that may have originated by gene duplication and independent evolution from a gene encoding the lysosomal enzyme.

P2–101 Prion protein – tubulin interaction sites K. Osiecka, H. Nieznanska, K. Skowronek, J. Karolczak, G. Schneider and K. Nieznanski Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, POLAND

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Microtubules are cytoskeletal structures composed of tubulin which are engaged in numerous cellular processes. An ability

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inhibitory properties of series of nucleoside phosphonic acids derivatives are tested and for the most promising compounds the enzyme-inhibitor structure will be determined to serve as a lead for structure-based drug design efforts.

P2–103 Molecular mechanism of the CtBP1-S/BARS- dependent fission process A. Pagliuso, C. Valente, G. Li, D. Corda and A. Luini Cell Biology and Oncology, Consorzio Mario Negri Sud, Chieti, ITALY

P2–105 Copper(I)-mediated protein-protein interactions result from suboptimal interaction surfaces A. Pavelkova1, L. Banci1,2, I. Bertini1,2, V. Calderone1, N. Della Malva1,3, I. C. Felli1,2, S. Neri1 and A. Rosato1,2 1Magnetic Resonance Center (CERM), Sesto Fiorentino, ITALY, 2Department of Chemistry, University of Florence, Sesto Fiorentino, ITALY, 3FIORGEN Foundation, Sesto Fiorentino, ITALY

Membrane fission is an essential event in intracellular membrane trafficking and it is required for the formation of all transport carriers. While several fissioning steps in membrane trafficking require the large GTPase dynamin, we have previously shown that several dynamin-independent pathways are controlled by C- (short)/brefeldin-A-ADP-ribosylated terminal-binding-protein-1 substrate (CtBP1-S/BARS). For instance, CtBP1-S/BARS is required for the fission and partitioning of the Golgi ribbon dur- ing mitosis, for the formation of basolaterally directed post Golgi carriers and COPI vesicles, and for fluid phase endocytosis. We have previously reported that CtBP1-S/BARS carries a lysophos- phatidic acid acyltransferase (AGPAT) activity when purified from bacteria. Later work, however, showed that this activity is not intrinsic to CtBP1-S/BARS, but it is due to a tightly associ- ated protein. We thus reasoned that CtBP1-S/BARS could inter- act with an AGPAT. Here we report that indeed CtBP1-S/BARS binds selectively to one of the nine known mammalian AGPAT isoforms, AGPAT 3, which localises on the Golgi complex. To investigate the involvement of AGPAT 3 in CtBP1-S/BARS- dependent membrane transport, we used COS7 cells expressing the temperature-sensitive mutant of VSVG as a cargo reporter. Both the injection of an anti- AGPAT 3 antibody, and the expression of a catalytically inactive AGPAT 3 mutant inhibit the formation of basolaterally directed post-Golgi carriers, indi- cating the involvement of AGPAT 3 in this BARS-dependent transport pathway. The homeostasis of metal ions in cells is the result of the contri- bution of several cellular pathways. Metal ions are transferred between proteins along these pathways, through the onset of transient, often weak, protein-protein interactions. Metal transfer typically implies the formation of adducts where the metal itself acts as a bridge between proteins, by coordinating residues of both interacting partners. Here we addressed the interaction between the human copper(I)-chaperone HAH1 and one metal- binding domain of one of its partners, namely the P-type copper- transporting ATPase ATP7A. The adduct was structurally char- acterized in solution, in the presence of copper(I), and through X-ray crystallography, upon replacing copper(I) with cad- mium(II). The existence of the cadmium(II) adduct in solution was confirmed by protein as well as 113Cd NMR spectra. We also applied molecular modelling techniques and site-directed muta- genesis to identify the features of the protein sequence/structure that determine the thermodynamic stability of the intermolecular, metal-mediated complex.

P2–104 Structure-based drug design of selective 5’-nucleotidases inhibitors P. Pachl1, J. Brynda1, I. Rosenberg2, M. Fa´ bry1 and P. Reza´ cova´ 1 1Institute of Molecular Genetics, Structural biology, Prague , CZECH REPUBLIC, 2Structural biology, Institute of Organio Chemistry and Biochemistry, Prague, CZECH REPUBLIC

P2–106 The influence of spin-labeled fluorene compounds on the structure, dynamics and toxicity of the ABeta peptide J. Petrlova1, H. S. Hong2, L. W. Jin2, J. Voss1 and K. Hideg3 1Biochemistry & Molecular Medicine, University of California Davis, Davis, CA, USA, 2School of Medicine, University of California Davis, Davis, CA, USA, 3Institute ofOrganic and Medicinal Chemistry, University Pe´cs, Pe´cs, HUNGARY

is by kinases cellular opposed nucleoside

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

The deposition and oligomerization of Ab peptide plays a key role in the pathogenesis of Alzheimer’s disease (AD). Ab peptide arises from a cleavage in the membrane-associated domain of the amyloid precursor protein (AAP) by b and c secretases. Several lines of evidence point to the soluble Ab oligomer (AbO) as the primary neurotoxic species in the etiology of AD. Recently (Hong et al, 2009, Neurobiol Aging; in press) we have demon- strated that a class of fluorene molecules specifically disrupts the AbO species. Here, to achieve a better understanding of the mechanism of action, we extend the application of electron para- magnetic resonance (EPR) spectroscopy of site-directed spin labels in the Ab peptide to investigate the binding and influence of fluorene compounds on AbO structure and dynamics. In addi- tion, we have synthesized a spin-labeled fluorene that provides both increased cell protection against AbO toxicity and a route to directly observe the binding of the fluorene to the AbO assem- bly. We also evaluate the ability of fluorenes to target multiple pathological processes involved in the neurodegenerative cascade, such as their ability to scavenge free radicals. Thus, the N-hydro- xyl- pyrroline and piperidine fluorene derivatives may be espe- The monophosphate 5’-nucleotidases belong to family of enzymes that catalyze the dephosphorylation of nucleoside monophos- phates. The ribonucleotides and deoxyribonucleotides could be synthesized de novo from low-molecular-weight precursors or by salvage from nucleosides or nucleobases coming from catabolism of nucleic acids. In this salvage pathway, nucleotides are phos- phorylated by nucleoside and nucleotide kinases. The phosphory- lation by 5’-nucleotidases that dephosphorylate ribo- and deoxyribonucleo- side monophosphates. Besides their role in the regulation of physiological dNTP pools, substrate cycles between ribonucleo- tidases and kinases may affect the therapeutic action of pyrimi- dine-nucleoside analogs used as anticancer and antiviral agents. Such compounds require the nucleoside kinases activity for phos- phorylation to their active forms. Results of clinical and in vitro studies propose that an increase in nucleotidase activity interfere with nucleoside analogue activation. The main goal of this pro- ject is the search for potent and selective inhibitors of mamma- lian 5’-nucleotidases based on nucleoside phosphonic acids and their derivatives and comparison of sensitivity of 5’-nucleotidases isolated from various sources toward individual inhibitors. We have prepared two types of human 5’-nucleotidase: cytosolic and mitochondrial by recombinant expression in Escherichia coli. The

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cially useful in that they posses both antioxidant and AbO dis- ruptive properties.

P2–107 Alternatively spliced variants of COUP-TF in deuterostomes I. I. Petta, A. Myrissa, and C. N. Flytzanis Biology, University of Patras, Patras, GREECE

sequence of membrane fusion proteins, several membranotropic segments exist which, under a concerted action, are essential for membrane fusion. Also in the two HCV envelope proteins, E1 (residues 192–383) and E2 (residues 384–746), several regions with potential fusion activity have been identified. Among these, combining multiple sequence analysis and secondary structure prediction, we have selected a peptide corresponding to E1 region encompassing residues 314–342. A preliminary conforma- tional analysis carried out by Circular Dichroism under different experimental conditions shows that the peptide conformation can be switched from beta to alpha by decreasing the solvent polarity. According to the CD data, we have also solved the 3D the structure of E1314–342 peptide in an apolar environment by standard 2D- NMR and found a partially ordered structure, characterized by a helix-break-helix motif, the two helical stretches encompassing residues 319–323 and 329–358, respec- tively. Furthermore, tryptophan fluorescence and ESR measure- ments indicate a strong interaction and deep penetration of the peptide into synthetic anionic and zwitterionic multilamellar lipo- somes, probably due to the largely apolar character of the pep- tide structure, the interaction with anionic phospholipids being among the strongest ever observed not only in comparison with the results obtained for the same peptide in presence of zwitter- ionic phospholipids, but also when considering the interactions of several other membrane active polypeptides with the same kind of phospholipids. Overall, the experimental data are in good agreement with a possible entrance of the E1314–342 peptide in the anionic double layer and support also the idea that this region of E1 might be involved in membrane destabilization and viral fusion, therefore it may represent a good target to develop anti-viral molecules.

this study indicate that

COUP-TF (Chicken Ovalbumin Upstream Promoter-Transcrip- tion Factor) is an orphan nuclear receptor, member of the ste- roid-thyroid-retinoic acid receptor superfamily. COUP-TF genes show extreme sequence conservation within all metazoan phyla. A characteristic COUP-TF splice site, just downstream of the DNA binding domain (DBD) and not within the DBD as in other nuclear receptors, is also conserved within metazoans. In the Mediterranean sea urchin Paracentrotus lividus, a small 63nt exon embedded within a larger than 10 kbp intron is alternatively spliced at this site generating two transcripts and consequently two protein products. The in frame insertion of the 21aa exon within the carboxyl terminal extension of the DBD, alters the DNA binding properties of the larger protein variant. Both pro- tein variants are found in embryonic and adult tissues of the sea urchin P. lividus. A pair of PCR primers was designed from the most conserved sequences of the DBD and LBD domains of the single P. lividus COUP-TF gene, such as the generated product to span the region where the alternative splicing occurs. Using these primers, RT-PCR reactions were carried out with substrate embryonic or adult tissue total RNAs from a variety of animal groups belonging to deuterostomes. Thus, the alternative splicing event was investigated in selected invertebrates such as echino- derms and their sister groups and vertebrates such as fish and mammals. In addition to the collected experimental data, a vast number of COUP-TF sequences (genomic or ESTs) from a pleth- ora of sequenced genomes was analyzed in silico, to determine either the presence of the small sea urchin exon in general, or the generation of COUP-TF splice variants at the particular splice site. The results of the presence of COUP-TF transcript variants is mostly restricted within the phy- lum of echinodermata.

P2–109 Aggregation studies of human septin 2 and its binding to phosphatidylinositol- 4,5-biphosphate. J. C. Pissuti Damalio, T. M. Nobre, W. Garcia, R. R. C. Garratt and A. U. Araujo Instituto de Fı´sica de Sao Carlos Universidade de Sao Paulo, Grupo de Biofı´sica Molecular ‘Se´rgio Mascarenhas’, Sao Carlos, BRAZIL

includes region, which generally

P2–108 A membrane active region of the E1 envelope glycoprotein of the HCV virus: structure and interactions D. Picone1, G. D’Errico1, E. Notomista2, M. Merola3 and R. Spadaccini4 1Department of Chemistry Paolo Corradini, Universita’ di Napoli Federico II, Naples, ITALY, 2Department of Structural and Func- tional Biology, Universita’ di Napoli Federico II, Naples, ITALY 3Department of Structural and Functional Biology, Novartis and Universita’ di Napoli Federico II, Siena, ITALY, 4Department of Biological and Environmental Sciences, Universita’ del Sannio, Benevento, ITALY

important side effects. A detailed knowledge of

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Septins are a conserved group of GTP-binding and filament- forming proteins. Originally discovered in yeast, they are involved in a variety of cellular processes, such as polarity determination, membrane dynamics, vesicles trafficking and exo- cytosis. All members of the septins family can be divided into three domains: a variable N-terminal, a GTPase domain, and a C-terminal characteristic sequences of coiled-coil. The function of Septin 2 (SEPT2) remains unclear; however, SEPT2 with SEPT1 and SEPT4 are accumulated in deposits known as neurofibrillary tangles and glial fibrils in Alzheimer’s disease. In this study, the three struc- tural domains of human SEPT2 (SEPT2, SEPT2-G and SEPT2- NG) were expressed in Escherichia coli and purified by affinity and size-exclusion chromatographies. Their products were ana- lyzed by rigth-angle light scattering and extrinsic fluorescence spectroscopy using ThioflavinT, as a fluorescent probe. In all the cases, the proteins form homodimers in vitro, suggesting that the GTPase domain (SEPT2-G) is enough to promote the oligomerization. Thermal unfolding of the recombinant products revealed the formation of aggregates under physiological condi- tions. These aggregates have the ability to bind a specific dye, Thioflavin-T, suggesting them to be an amyloidal fiber. Using Langmuir monolayers at the cell membrane lipid packing, SEPT2 and SEPT2-NG bound to the phospholipid phosphati- Hepatitis C virus (HCV) represents a major public health prob- lem worldwide, as it is a major cause of chronic hepatitis, cirrho- sis, and hepatocellular carcinoma. Currently there is no vaccine to prevent HCV infection and the available therapeutic agents not only have limited success against HCV, but also produce several the molecular basis of virus entry represents one of the most promis- ing approaches to develop new therapeutic strategies. However, the mechanism of virus fusion is very complex and, along the

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dylinositol 4,5-bisphosphate (PtdIns(4,5)P2), suggesting an asso- ciation with the plasma membrane and a role in septin regula- tion. Thus, the present study contributes for the knowledgment of the stability and the aggregation kinetic of the SEPT2, lead- ing to a better understanding of the septins role in neurodegen- erative disorders.

P2–110 Locally generated elastin peptides increase invasive potential of melanoma cells dominantly by galectin-3 P. Pocza, Z. Darvas and A. Falus Genetics Cell- and Immunobiology, Semmelweis University, Budapest, HUNGARY

and properties of a low molecular mass inhibitor. The inhibitor was isolated from disintegrated fungal mycelia. The cell homoge- nate was first passed through an SPE column and then applied to a rp-HPLC, where the inhibitory fraction was collected. The inhibitory activity against papain and different cathepsins was determined. Temperature and pH stability were also tested. The molecular mass determination was done by LC/MS-MS. The effect of isolated inhibitor on host immune system was estab- lished using a mouse model. BALB/c mice were immunized with antigen and treated with inhibitor. Their sera were used in Wes- tern blot analyses and harvested spleen cells were used for the measurements of cell proliferation and apoptosis. It was estab- lished that the inhibitor impairs proteolytic activity of papain and cathepsins B and L. The inhibitor was stable in a wide range of temperature (30–90 (cid:2)C) and pH (2–12). The effect of fungal inhibitor upon cell proliferation was negligible; on the other hand it appears that the inhibitor diminishes cell apoptosis. Based on the results of immunoblotting we came to a conclusion that fun- gal inhibitor can affect antigen presentation.

P2–112 Inteaction of the N-terminal myristic acid with matrix protein from Mason–Pfizer Monkey Virus J. Prchal1, J. Vlach2, M. Dolezal2, J. Lipov1, T. Ruml1 and R. Hrabal2 1Department of biochemistry and microbiology, Institute of Chemical Technology Prague, Prague, CZECH REPUBLIC, 2NMR laboratory, Institute of Chemical Technology Prague, Prague, CZECH REPUBLIC

they enhance the expression of (iii)

expression of they increase the (v)

Melanomas containing more elastin are associated with higher stages of the disease. The interaction between elastin-derived peptides and melanoma cells appears to play an important role in the progression of melanomas. The effects of the elastin- derived peptides VGVAPG and VAPG have been investigated on the migration, invasion, adhesion and angiogenesis of human melanoma cells of different invasive potential. Elastin, tropoelastin and VGVAPG peptide were demonstrated at the invasion site of melanoma using histochemistry and immunohis- tochemistry. Not only the VGVAPG elastin-derived peptide, which exhibits the XGXXPG consensus sequence in its primary structure, but also the shorter VAPG bind directly to three cell surface receptors: galectin-3, integrin avb3 and elastin-binding protein. Our results suggest that the increased levels of elastin- derived peptides facilitate the invasion of melanoma cells: (i) VGVAPG and VAPG elastin-derived peptides are chemotactic for melanoma cells; (ii) they can increase the migration of mel- anoma cells and the expression of CXCR-4 and CXCL-12 chemokines; the elastin- degrading MMP-2 and MMP-3; (iv) they increase the attach- ment of melanoma cells and the expression of different adhe- sion molecules; the lymphangiogenic VEGF-C and (vi) the galectin-3 receptor can mediate all these effects. Clinical and therapeutic aspects are also discussed.

P2–111 Low molecular mass inhibitor of cysteine proteinases produced by dermatophyte fungus Trichophyton mentagrophytes B. Premrov Bajuk1, T. Virant Celestina2, T. Malovrh3 and M. Drobnic Kosorok1 1Institute of Physiology Pharmacology and Toxicology, University of Ljubljana Veterinary Faculty, Ljubljana, SLOVENIA, 2Institute of Pathology Forensic and Administrative Veterinary Medicine, University of Ljubljana Veterinary Faculty, Ljubljana, SLOVENIA, 3Institute of Microbiology and Parasitology, University of Ljubljana Veterinary Faculty, Ljubljana, SLOVENIA

Polyprotein Gag plays a key role in formation and budding of retroviral particles. The N-terminal domain of Gag, the matrix protein (MA) interacts with the cytoplasmic membrane of infected cell through myristic acid which is covalently attached to the amino-terminal glycine. In this work we focus on the determi- nation of molecular basis of the phenotypic changes of M-PMV double-mutants T41I/T78I and Y28F/Y67F, which are unable to bud through the cytoplasmic membrane and rather accumulate on it. We determined the three-dimensional structures of unmyri- stoylated species of both mutants using NMR spectroscopy. Comparison of their structures with the structure of the wild type MA shows that the mutation caused only marginal changes of the structural motif. In both cases it increased the size and hydrophobicity of the protein interior. Isoleucines 41 and 78 in T41I/T78I and phenylalanines 28 and 67 are oriented to the pro- tein core in contrast to the original amino acid residues and they can influence the interaction of the matrix protein interior with the myristic acid. This finding supports a hypothesis that pheno- typic change of the mutant is caused by an enhanced interaction of the myristic acid with more hydrophobic protein core, which prevents its exposure and finally abrogates the interaction of immature viral particles with the cytoplasmic membrane. To prove the hypothesis we focus on the determination of structure of myristoylated MA proteins with the emphasis to the interac- tion of the myristoyl with protein core. Acknowledgements: Supported by the Czech Ministry of Edu- cation 1M6837805002, MSM 6046137305 and GACR #203/07/ 0872.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Dermatophytes are parasitic fungi that can cause skin, hair, and nails infections in humans or animals due to their ability to obtain nutrients from keratinized material. Proteolysis by cisteine proteinases has a key role in a wide range of cellular processes. Inhibitors represent an important way of regulating protease activity. We previously reported of a protein inhibitor from Trichophyton mentagrophytes, here we report of the purification

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Poster Presentations

P2–113 Digestive a-amylases of the flour moth Ephestia kuehniella -adaptation to alkaline environment and plant inhibitors J. Pytelkova1, J. Hubert2, M. Lepsik3, J. Sobotnik4, R. Sindelka5, I. Krizkova6, M. Horn1 and M. Mares1 1Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences, Biochemistry and Molecular Biology, Prague, CZECH REPUBLIC, 2Department of stored - food pest control, Research Institute of Crop Production, Prague, CZECH REPUBLIC, 3Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences, Computational Chemistry, Prague, CZECH REPUBLIC, 4Chemistry of Natural Products, Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences, Prague, CZECH REPUBLIC, 5Laboratory of Gene Expression, Institute of Biotechnology Czech Academy of Sciences, Prague, CZECH REPUBLIC, 6Mycology, Research Institute of Crop Production, Prague, CZECH REPUBLIC

filaments (4,5). Moreover, our group described that V10, a vana- date oligomeric species, may also prevent actin polymerization and therefore it may contribute to the effects observed for vana- dium in actin structure and function, besides other biological processes (6,7). We now show that incubation of F-actin with decavanadate inhibits up to 70% of the myosin ATPase activity stimulated by F-actin (IC50 = 0, 80 ± 0, 15 lM), while meta- vanadate and vanadyl have a much lower effect (only 25% inhi- bition for the maximum vanadium concentrations used, 50 lM). Decavanadate solution was titrated with G-actin, using 51V- NMR, and it was observed that broadening factor (F) is halfway between bottom (F = 0.91 ± 0.10) and top (F = 2.23 ± 0.07) for 25.42 ± 2.32 lM G-actin. However, due to viscosity of the final solution no kinetic constants could be calculated when deca- vanadate was titrated with the filamentous protein. It was also observed that in G- and F-actin, upon incubation with decavana- date, cysteine oxidation occurred, whereas no effects were detected upon incubation with monomeric vanadate. The con- comitant decavanadate reduction was observed by EPR. Finally, regarding the V(IV) interactions with F-actin, EPR studies indi- cate binding of 1 and 4 VO2+ ions per G- (Kd of 7.48 ± 1.11 lM) and F-actin (Kd = 43.05 ± 5.34 lM), respec- tively. References: 1. Lorinczy D, et al. Thermochimica Acta.1988; 322: 95. 2. Chasteen ND (1983) Structure and Bonding. Springer-Verlag, Berlin Heidelberg. 3. Chasteen ND 1981 Biological Magnetic Resonance. Vol. 3, (Berliner L & Reuben J, eds), p. 53. Plenum Press, NY.

4. Brener E, et al. Biol. Reprod 2003; 68: 837 5. Capella LS, et al. Tumor Biol. 2000; 21: 54. 6. Ramos S, et al. J. Inorg. Biochem.2006; 100: 1734. 7. Aureliano M & Crans DC. J. Inorg. Biochem. 2009; 103: 536.

P2–115 Antigenicity of recombinant fibronectin binding protein fragments in Lyme disease R. Ranka1, K. Brangulis1, V. Capligina1, V. Sondore2 and V. Baumanis1 1Laboratory of molecular microbiology, Latvian Biomedical Research and Study Centre, Riga, LATVIA, 2Laboratory of mole- cular microbiology, Infectology Center of Latvia, Riga, LATVIA

The digestive tract of lepidopteran insects is extremely alkaline. In the present work, molecular adaptation of amylolytic enzymes to this environment is investigated in the flour moth Ephestia ku- ehniella, an important stored-product pest. Three digestive a- amylases (EkAmy1 to 3) with an alkaline pH optimum were purified from larvae and biochemically characterized. These isoenzymes differ significantly in their sensitivity to a-amylase inhibitors of plant origin that are directed against herbivores as antifeedants. Such functional variability renders the amylolytic system less vulnerable to suppression by plant defensive mole- cules. Moreover, we found that expression of a-amylases is up- regulated in larvae feeding on a diet enriched with an a-amylase inhibitor. The a-amylases are secreted into the larval midgut by an exocytotic mechanism as revealed by immunogold microscopy. The cDNA sequence of EkAmy3 was determined, and a homol- ogy model of EkAmy3 was built in order to analyze structural features responsible for adaptation to alkaline pH. First, the overall fold was found to be stabilized by remodeling of ion pairs. Second, molecular simulations supported by activity mea- surements showed that EkAmy3 does not bind a chloride ion due to an Arg-to-Gln mutation in a conserved binding site. The chlo- ride-binding residues are in contact with the catalytic residues, and this change might help to fine-tune the catalytic pKa values to an alkaline pH optimum. We conclude that lepidopteran a- amylases are evolutionary adapted in terms of structure and expression dynamics for effective functioning in a unique diges- tive system.

P2–114 Decavanadate promotes actin cysteine oxidation trough vanadyl formation S. Ramos1,2, R. O. Duarte1, J. J. Moura1 and A. Alves2 1REQUIMTE/CQFB, Dpto. Quı´mica, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Caparica, PORTUGAL, 2CCMar, Dpto. Quı´mica e Bioquı´mica, Faculdade de Ciencias e Tecnologia, Universidade do Algarve, PORTUGAL

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Introduction:Borrelia burgdorferi, a spirochetal pathogen that causes Lyme disease, contains genes encoding numerous surface lipoproteins in its genome. Coordinated regulation of many of these lipoproteins is necessary to B. burgdorferi’s successful main- tenance in the enzootic life cycle. BBK32, a fibronectin-binding protein of B. burgdorferi,is one of such lipoproteins that are dif- ferentiallyexpressed by the Lyme disease spirochete at various stages ofits life cycle. The BBK32 protein binds to host extracel- lular ligand fibronectin and contributes to the pathogenesis of Borrelia burgdorferi. BBK32 was identified as an antigen that elicits an antibody response in infected mice as well as in Lyme disease patients. In this study recombinantly produced different BBK32 fragmentswas evaluated as antigens in the serology of Lyme disease. Methods:Recombinant fragments of BBK32 were cloned and expressed in Escherichia coli with six histidine tag. The expression of protein was proved by Western Blot analysis with anti-His antibodies. Obtained recombinant proteins were used as antigens in Western blot analysis using Lyme disease patient sera samples. Results:Antigenicy of different recombinant fibronectin binding protein BBK32 fragments was tested in this study. 84% of sam- ples were positive for at least one recombinant BBK32 protein The ability of actin monomers (G-actin) to polymerize into fila- mentous form (F-actin) is crucial in structural and motile func- tions of the cells. A major role of actin lies in the generation of force by striated and smooth muscle cells (1). The chemistry of vanadium is characterized by multiple oxidation states (+ 2 to + 5), being largely found in the + 4 and + 5 ones, both of which are readily accessible under physiological conditions (2). V(IV) and V(V) are known to bind to numerous proteins (3) and have been described as affecting agents of the formation of actin

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fragment. These findings indicate that the BBK32 proteins may serve as useful tool in Lyme disease pathogenesis studies. Acknowledgement: This work has been supported by Euro- pean Social Fund.

oppositely charged residues were juxtaposed, enabling a favorable electrostatic interaction that stabilized this conformation. In the X4 conformation, electrostatic repulsion between these residues forced a one register shift in the N-terminal strand of the V3. Despite this difference, both strains share a b-hairpin conforma- tion. Peptide immunogens, mimicking these V3 structures were designed, synthesized and administered to rabbits. These immu- nogens induced high titer antibody responses against gp120 and demonstrated neutralizing activity against different HIV strains. Our promising results suggest that our novel approach using V3 peptides constrained to, and thereby mimicking, the conforma- tions created in V3 results in more effective vaccine candidates than flexible V3 peptides. Our novel use of structural data is an important step forward in the design of HIV-1 vaccines.

P2–116 Study of the interactions between 14-3-3 protein and RGS domain of regulator of G protein signaling 3 (RGS3) L. Rezabkova1, E. Boura1, P. Herman2, J. Vecer2, L. Bourova3, P. Svoboda3, M. Sulc4 and T. Obsil1 1Physical and Macromolecular chemistry, Charles University Fac- ulty of Science, Prague, CZECH REPUBLIC, 2Physics, Charles University Faculty of Mathematics and Physics, Prague, CZECH REPUBLIC, 3Academy of Sciences of the Czech Republic Institute of Physiology, Membrane receptors, Prague, CZECH REPUB- LIC, 4Academy of Sciences of the Czech Republic Institute of Microbiology, Molecular structure characterization, Prague, CZECH REPUBLIC

P2–118 Activating receptors of mouse natural killer cells, mNKR-P1A/C are carbohydrate binding proteins with specificity similar to the rat NKR-P1A D. Rozbesky1, O. Vanek1, V. Zurek1, R. Hrabal2, P. Kolenko3, J. Dohnalek3, P. Pompach4 and K. Bezouska1 1Biochemistry, Charles University Faculty of Science, Prague, CZECH REPUBLIC, 2Biochemistry, Institute of Chemical Technology, Prague, CZECH REPUBLIC, 3Institute of Macromolecular Chemistry, Academy of Science of Czech Republic, Prague, CZECH REPUBLIC, 4Institute of Microbiology, Academy of Science of Czech Republic, Prague, CZECH REPUBLIC

funded

RGS proteins share conserved 125-amino-acid domain. They func- tion as GTPase-activating proteins for Ga-subunit of G-proteins. They bind to GTP-bound forms of Ga and stimulate GTP hydro- lysis. Their activity is regulated through various mechanisms including phosphorylation and interactions with other proteins. Several RGS interact with 14-3-3 proteins. It was suggested that 14-3-3 binding to selected RGS decreases their inhibitory effect on G protein signaling by blocking interaction between RGS and Ga- subunit. To investigate whether 14-3-3 protein binding affects con- formation of RGS domain, time-resolved fluorescence measure- ments of single-tryptophan mutants of RGS3 were performed. They revealed that phosphorylation of Ser264 itself induces signifi- cant structural changes in region surrounding nearby located Trp295 but not Trp424 located within RGS domain. Interaction between 14-3-3e and phosphorylated RGS3 induces significant structural changes in the vicinity of both tested tryptophan. More- over, experiments with isolated RGS domain suggest that this domain can interact with 14-3-3 protein in a phosphorylation-inde- pendent manner. We also solved crystal structure of RGS domain of RGS3 at 2.3 A˚ resolution. This structure suggests that 14-3-3- induced conformational change affects region within the Ga-inter- acting portion of the RGS domain. This can explain the inhibitory effect of 14-3-3 on GAP activity of RGS3. Acknowledgements: This work was by Grant IAA501110801 of the Grant Agency of the Academy of Sciences of the Czech Republic, by Research Project MSM0021620857 and Centre of Neurosciences LC554 of the Ministry of Education, Youth, and Sports of the Czech Republic, and by Research Project AV0Z50110509 of the Academy of Sciences of the Czech Republic.

NKR-P1A and NKR-P1C are important activation receptors of mouse NK cells but their cognitive ligands remain unknown so far. The latter receptor that corresponds to the NK1.1 alloantigen also represents one of the most important surface markers of mouse NK cells detected by monoclonal antibody PK136 in several mouse strains. In order to initiate ligand recognition experiments, we have obtained cDNA clones for both receptors by RT-PCR using spleens from B6/BL mice. DNA fragments coding for the extracellular ligand binding domains of the two receptors were transferred into pET-30 bacterial expression vectors. After induc- tion of protein production with IPTG, the protein precipitated into inclusion bodies, from which they could be easily refolded in vitro using standard refolding protocols. The quality of the refolding for both receptors was confirmed from 1H-15N-HSQC NMR spectra using uniformly 15N-labeled protein. The refolded protein appeared to be monomeric as revealed by gel filtration, dynamic light scattering, and analytical ultracentrifugation. Mass spectrom- etry has been used for the analysis of disulfide bonding which fitted into the pattern expected for C-type lectins. Both proteins bound specifically to microtiter wells coated with GlcNAcBSA neoglyco- protein, and the binding specificity was similar to the rat NKR- P1A. NKR-P1A had been crystallized, and its structure was solved using X-ray diffraction. The structure is unique, and represents a new fold within the C-type lectin family with two loops per recep- tor dimer forming the flaps involved in binding of the ligands, and lectin – lectin interactions typical for these receptors.

P2–117 HIV V3 region structures as novel HIV immunogens O. Rosen, A. Moseri and J. Anglister Weizmann Institute of Science, Structural Biology, Rehovot, ISRAEL

P2–119 Tinkering with acetylglutamate synthases V. Rubio, L. Ferna´ ndez-Murga, E. Sancho Vaello and S. De Cima Genomics and Proteomics, Instituto de Biomedicina de Valencia IBV-CISC, Valencia, SPAIN

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

N-Acetylglutamate synthase (NAGS) catalyzes the first step of arginine biosynthesis and is the maker in mammals of the essen- A major objective in HIV-1 vaccine development is identifying immunogens that can induce antibody response against epitopes that are shared among all or many virus strains. An important HIV-gp120 epitope is the third variable (V3) region which binds to chemokine receptors CCR5 (‘R5 viruses’) and CXCR4 (‘X4 viruses’). Based on our previous NMR structure determination we discovered that V3 peptides from both X4 and R5 strains adopt a b-hairpin conformation. In the R5 conformation, two

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preferentiality towards the GalR2 and opens new possibilities to clarify the galanin system as a putative drug target.

P2–121 In silico prediction of IKK-2 inhibition by natural phenolic compounds E. Sala1, J. Iwaszkiewicz2, V. Zoete2, A. Grosdidier2, L. Guasch1, S. Garcia-Vallve1, O. Michielin2 and G. Pujadas1 1Biochemistry and Biotechnology, Rovira i Virgili University, Tarragona, Spain, 2Molecular Modeling Group, Swiss Institute of Bioinformatics (SIB), Lausanne, SWITZERLAND

The NF-jb pathway plays an important role in regulating the expression of the cellular genes that control the immune and inflammatory response. IKK-2 is a serine-threonine protein kinase that belongs to the IKK complex and is critically involved in activating the transcription factor NF-jb in response to vari- ous inflammatory stimuli, hence the interest in synthesizing mole- cules that can inhibit IKK-2. The research interest of this study lies in the relationship between natural compounds and health, and particularly in how the phenolic compounds from red wine can prevent cardiovascular diseases. Our hypothesis is that phe- nolic compounds can inhibit the IKK-2 kinase because previous in vitro studies by our group have shown that procyanidins can inhibit NF-jb translocation to the nucleus. The same studies sug- gest that regulating upstream proteins such as IKK may inhibit degradation of IKb. The aim of the present work is to use a docking-based virtual screening approach to identify the inhibi- tors of IKK-2 in a database of natural phenolic compounds. Since no experimental structure of IKK-2 has been deposited in the public Protein Data Bank, the homology model of the IKK-2 was built using MODELLER 9v5 software. The molecular dock- ing studies were performed using EADOCK v2.0 and GLIDE v5.0. The results of this investigation are expected to elucidate the molecular background of natural phenolic compounds effect on human health.

tial urea cycle activator acetylglutamate. NAGSs are inhibited by arginine, except in land tetrapods, where arginine activates NAGS. Typically, NAGS is composed of an amino acid kinase (AAK)-type domain that binds the effector arginine, and a smal- ler C-terminal GNAT-type acetyltransferase domain that binds the substrates. The determination of the structure of a bacterial NAGS has shown that the enzyme is a ring-like hexamer (trimer of dimers) of AAK domains, with each subunit GNAT domain sitting on the AAK domain of another subunit. To better our understanding of the role of the AAK domain and of NAGS reg- ulation by arginine, we have cleaved the interdomain linker and isolated each domain of Pseudomonas aeruginosa NAGS. This has shed light into each domain function, into the mutual effects of AAK-GNAT domain association, and has highlighted the importance of the interdomain linker for arginine regulation, even reverting inhibition to activation by linker lengthening. We demonstrate also the paramount role of the hexameric architec- ture on NAGS function and regulation, by hexamer disassemby to dimers upon deletion of the N-terminal alpha-helix. In some microorganisms such as Mycobacterium tuberculosis NAGS has no AAK domain, and yet it is inhibited by arginine, indicating an alternative mode of arginine inhibition mediated by arginine binding to the GNAT domain. To get insight into this mode of inhibition, we have compared the arginine effects on M. tubercu- losis and P. aeruginosa NAGSs, identifying also, using site directe mutagenesis, potential residues of the mycobacterial arginine site. Yeasts have a two-domain NAGS, but this enzyme is degraded in the presence of N-acetylglutamate when expressed except kinase (NAGK), the next enzyme in the arginine biosynthesis route, forming with NAGK a complex considered a true metabo- lon. To get insight into this complex architecture and stoichiome- try, we have produced in and isolated from Escherichia coli the recombinant complex, dissociating and studying separately its components, and studying the impact on complex formation of NAGS and NAGK domain deletions and mutations of individual residues. Acknowledgements: Grant BFU2008-05021 from the Spanish Ministry of Science. The authors belong to CIBERER-ISCIII. We thank Nadine Gougeard for expert technical help.

P2–122 Abstract withdrawn

P2–120 Novel peptide agonists, favoring galanin receptor type 2 over galanin receptor type 1 and 3 J. Runesson1, I. Saar1, J. Ja¨ rv2 and U. Langel1 1Neurochemistry, Stockholm University, Stockholm, SWEDEN, 2Institute of Chemistry, University of Tartu, Tartu, ESTONIA

P2–123 Different domains regulate homomeric and heteromeric complex formation among type I and type II TGF-beta receptors K. E. Shapira1, M. Mouler Rechtman1, A. Nakaryakov1, M. Ehrlich2 and Y. Henis1 1Department of Neurobiology George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, ISRAEL, 2Department of Cell Research and Immunology George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, ISRAEL

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

The galanin peptide family and its three receptors have with com- pelling evidence been implicated in a variety of human disorders. The co-localization with other neuromodulators and the distinct up-regulation during and after pathological disturbances has drawn attention to this neuropeptide family although, so far, no therapeutics have emerged past the animal model stage. In the current study we present data on receptor binding and functional response from novel galanin receptor type 2 (GalR2) selective chi- meric peptides, including the M1145 peptide which show more than 90-fold higher affinity for galanin receptor type 2 over gala- nin receptor type 1 and a 76-fold higher affinity over galanin receptor type 3. Furthermore, the peptide produces an agonistic effect in vitro seen as an increase in inositol phosphate (IP) accu- mulation, both in the absence or the presence of galanin. The pep- tide design with a N-terminal extension of galanin(2–13), prevails new insights in the assembly of novel subtype specific ligands for the galanin receptor family. Preliminary data on peptides further exploring the usage of N-terminal extension shows even higher Transforming growth factor-beta (TGF-beta) binds to and sig- nals via two serine-threonine kinase receptors, type I (TbetaRI) and type II (TbetaRII). The oligomerization of TGF-beta recep- tors modulates ligand binding, receptor trafficking and may con- tribute to signal diversification. However, numerous features of the molecular domains that determine the homo- and hetero-olig- omerization of full-length receptors at the cell surface and the mode of these interactions remain unclear. Here, we address these questions through computerized immunofluorescence co- patching and patch/FRAP measurements of different combina- tions of epitope-tagged receptors and their mutants in live cells. We show that TbetaRI and TbetaRII are present on the plasma

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membrane both as monomers, homo- and hetero-oligomers. The homodimerization of TbetaRII depends on a cytoplasmic juxta- membrane region (amino acid residues 200–220). In contrast, the cytoplasmic domain of TRI is dispensable for its homodimeriza- tion. TRI/TRII hetero-oligomerization depends on the cytoplas- mic domain of TbetaRI and on a C-terminal region of TbetaRII (residues 419–565). TGF-beta1 elevates TbetaRII homodimeriza- tion to some degree and strongly enhances TbetaRI/TbetaRII heteromeric complex formation. Both ligand-induced effects depend on the region encompassed between residues 200–242 of TbetaRII. Furthermore, the kinase activity of TbetaRI is also necessary for the latter effect. All forms of the homo- and het- ero-oligomers, whether constitutively present on the membrane or formed upon TGF-beta1 stimulation, were stable in the time- scale of our patch/FRAP measurements. We suggest that the dif- ferent forms of receptor oligomerization may serve as a basis for the heterogeneity of TGF-beta signaling responses. toxic effect of paclitaxel, an anti-microtubule drug functioning via spindle assembly checkpoint in breast cancer. Here we pres- ent, an active formulation of ANK, a cell-permeable peptide (ANKtide) has been developed. Once inside the cancer cell, ANKtide is capable of breaking up SNCG-BubR1 association. The anti-microtubule drug action of paclitaxel in parallel with inhibition of SNCG activity causes spindle assembly checkpoint activation, leading to mitotic arrest and apoptosis. Flowcytome- try and cell-based experiments (proliferation assays and anchor- age independent colony forming assays) confirmed that the ANKtide enhances activity of microtubule inhibitors by three- fold in in vitro models. Xenograft studies using MCF-SNCG breast cancer cells in nude mice unequivocally demonstrated 125I cytotoxic and synergistic effects of ANKtide. Further, labelled ANKtide was used to demonstrate in vivo tumour cell penetration and tissue distribution of ANKtide. This peptide holds a significant promise as an adjuvant, chemopotentiating therapy for breast cancer treatment.

P2–124 Structural and functional studies of a phytotoxin-degrading enzyme I. Shin, J. H. Lee, W. S. Jung, I. Hwang and S. Rhee Agricultural Biotechnology, Seoul National University, Seoul, SOUTH KOREA

P2–126 Intrinsically unfolded CHOP regulates biological activity of colon cancer cells during calcium stress V. K. Singh1, I. Pacheco2, V. N. Uversky3, S. P. Smith1, R. J. MacLeod2 and Z. Jia1 1Department of Biochemistry, Queen’s University, Kingston, CANADA, 2Department of Physiology, Queen’s University, Kingston, CANADA, 3Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, IN, USA

Burkholderia glumae produces a toxoflavin, the phytotoxin which is essential for the pathogenicity and causing grain rot and seed- ling rot in rice. An enzyme catalyzing in vitro the degradation of toxoflavin has recently been identified and named as TflA (toxo- flavin lyase). Currently, we were able to determine a crystal struc- ture of TflA by using the mutant variants. In this crystal structure, the putative active site has been identified based on the binding site of a metal, and further site-directed mutagenesis pro- vided evidence that the metal-ligating residues play a crucial role in catalysis. Structure with a substrate analog, 8-azaxanthine, also revealed its binding mode in a close vicinity of the active site. Recent functional analysis suggested that two amino acids near the active site are likely to be involved in a toxoflavin- degrading activity. Subsequently, we were successful in crystalliz- ing one of those mutants. Further structural study will be carried out with this particular mutant enzyme, and kinetic study for the wild-type and mutant enzymes will be performed for better understanding of the mechanism of a toxoflavin degradation.

Intrinsically unfolded proteins are emerging as substantial func- tional constituents of mammalian proteomes. Although the abun- dance of these proteins has been established by bioinformatics approaches, the vast majority have not been characterized struc- turally or functionally. The C/EBP homologous protein (CHOP) is a proto-oncogene, traditionally shown as a dominant-negative inhibitor of C/EBPs and a transcriptional activator of activating protein-1. We report here the in vitro characterization of CHOP, where our computational analyses and experimental evidences show for the first time that CHOP is an intrinsically unfolded protein. Intrinsic fluorescence, NMR spectroscopy, and analytical size-exclusion chromatography studies indicate that CHOP con- tains extensive disordered regions and self-associate in solution. Interestingly, the disordered N-terminal region has a key role in the oligomerization of CHOP and is vital for its biological activ- ity. We report a novel mechanistic role of CHOP in the inhibi- tion of Wnt/TCF signaling and stimulation of c-Jun and sucrase- isomaltase reporter activity in intestinal colon cancer cells. These findings are discussed in the context of oligomerization of intrin- sically unfolded proteins as one of the mechanisms through which they exert their biological function.

P2–125 ANKtide: a cell-permeable peptide inhibitor of synuclein-gamma activity in anti-microtubule drug resistant breast cancer V. K. Singh and Z. Jia Department of Biochemistry, Queen’s University, Kingston, CANADA

P2–127 The insights into the autoinhibition of the full-length formin molecule N. Sinitsina1, I. Orchanskiy1, C. Gould2, B. Goode2 and O. Sokolova1 1Biology Department, Moscow State University, Moscow, RUSSIA, 2Biology Department, Brandeis University, Waltham, MA, USA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Formins are modular proteins containing a series of domains and functional motifs. They form stable dimers with each monomer comprising of the highly conserved FH2 domain, its neighboring Synuclein-gamma (SNCG) protein is a critical component of spindle assembly-checkpoint protein complex. Using a PAN-can- cer tissue microarray and immunohistochemistry study, we have demonstrated increased expression of SNCG in one in three can- cer cases irrespective of cancer types including breast, ovarian, prostrate, uterine, colon, lung, liver, pancreas, kidney, stomach, small intestine, thyroid, testicular, skin, bladder, sarcoma, & lym- phoma. Specifically, in breast cancer a significant correlation fo SNCG with cell proliferation, metastasis and development of resistance to microtubule inhibitors has been observed. We have developed a novel peptide (ANK) and demonstrated it to effec- tively bind to SNCG, resulting in a significant increase in cyto-

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P2–129 The effect of oxidative stress on fibrinogen and its physiological functions J. Stikarova1, J. Suttnar1, Z. Reicheltova1, T. Riedel1, P. Majek1, M. Maly2 and J. E. Dyr1 1Department of Biochemistry, Institute of Hematology and Blood Transfusion, Praha, CZECH REPUBLIC, 2Department of Cardi- ology, Motol University Hospital, Praha, CZECH REPUBLIC

proline-rich FH1 domain, which was predicted to be unstructured, surrounded by regulatory domains: N-terminal GTPase binding domain (GBD), an FH3 region that may be important for localiza- tion, and the DID and DAD domains. The FH2 domain has effect on actin nucleation and elongation of new actin filaments. The DID and DAD domains are responsible for autoinhibition (Bol- dogh et al, 2001). Deletion of the DAD domain results in a 20 000- fold decrease in inhibitory potency for the truncated constructs in vitro (Madania et al, 1999). On the other hand, the yeast formin Bni1 lacks the DID sequence and it was shown to be active. Here we show, using single particles electron microscopy and 3D recon- struction, that mammalian full-length formin mDia1 exists in solu- conformation, with N-terminal domains tion in ‘‘closed’’ inhibiting its C-terminal ‘donut’. We have also demonstrated that the larger number of molecules in ‘open’ conformation correlates with the increasing activity of the formin molecule. Using the steered molecular dynamics, we have identified two pairs of amino acid residues responsible for DID-DAD complex formation that have strong interactions between them. These include ionic interac- tions of positively and negatively charged groups of GLU178 and ARG 248, and hydrophobic interactions of a carbon atom of THR175 with an aromatic ring of PHE247.

P2–128 Effect of phosphorylation on interaction of human tau protein with 14-3-3 N. Sluchanko, A. Seit-Nebi and N. Gusev School of Biology/Department of Biochemistry, Lomonosov Moscow State University, Moscow, RUSSIA

The oxidation of proteins changes their chemical and physical properties due to cross linking, partial fragmentation, aggrega- tion and new groups forming, thus impairing physiological func- tions of protein molecules. Fibrinogen, one of the most abundant plasma proteins, belongs to the molecules sensitive to oxidative stress. It is a 340 kDa glycoprotein composed of 6 polypeptide chains linked by 29 disulfide bonds. On activation by thrombin, cleavage of the A and B peptides from N termini of the Aa and Bb chains initiates the polymerization process and clot formation. We studied both functional and structural properties of fibrino- gen in the presence of oxidative stress conditions. The oxidative modifications were induced by malondialdehyde or sodium hypo- chlorite or 3-morpholinosydnonimine. Structural changes of fibrinogen were analyzed using SDS–PAGE with imunochemical detection and by quantification of carbonyl groups. The analyzer ‘Cone and Plate’ was used for the measurement of the interac- tions of isolated platelets with modified fibrinogen under physio- logical conditions. The properties of fibrin network were examined by scanning electron microscopy and by the measure- ment of turbidimetric curves. The release of fibrinopeptides was evaluated using HPLC with UV-detection. All oxidative modifi- cations induced changes in the structure and function of fibrino- gen as compared with control fibrinogen. The obtained results contribute to understanding of fibrinogen polymerization, fibri- n(ogen)-platelets interactions and thus to possible role of oxida- tively modified fibrinogen in atherothrombosis. Acknowledgemnts: This work was supported by a grant of The Grant Agency of the Czech Academy of Sciences nr. KAN200670701 and by grant of MZd nr. MZ00002373601.

P2–130 Synthetic polyelectrolytes as a tool for artificial chaperons creation S. Stogov1, V. Muronetz1 and V. Izumrudov2 1School of Bioingeneering and Bioinformatics, Moscow State Uni- versity, Moscow, RUSSIA, 2School of Chemistry, Moscow State University, Moscow, RUSSIA

Tau proteins belong to the group of neuronal proteins that are responsible for microtubule organization and therefore affect axonal transport, morphology and neuronal outgrowth develop- ment. Deregulation of tau that is often followed by tau aggre- gation leads to different neurological diseases. It is supposed that tau functioning is regulated by phosphorylation. cAMP- dependent protein kinase phosphorylates several Ser residues of tau and among them Ser located in consensus sequence R(S/ X)XpSXP, that is recognized by universal adapter protein 14-3- 3. Since direct binding of tau to 14-3-3 was not analyzed in detail we investigated interaction of the shortest isoform of tau protein (s3) with human 14-3-3Z using native gel electrophore- sis, chemical cross-linking and size-exclusion chromatography. Phosphorylation by PKA (up to 2 mole of phosphate per mole of s3) strongly enhanced interaction of s3 with 14-3-3. Apparent KD of the complex formed by phosphorylated s3 and 14-3-3 was about 2 lM, whereas KD of unphosphorylated s3 was at least 10 times higher. The stoichiometry of complexes formed by phosphorylated s3 and 14-3-3 was variable and was different from 1:1. According to the data of crosslinking 14-3-3 decreased formation of high molecular mass crosslinked homooligomers of phosphorylated s3 and at the same time increased the probabil- ity of formation of crosslinked complexes containing both s3 and 14-3-3. Taking into account high content of 14-3-3 in the brain we suppose that interacting with phosphorylated tau 14-3- 3 might affect its oligomeric state and by this means regulate formation of neurofibrillar tangles, important markers of neuro- degenerative diseases. Acknowledgements: This work was supported by Russian Foundation for Basic Research (grant 07-04-00115).

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

We report the data obtained on studying thermoaggregation and thermodenaturation of enzyme glyceraldehyde-3-phosphate dehy- drogenase in the presence of polysulfoanions. Study of these model systems can be used for elaborating the criteria that the polyelectrolyte should meet to suppress thermoaggregation with- out a notable loss in the protein functional activity. The effi- ciency of action of all studied polysulfoanions proved to be significantly higher as compared with suppression of GAPDH thermoaggregation by carboxylic polyanions. The lengthening of the chains and rising in their content enhanced the ability to reduce thermoaggregation. It was shown that the presence of short charged chains consisted of one or two dozens of chain links led to greatest failure of GAPDH structure. Maximum de- stabilizing effect was observed using hydrophobic oligomers. The protein bound with polysulfoanions charged chain can be readily unbounded by the addition of external salt and/or by polycation. Based on the revealed results, it became possible to design the ‘best’ polyelectrolytes capable to prevent undesirable aggregation of the proteins and provide their pulsing liberation from the com-

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07-03-00228-a) CNTP and plex. That makes possible a sophisticated mimicking of the molecular chaperons action as the important step on the way of creation artificial chaperons. Acknowledgements: The work was supported by NATO (PDD(CP)-(CBP.NR.RIG 982779)) and RFBR (08-08-00540-a; 08-04-00231-a; (Russia, 02.512.11.2249).

case of the HIV-1 protease, catalysis cannot occur when the dim- mer is not formed. We performed computational Alanine-Scan- ning mutagenesis in all the amino acids of the interface of the two monomers both in a structure of the protease complexed with the substrate and in an uncomplexed one and observed the differences in the strength of the binding. The ??G obtained for each amino acid residue allowed us to identify warm and hot spots in the interface which can be related with the binding of the two monomers. These hot spots are usually strong targets for binding inhibitorial drugs and therapies.

P2–131 Burkholderia cenocepacia and its novel two domain superlectin O. Sulak1, M. Delia2, A. Imberty1 and M. Wimmerova´ 2 1CERMAV-CNRS, Glycobilogie moleculaire, Grenoble cedex, FRANCE, 2National Centre for Biomolecular Research, Masaryk University, Brno, CZECH REPUBLIC

P2–133 Identification of a signal peptide peptidase and its homologues in Arabidopsis thaliana T. Tamura1, T. Asakura1, T. Uemura2, T. Ueda2, K. Terauchi1, T. Misaka1 and K. Abe1 1Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, JAPAN, 2Graduate School of Science, The University of Tokyo, Tokyo, JAPAN

Burkholderia cenocepacia is a Gram-negative bacterium that can be found throughout the environment. It is a part of ‘Burkholde- ria cepacia complex’, which causes potentially deadly problems to patients suffering from cystic fibrosis and chronic granulomatous diseases. A goal of primary importance of this study is the under- standing of the molecular mechanisms, especially protein-carbo- hydrate interactions, which give pathogenic bacterium the ability to invade and colonize its host. Four genes coding protein homo- logues to the lectin PA-IIL from Pseudomonas aeruginosa have been found in the genome of B. cenocepacia. One of them, BclC, is a 28 kDa protein. Further sequence analysis showed two dis- tinct domains in the protein structure. The C-terminal part cod- ing lectin domain shows partial homology to the lectin PA-IIL and the N-terminal part displays the sequence similarity only with a hypothetical protein from Photorhabdus luminiscens. Detailed functional studies using glycan microarray, surface plas- mon resonance and isothermal titration calorimetry allowed to define binding properties of the lectin. The results indicated the unusual binding activity of the protein. The C-terminal domain recognizes D-mannosylated saccharides. Interestingly, the N-ter- minal domain also displays sugar-binding activity with a strong the Lewis type preference for L-fucosylated oligosaccharides, determinants. The structural basis of the protein affinity towards different saccharides could serve as a starting point in the design of new and efficient ligand analogs and inhibitors. Such studies may direct the conception of new strategies to fight against path- ogenic agents. Acknowledgements: This work has been supported by Minis- try of Education (MSM0021622413) and Grant Agency of Czech Republic (303/06/1168).

Signal peptide peptidase (SPP) is a multi-transmembrane aspartic proteinase mediating regulated intramembrane proteolysis (RIP), which hydrolyses a substrate within the plane of the cellular membrane. In vertebrates, it plays crucial roles in life processes such as differentiation, embryogenesis, cell signaling and immu- nological response, but no information is available on plant SPP. We first identified an ortholog of human SPP (AtSPP), and its five AtSPP homologs (AtSPPL1–AtSPPL5) in the Arabidopsis genome database (1). These clones were classified into three dif- ferent clusters: AtSPP into human SPP (HsSPP) orthologs, AtS- PPL1 into the HsSPPL3 family, and AtSPPL2-AtSPPL5 into the group of SPP-like proteins of plant origin. AtSPP, AtSPPL1 and AtSPPL2 were examined for their expression profiles by in situ hybridization. AtSPP was strongly expressed in both the shoot meristem of germinating seeds and the inflorescence meristem at the reproductive stage. On the other hand, AtSPPL1 and AtS- PPL2 were expressed in the shoot meristem of germinating seeds, though at very low levels in the shoot apex at the reproductive stage. The subcellular localization of AtSPP, AtSPPL1 and AtS- PPL2 was investigated using green fluorescent protein (GFP) fusion proteins in cultured ‘Deep’ cells. AtSPP-GFP localized to the endoplasmic reticulum (ER), and AtSPPL1- GFPand AtS- PPL2- GFP to the endosomes. These results suggest that AtSPP mediates the signal peptide cleavage in the ER membrane, as well as HsSPP, and also that AtSPPL1 and AtSPPL2 located in the endosomes have distinct roles in cells. Reference: 1. Tamura, et al. FEBS J. 2008; 275: 34–43.

P2–132 HIV-1 Protease: a computational mutagenesis analysis of the monomers interface B. Tamames, P. Alexandrino Fernandes, M. J. Ramos Faculty of Sciences of the University of Porto, Chemistry, Porto, PORTUGAL

P2–134 Enzymatic and functional characterization of human Procarboxypeptidase A4 S. Tanco1, X. Zhang2, C. Morano2, F. Aviles1, J. Lorenzo1 and L. Fricker2 1Instituto de Biotecnologia y Biomedicina, Protein engineering and enzymology, Barcelona, SPAIN, 2Department of Molecular Pharmacology, Albert Einstein College of Medicine, NY, USA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Human Procarboxypeptidase A4 (hPCPA4) is the zymogen of a new enzyme that belongs to the A/B metallocarboxypeptidases subfamily. This protein was found to be up-regulated in prostate cancer cell lines after treatment with histone deacetylase inhibi- tors and was thus proposed to have a function in cell prolifera- The enzyme protease is one of the enzymes of the HIV-1 virus, and is responsible the catalysis of one of the critical steps of its life cycle, the cleavage of the individual proteins from the syn- thesised polyproteins. When this enzyme is inactive, the infected cell produces immature virions incapable of attacking other cells. Thus, protease has been one of the most extensible studied tar- gets for therapy in the HIV-1 virus. The present work focuses on the interfacial surface between the two monomers of HIV-1 pro- tease. This study is important because the interface between monomers in enzymes is a prime target for inhibition, as its activities are greatly affected when in monomeric form. In the

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the biological differences between people that have the wild type receptor and those who have these polymorphisms.

P2–136 Mutagenic analysis of the conserved residues of a haloacid permease J. Tsang and Y. M. Tse School of Biological Sciences, The University of Hong Kong, Hong Kong, HONG KONG

tion and differentiation. Moreover, PCPA4 gene is located at the putative prostate cancer-aggressiveness locus, suggesting a possi- ble role of CPA4 in prostate cancer aggressiveness. In our labora- tory, hPCPA4 has been produced as a recombinant protein and its structure has been determined. In the present work, in order to investigate the biological role of this enzyme a thorough enzy- matic and functional characterization of this protein has been done. Enzymatic studies with a series of new carboxypeptidases substrates reveal that CPA4 has a preference for large hydropho- bic C-terminal aminoacids as well as short residues like Leucine, Methionine, Valine or Isoleucine. Its activity has been further tested using biologically relevant peptides and a peptidomics approach, confirming its preference for hydrophobic aminoacids in position P1 and P2 and revealing peptides like Angiotensin I or Met-Enkephalin as possible substrates. Finally, transfections of hPCPA4 in different cell lines showed its presence in the extra- cellular media as the zymogen form, suggesting that CPA4 may function outside the cell processing bioactive peptides.

P2–135 Theoretical study for elucidating ligand binding processes on human beta-1 adrenoceptor polymorphisms Gly49Ser and Gly389Arg M. A. Soriano-Ursu´ a1, J. Correa-Basurto2, F. J. Cipres-Flores2, L. H. Favila-Castillo3 and J. Trujillo-Ferrara2 1Escuela Superior de Medicina IPN, Fisiologı´a y Farmacologı´a, Mexico D.F., MEXICO, 2Escuela Superior de Medicina IPN, Bioquı´mica, Mexico D.F., MEXICO, 3Escuela Nacional de Ciencias Biolo´gicas IPN, Inmunologı´a, Mexico D.F., MEXICO

Haloacetic acids are common environmental pollutants and have been shown to be mutagenic. Burkholderia caribensis MBA4, which utilizes monobromoacetate as a growth substrate, was iso- lated from soil and has been shown to produce an inducible de- halogenase. By means of chromosome walking we have identified a haloacid transporter protein gene (deh4p) downstream of the dehalogenase gene. Comparative analysis showed that Deh4p is a major facilitator superfamily protein, which usually possesses twelve transmembrane segments (TMS). Recombinant DNA technique based on a PhoA-LacZ dual reporters system was used to analyze the topology of Deh4p and the results showed that the N- and the C-termini of the protein were located in the cyto- plasm. However, only eight TMS were detected. The first two and the last two putative TMS were not found. ClustalW align- ment of Deh4p with some closely related transporter classifica- tion TC2.A.1.6 proteins has identified the presence of many conserved residues. An assay system that examined the functional property of the haloacid permease was constructed and used for analysis of mutations on some of these conserved residues. An MBA4 mutant defective in the haloacid operon was used as the host and plasmids with a haloacid operon were transformed into the cell. Charged or potentially phosphorylated residues were selected for modification. A total of sixteen deh4p mutants were generated and tested. The results showed that six of these mutants impeded the growth of the bacterium in utilizing chloro- acetate as a growth substrate. Most of these critical mutations were located in the putative cytoplasmic loops of the protein.

P2–137 An efficient protocol for affinity purification of glutamate carboxypeptidase II from insect cells: one-step affinity purification via Avi-tag J. Tykvart, P. Sacha, J. Starkova and J. Konvalinka Biochemistry, Institute of Organic Chemistry and Biochemistry AS CR v.v.i., Prague 6, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Avi-tag is a 15 amino acid sequence tag serving for affinity purifi- cation of proteins. Avi-tag fusion proteins undergo specific bioti- nylation by E. coli biotin ligase (BirA) and subsequently are purified on the basis of reverse binding of biotin to monomeric streptavidin mutein. We establish a one-step purification protocol for two different proteins (glutamate carboxypeptidase II and its homolog, NAALADaseL) fused with Avi-tag and a tobacco-etch virus protease cleavage site, both expressed in insect cells. In order to explore the effect of BirA cellular localization and tune its in vivo biotinylation capacity, we constructed three stable transfectants of Drosophila S2 cells with different cellular locali- zation of BirA and investigated its influence on the level of the fusion protein biotinylation. Subsequently, the clone of insect cells with the most efficient BirA localization was transfected by The human b1 adrenoceptor (Hb1AR) is a protein with 477 amino acids (aa) codified from the 10q24-26 gene. Currently, sev- eral single nucleotide polymorphisms (SNPs) have been detected in this coding region. Whereas two have been confirmed (Gly49- Ser and Gly389Arg), all others (that occur with allele frequencies < 1–2%) have not. In recombinant cell systems have been dem- onstrated that the Gly49 Hb1AR mutation is much more suscep- tible to suffer down-regulation by an agonist ligand than Ser49 Hb1AR, while Arg389 b1AR is 33.5 times more responsive to agonist-evoked stimulation than Gly389 b1AR. Additionally, some studies have related the presence of these polymorphisms to cardiovascular pathological phenotypes. In the current contribu- tion, three Hb1AR 3-D models using the complete sequence of Hb1AR were built by I-TASSER server, which combines the methods of threading, ab initio modeling and structural refine- ment. One difference between these structures is an amino acid at positions 49 or 389. 3-D structures were minimized using CHARMM27 field with NAMD 2.6 program, whereas the ligand-binding was studied by docking simulations with Auto- dock 3.0.5. For this purpose, a set of ligands including known inverse, partial and full agonists and antagonists were tested. These compounds were built and geometrically optimized by GAUSSIAN 98 software at the B3LYP/6-31Gd,p level. Then, the ligands were docked on the previously built Hb1AR 3-D structures for determining the best free binding energy. The bind- ing sites on Hb1AR 3-D structures for the ligands in their mini- mal interaction energy were analyzed using AUTODOCK tools ver.1.4.5 software. Results showed differences in ligand affinity and recognition mode on each structure and a structural analysis showed different conformational disposition in extracellular and intracellular loops. These theoretical results may help to explain

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plasmids coding for the purified fusion proteins. Large-scale expression targeted the protein production into the cell media. The Avi-tag purification protocol was further optimized in order to increase the fusion protein yield and purity sufficiently for pro- tein crystallization experiments. The influence of tag sequence on the biochemical features of purified proteins and the ability to cleave the tag off by TEV protease treatment were additionally analysed. In conclusion, we present (i) a novel one-step purifica- tion protocol for GCPII, an intensively studied pharmaceutical target in prostate cancer and numerous neuro pathological disor- ders, and (ii) apply the same purification protocol on the study of NAALADaseL, a GCPII homolog of an unknown physiologi- cal function.

P2–139 Study of the interactions between 14-3-3 protein and DNA-binding domain of forkhead transcription factor FOXO4 P. Vacha1, J. Silhan2, J. Vecer3, P. Herman3, M. Sulc4, V. Obsilova2 and T. Obsil1 1Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Prague 2, CZECH REPUBLIC, 2Department of Protein Structure, Institute of Physiology, Acad- emy of Sciences of the Czech Republic, Prague 4, CZECH REPUBLIC, 3Department of Physics, Faculty of Mathematics and Physics, Charles University, Prague 2, CZECH REPUBLIC, 4Department of Molecular Structure Characterization, Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague 4, CZECH REPUBLIC

P2–138 Protein modeling program, fams-ace2 in CASP8, using the local consensus and 3D-1D quality assessment H. Umeyama, G. Terashi, H. Sakai, K. Kanou, T. Hirata and M. Takeda-Shitaka School of Pharmacy, Kitasato University, Tokyo, JAPAN

funded by

The forkhead family of transcription factors shares a highly con- served DNA-binding domain. The FOX-proteins play roles in physiological and pathological processes. The FOXO-class con- sists of FOXO1, FOXO3, FOXO4 and FOXO6.These play a cen- tral role in cell-cycle control, differentiation, metabolism control, stress response and apoptoss. Transcriptional activity of FOXO is regulated through insulin-phosphatidylinositol-3-kinase-AKT/ protein-kinase-B (PI3K-AKT/PKB) signaling pathway. The AKT/PKB-mediated phosphorylation triggers phosphorylation of additional sites and induces FOXO binding to 14-3-3.This pro- motes nuclear export of the complex and inhibits reimport of FOXO by interfering with its nuclear localization signal (NLS). The role of 14-3-3 in regulation of FOXO-transcription factors is two fold. Firstly, the 14-3-3 binding FOXO inhibits the interac- tion with DNA and secondly prevent its nuclear reimport by masking its NLS. We used fluorescence spectroscopy techniques to investigate the mechanism of 14-3-3-dependent inhibition of FOXO4/DNA binding properties. We labeled four sites within fork head domain of FOXO4 with extrinsic fluorophore 1,5-IAE- DANS and used time-resolved fluorescence spectroscopy to study interaction between FOXO4 and 14-3-3.We suggest that 14-3-3 physically interacts with tested regions of fork head domain thus mask the DNA-binding interface thus blocking the FOXO4 bind- ing to DNA. In addition, time-resolved tryptophan fluorescence indicates no significant 14-3-3 binding-induced conformational change of FOXO4 fork head domain. Thus 14-3-3 functions as a ‘molecular-hood’ that covers DNA-binding interface of FOXO4 and blocks its interaction with DNA. Acknowledgements: Work Grant/ was IAA501110801 of the Grant Agency of the Academy of Sciences of the Czech Republic, by Research Project/MSM0021620857 and Centre of Neurosciences LC554 of the Ministry of Educa- tion, Youth and Sports of the Czech Republic, and by Research Project/AV0Z50110509 of the Academy of Sciences of the Czech Republic.

P2–140 14-3-3c regulates CtBP1-S/BARS-mediated fission of post-golgi carriers C. Valente, G. Turacchio, A. Colanzi, A. Luini and D. Corda Department of cell biology and oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro, ITALY

In the CASP8, our fams-ace2 server participated in the 3D coor- dinate prediction category as a human expert group. We applied two different scoring functions for the fully automated prediction server of the protein modeling, fams-ace2: (i) the local consensus score; and (ii) the model quality score based on classification of the side-chain environment for each residue. The local consensus score was used as a filter to select the models which have locally similar structures comparing with the set of models. The model quality score by our program CIRCLE was then used for the final selection of the best model. The procedure of fams-ace2 can be summarized as the following four steps: (i) Obviously incor- rect models which have serious physical clashes or broken main- chain structures were removed. (ii) The top 10% of server models were selected in the order of the local consensus score. (iii) All of the server models, selected in step (ii), were refined and rebuilt utilizing our homology modeling program FAMS. (iv) The top five structures were selected, according to a model quality evalua- tion based on CIRCLE score. The coefficients of SS score in the circle which do not use the consensus method were changed in the fams-ace2. The fams-ace2 is a fully automated server and does not require human intervention. The parameters of fams- ace2 were optimized by the data set of previous CASP7. We used the GDT_TS as the quality of model compared to native. When we applied optimized fams-ace2 to CASP7 targets, fams-ace2 obtained the best results over all server groups. Moreover, in Template Based Modeling Targets, fams-ace2 also achieved best results over all groups including human groups. Although the advantage of fams-ace2 over other servers is slightly smaller than the results applying for CASP7 (123 domains), the intended results are accomplished. This small difference between CASP7 and CASP8 might be caused by the change of the distribution of target difficulty and performance of servers. When we calculate GDT_TS of CASP8 models, we did not consider the domain regions. Therefore the results of some targets will be changed. The advantages of fams-ace2 are the fully automated process, the lower calculation costs due to the decrease of the modeled num- ber in comparison with Fams-ace1, and a high accuracy similar to the top of human groups.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Membrane fission is required during formation of intracellular transport carriers, a central process in membrane trafficking. Some fission mechanisms involve dynamin, and we have shown a fundamental role for CtBP1-S/BARS (BARS) at the Golgi com- plex and in fluid-phase endocytosis, defining a novel dynamin- independent fissioning machinery. To identify BARS-interacting proteins and define their roles in BARS-induced fission, we used

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P2–142 Novel groups and classes of antimicrobial peptides from spider venoms A. Vassilevski, S. Kozlov and E. Grishin Laboratory of neuro receptors and neuro regulators, Institute of Bioorganic Chemistry, Moscow, RUSSIA

affinity chromatography, mass spectrometry and Western blot- ting. One such protein is the c isoform of the 14-3-3 proteins, which are known to interact with phosphatidylinositol 4-kinase IIIb (PI4KIIIb) in both mammals and yeast. PI4KIIIb and phos- phatidylinositol 4-phosphate are fundamental to the organisation of the Golgi complex and post-Golgi transport in mammalian cells. In pull-down assays, 14-3-3c binds both BARS and PI4KIIIb, although BARS does not bind PI4KIIIb directly. In COS7 cells expressing the GFP-tagged temperature-sensitive mutant of VSVG as a cargo reporter, RNA interference against 14-3-3c inhibited TGN-to-PM transport of VSVG (60%). Here, a decrease in post-Golgi transport carriers was accompanied by formation of long VSVG-containing tubules (5–30 lM) from the TGN, which elongated (and retracted) at 0.5 lM/sec, consistent with microtubule-based motility. Thus, 14-3-3c is involved in BARS mediation of TGN-to-PM transport through control of the fission/ formation of constitutive post-Golgi carriers. This is the first demonstration for 14-3-3c as an adaptor protein that acts by its dimerisation and binding to PI4KIIIb and BARS, thus providing a functional link from PI4KIIIb-dependent tubulation/ post-Golgi carrier formation to BARS-induced fission.

Due to extensive studies of spiders from the families Lycosidae, Oxyopidae and Zodariidae, an amazing versatility of antimicro- bial peptides (AMPs) contained in their venoms has been dis- closed. Most of the newly described peptides belong to the vast class of alpha-helical AMPs. However, none of them share high homology to the known peptide sequences and therefore repre- sent novel families of AMPs. An interesting peptide that has a short disulfide-stabilized loop resembling the ’Rana-box’ has been described. This is a striking example of evolutionary parallelism on the molecular level between amphibians and spiders. A new class of cyto-insectotoxins has been thoroughly studied. These molecules are exceptionally long (69 residues) linear cationic amphipathic peptides that exhibit equally important antimicro- bial and insecticidal activities. Cyto-insectotoxins are believed to have evolved from combining two modules of short AMPs in a single molecule due to a point mutation in the precursor’s pro- cessing motif. Other examples of modular AMPs with a different molecular design as well as proline-rich AMPs have been identi- fied in spider venoms and will be presented. We describe a wide variety of spider venom AMP precursor structures. The ‘‘simple’’ type of precursors corresponds to the archetypal prepropeptide arrangement. We also found ‘‘binary’’ precursors containing two homologous mature chains and ‘‘complex’’ precursors that get processed into multiple mature chains of different types. The cor- responding processing motifs that specify limited proteolysis have also been described for the first time.

P2–141 Studying NK cell lectin receptors and their interactions using HEK293T eukaryotic expression system O. Vanek1, P. Celadova1, P. Kolenko2, J. Dohnalek2 and K. Bezouska3 1Department of Biochemistry, Faculty of Science, Charles Univer- sity, Prague, CZECH REPUBLIC, 2Group of Structural Analysis of Molecules, Institute of Macromolecular Chemistry AS CR, Prague, CZECH REPUBLIC, 3Department of Immunology and Gnotobiology, Institute of Microbiology AS CR, Prague, CZECH REPUBLIC

repertoire has

P2–143 Surface mapping of plant oil-bodies’ proteins M. Vermachova1, Z. Purkrtova1, J. Santrucek1, P. Jolivet2, T. Chardot2 and M. Kodicek1 1Department of Biochemistry and Microbiology, Institute of Chemical Technology Prague, Prague 6, CZECH REPUBLIC, 2JRU 206 Chimie Biologique, Institut National de la Recherche Agronomique, Thiverval Grignon, FRANCE

rat and

receptor

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Natural killer cells are intensively studied part of immune system due to their ability to directly kill cancer cells. Recent research in their C-type lectin-like receptors shown that ligands of some of these previously orphan receptors are lying within their own family, describing a lectin–lectin interaction. This is the case for activation human KLRF1-AICL, inhibitory human NKRP1-LLT1 orthological and mouse NKRP1B/F-Clrb/g pairs of receptors. Moreover, in the rat this inhibitory NKRP1B-Clrb mutual system has been shown to be subverted by mouse cytomegalovirus protein RCTL, coding for viral version of Clrb, which serves as a decoy ligand for NK cells. Thus, this type of lectin receptor interaction seems to be important not only in immune surveillance but also in anti- infection immunity. We decided to study this system on a struc- tural level by means of protein crystallography and various bio- physical characterisation techniques. We present here our results using a eukaryotic expression system based on transient transfec- tion of human HEK293T cell line [1]. In contrary to bacterial recombinant production, proteins studied are obtained not in the form of inclusion bodies (requiring in-vitro refolding with unsatis- factory yield) but as a native glycosylated species. As the glyco- sylation might be important for lectin–lectin interaction, this is considered additional benefit of this system, aiming for a study of importance of the glycosylation itself. This work is supported by the European Commission (Integrated project SPINE2-COM- PLEXES, contract No. 031220). Reference: 1. Aricescu AR, Lu W & Jones EY. Acta Crystallogr D Biol Oil-bodies are plant lipoprotein particles used for lipid storage especially in the oil seeds. They are composed of neutral lipids surrounded by a phospholipid monolayer with several integrated proteins stabilizing their structure. However, only a little is known about steric arrangement and cross-interactions of these proteins. The main aim of this work is to identify the regions of the proteins that are in direct contact with aqueous surroundings of oil-bodies. In second part of study, our attention is focused on structure of these proteins; the native proteins are compared with those overexpressed in Escherichia coli. The surface mapping method is based on selective chemical modifications of certain amino-acid residues, namely arginine, lysine, tyrosine, trypto- phan, cysteine and both aspartate and glutamate. Only the amino-acid residues which are surface-accessible (i.e. exposed to water environment and not involved in any non-covalent interac- tions) can be modified. We have applied this method on oil- bodies isolated from the seeds of Arabidopsis thaliana. Integral proteins (modified or not) were resolved by SDS-PAGE and identified by trypsin or chymotrypsin cleavage. Modified residues were determined by characteristic mass shifts in MS-spectra. This method together with in silico modelling may pave the way for revealing the tertiary structure of this specific group of proteins. The obtained results confirmed the generally accepted model of Crystallogr 2006; 62:1243–1250.

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oil-bodies and allowed to discuss localisation of the protein com- ponents as well as their potential mutual interactions. size, identified by mass-spectrometry as

P2–144 Molecular modelling of a novel series of Dengue virus (typeII) helicase potential inhibitors, by a de novo structure-based drug design approach D. Vlachakis and M. Vlassi Biology, NCSR ‘Demokritos’, Athens, GREECE

inversely proportional

patients, the changes ranged from normal to strong decrease or even absence of urinary UMOD. In some cases protein of abnor- the mal molecular UMOD protein lacking C-terminal part, was found. The abnor- mal patterns normalized in all patients on enzyme replacement therapy (ERT) with recombinant GLA preparations (Fabrazyme or Replagal) but not in all of the patients treated with synthetic ceramid glucosyltransferase inhibitor Zavesca (Miglustat). The in situ immunohistochemical analysis of affected kidney sections revealed abnormal UMOD localization in TALH and collecting tubules and showed that UMOD expression in TALH epithelia was to the degree of Gb3 storage. Although the mechanisms how the enzyme defect and/or storage process lead to abnormal UMOD expression, processing and excretion remain unknown, our observations warrant evaluation of tubular functions in Fabry disease patients and suggest possi- ble way of ERT monitoring.

P2–146 Studies on yellow lupin (Lupinus luteus) gene encoding 5’-methylthioadenosine nucleosidase E. Wludzik, A. Guranowski and K. Nuc Department of Biochemistry and Biotechnology, Poznan University of Life Sciences, Poznan, POLAND

The Dengue virus is a member of the Flaviviridae family and is believed to be responsible for one of the most common arthro- pod-borne diseases in the world. Each year, 100 million people become infected with dengue virus. To date, there is no commer- cially available vaccine for Dengue. Helicase is a vital enzyme for the viral proliferation and transmission and offers a potential tar- get for the development of anti-dengue agents.In this study we are using a de novo structure-based drug design approach to design novel compounds as potential inhibitors of the Dengue virus (type II) helicase, towards the development of new drugs. Initially, a putative binding site was identified on the enzyme’s surface by analysing the known crystal structure. In order to gen- erate an initial set of compounds that could potentially bind to this site, the drug design software package, Ligbuilder, was used. The full population of the derived compounds, exceeding 20,000, was then combined using the MOE>Breed virtual combinatorial chemistry approach that narrowed them down to seven groups of ten compounds each. Subsequently, lead optimisation was carried out by docking the derived series of virtual compound libraries. The MOE>Dock module was used for this purpose. Finally, only the five top ranking compounds were subjected to extended molecular dynamics simulations, in order to further investigate their affinity and binding mode for the given target.Future work includes synthesis of the lead compounds and their in vitro bio- logical evaluation using an in-house developed enzymatic assay.

P2–145 Abnormal processing of uromodulin (UMOD) in Fabry disease patients is reversible by enzyme replacement therapy P. Vyletal1, H. Hulkova1, P. Novak2, L. Berna1, M. Zivna1, K. Hodanova1, M. Elleder1 and S. Kmoch1 1Institute of Inherited Metabolic Disorders, First Faculty of Medi- cine, Charles University Prague, Prague 2, CZECH REPUBLIC, 2Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, CZECH REPUBLIC

5’-Methylthioadenosine nucleosidase, MTAN (EC 3.2.2.16), cata- lyzes hydrolytic cleavage of 5’-methylthioadenosine to adenine and 5’-methylthioribose. It is the first step of the biochemical pathway, termed the MTA cycle, which leads to the reutilization of the methylthio- and four-carbon moieties of the 5’-methylthio- adenosine to methionine. The MTA cycle is important for plant physiology because it ensures high rates of ethylene or/and poli- amines synthesis without net consumption of methionine. MTAN from Lupinus luteus seeds was purified and kinetically characterized in 1981. Unlike bacterial and some plant MTANs the lupin enzyme does not cleave S-adenosylhomocysteine. In this study, in order to investigate molecular determinants of enzyme luteus cDNA activity and substrate specificity, we screened L. library for the MTAN cDNA. The obtained open-reading frame is 762 bp long and encodes a protein of 253 amino acids (FJ174792). Amino acid sequence of the lupin enzyme shows 62%, 61% and 66% identity to Arabidopsis thaliana MTAN1, A. thaliana MTAN2 and Oryza sativaMTAN, respectively. Resi- dues that were shown to be essential for catalysis in A. thaliana MTAN1, Glu 38, Glu 202 and Asp 225 are strictly conserved in L. luteus MTAN. The potential key residues for discriminating S- adenosylhomocysteine by L. luteus MTAN are Phe 134 and Leu 167. We also screened L. luteus genomic library for MTAN gene. The gene consists of 8 exons homologous to the previously char- acterized cDNA (FJ460365).

P2–147 Investigation of heme transport by porphyromonas gingivalis HmuY protein through the stability studies H. Wo´ jtowicz, M. Olczak and T. Olczak Biochemistry, Biotechnology, Wroclaw, POLAND

a gingivalis,

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Porphyromonas gram-negative black-pigmented anaerobic bacterium, is considered the main factor of chronic periodontitis. For successful establishment of an infection, the bacterium requires heme as a source of both iron and porphyrin ring. In P. gingivalis, heme is primarily acquired by heme-binding protein, HmuY, and its cognate outer-membrane receptor, HmuR. When bound to heme, HmuY is a tetramer whilst it is Fabry disease is an X-linked disorder caused by deficiency of lysosomal a-galactosidase A (GLA). Enzyme deficiency induces a massive storage of the enzyme substrate globotriaosylceramide (Gb3) in lysosomes of various cell types, which leads into multi- organ pathology. Gb3 storage in epithelial cells of the nephron leads to renal failure, which is a prominent feature of the disease. UMOD (Tamm-Horsfall protein) is a glycoprotein selectively expressed in the epithelial cells forming thick ascending limb of Henle’s loop (TALH), macula densa segment and distal convo- luted tubule. The protein is GPI anchored in the apical mem- brane of the cells from where it is continuously released into urine by not yet defined proteolytic mechanism. Defects in UMOD biology have been associated recently with several auto- somal dominant kidney diseases. Using Western blot analysis, we examined and found significant quantitative and qualitative changes in urinary UMOD excretion in 15 Fabry disease male patients at various clinical stages of the disease. In untreated

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Poster Presentations

P2–149 Crystal structure of aminoacylpeptidase dipeptidase from Vibrio alginolyticus, a metallopeptidase with dinuclear center T. K. Wu and J. Y. Chang Biological Science and Technology, National Chiao Tung University, Hsin-Chu, TAIWAN

dimeric in an apo-form. Since HmuY may switch between the apo- and holo-forms as part of its function, the biophysical prop- erties of both states are of biological relevance and may explain how heme binding and transport occur. Three tryptophan resi- dues present in HmuY sequence enable fluorimetric studies of local changes in the protein structure during unfolding. More- over, tryptophan 51 and 161 are located close to the heme cavity thus resulting in fluorescence quenching upon heme binding. Therefore, we expressed and purified single, double and triple tryptophan HmuY mutant proteins. Spectroscopic examinations of the mutated proteins that retained the native structure enabled in-depth analysis of the unfolding and heme binding – dissocia- tion process. Chemical and thermal unfolding studies indicated no significant thermodynamic differences in the stability of both HmuY forms. However, the mechanisms of unfolding are differ- ent, since holo-protein needs to release heme and dissociate into subunits to gain the initial conformation of apo-protein. Results obtained from these studies are crucial for understanding of the heme transport in P. gingivalis involving the HmuY protein.

Aminoacylhistidine dipeptidase (PepD, EC 3.4.13.3) is a member of the metallopeptidase family M20 from the metallopeptidase H (MH) clan. It catalyzes the cleavage and release of an N-terminal amino acid, usually neutral or hydrophobic residue, from Xaa- His dipeptide or degraded peptide fragments for amino acid utili- zation. We have cloned, over-expressed, and purified the wild- type protein and characterized its biochemical properties includ- ing substrate specificity, pH and temperature optima, and effects of metal ions substitution. Mutational analysis of the putative active-site residues supported the involvement of His80, Asp119, Glu150, Asp173, and His461 as well as Glu149, His219, Asn260, Arg369, and Gly435 in metal-binding and substrate recognition, respectively. Crystallization and X-ray structure determination of the PepD protein revealed a ‘catalytic domain’ and a ‘lid domain’, which are similar to some kinds of other dipeptidases. Two zinc ions, clammed by five conserved residues, are located in the catalytic domain and played important roles about sub- strate hydrolysis. The functional role of these residues on sub- strate binding and enzyme catalysis will be discussed.

P2–148 Structural interpretation of thermo- biochemical properties and thermostability of malate dehydrogenase from thermus thermophilus HB8 S. P. Wu1, S. H. Chou2, C. H. Hsu3 and T. S. Hwang2 1Department of Biotechnology, Yuanpei University, Hsinchu City, TAIWAN, 2Department of Medical Nutrition, I-Shou University, Kaohsiung County, TAIWAN, 3Department of Agricultural Chem- istry, National Taiwan University, Taipei City, TAIWAN

P2–150 Inhibition mechanisms of heavy metals on pinus brutia glutathione S-transferases C. Yilmaz1, E. Saatci2 and M. Iscan1 1Biological Sciences, Middle East Technical University, Ankara, TURKEY, 2Biology Department, Erciyes University, Kayseri, TURKEY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

by BAP- is Malate dehydrogenase (MDH, EC 1.1.1.37), an enzyme involved in TCA cycle and malate-aspartate shuttle, plays important roles in glucose metabolism and energy generation. MDH catalyzes a dehydrogenation reaction from malate to generate oxaloacetate, accompanying with the reduction of NAD to generate NADH. The overall reaction catalyzed by MDH is reversible; therefore, the activity of MDH can be easily measured by the decreasing amount of NADH which has an absorbance in wavelength 340 nm. The convenient measuring method make MDH be applied to coupling assays for measuring enzyme activity, such as activity assay of aspartate aminotransferase and aspartate oxi- dase. Here a thermophilic MDH from Thermus thermophilus HB8 was studied. Purified Thermus thermophilus MDH (TtMDH) was obtained from the recombinant E. coli harboring T. thermo- philus mdh gene. Characterization was performed and results showed the specific activity of TtMDH was 7 U/mg at 37ordm;C and increased according to the increasing temperature; however, the activity increased dramatically after 60ordm;Cand have an optimal temperature at 90ordm;C with a specific activity about 2,600 U/mg, more than 300-fold than at 37ordm;C. Investigation of TtMDH’s the stability showed it possessed a high stable char- acter because TtMDH still kept almost full activity after it was received heat-treatment for 30 minutes at different temperature below 90ordm;C. Structural simulation and energy minimization at high temperature was used to reconstruct structures from crys- tal structure of TtMDH. Inspecting the simulated structure, some little changes were occurred around the active site to make it more fit substrates when the temperature rose and resulting in the increase of activity, but the overall structures did not change a lot and kept all related positions, which made TtMDH had a high stability at high temperature. TtMDH is a rare stable MDH, suitable for using in industrial and academic application. Plants, especially trees have defensive mechanisms against many environmental stress factors, such as heavy metals which cause an increased production of reactive oxygen species. Glutathione S-transferases (GSTs) have roles in such stress conditions because of their ability to increase the solubility and further metabolic processing of such compounds in the cell upon conjugation of glutathione (GSH). The studies on the direct effects of heavy metals on plant GSTs are rare. Our aim is to investigate the effects of heavy metals on the activity of GSTs in the needle extracts of Pinus brutia, in vitro, which is a very important forest tree in Turkey and under the thread of pollution in most areas of its natural habitat. Cd+2, Zn+2, Hg+2 and Cu+2 were applied into the reaction mixture, at varying concentrations, seperately; while CDNB and GSH were varied between 0.5–1.25 mM. Zn+2 and Cd+2 combined effects were, also, investigated. The results of activity assays were analyzed by Dixon and Hill plots. The inhibition mechanism of Cd+2, Hg+2 and Cu+2 were mixed type with Ki values 171, 218 and 461 mM, respectively. Ki for Zn+2 was 68 mM with uncompetitive inhibition. Zn+2 and Cd+2 were combined as 1:2 (Zn+2:Cd+2), 1:1 (Zn+2:Cd+2) and 2:1 (Zn+2:Cd+2) mixtures and they exhibited mixed inhibition with 108 mM Ki; noncompetitive inhibition with 17 mM Ki; and uncompetitive inhibition with 52 mM Ki, respectively. Acknowledgement: This project supported DPT2002K120510.

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Abstracts

red shift in comparison to the PTAA-filaments interaction. This suggests that the insulin ‘noodle’-like filaments are formed by loose assembly of insulin molecules. References: 1. Nilsson et al., Adv Mat 2008; 20: 2639. 2. Chem Bio Chem 2006; 7:1096. 3. ACS Chem Biol 2007; 4: 553. 4. Sigurdson et al., Nature Methods 2007; 4: 1023.

P2–151 The role of flexible C-terminal region of Pseudomonas stutzeri L-rhamnose isomerase H. Yoshida1, K. Takeda2, K. Izumori3 and S. Kamitori1 1Life Science Research Center and Faculty of medicine, Kagawa University, Kagawa, JAPAN, 2Life Science Research Center and Faculty of Agriculture, Kagawa University, Kagawa, JAPAN, 3Faculty of Agriculture, Kagawa University, Kagawa, JAPAN

stutzeri L-rhamnose

P2–153 Fluorometric definition of primary amines based on their reaction with fluorescamine Z. Mustafaeva, Y. Budama Battal and S. Derman Bioengineering, Yildiz Technical University, Istanbul, TURKEY

L-rhamnose isomerase is homo tetrameric enzyme and can reversibly catalyze the isomerization between L-rhamnose and L-rhamnulose. Pseudomonas isomerase (P. stutzeri L-RhI) shows broad substrate specificity and is able to catalyze the isomerization between various aldoses and ketoses. In our previous study, we have determined the crystal structure of wild-type P. stutzeri L-RhI and reported the complex structures with bound substrates. On the basis of the active site structure of wild-type P. stutzeri L-RhI, the site-directed mutagenesis studies on P. stutzeri L-RhI have been done at the position of Ser329 on active site. While the C-terminal region on wild-type P. stutzeri L-RhI had been invisible due to the lack of electron density, the complex structures with bound substrates of newly constructed Ser329-substituted mutant for Phe or Leu showed the significant electron density map. The C-terminal region (GGGGIIGS + HHHHHH) in S329F/D-allose goes into the active site of the neighboring molecule and covers the active site, whereas the C- terminal region in S329F/L-rhamnose goes back to the active site of the same molecule. An increase in hydrophobicity on active site might stabilize the active site structure. The observation of the flexibility of C-terminal region suggests a flip-flop movement on inter-molecular surface of dimer-form. It is also supposed that the C-terminal region might stabilize the enzyme-substrate com- plex structure by covering the active site.

Introduction: Fluorescamine is known to react almost instanta- neously with primary amines (R-NH2). Fluorescamine itself is rapidly hydrolyzed to nonfluorescent products under reaction conditions. Fluorogenic reagents has been applied to biochemi- cally important determinations such as the detection of primary amines (amino acids, peptides, catecholamines, aminated sugars, etc.). In particular, fluorogenic reagents have been widely used in such determinations due to their weak or nonexistent fluorescent background. The advantage of the fluorescamine is that the for- mation of the fluorophore is almost instantaneous in neutral and alkaline solutions; moreover fluorescamine is a specific derivatizer for the amine function. Methods: The covalent conjugate of different proteins with co- polymers of acrylic acid (polyacrylic acid, PAA) were synthesized at different ratio of components (nProtein/nPolymer) and investi- gated the mechanism of condensation reaction by using different physicochemical analysis as HPLC, Fluorescence Spectroscopy and Fluorescamine assay. For determination of binding ratio of protein-polymer congutages, fluorescent measurements were per- formed in the presence of fluorescamine at the wavelengts of exci- tation 390 nm and emission 475 nm. Results And Conclusions: When conjugation reaction com- pleted, the amount of free amino groups had decreased and observed that Fluorescence Intensity had decreased and the esti- mated degree of primary amino group after conjugation, calcu- lated from fluorescence spectrums.

P2–152 Structure and cytotoxicity of novel insulin ’noodle’-like filamentous amyloids T. Zako1, T. Kobayashi2, M. Sakono1, M. Lindgren3, P. Hammerstrom4, P. Nilsson4 and M. Maeda1 1Bioengineering Laboratory, RIKEN Institute, Saitama, JAPAN, 2Graduate School of Frontier Sciences, The University of Tokyo, Chiba, JAPAN, 3Department of Physics, The Norwegian Univer- sity of Science and Technology, Trondheim, NORWAY, 4Depart- ment of Chemistry, Linkoping University, Linkoping, SWEDEN

P2–154 Three-dimensional structure of Pin protein from C. symbiosum: toward to cold-adaptation cis/trans isomerization and other cellular processes in psychrophilic archaea. I. Zhukov1, M. Jaremko2, L. Jaremko2, A. Ejchart2, J. Mueller3 and P. Bayer3 1Slovenian NMR Centre, National Institute of Chemistry, Ljublj- ana, SLOVENIA, 2Laboratory of Biological NMR, Institute of Biochemistry and Biophysics, Warsaw, POLAND, 3Centre for Medical Biotechnology – ZMB, Essen, Institute for Structural and Medicinal Biochemistry, GERMANY

cyclophilins, the

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Insulin is a small peptide hormone that is known to form protein assembly called amyloid fibrils under acidic conditions. We have previously shown that filamentous (‘noodle’-like) insulin amyloid which was morphologically different from fibrous (‘needle’-like) insulin amyloid was formed in the presence of a reducing agent, tris (2-carboxyethyl) phosphine hydrochloride (TCEP). The CD spectra showed that both of insulin fibrils and filaments contain a beta-sheet structure. Nevertheless, Thioflavin T (ThT) binding property was very different between them, suggesting a difference in inner structure. In this study, we examined their cell toxicities using two different cell lines with MTT assay, and also examined difference in their inner structures using novel luminescent conju- gated polyelectrolyte probes (LCPs)1–4. The cytotoxicity of the insulin filaments against rat PC12 and human HEK293 cell line was also extremely low while the fibrils were toxic, suggesting that the insulin filaments were generally nontoxic. This finding supports the idea that cell toxicity of amyloids correlates with their morphology. The fluorescence measurement in the presence of Polythiophene acetic acid (PTAA)1–4, one of the conformation sensitive LCPs, showed that PTAA weakly bound to the insulin filaments and that the insulin fibrils’ spectrum revealed a spectral The Pin proteins (Protein Interacting with NIMA-kinase) is a smallest parvulin-type PPIase founded in all eukaryotes and investigated up to now. Proteins belonged to parvulin family were described for all organisms from bacteria and eukarya up to human, but only one parvulin was isolated from archaeal origin. There are three structurally distinct PPIases subfamilies were characterized so far, the FK506-binding (FKBP’s), and the parvulins which catalyze cis/trans rotation around Xaa-Pro peptide bond in target proteins and named pept- isomerases (PPIases). Due to striking similarity to idyl-prolyl

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Poster Presentations

P2–155 Novel players in mitochondrial K+/H+ exchange L. Zotova and R. J. Schweyen Max F. Perutz Laboratories, Department of Genetics, Vienna Uni- versity, Vienna, AUSTRIA

human Pin1 and Pin14, the Pin protein from psychrophilic archa- eon C. symbiosum (CsPin) - the first and unique peptidyl-prolyl cis/trans isomerase from the Archaea Kingdom - constitute an interesting object to structural study of archaeal parvulins. In our work, the three-dimensional structure of CsPin protein was deter- mined with comparable resolution 2.1 A˚ on base 1798 conforma- tional restraints (1585 NOE-derived distance constraints, 38 hydrogen bonds, and 175 torsion angles constraints). The overall fold of CsPin protein - described as ’parvulin’ fold - comprise four-stranded antiparallel b-sheets wrapped around C-terminal a- helix and three a-helises stacking on the other side of the central b-sheet. The obtained spatial structure of Pin protein from C. symbiosum reveal structural similarity to other PPIases belonged to the same family, namely Par10 parvulin from Escherichia coli (pdb code 1jns) [1] and PrsA PPIase domain from Bacillus subtilis (pdb code 1zk6) [2] which have been solved previously by NMR spectroscopy. The detail structural analysis of CsPin in compare to E. coli and human PPIases shed of light on cold-adaptation mechanism of cis/trans isomerisation and other cellular processes within the Crenarchaeal endosymbiont. References: 1. Kuhlewein, Voll, Alvarez, Kessler, Fischer, Rahfeld & Gem- mecker. Prot. Sci 2004; 13: 2378–2387. 2. Tossavainen, Permi, Purhonen, Sarvas, Kilpelainen & Seppala. FEBS Lett 2006; 580: 1822–1826.

these mutants that

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

The inner mitochondrial membrane is impermeable to cations. Therefore ion transporters are assumed to exist in this membrane and to be essential for normal function of the mitochondria. Dis- ruption of the YOL027c in yeast cells was shown earlier to result in strongly reduced mitochondrial K+/H+ exchange activities and growth defect on non-fermentable media. Yol027 has homo- logues in various eukaryotic organisms, including Mrs7 in yeast and LETM1 in mammalian cells. Deletion of human LETM1 is supposed to cause Wolf-Hirschhorn syndrome. Phenotypes of the yol027D disruption can be suppressed by over-expression of its yeast homologue Mrs7 or by unrelated yeast protein Ydl183. All three proteins are integral proteins of the inner mitochondrial membrane with a single transmembrane domain. The single, dou- ble and triple mutants lacking these proteins most likely differ only by the degree to which they have lost K+/H+ exchange activity. The triple mutant yol027D mrs7D ydl183D have much stronger synthetic phenotype than single and double mutants, e.g. completely loss of K+/H+ exchange activities and signifi- cant reduction of the cell viability. Expression of Yol027, Mrs7, Ydl183 or human LetM1 in the triple mutant yol027D mrs7D ydl183D can rescue its growth and activate the K+/H+ exchange reaction. The triple mutant phenotype can be efficiently suppressed by nigericin, a synthetic K+/H+ ionophore, that indicates lacked this exchange activity. Accordingly, the homologues Mkh1, Mrs7 and LetM1 as well as indirectly the apparently unrelated Ydl183 are directly or involved in the formation of an active mitochondrial K+/H+ exchange system.

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Abstracts

P3 Metabolites in Interactions

as controls (11.3 ± 5.7 compared with

P3–1 Preparation of apo-cytochrome b5 by its heterologous expression in Escherichia coli D. Aimova, V. Kotrbova, M. Ingr, L. Borek-Dohalska, V. Martinek and M. Stiborova Charles University Faculty of Science, Biochemistry, Prague, CZECH REPUBLIC

NAG synthase (NAGS) activity was measured after incubation of sonicated mouse liver mitochondria (MLM) with the sub- strates and VPA or valproyl-CoA. Both OrA and NAG were quantified by LC-MS/MS with MRM acquisition mode using [15N2]-OrA and [13C5]-NAG as respective internal standards. Results: The amount of NAG in livers of VPA-treated rats was decreased and 16.8 ± 5.7 nmol/g wet weight, respectively). Additionally, uri- nary OrA levels were slightly decreased after VPA administra- tion. In MLM valproyl-CoA was found to reduce NAGS activity in higher extent than VPA, and the characterization of the inhibi- tion using acetyl-CoA as substrate, reveals a mixed inhibition mechanism. Discussion: The results clearly demonstrate: (1) an inhibition of NAGS by valproyl-CoA; (2) an in vivo reduced bioavailability of NAG, together with a reduced OrA excretion which suggest a decrease of mitochondrial carbamoyl-phosphate synthesis. These effects would account to a reduced flux through the urea cycle explaining the potential VPA-associated hyperammonemia. Acknowledgement: Supported by FCT, SFRH/BD/22420/ 2005.

P3–3 Energy metabolism assessment during heart preservation in Celsior and Histidine buffer solution by 1H-NMR spectroscopy M. Alves1, F. O. Martins1, P. J. Oliveira1 and R. A. Carvalho2 1Centre for Neuroscience and Cell Biology, Zoology Department, Coimbra, PORTUGAL, 2Centre for Neuroscience and Cell Biology, Biochemistry Department, Coimbra, PORTUGAL

Cytochrome b5 (b5) has been shown to modulate several cyto- chrome P450 (CYP)-mediated reactions. In order to elucidate the mechanism of such modulations it is neccessary to evaluate not only the effect of native b5, but also that of the apo-cytochrome b5 (apo-b5), on CYP-catalyzed reactions. Therefore here, the apo- b5 protein was prepared using a heterologous expression in Esc- herichia coli. The gene for rabbit b5 was prepared from synthetic oligonucleotides using polymerase chain reaction (PCR), cloned into pUC19 plasmid and amplified in DH5a cells. The gene sequence was verified by DNA sequencing. The sequence coding b5 was cleaved from pUC19 by NdeI and XhoI restriction endo- nucleases and re-cloned to the expression vector pET22b. This vector was used to transform E. coli BL-21 (DE3) Gold cells by heat shock. Expression of b5 was induced with isopropyl b-D-1– thiogalactopyranoside (IPTG). The b5 protein, produced predom- inantly in its apo-form, was purified from isolated membranes of E. coli cells by chromatography on a column of DEAE–Sepha- rose. Using such procedures, the homogenous preparation of b5 protein was obtained. Oxidized and reduced forms of the apo-b5 reconstituted with heme exhibit the same absorbance spectra as native b5. The prepared recombinant apo-b5 reconstituted with heme can receive electrons from NADPH:CYP reductase, being reduced by this enzyme. The reconstituted apo-b5 is also fully active to increase the CYP3A4 enzymatic activity measured with 1-phenylazo-2-hydroxynaphthalene (Sudan I) as a substrate. Acknowledgements: Supported by GACR (303/09/0472 and 203/09/0812), GAUK (1272080) and the Czech Ministry of Edu- cation (MSM 0021620808).

P3–2 Quantitative analysis of N-acetylglutamate and orotic acid by MS–MS: underlying mechanism of valproate effect on mitochondrial detoxification of ammonia C. Aires1, A. van Cruchten2, J. Ruiter2, L. IJlst2, I. Tavares de Almeida1, M. Duran2, R. J. A. Wanders2 and M. F. B. Silva1 1iMed.UL - Research Institute for Medicines and Pharmaceutical Sciences, Metabolism and Genetics, Lisboa, PORTUGAL, 2Academic Medical Center University of Amsterdam, Laboratory Genetic Metabolic Diseases, Amsterdam, THE NETHERLANDS

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Valproic acid (VPA)-induced hyperammonemia occurs in approx- imately 45% of patients after treatment with the drug. Previous results suggested an interference of VPA with mitochondrial urea cycle as possible underlying cause. Aims: To elucidate the pathogenic mechanisms causing hyper- ammonemia, namely the interference of VPA and its metabolite valproyl-CoA with the biosynthesis of N-acetylglutamate (NAG) and of carbamoyl-phosphate, evaluating the levels of orotic acid (OrA). Methods: NAG was quantified in livers of control and VPA- treated rats (100 mg/Kg, 15 days, n = 12). Urines collected (for 24 hours at day1 and day15) were used to quantify OrA. The The myocardium is capable of consuming a variety of fuels including fatty acids, glucose, lactate, amino acids and ketone bodies. During heart preservation, severe ischemia occurs and energy metabolism is readapted according to substrate availabil- ity. We aimed to compare and follow, during different ischemic periods, energy metabolism throughout heart preservation in Cel- sior (Cs), Histidine Buffer (HBS) and Krebs-Henseleit (K-H) solutions. Male Wistar rat hearts were kept at 4 ?C in the preser- vation solutions and samples were collected at 30, 60, 120, 240, 300 and 360 minutes. Proton Nuclear Magnetic Ressonance (1H- NMR) spectra were acquired in a 600 MHz spectrometer. Metab- olite consumption and/or production were measured. Heart pres- ervation in all solutions highly increased lactate production however, in HBS, this increase was more pronunced: 3.1 mM against 1.8 mM of the other solutions. Heart preservation in K- H and HBS increase glucose consumption while in Cs lactobionic acid concentration decreases. K-H preservation also promoted pyruvate consumption by 0.2 mM. In Cs preservation, glutamate and glutathione are increased about 3.5 mM and 0.90 mM respectively, after 360 minutes. Energy metabolism is differently affected in Cs, K-H and HBS heart preservation. The lack of oxygen during ischemia induces a shift to anaerobic metabolism, glucose uptake, glycogenolysis and glycolytic flux. HBS is more effective in enhancing anaerobic metabolism and lactate produc- tion while Cs is more effective in preventing oxidative damages with glutathione and glutamate production. Evaluation of HBS and Cs preservation will be discussed in detail. Acknowledgement: Supported by FCT (SFRH/BD/31655/ 2006, POCI/SAU-OBS/55802/2004).

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Poster Presentations

stages (lactation, dry and pregnancy)

of mean obtained concentrations

P3–4 Carbohydrate protein interactions: recognition of the N-glycan core by hevein A. Arda Freire1, J. J. Herna´ ndez-Gay1, S. Mezzato2, D. Varo´ n2, F. J. Canada1, B. Leeflang3, J. P. Kamerling3, C. Unverzagt2 and J. Jime´ nez-Barbero1 1CIB-CSIC, Protein Science, Madrid, SPAIN, 2Universita¨t Bayreuth, Bioorganic Chemistry, Bayreuth, GERMANY, 3Bijvoet Center for Biomolecular Research, Bioorganic Chemistry, Utrecht, THE NETHERLANDS

triacylglycerols

cal in autochthonic Pramenka sheep – type Dubska, during, 2008, using enzymatic in average three methods. For this purpose 20 adult, healthy, years old sheep, regularly labeled, were conducted during study from April to October, 2008. The blood samples were collected from vein jugularis in period of lactation, dry and pregnancy. The cholesterol (1,9 ± 0,32 mmol/l), were very high significant (p £ 0.001) in dry compared to lactation (1,6 ± 0,22 mmol/l). Also, very high dif- ference (p < 0.001) were obtained in level of cholesterol in preg- nancy (1,9 ± 0,27 mmol/l), comparing to lactation. The obtained mean concentrations of (0,31 ± 0,08 mmol/l) were very high significant (p < 0.001) comparing with concentra- tions of this parameters (0,19 ± 0,06 mmol/l) in stages of lacta- tion. Comparing the contents triacylglycerols between sheep in pregnancy and lactation, received differences were statistically very significant (p < 0.01). There was not statistically signifi- cance in level triacylglycerols between dry and sheep in preg- nancy. It was concluded that lowest concentrations of these parameters were in lactation, and there was not obtained signifi- cant difference between dry and pregnancy cholesterol and tria- cylglycerols.

P3–6 Ochratoxin A activates human pregnane X receptor, constitutive androstane receptor and Aryl hydrocarbon receptor in primary cultures of human hepatocytes I. Ayed1, W. Hassen1, P. Maurel2, J. M. Pascussi2 and H. Bacha1 1Faculty of Dentistry, Biochemistry, Monastir, TUNISIA, 2INSERM, U632, Montpellier, FRANCE

Molecular recognition by specific targets is at the heart of key life processes. In recent years, it has been shown that the interactions between proteins (lectins, enzymes, antibodies) and carbohydrates mediate a broad range of biological activities, starting from fertil- ization, embryogenesis, and tissue maturation, and extending to pathological processes. The elucidation of the mechanisms that govern how sugars are accommodated in the binding sites of these receptors is currently a topic of major interest. Among the processes related to carbohydrate recognition, it has been recently shown that UDA, Urtica dioica lectin, binds to the major histo- compatibility complex (MHC) molecule by recognition of the gly- in contrast to the interactions reported for can components, other superantigens, which recognize MHC protein domains in regions outside the peptide-binding groove. Indeed, chitooligosac- charides (b1-4 linked-oligomers of N-acetyl-D-glucosamine) block the interaction between UDA and MHC, and the subsequent T cell activation, suggesting that the superantigenic activity of this lectin is related to its attachement to glycosylated receptors. From the chemical and structural viewpoint, UDA contains 89 aminoacids, comprising two hevein-like domains, while affinity measurements have suggested that possesses two binding sites with significantly different affinity constants. In the past years, we have deeply studied the recognition properties of hevein domains and, on this basis, and in order to gain insights into the recognition process between UDA and the N-glycan chains of the glycosylated receptors, we have studied the interaction of he- vein with two N-glycan core (b-D-Manp-1 fi 4-b-D-GlcpNAc- 1 fi 4-b-D-GlcpNAc-Asn) derivatives. In particular, we have focused on how the presence of one mannose residue, at the non reducing end, and one aminoacid moiety, at the reducing end of chitobiose, may affect the binding of chitooligosaccharides by he- vein domains. 1H NMR titration experiments were performed at different temperatures in order to extract the corresponding Ka values. Further evidence of the complex formation was obtained by 2D NOESY experiments that allowed identifying different sugar-protein intermolecular contacts. Besides, the stability and structural features of the complexes were studied by docking cal- culations and solvated molecular dynamics.

P3–5 Investigation of cholesterol and triacylglycerols levels in sheep in dependence of physiological stages (lactation, dry and pregnancy) Z. Asimovic1, A. Salkic2, M. Brka3 and L. Orucevic1 1Faculty of Agriculture and Food Science, Biochemistry, Sarajevo, BOSNIA AND HERZEGOVINA, 2Veterinary Practice, Veteri- nary medicine, Travnik, BOSNIA AND HERZEGOVINA, 3Faculty of Agriculture and Food Science, Zootehnic, Sarajevo, BOSNIA AND HERZEGOVINA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Background: Ochratoxin A (OTA) is a mycotoxin produced by fungi of two genera: Penicillium and Aspergillus. OTA has been shown to be nephrotoxic, hepatotoxic, teratogenic and immuno- toxic to several species of animals and to cause kidney and liver tumours in mice and rats. Biotransformation of OTA has not been entirely elucidated. At present, data regarding OTA metab- olism are controversial. Several metabolites have been character- ized in vitro and/or in vivo, whereas other metabolites remain to be characterized. Several major mechanisms have been shown as involved in the toxicity of OTA: inhibition of protein synthesis, promotion of membrane peroxidation, disruption of calcium homeostasis, inhibition of mitochondrial respiration and DNA damage. The contribution of metabolites in OTA genotoxicity and carcinogenicity is still unclear. Objective: The aim of this study was to investigate the cytochr- oms P450 induced by OTA in human cultured hepatocytes and to determine if OTA can activate nuclear receptors, pregnane X receptor (PXR), constitutive androstane receptor (CAR) and the Aryl hydrocarbon receptor (AhR). Methods: we looked, firstly, on mRNA expression of some cyt- ochroms known as target genes of these receptors and then, on receptors mRNA level using real-time quantitative reverse tran- scription-polymerase chain reaction (RT-QPCR). Results: Our results showed for the first time that, treatment of primary cultured hepatocytes with increasing concentrations of OTA for 24 hours, caused a significant up-regulation of CYP3A4, CYP2B6 and in a lesser extent CYP3A5 and CYP2C9, while, PXR and CAR mRNA expression were not affected. OTA was found also to induce an over expression of CYP1A1 and The aim of this study was to investigate the serum total choles- terol and triacylglicerols levels in dependence of three physiologi-

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CYP1A2 accompanied by an increase in AhR mRNA expression. These findings suggest that these nuclear receptors could be involved in metabolic activation and toxicity mediated by OTA. Conclusions: Our results support the presence of new transduc- tion pathways, the PXR and/or CAR and AhR pathway. Both the PXR and/or CAR and the AhR pathways are activated by OTA within a similar range of concentrations. The observations raise the question of OTA toxicity in the liver, the major side of expression of the promiscuous human PXR. More attention needs to be paid to effects of OTA in reproductive organs that mainly express AhR and ER. Further studies will be needed to recognize the molecular mechanism of interaction of OTA with nuclear receptors.

P3–8 Tanniniferous oak (Quercus hartwissiana) leaves do not affect plasma levels of leptin, IGF-I and LH in lambs M. Cenesiz1, S. Yildiz2, M. Kaya1, F. Onder2, O. Ucar3, M. Uzun2, M. Blackberry4, D. Blache4, I. KayaI5, Y. Unal5, A. Oncuer5 and G. Martin4 1Ondokuz Mayis University Veterinary Faculty, Physiology, Sam- sun, TURKEY, 2Kafkas University Veterinary Faculty, Physiology, Kars, TURKEY, 3Ataturk University Veterinary Faculty, Artificial Insemination, Erzurum, TURKEY, 4University of Western Austra- lia, Department of Agriculture, Perth, AUSTRALIA, 5Kafkas University Veterinary Faculty, Animal Nutrition, Kars, TURKEY

P3–7 Fluorescence microscopy of the viper venom‘s interaction with mixed-lipid giant unilamellar vesicles N. Ayvazyan, N. Zaqaryan and N. Ghazaryan Yerevan State University, Biophysics, Yerevan, ARMENIA

Aim of the current study was to evaluate the effects of diets con- taining different levels of tanniniferous oak (Quercus hartwissi- ana) leaves in the absence and presence of a tannin binding substance, polyethylene glycol, on plasma leptin, IGF-I and LH levels in ewe-lambs. Lambs (n = 42) were kept in individual metabolism cages and a total of 7 groups were formed. All groups were given 272 g concentrate and varying amounts of hay in a way that the amount of roughage was equal to 645 g. The diets were isonitrogenous and isoenergetical and the experiment continued for 60 days. Blood samples were collected fortnightly for the measurement of leptin and IFG-I. Additionally, on day 55 post-prandial rhythm of leptin was assessed with 30 minutes intervals for 8 hours and LH response to naloxone was assessed for 2 hours with 15 minutes intervals. For the determination of LH pulsatility, blood samples were collected on day 45 of the experiment with 15 minutes intervals for 6 h. IGF-I levels, fort- nigtly leptin secretion, LH pulsatility and LH response to nalox- one did not differ among the groups. Post-prandial leptin secretion appeared to be episodic but it was not affected by die- tary treatments (p > 0.05). Both leptin and IGF-I concentrations were positively correlated to LH pulse frequency (R2 = 0.235, p = 0.027 and R2 = 0.248, p = 0.006, respectively).

P3–9 Grape seed proanthocyanidin extract modulates amino acid metabolism in rats fed with lard S. Dı´ az, H. Quesada, D. Pajuelo, A. Ferna´ ndez, M. J. Salvado´ , C. Blade´ and L. Arola Rovira i Virgili University, Biochemistry and Biotechnology, Tarragona, SPAIN

Because of their unique biological effect, many types of snake’s venom have been utilized as valuable pharmacological reagents for studies on the interaction of their content and organized lipid interfaces, like as BLMs, LUVs, SUVs MLVs etc. But usually because of their particular characteristics (size and lamellari- ty)these model membrane systems are not necessarily accurate de- scriptionsof cell membranes. Giant unilamellar vesicles (GUVs) with a mean diameter of 30 lm have a minimum curvature and mimic cell membranesin this respect; and could be ideal for studying lipid/lipid and lipid/proteininteractions using micros- copy techniques with membrane fluorescence probes. GUVs were formed from the total lipid fraction from bovine brain by the electroformation method, developed by Angelova and Dimitrov (1987). Vipera lebetina obtusa venom was added to the sample- chamber before the vesicles were formed. Themembrane fluores- cence probes, ANS and pyrene, were used to assess the state of the membrane and specifically mark the phospholipid domains. These probes were added to the samplechamber after the vesicles were formed (shaking for 1 minutes in 25 ?C). Fluorescent spec- tra were acquired on a Varian fluoremeter instrument; the excita- tion wavelengthused for the pyrene monomers and dimeres are 286 nm and 334 nm (Lemission are 395 nm and 470 nm respec- tively); the excitation and emission wavelengthsused for ANS are 360 nm and 490 nm respectively. ANS and pyrene allows us to quantify the fluidity changes in themembrane by measuring of the fluorescence intensity. The presence of viper venom in GUVs media lead to a noticable decreasing of membrane fluidity com- pare the control, while the binding of fluorophores with GUVs modified by venom has shown the co-existence of liquid regions and solid-phase packing lipid domains in the bilayer. It was rec- ognized early (Sanchez S., Bagatolli L. et al. 2002) that the vipers venom components preferred an organized lipid substrate near the lipid’sphase transition and were particularly active against micellarlipids. Thesestudies also emphasize the importance of a membranesurface curvature for its interaction with enzymatic components of venom.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Obesity and dyslipemia are key factors in the development of car- diovascular diseases. Consumption of flavonoids reduces the risk of developing pathologies associated to obesity largely because of their antioxidant properties. Previous studies have shown that proanthocyanidins affect the metabolic function in a postprandial situation by significantly inhibiting the increase of plasmatic tri- glycerides. The aim of this study, then, is to dilucidate not only how gluconeogensis works but also to what extent substrates are available and how they are used. For this reason, plasma levels of lactate, urea, glycerol and free amino acids, were analysed, as well as the activity in liver of the main enzymes that control the amino acid metabolism. Male Wistar rats were deprived of food for 14 hours before the experiment. A lard oil (2.5 ml/Kg) with or without grape seed proanthocyanidin extract (GSPE) (250 mg/ Kg) was administered orally. The animals were sacrificed 5 hours after the treatment, and blood and liver were collected. Our results show a significant decrease in plasma lactate and urea, but only a slight decrease in glycerol that is not statistically sig- nificant. None of the enzymes analysed (glutamate oxaloacetate

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transaminase, glutamate pyruvate transaminase, glutamine syn- thetase and glutamate dehydrogenase) are modified although GPT undergoes a slight increase. The aminograms show that some amino acids are significantly affected. aspartate, glutamate and tyrosine decrease while histidine increases. In conclusion, these results suggest an induction of the gluconeogenic pathway, which indicates that proanthocyanidins are powerful agents for activating alternative pathways to obtain energy. Acknowledgement: This work was supported by the Spanish Government (grant AGL2005-04889).

P3–10 Microbial dechlorination of polychlorinated biphenyls V. Dudkova´ 1, K. Demnerova´ 1 and D. L. Bedard2 1Institute of Chemical Technology Prague, Biochemistry and microbiology, Prague 6, CZECH REPUBLIC, 2Rensselaer Polytechnic Institute, Department of Biology, NY USA, USA

is

ing their remarkable immunogenic properties, synthesis of both pure oligosaccharides and glycoconjugates have become of increasing importance in the development of new diagnostic and therapeutic strategies. As an alternative to chemical synthesis, the use of enzymes is of growing interest. Here we report a chemo- enzymatic method for the synthesis of bioactive glycoconjugates. By exploiting the transglycosylation activity of microbial glyco- sylhydrolases, we succeeded in assembling of furanosyl-contain- ing oligosaccharides with different glycosidic linkages between single monosaccharides. Hexofuranosyl residues linked (1 fi 2), (1 fi 3), (1 fi 5) and (1 fi 6), each of them already found to be specific for some pathogenic species, were synthesised, puri- fied and identified by NMR and MS analyses. With the assis- tance of glycosylhydrolases, non-carbohydrate compounds were transferred onto furanosyl entity as well. Thus a number of hap- tens mimicking extracellular structural segments of some patho- gens have been prepared. An effect of the substrate structure modification on the enzyme regioselectivity was examined by means of molecular modelling and the consistency with reaction yields is discussed. Regarding progressive requirements for envi- ronmentally friendly alternatives, ionic liquids were tested as sol- vents for these enzymatic reactions and the influence on reaction yields and kinetics was evaluated. supported by MSˇ MT: Acknowledgements: This project MSM 6046137305 and the Ministe` re des Affaires Etrange` res of the French government.

P3–12 Evaluation of toxicity of organochlorine pesticides and benzonitrile herbicides P. Janu, P. Lovecka´ , Z. Knejzlı´ k, M. Mackova´ and K. Demnerova´ Biochemistry and Microbiology, Institute of Chemical Technology, Prague, CZECH REPUBLIC

Polychlorinated biphenyls (PCBs) are organic xenobiotics con- taminating environment for at least 50 years. The extensive appli- cation of the PCBs resulted in their wide distribution in the environment. PCBs are actually a large family of 209 congeners that all share the biphenyl backbone but differ by the number and the position of the chlorine atoms on the biphenyl ring. The degree of chlorine substitution influences their biodegradability which decreases with increasing chlorination. PCBs should be eliminated from environment by the activity of various organisms under different conditions. Our work is focused on the PCBs bio- degradation under anaerobic conditions. There are the suitable high chlorinated biphenyls converted to the chlorinated biphenyls with lower rate of chlorine. PCB determination was done by gas chromatography coupled with electron capture detector. Suitable microbial consortium was isolated from sediment of Stra´ zˇ sky´ canal (located near by plant producing PCBs in the past). This consortium was able to dechlorinate polychlorinated biphenyls under anoxic conditions. The effectiveness of this process was tested under different cultivation temperature (4 ?C and 28 ?C) and by addition of dechlorination inducers: 2,6- or 4–4 dibromo- biphenyls. Transfered bacterial medium was amended by indus- trial used mixture of PCBs (Aroclor 1248 or Aroclor 1260 or Delor 103 or Delor 106). For further dechlorination improve- ment pyruvate or lactate or acetate as electron donors were added. In all cases dechlorination proceeds at para- position. Acknowledgement: The authors thank for the support of the grants: Centrum 1M06011, FRVS G4/943/2009, NPV II 2B06156 and VZ MSM 6046137305.

P3–11 Neoglycoconjugates – Mimics of immunogenic pathogen structures I. Chlubnova1, I. Chlubnova2, R. Daniellou2, V. Spiwok1, C. Nugier-Chauvin2, H. Dvorakova3, B. Kralova1 and V. Ferrieres2 1Biochemistry and Microbiology, Institute of Chemical Technology, Prague, CZECH REPUBLIC, 2Ecole Nationale Supe´rieure de Chimie de Rennes, CNRS UMR 6226, Rennes Cedex 7, FRANCE, 3Central Laboratories, Institute of Chemical Technology, Prague, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Organochlorine pesticides often used in agriculture are substances that accumulate in the environment and represent one of the sig- nificant threats to the whole ecosystem. Our study investigates the toxicity of these compounds (4,4’-DDT, 2,4’-DDT, d-HCH, c-HCH and HCB) and benzonitrile herbicides (dichlobenil, brom- oxynil, ioxynil) and their metabolites and is focused on mecha- their action. As a prokaryotic model system the nisms of luminescent bacteria Vibrio fischeri was chosen together with the analysis of growth and viability of four different bacterial species. The eukaryotic model was represented by the seeds of Lactuca sativa, var. capitata and by ‘hairy root’ culture of Solanum ni- grum. Mutagenicity was tested using the Ames test with bacteria Salmonella typhimurium His-. As other eukaryotic model system was chosen ‘Hek 293 T cells’ (human embryonic kidney cells) mammalian tissue cultures. DNA damage of these cells is prepar- ing to be tested by comet assay. The pesticide 4,4’-DDT was sig- nificantly toxic for all examined bacterial species. The test with the luminescent bacteria Vibrio fischeri proved high toxicity of the herbicides, but the toxicity of metabolites did not exhibit so high values. For the eukaryotic model represented by the seeds of Lactuca sativa, var. capitata was the toxicity of each single insecticide comparable. Herbicides were more toxic than insecti- cides and much more toxic than their metabolites. It was not proven that the tissue culture ‘hairy-root’ is more sensitive to toxic substances than the seeds. Mutagenicity was proved only for 4,4’-DDT by means of the Ames test. The tests examining both toxicity and mutagenicity were applied using either water or organic extracts acquired from real contaminated soil. Together with defining the risk of individual compounds, this approach allows evaluation of the toxic effect of each compound in differ- ent levels of ecosystem. By investigating the toxicity of these Numerous microbes, many of which are highly pathogenic for humans, bear in their cellular matrixes glycoconjugates with sugar functionalities in a furanose – five-membered ring form. These structures are completely absent in mammals and play a critical role in the pathogenicity of invading organisms. Consider-

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compounds and the mechanisms of their action we can reduce the impacts on the environment and analyze and prevent poten- cial hazards. Acknowledgements: The authors thank for the support of grants TANDEM FT-TA5/043, TANDEM FT-TA4/101 and MSM 6046137305.

P3–13 Cytochrome b5 potentiates participation of cytochrome 1A1 and 1A2 in oxidation of anticancer drug ellipticine to pharmacologically more efficient metabolites V. Kotrbova1, B. Mrazova1, E. Frei2 and M. Stiborova1 1Biochemistry, Charles University Faculty of Science, Prague, CZECH REPUBLIC, 2Department of Molecular Toxicology, German Cancer Research Center, Heidelberg, GERMANY

they can modulate the activity of xenobiotic-metabolizing enzymes involved in the activation and detoxification of food and environmental carcinogens. Thus, their potential negative effects should be examined. The objective of the present study is to investigate the effects of chemopreventive compounds on cyto- chromes P450 (CYP1A and CYP2B), namely on their induction and metabolic activities in small intestine of rat model organism. The induction effects of selected chemopreventive compounds, administered per orally to rats, on CYP1A and 2B were deter- mined in small intestine using Western blotting and specific meta- bolic activity assays. Comparing CYPs expression along small intestine, the highest induction was observed in the proximal part near pylorus with rapid decrease towards the distal part. In response to chemopreventive compounds, the induction of CYP1A1 and CYP2B1 in small intestine was observed after b- naphthoflavone, diallyl sulphide and curcumin treatment. The results of Western blotting detection of CYPs correlate well with their specific enzymatic activities. Acknowledgements: This work was supported by the Czech Science Foundation (Grant No. 305/09/H008) and by the Minis- try of Education, Youth and Sports of the Czech Republic (Grant No. MSM 0021620808).

P3–15 Cytochromes P450 oxidize carcinogenic aristolochic acid I forming its detoxication metabolite and decreasing levels of AA-DNA adducts K. Levova1, J. Sistkova1, E. Frei2, V. M. Arlt3, D. H. Phillips3, H. H. Schmeiser2 and M. Stiborova1 1Biochemistry, Charles University Faculty of Science, Prague, CZECH REPUBLIC, 2Molecular Toxicology, German Cancer Research Center, Heidelberg, GERMANY, 3Section of Molecular Carcinogenesis, Institute of Cancer Research, Sutton, UK

Ellipticine is an alkaloid exhibiting significant antineoplastic activities. Its mode of action is based mainly on DNA intercala- tion, inhibition of topoisomerase II and cytochrome P450 (CYP)- mediated formation of covalent DNA adducts. Many CYP- dependent reactions have been shown to be stimulated by another microsomal protein, cytochrome b5 (b5). Two hypothe- ses trying to explain this effect are an increase in efficiency of processes connected with electron transfers and an induction of some conformational changes potentiating the CYP-mediated oxidation. Five ellipticine metabolites, 9-hydroxy-, 12-hydroxy-, 13-hydroxy-, 7-hydroxyellipticine and the ellipticine N2-oxide, are generated by CYP1A1/2. The patterns and amounts of ellipti- cine metabolites vary significantly when b5 is present in the incu- bations. The formation of detoxication products of its oxidation (7-hydroxy- and 9-hydroxyellipticine) is decreased, while genera- tion of 13-hydroxy- and 12-hydroxyellipticine, the metabolites responsible for formation of DNA adducts, is increased signifi- cantly. The enhanced generation of ellipticine-DNA adducts in the presence of b5 in the system was confirmed by 32P postalbel- ing. Other proteins with or without heme in their molecules (apo- b5, myoglobin, HSA) or hemin are without such effects on ellip- ticine oxidation. Cytochrome b5 affects also the kinetics of ellipti- cine oxidation by CYP1A1/2. In the case of the CYP1A1, the presence of b5 changes the kinetics of ellipticine oxidation to 12- hydroxy- and 13-hydroxyellipticine from hyperbolic to sigmoidal, whereas no cooperativity was observed with CYP1A2. Acknowledgement: Supported by GACR (203/09/0812, 303/ 09/0472), the Czech Ministry of Education (MSM0021620808) and GAUK (1272080).

P3–14 Effects of chemopreventive compounds on cytochromes P450 J. Krizkova, K. Burdova, P. Hodek and M. Stiborova Biochemistry, Charles University Faculty of Science, Prague, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Aristolochic acid (AA) causes the development of aristolochic the Balkan endemic nephropathy acid nephropathy (AAN), (BEN) and urothelial cancer. One of the common features of AAN and BEN is that not all individuals exposed to AA suffer from these diseases. We suggested that one cause for these differ- ent responses may be individual differences in the activities of enzymes catalyzing the detoxication and/or activation of AA. Using a HRN [Hepatic Cytochrome P450 (CYP) Reductase Null] mouse line, we investigated AAI detoxication in vivo and in vitro. We found that hepatic microsomes of wild-type (WT) mice oxi- dize AAI in vitro to detoxication metabolite, AAIa, while those of a HRN line were without this effect. Levels of AA-DNA ad- ducts in livers and kidneys of WT mice exposed to AAI were more than five-fold lower than in those of HRN mice. These results suggest that hepatic CYPs decrease the actual concentra- tion of AAI both in liver and kidney, thereby protecting its acti- vation to AA-DNA adducts. To define the role of CYP enzymes in AAI oxidation, we used besides hepatic microsomes of these mouse models, also those of human and rat. Furthermore, we investigated the modulation of this reaction by specific inducers and selective inhibitors of these enzymes. The efficiency of human recombinant CYPs to oxidize AAI was also tested. The results demonstrate a major role of hepatic CYPs in AAI detoxication in vivo and that of CYP1A1/2 in this reaction in vitro. Acknowledgements: Supported by GACR (303/09/0472, 305/ 09/H008) and Ministry of Education (MSM 0021620808). Cancer is worldwide a life-threatening disease with one of the highest mortalities. Dietary composition is one of the factors rais- ing the probability of the carcinoma incidence. Since flavonoids and other chemopreventive compounds exert beneficial effects on human health, their consumption rapidly increases. However,

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P3–16 Potential interference of valproic acid with the biotin-dependent carboxylase of leucine metabolism P. Luı´ s1, I. Tavares de Almeida1, M. F. B. Silva1, J. P. Ruiter2, L. Ijlst2, M. Duran2, R. J. A. Wanders2, L. Diogo3 and P. Garcia3 1CPM- iMED.UL, Universidade de Lisboa, Lisboa, PORTUGAL, 2AMC, Department of Clinical Chemistry and Pediatrics, Amster- dam, THE NETHERLANDS, 3Hospital Pedia´trico de Coimbra, Coimbra, PORTUGAL

groups of rats were studied: 8 hour overnight fasted (n = 6) and 6 hours daily fasted (n = 6). 13C and 2H NMR isotopomer anal- ysis were performed in the monoacetone derivative of glucose produced by the isolated perfused liver. Gluconeogenesis (GNG) determined from the 13C isotopomer analysis of carbon 3 (GNG3) and carbon 4 (GNG4) was significantly smaller than GNG determined from the deuterium 2H5/2H2, for both groups, suggesting an underestimation of gluconeogenesis by 13C analysis (8 hour overnight fasted: GNG3 = 27.0±8.0; GNG4 = 32.7± 2H5/2H2 = 54.0±13.9; 6 hours daily fasted: GNG3 = 10.2; 9.5±1.8; GNG4 = 14.0±2.3; 2H5/2H2 = 18.2±3.2). Differences in GNG3 and GNG4 did not reach statistical significance, sug- gesting that transaldolase exchange of F6P carbons 4–6 and glyc- 13C enrichment of carbon 4 eraldehyde-3-P, which causes independently of gluconeogenic activity, was not significant. Also interesting is the variation in GNG within the 8 hours overnight fasted animals, suggesting that much care should be taken when analyzing data from intermediate fasting durations.

P3–18 Interaction of HPMA copolymers – doxorubicin conjugates with human liver microsomal P450 V. Masek1, E. Anzenbacherova2, T. Etrych3, V. Subr3, K. Ulbrich3 and P. Anzenbacher1 1Department of Pharmacology, Faculty of Medicine and Dentistry Palacky University, Olomouc, CZECH REPUBLIC, 2Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry Palacky University, Olomouc, CZECH REPUBLIC, 3Department of Biomedicinal Polymers, Institute of Macromolecu- lar Chemistry v.v.i. AS CR, Prague, CZECH REPUBLIC

biotin-dependent a

The potential hepatotoxicity associated with valproic acid (VPA), an important anticonvulsant drug, has been unequivocally associ- ated with mitochondrial dysfunction and the oxidative metabo- lism of fatty acids or branched-chain amino acids intermediates. Aims: To elucidate the effect of valproate on the leucine path- way using a metabolomics approach first, and then to evaluate the potential inhibitory effect of valproate on the activity of bio- tinidase. Methods: The Organic acids (OA) profile and respective glycine conjugates were accessed in urine samples of VPA-treated patients and healthy individuals using gas-chromatography/mass spectrometry. Biotinidase activity was determined in the respec- tive plasma samples, using an optimized spectrophotometric assay. Intact rat liver mitochondria (RLM) isolated from control rats and from rats after administration of a single dose or daily doses of VPA for 15 days, were also used to study OA profile ex vivo. Results: Specific intermediary metabolites of leucine pathway namely 3-hydroxyisovaleric acid (3OH–IVA) and 3-methylcroto- nylglycine were significantly excreted in higher levels, in humans after VPA treatment. RLM isolated from VPA-treated rats also revealed an increase of 3OH–IVA. Biotinidase activity in plasma samples ranged from very low, to normal levels in patients under therapy as compared with controls. Discussion: The VPA-induced impairment of biotinidase activ- ity leads to a subsequent interference on the activity of 3-meth- ylcrotonyl-CoA carboxylase, enzyme, demonstrated in vivo by the increased excretion of 3-methylcroto- nylglycine. Further studies are necessary to elucidate the effect of VPA on the activity of other biotin-dependent carboxylases, which are key enzymes on the mitochondrial metabolism. Acknowledgement: Supported by FCT, SFRH/BD/25913/2005.

P3–17 Hepatic glucose metabolism by 13C and 2H–NMR isotopomer analysis during two intermediate fasting periods F. O. Martins, P. M. Nunes, J. G. Jones and R. A. Carvalho NMR unity, Centre for Neuroscience and Cell Biology, Coimbra, PORTUGAL

inhibition of

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Interaction of nine forms of human hepatic cytochromes P450 (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) with two HPMA-based doxo- rubicin (DOX) conjugates designed for passive tumor targeting was com- studied using pooled human microsomes. The pounds used in this study were two high-molecular-weight N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers bear- ing doxorubicin attached to the polymeric carrier by (A) hydrazone bond enabling intracellular pH-controlled drug release; or (B) amide bond through enzymaticaly cleavable tet- rapeptide Gly-Phe-Leu-Gly spacer. Both polymeric conjugates differing in mechanism of their anti-tumour activity and the free doxorubicin as control were tested for potential inhibition activ- ity. Cytochromes P450 are the most important enzymes of drug biotransformation. Potential drugs are tested whether they inter- act with the P450 enzymes and may eventually cause unwanted drug interactions. Among nine cytochrome P450 forms studied, no HPMA copolymer with bound DOX caused an inhibition of potential clinical significance. The extent of inhibition of enzy- matic activities of the cytochrome P450 (CYP) forms studied was negligible with exception of CYP1A2 and CYP2B6 and was apparently caused by DOX as no inhibition was observed with polymers alone and the extent of inhibition by the com- plex corresponded to this of the free DOX at the same concen- tration. In conclusion, the polymers as well as their conjugates with DOX appear to be relatively safe at least in this respect, i.e. of the liver microsomal drug metabolizing enzymes. Acknowledgment: Financial support through the grants from Academy of Sciences of the Czech Republic (KAN 200200651) and from the MSMT 6198959216 project is gratefully acknowl- edged. The liver has a key role in regulation of glucose metabolism hence measurements of hepatic glucose fluxes and the main sources (glycogenolysis and gluconeogenesis) are crucial compo- nents for evaluating the hepatic contribution to glucose levels. Maintenance of glucose homeostasis during the fed to fasted transition involves significant changes in hepatic glucose fluxes. Methodologies based on the administration of stable isotope tracers are currently under development and several approaches have been described. In this study we compared [1-13C] lactate and 2H2O tracers for discriminating the gluconeogenic contribu- tion to endogenous glucose production. Perfused livers from two

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3-ABA is oxidized by peroxidases. Of peroxidases tested, the highest efficiency to oxidize 3-ABA was detected with horseradish peroxidase, followed by myeloperoxidase and lactoperoxidase. Acknowledgements: Supported by GACR (303/09/0472, 203/ 09/0812 and 305/09/H008) and the Czech Ministry of Education (MSM 0021620808).

P3–19 New insights in PGPR – soybean plant interactions M. Mihasan, M. Stefan, L. Gorgan, L. Raus, D. Topa and S. Dunca Biochemistry and Molecular Biology Department, Alexandru Ioan Cuza University, Iasi, ROMANIA

P3–21 Characterization of adduct generated by 13-hydroxyellipticine with deoxyguanosine in DNA M. Moserova1, V. Kotrbova1, E. Frei2 and M. Stiborova1 1Biochemistry, Charles University Faculty of Science, Prague, CZECH REPUBLIC, 2Molecular Toxicology, German Cancer Research Center, Heidelberg, GERMANY

Although the beneficial effects of Plant Growth Promoting Rhi- zobacteria (PGPR) on soybean growth are widely accepted, the mechanisms by which plant development is induced are not yet fully understood. The main challenge of understanding PGPR – soybean plants interactions is related to the economical impor- tance of rhizobacteria utilization as bio-fertilizers in a free of fer- tilizers and pesticides agriculture. In this context, the impact of several bacterial strains [isolated from Glycine max (L.) Merrrhiz- osphere] on soybean in vitro germination was studied. The dynamics of total soluble proteins,soluble carbohydrates, and lip- ids content were analyzed during in vitro germination of soybean seeds. PGPR treated seeds showed lowered metabolic rates, as well as a diminished germination yield compared to the control. Using catalase, peroxidase and superoxid-dismutase as oxidative- stress markers, we concluded that the inhibitory effect on in-vitro soybean germination is not induced by a PGPR-generated stress. The differences between the treated and un-treated seeds are due to a possible nutrient competition that takes place in the in-vitro conditions employed. A question is still open regarding this inhi- bition: does it also takes place in the normal, field conditions, where the bacteria may have use some other nutrients sources?

P3–20 Cytochromes P450 and peroxidases oxidize 3-aminobenzanthrone, the human metabolite of carcinogenic 3-nitrobenzanthrone J. Mizerovska1, H. Dracinska1, V. M. Arlt2, H. H. Schmeiser3, E. Frei3 and M. Stiborova1 1Biochemistry, Charles University Faculty of Science, Prague, CZECH REPUBLIC, 2Section of Molecular Carcinogenesis, Insti- tute of Cancer Research, Surrey, UK, 3Division of Molecular Toxi- cology, German Cancer Research Center, Heidelberg, GERMANY

Ellipticine and some its derivates are used in the therapy of breast cancer and leukemia and have multiple cellular targets. We found that ellipticine forms covalent DNA adducts and that the formation of the major adduct is dependent on the activation of ellipticine by cytochromes P450 (CYP) and peroxidases. 13- Hydroxyellipticine formed by CYP3A4 was identified to be bound to deoxyguanosine in DNA, generating the major DNA adduct. To characterize this adduct, we investigated the effect of pH and the phase II enzymes, sulphotransferases (SULTs) and N,O-acetyltransferases (NATs) on efficiency of 13-hydroxyellipti- cine to form this DNA adduct. 13-Hydroxyellipticine incubated with DNA in vitro generates the major deoxyguanosine adduct, which was detected and quantified by the 32P-poslabeling tech- nique. HPLC was used to isolate the adduct formed in the reac- tion mixture from deoxyguanosine. The levels of this DNA adduct were significantly increased by presence of PAPS and SULTs expressed in the target tumors for ellipticine action. Like- wise, acetyl-coenzyme A and NAT1/2 stimulated the formation of this adduct. The results shown here allow us to proposed the mechanism of the reaction generating this deoxyguanosine adduct, found to be formed from either 13-hydroxyellipticine or from ellipticine by CYPs and in rats treated with this drug. We predict that ellipticine is bound to deoxyguanosine by its 5- methyl group, which is activated after hydroxylation due to CYP-mediated oxidation to alcohol (13-hydroxyellipticine). The study targeted to confirm this suggestion is underway in our lab- oratory. Acknowledgements: Supported by GACR (303/09/0472, 305/ 09/H008) and Czech Ministry of Education (MSM 0021620808 and 1M0505).

P3–22 Preparation of apo-cytochrome b5 utilizing apo-myoglobin B. Mrazova1, M. Martinkova1, V. Martinek1, E. Frei2 and M. Stiborova1 1Biochemistry, Charles University Faculty of Science, Prague, CZECH REPUBLIC, 2Division of Molecular Toxicology, German Cancer Research Center, Heidelberg, GERMANY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Cytochrome b5 (cyt b5), a component of endoplasmic reticulum membrane, plays a role in modulation of activity of some cyto- chromes P450 (CYP). To elucidate the mechanism of such modu- lations it is necessary to evaluate not only the effect of native cyt The aromatic compound 3-nitrobenzanthrone (3-NBA) is one of the most potent mutagens and suspected human carcinogen iden- tified in diesel exhaust, ambient air particulate matter, in surface soil and rainwater. The major metabolite of 3-NBA, 3-aminoben- zanthrone (3-ABA), was detected in the urine of salt mining workers occupationally exposed to diesel emissions, demonstrat- ing that exposure to 3-NBA can be significant and is detectable. The enzymes activating 3-ABA to form DNA adducts in vitro and in vivo have already been identified. The cytochromes P450 (CYP) 1A1 and 1A2 enzymes are the principal enzymes forming DNA adducts from 3-ABA in livers. In this study, we investi- gated the oxidative metabolism of 3-ABA catalyzed by human and rat hepatic CYP in vitro, identified the 3-ABA metabolites, characterized kinetics of their formation and determined the CYP enzymes responsible for 3-ABA metabolite formation. Oxi- dation of 3-ABA was studied using Supersomes?, microsomes containing human and rat recombinant CYP enzymes. CYP enzymes of both species oxidize 3-ABA up to three metabolites. Using co-chromatography with synthetic standards, two of them were identified to be oxidative metabolites of 3-ABA, N-hydroxy- 3-ABA and 3-NBA, while the structure of the third 3-ABA metabolite remains to be characterized. We also found that

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Poster Presentations

P3–24 Metabolomic analysis of the fission yeast Schizosaccharomyces pombe T. Pluskal1, T. Nakamura2, A. V. Briones1, K. Nagao1 and M. Yanagida1 1G0 Cell Unit, Okinawa Institute of Science and Technology, Uruma, JAPAN, 2Graduate School of Biostudies, Kyoto University (CREST Research Program), Kyoto, JAPAN

compounds (MW 100–1000 Da)

b5, but also that of apo-cyt b5. The effect of apo-cyt b5 on this enzymatic system has not been investigated in details, because preparation of cyt b5 as a pure protein failed. To prepare the native apo-cyt b5 in a large scale we utilized a protein with higher affinity toward the heme, the apo-myoglobin from the equine skeletal muscle. First, we extracted heme moiety from the native myoglobin by butanone extraction. Than the effect of pH on spontaneous heme release from both proteins was investigated: purified rabbit cyt b5 as well as equine skeletal muscle myoglobin. The prepared apo-myoglobin was incubated with the cyt b5 and heme transfer was monitored. The optimal pH range for heme transfer from cyt b5 into apo-myoglobin was between 4.2 and 5. Native apo-cyt b5 was separated from myoglobin on a column of DEAE-Sepharose. The apo-cyt b5 reconstituted with heme reveals the same oxidized and reduced absorbance spectrum as native cyt b5 and was found to be reduced also with NADPH:CYP reduc- tase. The experiments investigating the effect of the purified native cyt b5 and apo-cyt b5 on oxidation of xenobiotic substrates of CYP3A4 and 1A are under way in our laboratory. Acknowledgements: Supported by GACR (303/09/0472, 203/ 09/0812, 305/09/H008) and Ministry of Education (MSM 0021620808, 1M0505).

P3–23 Formation and characterization of deoxyguanosine adducts generated by carcinogenic o-anisidine and o-nitroanisole K. Naiman1, D. Martin1, M. Marketa1, D. Helena1, M. Vaclav1, S. Martin2 and S. Marie1 1Biochemistry, Charles University Faculty of Science, Prague, CZECH REPUBLIC, 2Organic Chemistry, Charles University Faculty of Science, Prague, CZECH REPUBLIC

large-scale metabolomic

The fission yeast Schizosaccharomyces pombe is an excellent model organism for the control of cell division cycle, signal trans- duction and chromosome segregation. Whole genome has been sequenced and transcriptomic analyses were conducted. However, metabolic profiling was hardly done except for several specific compounds. We here report the first global semi-quantitative analysis of S. pombe metabolome using liquid chromatography high-resolution mass spectrometry. The procedures to obtain from S. pombe metabolic extracts were established. One hundred and seventeen compounds were identified while several thousand peaks were observed. A software system was developed in order to visualize the semi- quantitative metabolome data using a dynamically generated scatter plot. We examined the metabolome of S. pombe cells exponentially grown at 26 and 36ordm;C in a synthetic culture medium. Their profiles were similar except for varying amounts of certain amino acids and significant increase of the following compounds at 36(cid:2)C: trehalose (200-fold), ribulose (8-fold), arabi- tol (16-fold) and ophthalmic acid (5-fold). The reproducibility of data is quite high, as exemplified by the fact that the deletion ferrichrome synthetase sib1 showed no significant mutant of change except the disappearance of ferrichrome. To take advan- tage that metabolomic profiles at 26 and 36(cid:2)C were rather simi- lar, we undertook to analyze temperature-sensitive (TS) mutants that may alter metabolism. We present results using hcs1 mutant defective in the HMG-CoA synthase. An expected decrease of HMG-CoA was discovered. In addition, complex metabolic changes that included urea cycle intermediates and acetylated compounds were observed. Our results prove the applicability of our approach to monitor changes in response to environment conditions, as well as genetic perturbations.

P3–25 Segregational instability of expression plasmids carrying the human interferon gamma gene in E. coli M. Popov1, S. Petrov1, M. Receb1, I. Ivanov1, G. Nacheva1 and U. Reichl2 1Gene Regulations, Institute of Molecular Biology ‘Roumen Tsa- nev’, Sofia, BULGARIA, 2Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, GERMANY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Human interferon gamma (hIFNc) is a cytokine endowed with multiple biological activities and wide pharmaceutical applica- tion. It is produced in minute concentrations in human blood and its deficiency is sometimes related with different pathological conditions. In these cases the treatment with recombinant hIFNc has curative effect. The segregation of the expression plasmids leading to loss of recombinant DNA from the cells is due to irregular distribution of the plasmid copies during cell division. Under non-selective conditions segregational instability results in a heterogeneous population of cells, where the non-productive plasmid-free cells overgrow the plasmid-bearing cells and the fer- mentation productivity decreases. Factors affecting segregational 2-Methoxyaniline (o-anisidine) and 2-methoxynitrobenzene (o-ni- troanisole) are industrial and environmental pollutants causing tumors of urinary bladder. The International Agency for Research on Cancer has classified both compounds as carcino- gens, which are possibly carcinogenic to humans. Besides their carcinogenicity, they exhibit other toxic effects, including hemato- logic changes, anemia and nephrotoxicity. We found that both carcinogens are oxidatively activated by cytochromes P450 (CYP) to species binding to DNA in vitro and in vivo. The same adducts as found in DNA incubated with o-anisidine and o-nitro- anisole and human microsomes containing CYP enzymes in vitro were detected in urinary bladder, the target organ, and to a lesser extent, in liver, kidney and spleen of rats treated with both these carcinogens. The DNA adducts generated by these carcinogens were identified as deoxyguanosine adducts formed from their common metabolite, N-(2-methoxyphenyl)hydroxylamine. Three deoxyguanosine adducts prepared by the reaction of this deoxy- nucleoside and N-(2-methoxyphenyl)hydroxylamine were sepa- rated by HPLC and their structures were evaluated by mass- and/or NMR-spectrometry. The structure of the major deoxygu- anosine adduct was identified to be N-(deoxyguanosin-8-yl)-2-me- thoxyaniline. Using co-chromatography on HPLC, we confirmed that this major adduct is identical with that detected by the 32P- postlabeling assay, found to be formed in vitro and in vivo. On the contrary, the structures of two minor adducts formed during reactions of N-(2-methoxyphenyl)hydroxylamine with deoxygu- anosine await further investigation. Acknowledgements: Supported by GAUK (7418/2007), GACR (303/09/0472) and Czech Ministry of Education (MSM 0021620808).

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tion relative to biphenyl bond is preferred for methylation. All plant species growing on real PCB contaminated soil (dichloro- up to nonachlorobiphenyls) demonstrated that PCBs were trans- located into different plant tissues, even fruits and flowers, and hydroxy-PCBs (dichloro- up to pentachloro-) commonly occurred in these tissues, too. Methoxy-PCBs (dichloro- up to hexachloro- ) were found among all tested species, too. Plants are able to metabolize wide range of PCBs, even those with higher level of chlorination via hydroxylation and possibly subsequent methyla- tion (or another type of conjugation reaction). Acknowledgements: The authors thank for the support of the research projects Z 40550506 and GACR 525/09/1058.

instability are plasmid design, plasmid copy number, host cell genotype, level of protein expression, fermentation conditions, etc. The gene expression control elements (promoters, ribosome binding sites, terminators) strongly affect the production of the recombinant protein and therefore segregational instability of the plasmid. The aim of this work is to investigate the effect of the efficiency of the constitutive hIFNc gene transcription and trans- lation on plasmid segregation in a chemostat culture. To this end a number of expression plasmids were constructed bearing con- trol elements with different transcription and translation effi- ciency. To describe the bacterial cell growth and production of hIFNc, the mathematical model of Lee, Seressiotis and Bailey (1985) was used. This is the first application of this model for analysis of plasmid segregational instability based on real experi- mental data derived from both batch and continuous cultivation. According to the obtained results, the plasmid instability in our experimental system is strongly dependent on the level of hIFNc expression. The model simulation indicates an optimal copy num- ber of 80–100 plasmids per cell for maximum yield of hIFNc. These results will be used for construction of expression plasmids maintaining the optimal copy number required for efficient pro- duction of recombinant hIFNc. Acknowledgments: Supported by NSF, grant D01–1171/07.

P3–27 Beta-N-Acetylhexosaminidases accept 4-deoxy- N-acetylhexosaminides as substrates K. Slamova1, P. Bojarova1, R. Gazak1, A. Tramice2, N. Kulik3, M. Mackova4 and V. Kren1 1Laboratory of Biotransformation, Institute of Microbiology, Pra- gue 4, CZECH REPUBLIC, 2Istituto di Chimica Biomolecolare CNR, Istituto di Chimica Biomolecolare, Napoli, ITALY, 3Labora- tory of High Performance Computing, Institute of Systems Biology and Ecology AV CR, Nove Hrady, CZECH REPUBLIC, 4Department of Biochemistry and Microbiology, Institute of Chemical Technology, Prague 6, CZECH REPUBLIC

P3–26 Newly identified plant metabolites of polychlorinated biphenyls J. Rezek1, J. Rezek2, J. Doubsky1, M. Mackova2, J. Triska3, T. Macek1 and T. Macek2 1ICT and IOCB joint laboratory, Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences, Prague, CZECH REPUBLIC, 2Department of Biochemistry and Microbiology, Faculty of Food and Biochemical Technology Institute of Chemical Technology Prague, Prague 6, CZECH REPUBLIC, 3Department of Analytical Chemistry, Institute of Systems Biology and Ecology Czech Academy of Sciences, Ceske Budejovice, CZECH REPUBLIC

b-N-Acetylhexosaminidases (EC 3.2.1.52, CAZy GH20) possess so called wobbling specificity, which means that they cleave sub- strates both in gluco- and galacto- configurations, with the activ- ity ratio depending on the enzyme source. Here we present a new finding that fungal b-N-acetylhexosaminidases are also able to hydrolyze and transfer 4-deoxy-N-acetylhexosaminides. This fact clearly demonstrates that the 4-hydroxy moiety at the substrate pyranose is not essential for the binding of the substrate to the enzyme‘s active site, which was also confirmed by molecular docking of the tested compounds into the model of the active site of b-N-acetylhexosaminidase from Aspergillus oryzae. A set of four 4-deoxy-N-acetylhexosaminides was prepared via chemical synthesis and screened against a panel of b-N-acetylhexosaminid- ases from various types of sources (fungal, human, animal, plant and bacterial representatives) for hydrolysis. The results of this screening as well as structures of 4-deoxy disaccharides prepared by transglycosylation reaction using b-N-acetylhexosaminidase fromTalaromyces flavus are reported. This structure-function rela- tionship study will be used in the design of new b-N-acetylhex- osaminidase inhibitors and for the synthesis of novel type of glycomimetics. Acknowledgements: Support by the GA CR 305/09/H008 grant, MSMT LC06010 grant, GA CR 203/09/P024 grant and research concept AV0Z50200510 is gratefully acknowledged.

P3–28 Chronic food restriction increases 11-beta- hydroxysteroid dehydrogenase type I gene expression in white adipose tissue of rat T. Sledzinski1, J. Turyn2, J. Klimek1 and J. Swierczynski2 1Department of Pharmaceutical Biochemistry, Medical University of Gdansk, Gdansk, POLAND, 2Department of Biochemistry, Medical University of Gdansk, Gdansk, POLAND

dehydrogenase type

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

With developing phytoremediation technologies that use plants to help cleaning of polluted soils we aim to characterise metabo- lites of polychlorinated biphenyls (PCBs) produced by plant cells. Tobacco (Nicotiana tabacum) callus culture WSC-38 and black nightshade (Solanum nigrum) hairy root culture SNC-9O were used for in vitro experiments. Twenty one plant species harvested in contaminated locality were used to confirm the presence of metabolites in vivo. Plant tissues (in the case of cell cultures after cultivation with different PCB congeners) were homogenised and extracted using modified Bligh-Dyer procedure. Metabolites were identified based on the mass spectra characteristics after gas chromatography separation (GC·GC/TOFMS LECO Pegasus 4D). Hydroxylated standards of PCB 4 and PCB 10 were synthe- sized newly and used for identification of metabolites. Expected hydroxylated PCB metabolites were found among plant cell cul- tures cultivated with individual PCB congeners (dichloro- up to pentachlorobiphenyls). SNC-9O hairy root culture gave wider range of metabolites comparing to WSC-38 callus culture. Num- ber of metabolites decreased with increasing chlorination of PCB molecule. Dichlorinated PCBs lead always to at least two metab- olites, trichlorinated usually to lower number of metabolites but tetrachlorinated and pentachlorinated PCBs mostly didn’t give any metabolites. PCB 4 and PCB 10 demonstrated that 4 or 4’ position is preferred for hydroxylation attack; hydroxy-PCBs appear in its free and also in conjugated form. New type of PCB metabolites was commonly found among the samples. These metabolites were characterised as methoxy-PCBs and hydroxy- methoxy-PCBs. PCB 4 and PCB 10 demonstrated that meta posi- Introduction: 11-b-hydroxysteroid I (11bHSD1) in rats white adipose tissue (WAT) catalyses conver- sion of less active glucocorticoids, 11-dehydrocorticosterone to

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Poster Presentations

P3–30 Rhizosphere bacteria and their role in degradation of PCB P. Stursa1, O. Uhlik1, O. Uhlik2, V. Kurzawova1, L. Kochankova3, T. Macek2, M. Mackova2 and M. Mackova1 1Department Biochemistry and Microbiology, Institute of Chemical Technology, Prague, CZECH REPUBLIC, 2IOCB & ICT Joint Laboratory, Institute of Organic Chemistry and Biochemistry CAS, Prague, CZECH REPUBLIC, 3Department of Environmental chemistry, Institute of Chemical Technology, Prague, CZECH REPUBLIC

more active corticosterone. Locally formed corticosterone acts on adipose tissue promoting adipocytes differentiation. Mice with overexpression of 11bHSD1 gene display enhanced adipocytes differentiation, and have higher adipose tissue mass. The increase of adipose tissue mass is a result of increased triacylglycerols accumulation in adipocytes. Our previous studies have shown that chronic food restriction, which is often practiced by obese subjects trying to lose body weight, leads to increased expression of lipogenic enzyme genes in rat adipose tissue. The aim of the present study was to investigate whether the expression of 11bHSD1 and some lipogenic enzyme genes (fatty acid synthase and malic enzyme) is synchronously regulated in WAT of rats by prolonged food restriction. Methods: Two months old rats were divided into control and food restricted groups. Food restricted rats obtained 50% of food consumed by control rats for 30 days. 11bHSD1 and lipo- genic enzyme mRNA levels in perirenal WAT were analysed by real-time PCR and enzymes activities were measured. Results: Chronic food restriction caused a significant increase of both 11bHSD1 and lipogenic enzyme genes expression in WAT of rats. Conclusions: The study shows coordinated induction of expres- sion of 11bHSD1 and lipogenic enzyme genes in WAT of rats by chronic food restriction. It is not excluded that local synthesis of glucocorticosteroids may play important role in the regulation of lipogenesis in white adipose tissue and consequently contribute to obesity.

P3–29 Modulation of heme oxygenase-1 affects GM1 ganglioside density in rat livers V. Smid1, T. Petr1, J. Smidova2, H. Hulkova3, L. Muchova1, L. Vitek1 and F. Smid1 1Institute of Clinical Biochemistry and Laboratory Diagnostics, 1st Faculty of Medicine Charles University, Prague, CZECH REPUBLIC, 2Institute of Histology and Embryology, 1st Faculty of Medicine Charles University, Prague, CZECH REPUBLIC, 3Institute of Inherited Metabolic Disorders, 1st Faculty of Medicine Charles University, Prague, CZECH REPUBLIC

Decrease of polychlorinated biphenyls (PCBs) concentration in real contaminated soil, planted with two species (tobacco and black nightshade), was followed. At first experiment was focused on influence of bioaugmentation by bacterial strain Pseudomonas pseudoalcaligenes JAB1 (PCB degrading) on PCB degradation in contaminated vegetated soil. As abiotic factor potentially affect- ing final result of the experiment, treatment with brassinosteroids was used. In the case of tobacco and black nightshade the posi- tive stimulation of plants by brassinosteroids, which thrived bet- ter, was observed but the positive influence on microbial degradation was not confirmed. In the second part of the experi- ment, addition of different microbial strains originally isolated directly from rhizosphere of plants growing in contaminated soil and the differences in degradation efficiency of various plant-bac- teria pair was determined. The aim of this experiment was to find an optimal plant-bacteria pair for efficient PCBs removal. Total number and number of microorganisms capable of growth on mineral medium with biphenyl as a sole source of carbon were determined by traditional cultivation methods. The bacterial strains were isolated from real contaminated soil vegetated with plant species given above and isolated bacteria potentially capa- ble of PCBs degradation were detected with the help of dibenzo- furan. The presence of bphA1 gene was detected and confirmed in 13 isolates in tobacco and black nightshade soil by PCR. The comparison of PCBs removal efficiency showed that degradation is higher in soil vegated by tobacco. But in this case it was found that tobacco acumulates PCBs much more in its tissue than black nightshade and therefore it was impossible to determine whether PCBs were degraded or just transported into the plant tissue. In the last part of this experiment microbial diversity was analysed by molecular biological methods after total DNA isolation. Higher presence of microbial species in vegetated than in non- vegetated soil was confirmed using temporal temperature gel gra- dient elctrophoresis (TTGE). In the case of two isolated strains identified after 16 S rDNA sequencing the same microbial strains as originally added to the soil were determined. We assume that these microbial strains survived and adapted for given condi- tions. Acknowledgement: The work was sponsored by the grants NPVII 2B06156, GACR 525/09/1058, MSM 6046137305.

P3–31 Oxidation of 2-nitrophenol, a metabolite of carcinogenic 2-nitroanisole, by cytochromes P450 – similarity between human and rat enzymes M. Svobodova, M. Martinkova, H. Dracinska and M. Stiborova Biochemistry, Charles University Faculty of Science, Prague, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

2-Nitrophenol (2-NP) is the main detoxication metabolite of 2-ni- troanisole (2-NA) that is an important industrial pollutant and a potent carcinogen for rodents. The aim of this study was to Gangliosides are highly enriched in the outer leaflet of plasma membrane and their increased amount in membrane stabilizes the membrane against detergent effect of bile acids which can lead to apoptosis. Induction of heme oxygenase-1 (HO-1), the rate limiting enzyme in heme catabolic pathway, has been shown to protect liver against oxidative stress, apoptosis and inflamma- tion. Adult Wistar female rats were pretreated with 15 lM hemin i.p. (HO-1 induction) or 15 lM Sn mesoporfyrin i.p. (HO-1 inhi- bition) followed by treatment with ethinylestradiol (5 mg/kg body weights.c.) or propanediol (vehicle) for 5 days. GM1 ganglioside in liver sections was detected using peroxidase labeled cholera toxin B-subunit and final color reaction with diaminobenzidine tetrahydrochloride. Image analysis was used for evaluation of GM1. Inhibition of HO-1 resulted in significant increases in opti- cal density of GM1 in both vehicle treated and cholestatic liver sections respec- (146% ± 8% and 139% ± 7%, p < 0.001, tively) compared to controls. Activation of HO-1 had not signifi- cant effect on GM1 density. We conclude that the increase in GM1 density after HO-1 inhibition is an adaptive response of the liver to the loss of hepatoprotective effect of heme oxygenase-1 in both control and cholestatic animal models. Acknowledgements: Supported by grant IGA–MZ 9366–3.

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incubation conditions assessed and optimized, and drug metabo- lites extracted and then detected using LC–MS. Celecoxib was also subjected to a horse administration trial, and the urine ana- lyzed for metabolites. Phase I metabolites pyrazole acid, N-deme- thylated tepoxalin and hydroxyl pyrazole acid were detected in the tepoxalin in vitro study. In the celecoxib in vitro microsomal study, carboxycelecoxib was the major metabolite, with the par- ent drug still evident. In the parallel in vivo study, celecoxib was highly metabolized with the major metabolite carboxycelecoxib being detected after 6 hours post administration. A minor metab- olite, 4-hydroxycelecoxib, was also detected. A good correlation of the major metabolites was observed between the in vitro and in vivo studies.

P3–33 Response of antioxidant enzymes after biogenic amines administration N. Zamosteanu1, C. Filip1, I. Albut1 and R. Cuciureanu2 1Biochemistry, Gr.T.Popa University of Medicine and Pharmacy, Iasi, ROMANIA, 2Department of Environment and Food Chemistry, Gr.T.Popa University of Medicine and Pharmacy, Iasi, ROMANIA

investigate the efficiency of rat hepatic CYPs to metabolize 2-NP, to determine the metabolites formed during such a metabolism and to compare the efficiencies of rat CYPs with those of human. 2-NP is oxidized by rat liver microsomes to one metabolite, 2,5- dihydroxynitrobenzene (2,5-DNB). To define the role of CYPs in 2-NP oxidation we investigated the modulation of this reaction by specific inducers and selective inhibitors of these enzymes. Most of inhibitors of individual CYPs tested in this study, a- naphtoflavone (for CYP1A), diamantane (for CYP2B), sulfaphe- nazole (for CYP2C), quinidine (for CYP2D), diethyldithiocarba- mate (for CYP2E1) and ketoconazole (for CYP3A) influenced the 2-NP metabolism. Diethyldithiocarbamate, quinidine, ketoco- nazole and diamantane inhibited generation of 2,5-DNB, whereas a-naphtoflavone and sulfaphenazole were almost without this effect. Using microsomes from Baculovirus transfected insect cells expressing recombinant rat and human CYP enzymes, we found that rat recombinant CYP2E1, 2C11, 2B1, 1A2, 1A1 and 3A4 were the most effective to oxidize 2-NP. Similarly, human CYP2E1, followed by CYP2A6, 2C6, 3A4 and 2D6 were the most efficient to oxidize 2-NP. The present study shows the simi- larity between human and rat hepatic enzymes metabolizing 2- NP to 2,5-DNB and indicate that rat might serve as a suitable model to mimic the fate of 2-NP in human. Acknowledgements: Supported by GACR (303/09/0472) and the Czech Ministry of Education (MSM 0021620808).

P3–32 In vitro and in vivo analysis of drug metabolism in liver microsomes and from a horse administration trial B. Wilhelmi1, K. Naicker1 and S. De Kock2 1Biochemistry Microbiology and Biotechnology, Rhodes University, Grahamstown, SOUTH AFRICA, 2The Laboratory, The National Horseracing Authority of Southern Africa, Johannesburg, SOUTH AFRICA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Reactive oxygen species (ROS) are known to determine redox balance alteration, oxidative stress and carcinogenicity. Many diseases, including cancer and other pathologies associated like arteriosclerosis and cataracts, are presented to mitochondrial dys- functions provoked by reactive oxygen species. Biogenic amines are compounds synthesized in human body which in high concen- tration become toxic and lead to a wide range of symptoms as hypertension, nausea, and headache. In this study, the activities of several enzymes involved in antioxidantive processes, glutathi- one peroxidase (GPx), superoxide dismutase (SOD), catalase were measured in blood after intra peritoneal (i.p.) administra- tion of histamine (10 mg/kg body), tyramine (5 mg/kg body) and cadaverine (5 mg/kg body), single dose in Whistar rats. Blood levels of these enzymes were determined in blood, at 24 and 72 hours after biogenic aminesadministration , using RANDOX kits for manual use. Our data show that SOD activity presented a significant decrease after 72 hours after amines administration as compared with those obtained after 24 hours. Meanwhile the GPx activity present a significant increase in after 72 hours after amines administration as compared with those obtained after 24 hours. As concern for catalase activity, there were no signifi- cant modification neither at 24 hours nor at 72 hours after amines administration. We assume that administered amines gen- erates an increased amounts of reactives species of oxygen, which disturb the balance in antioxidative processes. The non steroidal anti-inflammatory drugs tepoxalin and celecox- ib are potential drugs of abuse in the horse racing industry due to their ability to mask pain prior to racing or sales. Tepoxalin is a cyclooxygenase and 5-lipoxygenase inhibitor, with a number of phase I and phase II metabolites reported from animal trials. Celecoxib is a cyclooxygenase inhibitor, with the major route of metabolism via cytochrome P450 through methyl hydroxylation and oxidation to carboxylic acid. The ability to perform in vitro microsomal assays on potential drugs of abuse may reduce the need for invasive animal trials, and provide a source of metabolic standards for use in doping control. In this investigation, a liver microsomal system was established to study steroidal anti-inflam- matory drug metabolism. Bovine liver microsomes were isolated,

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P4 Cellular and Subcellular Biochemistry

P4–1 Pro-apoptotic and differentiation-inducing effects of genistein and functionally related agents in leukemia cells. Regulation by mitogen-activated protein kinases Y. Sa´ nchez1, C. Calle2, E. de Blas1 and P. Aller1 1Department of Cellular and Molecular Physiopathology, Centro de Investigaciones Biolo´gicas Consejo Superior de Investigaciones Cientı´ficas, Madrid, SPAIN, 2Department of Biochemistry and Molecular Biology, Facultad de Medicina Universidad Complu- tense, Madrid, SPAIN

Obtained results revealed a gradual rise in enzymes activity with a peak at 3 hours (1.5 times). Targets of investigated enzymes are cytoskeleton proteins which consecutive degradation realizes in compromise of membrane integrity, its blebbing and externaliza- tion of phosphatidylserine (PS) on the cell surface. The PS trans- location was estimated directly by the binding of annexin V- FITC according to the Apoptosis Detection kit (BD Bioscience). We demonstrated the enhance of annexin V-FITC positive cells by 1.2 and 2.3 times 30 minutes and 3 hour after X-ray exposure. This process is under control of some translocases among them the most prominent is scramblase which is selectively cleaved and activated by PKC d in caspase-3-dependent manner. Our findings are to be useful for further elucidation of apoptotic mechanisms in lymphocytes under pathological condition.

P4–3 The role of GLP-2 on oxidant-antioxidant system at an in vivo mouse model of intestinal injury induced by TNF-alpha/Act D P. Arda Pirincci and S. Bolkent Department of Biology, Istanbul University, Faculty of Science Istanbul, TURKEY

acid (ATRA) retinoic

The reported capacity of genistein to act as a chemo-sensitizing agent, lead us to analyze the action of this isoflavone and func- tionally related agents (tyrosine kinase inhibitors, estrogens, DNA topoisomerase II inhibitors) in leukemia cells. The results were as follows: (a) Co-treatment with the tyrosine kinase inhibi- tors genistein, herbimycin A and epigallocathechin-3-gallate, and the tyrphostin adaphostin, potentiated apoptosis induction by the antileukemic agent arsenic trioxide (ATO, TrisenoxTM) in U937 promonocytes and other leukemia cell lines. By contrast, no potentiation was obtained using 17-b-estradiol or the DNA topo- isomerase II poison etoposide, although etoposide caused G2/M cycle arrest, as genistein. (b) The potentiaton of ATO-provoked apoptosis by tyrosine kinase inhibitors was associated to reactive oxygen species (ROS) production and p38-MAPK activation, attenuated by antioxidants (N-acetyl-L-cysteine, butylated hy- droxyanisole) and by p38-MAPK inhibitor (SB203580), but enhanced by MEK/ERK inhibitor (PD98059). (c) Unlike the other agents, adaphostin down-regulated ERK phosphorylation and potentiated apoptosis by antitumour drugs other than ATO (the proteasome inhibitor MG-132 and 2-methoxyoestradiol). (d) In addition to the pro-apoptotic action, genistein induced differ- entiation in acute myeloid leukemia cells (HL60, NB4) and coop- erated with all-trans in inducing differentiation. This response was mimicked by etoposide but not by other tyrosine kinase inhibitors, and was apparently depen- dent on MEK/ERK activation. Thus, on the ground its multiple signalling activities, genistein might be a clinically useful agent in the design of both apoptosis- and differentiation-based anti-leu- kemic therapies.

P4–2 Role of proteolytic enzymes in rat’s lymphocytes apoptosis under x-ray exposure T. Andriichuk, N. Raksha and L. Ostapchenko Biochemistry, Kyiv Taras Shevchenko National University, Kyiv, UKRAINE

TNF-a is a multifunctional cytokine, which has been implicated in the regulation of many inflammatory processes. Blocking the synthesis of protective proteins through an transcriptional inhibi- tor such as actinomycin D (Act D) sensitizes many cell types to TNF-a toxicity. Glucagon-like peptide-2 (GLP-2) is a progluca- gon-derived peptide hormone, which has intestinotrophic effects. In this study, we aimed to investigate the effects of GLP-2 on oxidant-antioxidant system at intestinal injury induced by TNF- a/Act D. BALB/c mice used in this study were divided into six groups. Group I: Control animals, Group II: Animals injected 15 lg/kg TNF-a, Group III: Mice injected 800 lg/kg Act D, Group IV: Animals receiving Act D and TNF-a at the same doses, Group V: Animals injected 200 lg/kg h[Gly2]GLP-2 at every 12 hour for ten consecutive days. Group VI: Animals given Act D and TNF-a after receiving h[Gly2]GLP-2 for 10 days at the same doses. Malondialdehyde (MDA) formation as index of lipid peroxidation and the parameters of antioxidant system such as glutathione (GSH), glutathione peroxidase (GPx), catalase (CAT), and superoxide dismutase (SOD) were examined in the intestinal homogenate by spectrophotometry. Administration of TNF-a/Act D caused an insignificant increase in MDA and GSH levels, GPx and SOD activities; a significantly decrease in CAT activity. GLP-2 pretreatment prevented the TNF-a/Act D- induced oxidative injury by a significant decrease in MDA and GSH levels, GPx and SOD activities; a markedly increase in CAT activity. In conclusion, GLP-2 has a protective effect against intestinal injury induced by TNF-a/Act D.

P4–4 The antioxidant effect of EGF in protection against intestinal injury induced by ischemia/ reperfusion in rats P. Arda Pirincci and S. Bolkent Department of Biology, Istanbul University Faculty of Science, Istanbul, TURKEY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Intestinal injury induced by ischemia/reperfusion in experimental animals is a model commonly used in researches on pathogenesis of systemic inflammation, respiratory failure and multiple organ Apoptosis or programmed cell death, involves a cascade of regu- latory events resulting in activation of specific proteases (caspas- es, calpains etc). Being activated these proteases eventually mediate development of some biochemical and morphological changes associated with apoptosis. Nevertheless proteases are the key effector molecules in different apoptotic pathways their exact role in radiation-induced apoptosis remains obscure. Main execu- tors of apoptotic machinery are members of caspase family – cas- pase-3, 6 and calpains. Our data (measured by Caspase Colorimetric Kit, BioSourse) showed increase of caspase-3 activ- ity by 1.2 and 2.8 times, caspase-6 activity by 1.3 and 2 times respectively 30 minutes and 3 hours after irradiation. Calpains substrate. activity was determined by using SLT-AMC as

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Abstracts

P4–6 Characterizing hypoxia-induced autophagy: the role of BNIP3 and reacitve oxygen species M. Azad, Y. Chen, E. Henson and S. Gibson Manitoba Institute of Cell Biology, University of Manitoba, Winnipeg,MB, CANADA

failure, and forming oxidative damage on them. It is known that epidermal growth factor (EGF), a mitogenic peptide, behaves as a cytoprotective and trophic agent for gastrointestinal epithelium. This study aimed to investigate whether or not EGF has an anti- oxidant effect on intestinal injury induced by ischemia/reperfu- sion in rats. Male Sprague–Dawley rats were divided into the five groups. Group I: Animals injected 150 lg/kg EGF 30 minutes prior to intestinal ischemia/reperfusion; Group II: Rats injected 10 mM acetic acid 30 minutes prior to intestinal ischemia/reper- fusion; Group III: Animals administered EGF; Group IV: Con- trol animals administered acetic acid; Group V: Sham-operated animals. Intestinal ischemia/reperfusion injury was produced by causing complete occlusion of the superior mesenteric artery for 60 minutes followed by a final 60 minutes period of reperfusion. Spectrophotometric methods were used to evaluate the effects of EGF on intestinal lipid peroxidation and glutathione levels, and glutathione peroxidase, catalase and superoxide dismutase activi- ties. Administration of EGF prior to intestinal ischemia/reperfu- sion increased these antioxidant enzyme activities and decreased lipid peroxidation level according to the ischemia/reperfusion group. As a result, EGF has an antioxidant effect on oxidative injury of small intestine induced by ischemia/reperfusion in rats.

P4–5 Ceramide production at the plasma membrane from glycolipids M. Aureli, N. Loberto, A. P. Masilamani, G. Illuzzi, S. Sonnino, V. Chigorno and A. Prinetti Department of Medical Chemistry Biochemistry and Biotechnol- ogy, University of Milan, Milan, ITALY

Objectives: Autophagy is a regulated degradation pathway functioning in both cell survival and cell death. Its role in cancer is controversial since autophagy can be protective or destructive to tumor cells, depending on cell type and conditions. Hypoxia is common in solid tumors, correlating with poor prognosis. Reac- tive oxygen species (ROS) are important signaling molecules in hypoxia, and are required for starvation-induced autophagy. This study investigates autophagy in hypoxic cancer cells and exam- ines the role of ROS and the hypoxia-inducible protein, BNIP3. Methods: We analyzed multiple cancer cell lines for response to etoposide and hypoxia (< 1% O2). Cell death was measured by membrane permeability assay. Apoptosis was quantified by cas- pase activity, nuclear condensation and phosphorylation of his- tone H2A.X. Autophagy was assayed by LC3-GFP distribution, electron microscopy, acidic vacuole formation, and siRNA knock-down of autophagy genes. Inhibitors of autophagy (3- methyladenine) and apoptosis (zVAD-fmk) were employed to dif- ferentiate the two types of cell death. BNIP3 was over-expressed by transient transfection or inhibited using siRNA. ROS were measured using redox-sensitive dyes. Results: Chronic hypoxia induced autophagic cell death in apoptosis-competent cancer cells, and BNIP3 was involved in this process. BNIP3 itself induced autophagic cell death, and BNIP3 knock-down protected against hypoxia-induced autophagy and cell death. The precise mechanism of BNIP3’s involvement is unknown, but we hypothesize that BNIP3 mediates hypoxia- induced autophagic cell death by inducing mitochondrial ROS production. Preliminary results indicate that hypoxia induces ROS and further experimentation with BNIP3-/- cells will deter- mine how ROS and BNIP3 may cooperate to regulate hypoxia- induced autophagy.

P4–7 Visfatin enhances ICAM-1 and VCAM-1 expression through ROS-dependent NF-jB activation in endothelial cells M. K. Bae1, K. Su-Ryun1 and B. Soo-Kyung2 1Department of Oral Physiology, School of Dentistry, Pusan National University, YangSan, SOUTH KOREA, 2Department of Physiology, School of Medicine, Pusan National University, YangSan, SOUTH KOREA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Visfatin has recently been identified as a novel visceral adipokine which may be involved in obesity-related vascular disorders. However, it is not known whether visfatin directly contributes to endothelial dysfunction. Here, we investigated the effect of visfa- tin on vascular inflammation, a key step in a variety of vascular diseases. Visfatin induced leukocyte adhesion to endothelial cells and the aortic endothelium by induction of the cell adhesion mol- ecules, ICAM-1 and VCAM-1. Promoter analysis revealed that visfatin-mediated induction of CAMs is mainly regulated by nuclear factor-kB (NF-jB).Visfatin stimulated IkBa phosphoryla- tion, nuclear translocation of the p65 subunit of NF-jB, and NF-jB DNA binding activity in HMECs. Furthermore, visfatin increased ROS generation, and visfatin-induced CAMs expres- sion and NF-jB activation were abrogated in the presence of the direct scavenger of ROS.Taken together, our results demonstrate that visfatin is a vascular inflammatory molecule that increases Several studies have proposed the presence of glycohydrolases on the plasma membrane (PM) where they can be involved in the modifications of the cell surface glicosphingolipids or of glyco- proteins. In human fibroblasts we observed a plasma membrane production of ceramide associated to plasma membrane enzymes. The over-expression of the membrane-associated sialidase Neu3 yielded an increase of ceramide production. Ceramide is a key lipid molecule necessary to regulate some cellular processes, including apoptosis and cell differentiation. The ceramide pro- duction from GM3 has been observed in fibroblasts even under experimental conditions able to block endocytosis or lysosomal activity, suggesting that galactosidase and glucosidase enzymes are also available at the plasma membrane. In these cells we found that stable over-expression of Neu3 induces an increase of the total cell galactosidase and glucosidase activities, resulted in reduced cell growth and increased apoptosis accompanied by the augmented production of ceramide levels. In order to evaluate the presence of galactosidase and glucosidase on the plasma membrane, cell surface protein biotinylation was carried out on normal and Neu3 over-expressing fibroblasts. The biotinylated proteins were then purified by avidin affinity chromatography and isolated fractions were subjected to the galactosidase and glucosidase activity assays, using both artificial and natural sub- strates. In the Neu3 over-expressing cells we found a dramatic increase of the activities of membrane-associated enzymes b- galactosidase and b-glucosidase respect to control cells. We can thus conclude that the increased expression and activity of the Neu3 sialidase is followed by an increase of the activity of other two membrane-associated enzymes involved in the catabolism of the glycosphingolipid: b-galactosidase and b-glucosidase. In addi- tion to this, preliminary experiment suggesting a plasma mem- brane activity on membrane associated lactosylceramide, with no addition of detergent or activator proteins.

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Abstracts

Poster Presentations

expression of the inflammatory CAMs, ICAM-1 and VCAM-1, through ROS-dependent NF-jB activation in endothelial cells.

expressed using pET28a bacterial expression system, and, sub- jected to IMAC purification and extensive dialysis. The dialysed recombinant individual co-activator was coated on each well of 96-well plate before incubating it with mixed recombinant PPARc and plant compounds. The presence of the PPARc was subsequently detected using ELISA system. The preliminary result has indicated stable assay for identifying compounds with PPARg ligand characteristic. Therefore, this assay can be used to determine the active ligand for PPARc from natural products.

P4–8 Upregulation of fibroblast growth factor-2 by visfatin that promotes endothelial angiogenesis Y. H. Bae1, M. K. Bae2, S. R. Kim3 and S. K. Bae1 1Department of Physiology, Pusan National University YangSan, SOUTH KOREA, 2Department of Oral Physiology, School of Dentistry, Pusan National University, YangSan, SOUTH KOREA, 3Department of Molecular Biology, Medical Research Center for Ischemic Tissue Regeneration, Pusan National University, Pusan, SOUTH KOREA

P4–10 Putative phosphatidic acid phosphatases are involved in pollen tube polar growth R. Bezvoda1, M. Potocky2, P. Pejchar3, J. Martinec3 and V. Zarsky1 1Department of Plant Physiology, Charles University Faculty of Science, Prague, CZECH REPUBLIC, 2Laboratory of cell biol- ogy, Institute of Experimental Botany, Prague, CZECH REPUB- LIC, 3Laboratory of signal transduction, Institute of Experimental Botany, Prague, CZECH REPUBLIC

Adipokines have been known to act as angiogenic regulators in the process of angiogenesis. Recently we have demonstrated that visfatin, a novel adipokine, has angiogenic activity. However, lit- tle has been reported on the underlying mechanism of visfain- induced angiogenesis. In this study, we report that visfatin- induced angiogenesis is mediated by endothelial fibroblast growth factor 2 (FGF-2). Visfatin increased the levels of FGF-2 mRNA and protein in human endothelial cells. The enhancement in FGF-2 expression was prevented by an inhibitor of the extracel- lular signal-regulated kinase 1/2 (Erk1/2) pathway. Furthermore, visfatin-induced angiogenesis was reduced by inhibition of FGF- 2 receptor kinase or by neutralization of FGF-2 function. Taken together, our results indicate that visfatin-induced endothelial angiogenesis is composed largely of two sequential steps: the induction of Erk1/2-dependent FGF-2 gene expression by visfatin and the subsequent FGF-2-induced angiogenesis. These data fur- ther suggest an integral role for visfatin-FGF-2 signaling axis in modulating endothelial angiogenesis.

supported by projects Membrane phospholipids serve as both structural and signalling molecules in plant cells. Phospholipase D (PLD) and its product, phosphatidic acid (PA), is known to regulate both stress signal- ling, but also cell expansion and developmental processes. We looked into the role of possible negative regulators of PLD/PA pathway, lipid phosphate phosphatases (LPPs) in polar expansion of tobacco pollen tube. We utilised three pharmacological inhibi- tors of LPPs that were previously used in mammalian cell biol- ogy. All three drugs had similar effects on growth kinetics of tobacco pollen tubes. This effect is in agreement with hypothes- ised role of LPPs as negative regulators of PLD pathway. As PA is important for actin dynamics, the modulation of PA level by those inhibitors is able to modify the effect of latrunculin B on pollen tubes. We cloned and sequenced a full length cDNA of LPP from tobacco pollen. It is a clear member of plant LPP fam- ily with homology to its yeast counterpart. Furthermore, we designed and utilised antisense oligonucleotides (aODNs) for gene-specific knock down of corresponding protein in pollen tubes. Currently, we are evaluating the effect of drugs that possi- bly modulate LPP activity on phospholipid content and on cyto- skeletal dynamics in growing pollen tubes. Acknowledgements: The work is GAAV-IAA601110916, MSM-0021620858 and MSM-LC06034.

P4–9 Development of cell-free assay for the screening of PPARg ligand from natural products V. Balakrishnan1, W. K. Lau2, M. F. Hamzah3, A. S. C. Chong4 and T. S. Tengku Muhammad5 1Institute for Molecular Medicine, Universiti Sains Malaysia, Pulau Pinang, MALAYSIA, 2School of Biological Sciences, Universiti Sains Malaysia, Pulau Pinang, MALAYSIA, 3Drug Discovery, Insitute of Pharmaceutical and Nutraceutical Malaysia, Pulau Pinang, MALAYSIA, 4Drug Discovery, Institute of Pharmaceutical and Nutraceutical Malaysia, Pulau Pinang, MALAYSIA, 5Faculty of Science and Technology, Universiti Malaysia Terengganu, Terengganu, MALAYSIA

the nuclear

P4–11 Microtubule dynamics modulation in chemotherapy-induced neurotoxicity : a protective role of olesoxime A. Rovini1, M. Carre1, T. Bordet2, N. McKay1, R. Pruss2 and D. Braguer1 1INSERM UMR911, Universite de la Mediterranee, Marseille, FRANCE, 2Trophos, Parc Scientifique de Luminy, Marseille, FRANCE

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Microtubules play a central role in processes such as mitosis completion, cell spreading, and organelle trafficking. They consti- tute the primary target of Microtubule-Targeting Agents (MTAs) used as cancer chemotherapeutics. However, peripheral neuropa- thy is often a dose-limiting side effect of MTAs, and the mecha- nism for this neurotoxicity is still poorly understood. To date, Peroxisome proliferator activated receptor gamma (PPARc) is ligand-activated transcription factor of receptor superfamily. The activation of PPARc by its specific ligands induces the recruitment of the co-activator proteins, which in turn, leads to the binding of the ligand-bound PPARc to the pro- moter of the target genes. Interestingly, a group of PPARg ligands known as thiazolidinediones are drugs against type II dia- betes. It has been postulated that the PPARg-activated genes are involved in glucose uptake in adipocyte and muscle cell. There- fore, the main aim of this study is to develop the high-through- put screening system to screen for PPARg ligand from natural products. Thus, the ligand binding domain of PPARc and the PPARc binding domain of co-activator (P300 and NCoA1) were

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Poster Presentations

Abstracts

P4–13 Transcriptional regulation of MTERF family genes in human and Drosophila F. Bruni1, M. A. Ferna´ ndez-Moreno2, P. Loguercio Polosa1, R. Garesse2, P. Cantatore1, M. N. Gadaleta1 and M. Roberti1 1Dipartimento di Biochimica e Biologia Molecolare ‘Ernesto Quagliariello’, Universita degli Studi di Bari, Bari, ITALY, 2Departamento de Bioquı´mica Instituto de Investigaciones Biomedicas ‘Alberto Sols’, Universidad Autonoma de Madrid, Madrid, SPAIN

there are no approved therapies for prevention or treatment of neuropathies triggered by MTAs. Here, we investigated the neu- rotoxic effect of MTAs in vitro and the protective effect of oles- oxime (TRO19622), a new drug candidate that has promising neuroprotective properties in vivo. We first showed that MTAs induced loss of neurite outgrowth in rat and human differenti- ated neuronal cells in vitro and that olesoxime prevented this effect. In parallel, olesoxime did not alter cytotoxic properties of MTAs in human tumor cell lines, a necessary prerequisite to fur- ther study such a combination. Since regulation of microtubule dynamics is critical for neuron organization, we analysed the dis- tribution of proteins that specifically promote microtubule plus- ends growth. Interestingly, MTAs triggered EB1 and EB3 dissoci- ation from microtubules to cytosol. Concomitant treatment with olesoxime preserved normal EBs distribution while maintaining neurite outgrowth, specifically in differentiated neuronal cells, suggesting a tight link between microtubule dynamics disruption and neurite architecture loss. Videomicroscopy analysis showed that olesoxime significantly increased the growing rate of micro- tubules and decreased the attenuation mean duration in neuro- nally differentiated cells. All these data indicate that olesoxime neuroprotection results from its ability to specifically protect the microtubule network against MTAs in differentiated neuronal cells.

P4–12 Histone deacetylase inhibitors augment actinomycin D-induced ROS generation in normal peripheral blood lymphocytes B. Brodska´ , P. Otevrelova´ and I. Kalousek Cellular Biochemistry, Institute of Hematology and Blood Transfusion, Prague 2, CZECH REPUBLIC

Background: In the recent years, a considerable effort has been devoted to uncover the transcriptional mechanisms that orches- trate the mitochondrial biogenesis. In human, the transcription factors NRF-1 and -2 regulate the expression of many nuclear genes including those encoding proteins involved in mitochon- drial biogenesis and function. In Drosophila, DREF controls the expression of genes involved in nuclear DNA replication and cell cycle control as well as in mitochondrial DNA maintenance. Objectives: We intend to contribute to the study of mitochon- drial biogenesis in human and Drosophila. In particular, we focused our attention on the regulation by human NRF-2 and Drosophila DREF of genes encoding mitochondrial proteins belonging to the MTERF family, which includes the repressor and the termination factor of mitochondrial transcription. Methods: Transcription factor binding sites were predicted by using MAPPER and TFsearch. Protein binding was tested in vitro by EMSA and in vivo by ChIP experiments. Transient transfection assays and RNAi experiments were carried out in Drosophila S2 cells. Results: EMSA and ChIP assays demonstrated the functionality of in silico predicted binding sites for NRF-2 and DREF in MTERF gene promoters. Transient transfection assays showed that DRE sequences are required for promoter activation; RNAi- mediated depletion of DREF caused a remarkable decrease of mRNA level of the analysed MTERF genes. Moreover, a preli- minary bioinformatic analysis indicated the existence of putative binding sites of hDREF, the human orthologue of the Drosophila factor, and D-elg, the Drosophila orthologue of human NRF-2, in MTERF gene promoters. Conclusion: We demonstrate that the expression of some mito- chondrial genes encoding MTERF family proteins is positively controlled by NRF-2 in human and by DREF in Drosophila. Moreover, our results provide interesting clues on the existence of common regulatory patterns of mitochondrial biogenesis in human and Drosophila.

P4–14 Bordetella adenylate cyclase toxin exploits lipid rafts for translocation into cell cytosol L. Bumba1, J. Masin1, R. Fiser2 and P. Sebo1 1Biology of Bacterial Pathogens, Institute of Microbiology, Prague 4, CZECH REPUBLIC, 2Department of Microbiology, Faculty of Sciences Charles University, Prague 2, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Bordetella pertussis produces an adenylate cyclase toxin (CyaA) that targets myeloid phagocytic cells expressing the CD11b/CD18 integrin receptor. CyaA penetrates cellular membranes, forms small cation-selective pores, and delivers directly across the cyto- plasmic membrane a calmodulin-activated adenylate cyclase (AC) domain that catalyzes unregulated conversion of cytosolic ATP into cAMP. Translocation of the AC domain across plasma mem- brane is accompanied by entry of extracellular calcium ions into cells and yields elevation of cytosolic calcium concentration ([Ca2+]i). We show that the capacity of CyaA to promote calcium entry is indispensable for efficient translocation of the AC enzyme into cell cytosol. The [Ca2+]i increase activated the Ca2+-depen- dent protease calpain for proteolysis of the cytoskeletal protein Cytotoxic drugs provoke an induction of apoptosis process blocked in cancer cell lines, but the effect of apoptosis induction also occurs in normal cells. Combination of two or more antican- cer agents often leads to higher and more specific therapeutic effect. In our study, we examined the impact of combination of actinomycin D (ActD) and histone deacetylase inhibitors (HDACi) on apoptotic features of normal peripheral blood lym- phocytes. Cytostatic agent ActD causes an arrest of rRNA syn- thesis and subsequent inhibition of MDM2 ligase activity provokes p53 stabilization. p53-upregulation of Puma mRNA and protein levels then activates mitochondrial way of apoptosis. ActD causes broad ROS generation. HDACi n-butyric acid or SAHA also induce the inner way of apoptosis, but the mitochon- dria permeabilization is mediated via Bim and Bmf2 upregulation followed by activation of Bak and Bax proteins. Moreover, HDACi-induced apoptosis in normal lymphocyte cells is regu- lated by variations in p21 and c-myc expressions. ROS genera- tion slightly occurs in n-butyric acid treatments, but there is no ROS induction caused by SAHA. ActD+SAHA treatment pro- vokes p53 protein stabilization like ActD alone, but p53 mRNA decreases. C-myc variations occur as well as with HDACi, but we did not detect any increase in BIM levels, and p21 expression gradually decreased in both mRNA and protein. ROS generation markedly increases in comparison with pure ActD action, and mitochondrial membrane depolarization, executive caspases acti- vation as well as the viability test also indicate, that ActD and SAHA cooperate in their apoptosis-inducing activities. Acknowledgement: Supported by grant IGA NS 9637-4 from the MHCR.

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Poster Presentations

talin, the cleavage of which releases CD11b/CD18 from cytoskele- tal constraints, thus allowing lateral movement of the CyaA- CD11b/D18 complex in the membrane and mobilization of the toxin-receptor complex into cholesterol-rich membrane microdo- mains known as lipid rafts. Depletion of cholesterol from plasma membrane as well as inhibition of calpain by calpeptin strongly inhibited the capacity of CyaA to deliver the AC domain into cyto- sol of cells. These results show that CyaA hijacks intracellular Ca2+ signaling for promoting mobilization of the toxin molecule into the cholesterol-rich membrane regions, where appropriate lipid environment allows passage of the 40 kDa AC enzyme domain across the lipid bilayer of cell membrane.

taraxifolius (M.Bieb) DC. var. taraxifolius, in this wise approach- ing to solve problems related to taxonomy and aiding in the revi- sion studies. Methods: The structure of achene was studied by Transmission Electron Microscopy (TEM). Key Results: Achene fruit wall was wavy and had thick cell walls. There was a thick cuticula layer on the epidermis cells. Below the epidermis, there was a 5–6 layer of sclerenchymatic cells. Below the sclerenchymatic cells, there was 3–5 layer of cells, which has thick cell walls and stained darkly. Below this cells, there were 1–2 layer of parenchyma cells, which was long and rectangular. Innermost there were endosperm. Endosperm cells was polygon shaped and filled with nutrient. Conclusions: Taxa of Senecio will be studied by Transmission Electron Microscopy (TEM) and morphological and cytological differences will be determined, so this study aid for the knowl- edge of the genus as taxonomical and phylogenetical. In the flora of Turkey, more than 30 synonim and more than 15 taxa can be revisied for the concerned genus.

P4–15 Activation of mitochondrial glycerol-3- phosphate dehydrogenase from rat liver and brown adipose tissue by idebenone (hydroxydecyl-ubiquinone) H. Rauchova1, Z. Drahota2, T. Soukup3, B. Christian4 and R. Fato4 1Institute of Physiology AS CR v.v.i., Experimental hypertension, Prague 4, CZECH REPUBLIC, 2Institute of Physiology AS CR v.v.i., Bioenergetics, Prague 4, CZECH REPUBLIC, 3Institute of Physiology AS CR v.v.i., Functional Morphology, Prague 4, CZECH REPUBLIC, 4University of Bologna, Biochemistry, Bologna, ITALY

P4–17 Achene Structure of Senecio bicolor (Willd.) Tod. ssp. bicolor var. bicolor H. N. Bu¨ yu¨ kkartal1, H. Colgecen2, N. Erdogan3, N. M. Pinar1 and U. Budak4 1Biology, Faculty of Science, Ankara University, Ankara, TUR- KEY, 2Biology, Faculty of Arts and Science, Zonguldak Karaelmas University, Zonguldak, TURKEY, 3Department of Biology, Fac- ulty of Arts and Science, Mehmet Akif Ersoy University, Burdur, TURKEY, 4Department and Biology, Faculty of Arts and Science, Bozok University, Yozgat, TURKEY

Mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) located on the outer surface of the inner membrane forms one of the branches of the respiratory chain. Together with its cytosolic counterpart operates in the glycerolphosphate shuttle, which con- nects glycolysis with the respiratory chain. In our previous paper (J Bioenerg Biomembr 2008, 40: 85–93) we found that oxidation of mGPDH from brown adipose tissue of cold-adapted hamsters is activated by idebenone (IDE), a synthetic analog of coenzyme Q. On the other hand, oxidation of succinate is not changed signifi- cantly and oxidation of NADH-dependent substrates is strongly inhibited. In the present communication we show additional data on the similar activating effect of IDE on mGPDH from rat liver and brown adipose tissue from newborn rats. We compare liver mitochondria from control and triiodothyronine-treated animals where expression of mGPDH is stimulated and the enzyme activity increases 3–5 folds. In both groups the activation of mGPDH by IDE is pronounced. Moreover, we check mGPDH from brown adipose tissue of newborn rats and we confirm that the rate of gly- cerol-3-phosphate oxidation increases within the concentration range of 5–25 lM IDE. Presented data also demonstrate the lower inhibitory effect of free fatty acids on glycerol-3-phosphate oxida- tion in the presence of IDE. Our data further support the idea that the partial release of endogenous fatty acids can be involved in the activating effect of IDE on mGPDH. Background and Aims: The aim of this study was to determine the cytological structure of the layers of achene of Senecio bicolor (Willd.) Tod. ssp. bicolor var. bicolor, in this wise approaching to solve problems related to taxonomy and aiding in the revision studies. Methods: The structure of achene was studied by Transmission Electron Microscopy (TEM). Key/Results: Achene fruit wall was wavy and epidermis had thick cell walls. There was a thin cuticle layer on the epidermis cells. Below the epidermis, there were 5–6 layers of sclerenchy- matic cells. Below the sclerenchymatic cells, there were 3–5 layer of cells, which have thin cell walls and look like middle layer cells. Below these cells, there were 1–2 layers of parenchyma cells, which was long and rectangular. Innermost there were endo- sperm cells. Endosperm was rich of nutrients. Conclusions: Through the findings gained from Senecio taxa, a comprehensive and literal revision will be achieved.

P4–18 Isolation of cystathionine gamma-lyase in calf lens E. Calvani, R. Moschini, M. Cappiello, A. Del Corso and U. Mura Biology, University of Pisa, Pisa, ITALY

P4–16 Acen Structure of Senecio taraxifolius (M.Bieb) DC. var. taraxifolius H. N. Bu¨ yu¨ kkartal1, N. M. Pinar1, H. Colgecen2, N. Erdogan3 and U. Budak4 1Biology, Faculty of Science, Ankara University, Ankara, TUR- KEY, 2Biology, Faculty of Arts and Science, Zonguldak Karaelmas University, Zonguldak, TURKEY, 3Department of Biology, Fac- ulty of Arts and Science, Mehmet Akif Ersoy University, Burdur, TURKEY, 4Department and Biology, Faculty of Arts and Science, Bozok University Yozgat, TURKEY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Background and Aims: The aim of this study was to determine the layers of achene of Senecio the cytological structure of Glutathione (GSH) levels play a key role in maintaining lens trans- parency. Cysteine (Cys) is the limiting amino acid for the synthesis of GSH and its availability is controlled by the reverse transsulfu-

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P4–20 The channel of the mitochondrial inner membrane protein translocase TIM22: structure-function correlations and insights into the biogenesis the complex P. M. Peixoto1, T. d .J. Roy2, I. Corraliza2 and M. L. Campo2 1Biochemistry and Molecular Biology, University of Extremadura and New York University, Ca´ceres-New York, SPAIN, 2Biochemistry and Molecular Biology, University of Extremadura, Ca´ceres-New York, SPAIN

ration pathway which converts methionine into Cys. Cystathionine gamma-lyase (CGL) is a pyridoxal phosphate dependent enzyme that catalyses the rate limiting step of the reverse transsulfuration pathway, i.e. the conversion of L-cystathionine into L-cys. In liver and kidney CGL has been shown to be essential for an adequate supply of Cys for GSH synthesis, in fact Cys deficiency plays a key role in several physiological and pathological conditions related to GSH depletion. Recently it has been shown the existence of reverse transsulfuration pathway also in the lens and its up regulation in oxidative conditions, disclosing the hypothesis that CGL could be relevant for cysteine availability in aging lenses. CGL appeared almost undetectable in crude extracts obtained from calf lens, both as active enzyme (through enzymatic assay) and as protein (through Western Blot analysis). However, partial purification of the crude extract upon hydrophobic chromatography, revealed the presence of the enzyme. CGL, on the basis of Western Blot, and gel filtration chromatography analysis, displayed an homotetra- meric structure of approximately 160 kDa. When cultured bovine lens epithelial cells were used as source for CGL, a specific activity two order of magnitude higher than those measured in the whole lens was observed. The purification of the CGL from lens epithelial cells is going to be accomplished in order to define its kinetic fea- tures and modulability.

P4–19 Mitochondrial Hexokinase Activity Modulates Reactive Oxygen Species Production in Potato Tuber Mitochondria J. Camacho-Pereira, L. Evangelista Meyer, L. Bender Machado, M. Fernandes Oliveira and A. Galina Institute of Medical Biochemistry – Federal University of Rio de Janeiro, Universidade Federal do Rio de Janeiro, Rio de Janeiro, BRAZIL

Translocases are multisubunit complexes that arbitrate the traf- ficking of mitochondrial proteins in and across their two distinc- tive membranes. Aqueous channels are an essential part of the outer (TOM) and inner (TIM) membrane translocases. Though the existence in organello of a channel activity associated to the TIM22 translocase has been questioned, as its function relates to the insertion of multispaning proteins into the inner membrane, we have previously reported the conditions to induce the channel activity associated to the TIM22 complex. Our results portray Tim22 as a dynamic channel solely active in the presence of its cargo proteins. Using electrophysiological techniques, applied to the inner membranes of mitochondria, isolated from yeast cells lacking and/or over-expressing some of the defined components of TIM22, we have obtained some insights into the structure- function correlations of the channel present in this inner mem- brane translocase. The molecular dissection studies carried out indicate that Tim54p does not participate in the structure or functioning of the TIM22 channel, whereas Tim22p is its core component. In addition, we imply Tim18p as a putative receptor for internal signal peptides that triggers the channel activity. Also, the structure of the TIM22 channel is compatible with that of two identical pores that cooperatively gate, and the estimated pore size may accommodate up to four transmembrane segments. Remarkably, and despite their different composition, the chan- nels of TOM, TIM23 and TIM22, the three most studied mito- chondrial translocases, are noteworthy similar. Most likely these results are in tenant with the common task of these channels as membrane conduits transiently clapping the proteins in route to their final destination inside mitochondria. Acknowledgements: Supported by Grants PRI 07A072 from Junta de Extremadura and BFU2008-00475 from Spanish Minis- try of Science and Innovation.

P4–21 A weak link in metabolism: humans cannot synthesize enough glycine to meet metabolic requirements M. L. Cardenas1, E. Melendez Hevia2, P. de Paz Lugo2 and A. Cornish Bowden1 1CNRS, Bioenergetique et Ingenierie des Proteines, Marseille, FRANCE, 2ASIMC, Centro de Investigacion, La Laguna Tenerife, SPAIN

Potato tuber mitochondria (PTM) have a tightly bound hexoki- nase (mt-HK). The relationship between mitochondrial mem- brane potential (DYm) and the reactive oxygen species (ROS) production with the mt-HK activity was evaluated in PTM. The mt-HK activity is inhibited by the ADP produced from the reac- tion. This inhibition can be reversed when the PTM respiration is activated. Oxygen consumption rate is stimulated by glucose after reaching the state 4 rate. A small depolarization in DYm (<3%) was detected and ROS formation was completely abol- ished. The mt-HK inhibitors, mannoheptulose or N-acetylglucos- amine stimulated ROS production by PTM in the presence of glucose. The blockage of ROS production by mt-HK activity is comparable to that of plant uncoupling protein (PUMP) acti- vated by linolenic acid. The localization of mt-HK is fifty times more efficient to impair the H2O2 production than the yeast HK. Mt-HK reaction stimulates respiration using succinate, NADH or pyruvate/malate (PM) as substrates. However, glucose prevents H2O2 accumulation by 90% with succinate and 50% with NADH as respiratory substrates. The rate of H2O2 produc- tion and O2 consumption in potato tuber slices were also modu- lated by glucose and mt-HK inhibitors. Together, these results indicate that mt-HK activity is involved in the local mitochon- drial ADP recycling mechanism, leading to a decrease in DYm and ROS production acting as a preventive antioxidant defense in plants. Acknlowdegement: Support: FAPERJ, CNPq.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Various nutritional studies have suggested that glycine should be reclassified as an essential amino acid in some conditions, even though the reaction catalysed by glycine hydroxymethyltransfer- ase allows it to be synthesized from serine. However, the amount of glycine produced in this reaction must be equimolar with the amount of tetrahydrofolate-C1, and although glycine can be con- verted to tetrahydrofolate-C1 the reaction is irreversible, and no known process can convert tetrahydrofolate-C1 into glycine. The capacity to synthesize glycine is thus constrained by the demand for C1 units and not by the demand for glycine (Melendez-Hevia

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unprecedented proteomic analysis that identifies BiP as a key- protein in the death programme induced by AEA in human neu- ronal cells.

P4–23 Histochemical and morphological properties Of primary flight muscles in miniopterus schreibersi S. Cebesoy Biology, Faculty of Science, Ankara University, Ankara, TURKEY

and de Paz-Lugo (2008) Branch-point stoichiometry can generate weak links in metabolism: the case of glycine biosynthesis, J. Bio- sci. 35, 771–780). To evaluate the practical consequences of this constraint, we have made a detailed assessment of the amount of glycine synthesized from serine and available all other sources, and compared it with the amount needed for collagen mainte- nance and other metabolic purposes. A 70 kg adult has about 3 g/day of glycine available from all sources, but needs about 1.5 g/day for metabolic functions other than collagen and a lar- ger amount for collagen synthesis, leaving a significant daily defi- cit even when recycling of glycine is taken into account. This result explains not only the nutritional observations, but also the fact that collagen-related diseases such as arthrosis are common in humans and are among the rare diseases seen in large animals in the wild. Furthermore, they are also found in fossil remains of prehistoric humans.

P4–22 Endocannabinoid system in human neuronal cells, and proteomic analysis of anandamide- induced apoptosis G. Catanzaro1, N. Pasquariello1, V. Marzano2, D. Amadio3, S. Oddi1, G. Federici2, A. Urbani4 and M. Maccarrone1 1Biomedical Sciences, University of Teramo, Teramo, ITALY, 2Internal Medicine, University of Rome ‘Tor Vergata’, Rome, ITALY, 3Experimental Medicine and Biochemical Sciences, University of Rome ‘Tor Vergata’, Rome, ITALY, 4Biomedical Sciences, University ‘G. D’Annunzio’ of Chieti, Chieti, ITALY

Two primary flight muscles of Miniopterus schreibersi were stud- ied using morphological and histochemical analysis. All animals were killed with an overdose of sodium pentabarbital adminis- tered intraperitoneally. Muscles were dissected free, cleaned of excess fasia, blotted dry and weighed. Their proximal and distal portions were mounted in gum tragacanth on cork, quick-frozen by immersionin isopentane cooled to about -160 (cid:2)C. Transverse serial sections (10–12 lm thickness) were obtained with a freezing cryostat. Sections from each muscle were stained with nicotin- amide adenine dinucleotide tetrazolium reductase (NADH-TR) to assess oxidative capacity. The method was used demonstate succinate dehydrogenase (SDH) activity. Sections of each muscle were stained using myosin adenosine triphosphatase (mATPase). Two fast-twitch fiber types are histochemically identified in pec- toralis muscles of M. schreibersi. These were classified as type II a and type II b according to glycine-calcium-formalin preincuba- tion staining protocol for myosin ATPase. The primary flight muscles, serratus ventalis included type I, type II a and type II b fibers. Type I fibers in serratus ventralis were highly oxidative, as stained darkly for NADH-TR. All type II a fibers exhibited rela- tively intense staining properties for NADH-TR and SDH, sug- In M. schreibersi gesting a intermediate oxidative capacity. primary flight muscles, type II b fibers were low oxidative, as indicated by light reaction for NADH-TR. Fiber ratios of M. schreibersi for the pectoral muscle are fiber type IIa 87% and fiber type IIb 13%. Fiber ratios for serratus ventralis are fiber type I 14%, fiber type IIa 70% and fiber type IIb 16%.

P4–24 RRN3 5’UTR contains cryptic promoter J. Cerny, T. Masek, J. Dubska and M. Pospisek Genetics and Microbiology, Faculty of Science, Charles University Prague, Prague 2, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

The human neuroblastoma SH-SY5Y cell line is an experimental model for neuronal cell damage and death, as well as major human neurodegenerative disorders. Endocannabinoids represent a new class of lipid mediators which includes amides, esters and ethers of long chain poly-unsaturated fatty acids, isolated in many tissues and implicated in different biological actions. The main members of this group are anandamide (N-arachidonoyleth- anolamine, AEA) and 2-arachidonoylglycerol (2-AG), which acti- vate type 1 and type 2 cannabinoid receptors (CB1R and CB2R). Cannabinoid receptors, their agonists and the enzymes responsi- ble of the synthesis, transport and hydrolysis of AEA, 2-AG and congeners compose the ‘‘endocannabinoid system’’ (ECS). In this study, we report the biochemical, morphological and functional characterization of the ECS (CB1R and CB2R; transient receptor potential vanilloid 1, TRPV1; N-acyl phosphatidylethanolamine- specific phospholipase D, NAPE-PLD; fatty acid amide hydro- lase, FAAH; diacylglycerol lipase, DAGL; and monoacylglyce- ride lipase, MAGL) in SH-SY5Y cells. We also show that AEA dose-dependently induces apoptosis of SH-SY5Y cells, and that the CB1R antagonist SR141716 minimized this effect. By means of proteomic analysis, we further demonstrate that AEA-induced apoptosis was paralleled by a 33 to 35-fold up-regulation or down-regulation of five genes: the three genes that were up-regu- lated (i.e., BiP/GRP78, stress-induced phosphoprotein-1, and tri- osephosphate isomerase-1) are known to act as anti-apoptotic agents, whereas of the two genes that were down-regulated (i.e., actin-related protein 2/3 complex subunit 5, and peptidylprolyl isomerase-like protein 3 isoform PPIL3b) the first is required for actin network formation and the second is still function-orphan. Interestingly, only the effect of AEA on BiP was reversed by SR141716, and silencing or over-expression of this gene increased or reduced, respectively, AEA-induced apoptosis of SH-SY5Y cells, as well as the expression of the apoptotic marker genes p53, PUMA and caspase-8. In conclusion, we show a complete and fully functional ECS in human SH-SY5Y cells, and report an Protein product of RRN3 possesses an important role in the tran- scriptional regulation of rRNA synthesis in Saccharomyces cerevi- siae. Mechanism by which Rrn3p controls transcription of rRNA has already been explored in details. However, mechanism of RRN3 expression itself is still unclear except some recent data showing a tightly regulated RRN3 expression in response to vari- ous external stimuli. Our experiments revealed that RRN3 tran- script belongs among mRNAs that are localized on polysomes under conditions when classical cap-dependent translation is blocked. This indicates that RRN3 can be translated by an alter- native mechanism. We inserted putative 5’ untranslated region of RRN3 transcript into intercistronic position of various bicistronic plasmids and measured enzymatic activity of reporter proteins coded both by the first and the second cistrons. Obtained results indicate that RRN3 translation could be initiated via internal ribosome entry site. To confirm that and to exclude possible

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influence of the cryptic transcription, we examined presence of the cryptic start of transcription by employing, promoterless vec- tors, vectors bearing inducible promoter and qRT-PCR technol- ogy. On the basis of obtained results we concluded that an examined part of the RRN3 gene contains a cryptic promoter. We will speculate about the possible physiological function of the the RRN3 newly described alternative transcription start of mRNA.

carcinogens, like environmental chemical contaminants, act as tumor promoters or genotoxic initiators by modulating gap junc- intercellular communication (GJIC) We investigated the tional effects of 24 hours treatment of 10 nM HgCl2, a well docu- mented carcinogen, on cultured human keratynocytes (HK), obtaining the results summarized in the following: 30%–40% decrease of the GJIC (tested by Lucifer Yellow microinjection assay) with no change in cell viability; Marked depletion of free- SH groups (tested by C5 maleimide) intracellular content indicat- ing a change in the cell redox state; Increase of H2O2 (tested by DCF) and O2- (tested by Mitosox) of mitochondrial origin; Over- expression of the transcripts and protein level of C·26, C·32 and C·43 connexins isoforms (tested by RT-PCR and immunoblot- ting respectively). In addition an enhanced phosphorylation state of C·43 was observed. Rescue of the mitochondrial reactive oxy- gen species (ROS) overproduction and full recovery of the GJIC after treatment of HgCl2-exposed HK cells with all-trans retinoic the acid (ATRA) or dibutirryl-cAMP. These results support involvement of Hg-induced mitochondrial dysfunction in a chain of events triggered by the tumor promoter which, by changing the intracellular redox signaling, leads to inhibition of GJIC and possibly to carcinogenic priming.

P4–25 Expression of endothelial selectin ligands on human leukocytes following dive V. Cikes Culic1, D. Glavas2, A. Markotic1, Z. Valic3, N. Kovacic4, I. Palada3, R. Martinic5, T. Breskovic6, D. Bakovic6, A. O. Brubakk7 and Z. Dujic3 1Department of Medical Chemistry and Biochemistry, University of Split School of Medicine, Split, CROATIA, 2Department of Cardiology, University of Split Hospital Center, Split, CROATIA, 3Department of Physiology, University of Split School of Medicine, Split, CROATIA, 4Department of Anatomy and Clinical Anatomy, University of Zagreb School of Medicine, Zagreb, CROATIA, 5Department of Pathophysiology Laboratory for Tissue Typing, University of Split Hospital Center, Split, CROATIA, 6Depart- ment of Physiology, University of Split Scool of Medicine, Split, CROATIA, 7Department of Circulation and Medical Imaging, Norwegian University of Science and Technology, Trondheim, NORWAY

P4–27 Molecular identification of albumin and Hsp70 as cytosolic anandamide-binding proteins C. De Simone2,3, S. Oddi1,2, F. Fezza2,3, N. Pasquariello1, A. D’Agostino1, G. Catanzaro1,2, C. Rapino1, A. Finazzi-Agro3 and M. Maccarrone1,2 1Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Roma, ITALY, and Department of Biomedical Sciences, University of Teramo, Teramo, ITALY, 2Biomedical Sciences, University of Teramo, Teramo, ITALY, and European Center for Brain Research (CERC)/IRCCS, S. Lucia Foundation, Rome, ITALY, 3Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Rome, ITALY

The fact that impaired endothelial-dependent vasodilatation after scuba diving often occurs without visible changes in the endothe- lial layer implies its biochemical origin. Since Lewis x (CD15) and sialyl-Lewis x (CD15s) are granulocyte and monocyte carbo- hydrate antigens recognized as ligands by endothelial selectins, we assumed that they could be sensitive markers for impaired vasodilatation following diving. Using flow cytometry, we deter- mined the CD15 and CD15s peripheral blood mononuclear cells of eight divers, 30 minutes before and 50 minutes after a single dive to 54 m for 20 minutes bottom time. The number of gas bubbles in the right heart was monitored by ultrasound. Gas bubbles were seen in all eight divers, with the average number of bubbles/cm2 1.9 ± 1.9. The proportion of CD15 + monocytes increased 2-fold after the dive as well as the subpopulation of monocytes highly expressing CD15s. The absolute number of monocytes was slightly, but not significantly, increased after the dive, whereas the absolute number of granulocytes was markedly elevated (up to 61%). There were no significant correlations between bubble formation and CD15 + monocyte expression (r = 0.56; p = 0.17), as well as with monocytes highly express- ing CD15s (r = 0.43; p = 0.29). This study suggests that bio- chemical changes induced by scuba diving primarily activate existing monocytes rather than increase the number of monocytes at a time of acute arterial endothelial dysfunction.

Anandamide (arachidonoylethanolamide, AEA) acts as endoge- nous agonist of both cannabinoid and vanilloid receptors. Its cel- lular uptake and intracellular synthesis and degradation are crucial steps for controlling its extracellular level and the dura- tion of its activity. Therefore, they represent potential targets for the development of therapeutic strategies aimed at modulating the level of AEA in several diseases. Metabolic pathways and biological activity of AEA have been deeply investigated and well-characterized. In contrast, the effective nature and mecha- nisms underlying AEA transport remain controversial and still unsolved issues. Here we investigated the presence of a potential carrier-mediated trafficking of AEA within the cytosol using a biotinylated analogue (biotin-AEA) that has the same lipophilici- ty of the parent compound and that could be used as a ligand to catch, by affinity chromatography, AEA-interacting proteins. The identity of two of these AEA-binding proteins, Hsp70 and albumin, was determined by mass spectrometry, confirmed by Western blotting and confocal microscopy, and further validated through an AEA binding assay. These findings suggest that the trafficking of AEA from the plasma membrane to the internal compartments of a cell may occur, at least in part, via a nonve- sicular mechanism mediated by cytosolic protein carriers.

P4–26 Gap junctional intercellular communication, carcinogenesis and mitochondria A. D’Aprile1, C. Piccoli1, M. Ripoli1, G. Quarato1, R. Zefferino2, D. Boffoli1 and N. Capitanio1 1Biomedical Sciences, University of Foggia Medical School, Foggia, ITALY, 2Occupational and Medical Sciences, University of Foggia Medical School, Foggia, ITALY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Gap junctions are membrane channels permitting intercellular communication. Their loss of function, described in cancer cells, has been hypothezised to be involved in carcinogenesis. Many

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P4–28 Involvement of mitochondrial Mn-SOD in the defence of diclofenac-induced apoptosis in the SH-SY5Y neuroblastoma cell line E. De Vendittis1, C. Zappelli1, F. Cecere1, A. Iuliano1, I. Castellano1, P. Grimaldi1, M. Masullo2 and M. R. Ruocco1 1Dipartimento di Biochimica e Biotecnologie Mediche, Universita’ degli Studi di Napoli Federico II, Naples, ITALY, 2Dipartimento di Scienze Farmacobiologiche, Universita’ degli Studi Magna Grae- cia di Catanzaro, Roccelletta di Borgia (CZ), ITALY

Mitochondrial Mn-SOD is encoded by the nuclear genome as a precursor headed by a signal peptide spanning 24 amino acid res- idues; this leader peptide is then removed for the entry of the mature enzyme in mitochondria. The rat mitochondrial Mn-SOD (ratSOD2), sharing 93% amino acid identity with the human counterpart, is an appropriate model to study the molecular and functional properties of this key mitochondrial enzyme. This investigation regards the role of some crucial amino acid posi- tions of ratSOD2 regulating catalysis, reactivity and thermal resistance of the enzyme. In particular, the role of three amino acid residues, namely Q143, Y34 and S82, has been investigated. The study was carried out through the heterologous production of the mature form of ratSOD2 and its mutants obtained by site- directed mutagenesis on the corresponding gene. Six recombinant forms of the enzyme were produced, carrying the Q143 or H143 residue with or without the Y34F or S82A replacement. All pro- teins bound manganese and were organized as homotetramers. A six-fold reduction of the activity was observed in ratSOD2 forms containing the H143 variant; on the other hand, the Y34F and S82A substitutions only caused a modest reduction of enzymatic activity, compared to the Q143 form. Heat inactivation studies showed the high thermo-tolerance of ratSOD2 and allowed an evaluation of the related activation parameters of inactivation. Compared to the Q143 variant, the H143 counterpart was signifi- cantly less heat stable and displayed moderately lower enthalpic and entropic factors; the Y34F substitution caused a moderate reduction of heat stability, whereas the S82A replacement slightly improved the thermo-tolerance of the Q143 variant; both substi- tutions significantly increased the enthalpic and entropic factors of heat inactivation, the greatest effect being observed with the S82A substitution. All recombinant forms of ratSOD2 were glu- tathionylated in Escherichia coli, a feature pointing to the high reactivity of ratSOD2 towards glutathione. Moreover, the S82 position of the enzyme was found phosporylated in an in vitro system containing human mitochondrial protein extracts as source of protein kinases. These data highlight the role played by some critical residues of ratSOD2 and suggest fine levels of regu- lation occurring in vivo.

Manganese superoxide dismutase (Mn-SOD) is a mitochondrial enzyme that dismutates two superoxide radicals into hydrogen peroxide and molecular oxygen. This enzyme is crucial for the defence against cellular reactive oxygen species (ROS), function- ing as an essential anti-oxidant enzyme protecting critical targets from superoxide modification. Diclofenac is a non-steroidal anti- inflammatory drug (NSAID) frequently used as an analgesic and in the treatment of rheumatic diseases; more recently, a number of experimental and clinical studies suggested its possible usage as an anticancer agent. Many reports have shown that diclofenac, as well as other NSAIDs, induce apoptosis in a variety of cell lines such as hepatic, gastric and renal, thus influencing their cel- lular redox state. On the other hand, a few data are available regarding the effects of these drugs on neuronal cells.Here we investigate diclofenac-induced apoptosis in the neuroblastoma cell line SH-SY5Y and the possible involvement of Mn-SOD in this process. Flow cytometric analysis of SH-SY5Y cells treated with diclofenac revealed a time- and dose-dependent increase of apoptotic nuclei. Moreover, the treatment of SH-SY5Y with dic- lofenac induces an increase in cellular ROS levels, as measured by oxidation-sensitive fluorescence probes. To evaluate the involvement of Mn-SOD in the cytotoxic effect induced by dic- lofenac, both protein level and enzyme activity have been evalu- ated in protein extracts obtained from SH-SY5Y cells grown in the absence or in the presence of diclofenac. Western blotting analysis showed that diclofenac decreases the levels of Mn-SOD; concomitantly, its enzymatic activity is reduced, as measured by a colorimetric assay on non-denaturing polyacrylamide gels. However, diclofenac does not affect the mRNA levels of Mn- SOD, as determined by RT-PCR experiments. When SH-SY5Y cells were cultured in the presence of a recombinant thioredoxin from the hyperthermophilic archaeon Sulfolobus solfataricus, a marked attenuation of the diclofenac-induced apoptosis was observed, together with an increase of the Mn-SOD levels. Fur- thermore, diclofenac induces a reduction of the mitochondrial membrane potential and a release of cytocrome c from mitochon- dria. These data suggest that mitochondria are involved in the diclofenac-induced apoptosis in SH-SY5Y neuroblastoma cell line and point to a possible role of Mn-SOD in this process.

P4–30 Decreased mitochondrial GSH level – an early indicator of manganese (II) induced apoptosis in HUH7 cell line A. Dinischiotu1, M. C. Munteanu1, A. Hermenean2 and M. Costache1 1Biochemistry and Molecular Biology, University of Bucharest, Bucharest, ROMANIA, 2Histology, Vasile Goldis Western University of Arad, Bucharest, ROMANIA

P4–29 Key amino acid positions involved in activity, heat stability and covalent modification of rat mitochondrial manganese superoxide dismutase E. De Vendittis1, I. Castellano1, A. De Vendittis1, F. Cecere1, R. Cotugno1, A. Chambery2, A. Di Maro2, M. Masullo3 and M. R. Ruocco1 1Dipartimento di Biochimica e Biotecnologie Mediche, Universita’ degli Studi di Napoli Federico II, Naples, ITALY, 2Dipartimento di Scienze della Vita, II Universita’ di Napoli, Caserta, ITALY, 3Dipartimento di Scienze Farmacobiologiche, Universita’ degli Studi Magna Graecia di Catanzaro, Roccelletta di Borgia/CZ), ITALY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Manganese (II) is a naturally occurring trace metal, both nutri- tionally essential and potentially toxic, commonly found in the environment. It can be concentrated in hepatocytes due to long parenteral nutrition, lack of iron or an excessive consumption of vitamins and mineral tablets. Manganese (II) can trigger mito- chondrial dysfunction. We have studied the effect of 5 mM man- ganese (II) on the apoptosis in the hepatocarcinoma cell line (HUH7). The total and mitochondrial GSH concentrations were measured by a spectrophotometric method. The cytochrome C release was evidenced by Western blot analysis. The expression of Bcl-2 and Bax mRNAs was analyzed through quantitative real time PCR (qRT-PCR). A decrease in the level of GSH by 25% and 70% in the mitochondrial fraction was noticed after 30 min- utes and 6 hours of exposure, respectively, compared to control,

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whereas the total GSH level increased.The cytochrome C release in the cytosol occurred simultaneously with the decrease of the mitochondrial GSH level in the manganese (II) treated HUH7 cells. The Bcl-2 mRNA expression decreased with 58% compared to Bax one after 30 minutes of exposure. Manganese (II) induced degradation of the nuclear DNA in an internucleosomal pattern. Our results suggested that manganese (II) cytotoxicity in HUH7 cells is based on the induction of mitochondrial apoptosis and the mitochondrial GSH decrease is an apoptosis promoting fac- tor. The mitochondrial GSH decrease had a key role in the ROS induced injury and acts as an early activator of apoptosis induced by manganese (II).

Bcl2/Bax gene expression were investigated in Hep G2 cell line. The concentration of superoxide, the specific activities of super- oxide dismutase and catalase were assayed after 24, 48 and 72 hours of treatment. The citocrom C presence in the citosol was evidenced by Western blot analysis. The gene expression of Bcl2 and Bax was analyzed by qRT-PCR after 6, 12 and 24 hours of exposure. The levels of caspase 3 and p53 were esti- mated by immunocitochemical methods. The quantity of super- oxide increased after 48 and 72 hours simultaneously with the specific activity of SOD, whereas CAT presented a diminution of activity. The citocrom C level was not enhanced in the first 8 hours of exposure butthe Bcl2/ Bax mRNA levels were suprau- nitary starting with 6 hours of treatment. The positive reaction for p53 appeared in the nuclei with normal and altered shapes after 48 and 72 hours and the active caspase 3 was present in nuclei after 72 hours. According to our results, OTA induced oxi- dative stress in HepG2 cells might represent an important factor in the chain of cellular events leading to apoptosis.

P4–31 Action of SiO2 nanoparticles on mRNAs of Hsp27, Hsp60, Hsp70 and Hsp 90 in MRC5 lung fibroblasts A. Dinischiotu1, M. C. Munteanu1, C. Sima2, O. Zarnescu3, M. Costache1 and C. Grigoriu2 1Biochemistry and Molecular Biology, University of Bucharest, Bucharest, ROMANIA, 2Laser, National Institute of Laser Plasma and Radiation Physics, Bucharest, ROMANIA, 3Animal Biology, University of Bucharest, Bucharest, ROMANIA

P4–33 Mitochondrial structure and function analysis in human hepatocarcinoma cell lines with different growth/differentiation characteristics R. Domenis, M. Comelli and I. Mavelli Dipartimento Scienze e Tecnologie Biomediche, Universita degli Studi di Udine, Udine, ITALY

Concern about the potential health effects of environmental nanoparticles has become apparent in recent years. SiO2 nano- particles are photosensitive and produce reactive oxygen species in the presence of light. In our study the primary amorphous nanoparticle size distribution was a lognormal function, in the range 3–14 nm, most of the nanoparticles being of 5–8 nm. We have investigated the expression of Hsp 27, 60, 70 and 90 kD in the MRC 5 cells exposed to nanoparticles at translational level by qRT-PCR. The MTT test was used to assess cell viability after MRC5 cell treatment with 6.3·106 SiO2 particles per indi- vidual cell for 24, 48 and 72 hours. The number of the MRC5 cells cultivated in the presence of SiO2 nanoparticles was lower compared to control at all three time intervals. The treated cells presented cytoplasmic vacuolization. The expression of Hsp27 at the transcriptional level was almost unchanged after 24 and 48 hours and significantly down regulated after 72 hours of treat- increase of Hsp60 mRNA expression ment. A significant occurred after 48 and 72 hours of exposure with 74% and 86.5%, respectively. The level of Hsp70 mRNA significantly decreased after 72 hours, whereas that of Hsp90 was not affected by the SiO2 nanoparticles. Our results suggest that these nano- particles specifically affected the protein folding in mitochondria and could activate the intrinsic pathway of apoptosis.

P4–32 Biochemical and molecular studies on ochratoxin A exposed HepG2 cells A. Dinischiotu, M. C. Munteanu, L. Postolache, D. Marinescu, O. Zarnescu and M. Costache Biochemistry and Molecular Biology, University of Bucharest, Bucharest, ROMANIA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

The renaissance of mitochondria structure and function studies in the tumor field is related to some main factors: (i) the inte- gration of mitochondrial bioenergetics with the execution of cell death, (ii) the finding that mitochondria from different cell types and tissues of mammals differ in their molecular composition, (iii) the recent genetic and proteomic data validating the changes in metabolic regulation occurring during tumorigenesis known as Warburg effect. This is the cell shift to a glycolytic phenotype with impaired mitochondrial OXPHOS and is consid- ered as the metabolic hallmark of cancer. Nevertheless, there are still numerous questions that merit investigation, including the extension of the phenomenon since not all tumor cell types depend exclusively on glycolysis for ATP supply, and the defini- if any, between the altered metabolic phenotype tion of link, and the peculiar characteristics of tumor cells, such as their rate of growth. We investigated mitochondrial structure and function in two human hepatocellular carcinoma (HCC) cell lines, HepG2 and JHH-6 that were assigned to a high and low hepat- ocytic differentiation grade (albumin synthesis). JHH-6 showed undifferentiation cell morphology with a low doubling time (16 versus 31 hours of HepG2). Mitochondria were analysed both in situ and after isolation. Cells were examined by confocal microscopy using Mitotracker Red demonstrating that mito- chondrial mass of JHH-6 is about 50% of that of HepG2 (data confirmed by the yield of isolated mitochondrial fraction with respect to total cell homogenate). The expression levels of the OXPHOS complexes were evaluated by immunoblotting analysis on isolated mitochondria. Increased expression were observed for complex I, IV and V in HepG2 with respect to JHH6, while complex III was equal. The expression of F1F0 ATP synthase inhibitor protein IF1 was also evaluated and (complex V) related to the ATPase activity of the enzyme. The results showed that IF1 expression was lower in mitochondria from highly differentiated HepG2 with respect to JHH-6 in accor- dance with a higher ATPase activity. These data suggest a less diminished mitochondrial metabolic capacity in the more differ- entiated human HCC cell line, in which mitochondrial biogene- sis and expression of most OXPHOS complexes are less Ochratoxin A (OTA) is a mycotoxin contaminating feed and food, known for its carcinogenicity, nephro-,geno- and immuno- toxicity, which interferes with cell oxidative status as well as other processes. In this study, induction of oxidative stress medi- ated by 5 lM OTA, citocrom C presence in the citosol, modula- tion of p53 and caspase 3 protein expression and modification of

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diminished, while down-regulation of F1F0 ATPase activity by IF1 may be of minor extent. Acknowledgement: Supported by Italian MIUR grant (Italian Human ProteomeNet Project 2007).

observed that at acidic pH AnxA6 exhibits the highest affinity to lipid monolayers containing the highest amount of cholesterol. Such interactions might be involved in the organization of choles- terol-enriched lipid rafts. Experiments with cholesteryl acetate provided evidence that the hydroxyl group of cholesterol plays a role in AnxA6-lipid interactions.

P4–34 Biochemical characterization of the hydrolytic exoribonucleases from the human pathogens Salmonella typhimurium and Streptococcus pneumoniae S. Domingues, R. Goncalves Matos, F. Pereira Reis, A. Mendes Fialho, A. Barbas and C. M. Arraiano ITQB, Controlo Expressao Ge´nica, Lisboa, PORTUGAL

P4–36 Metformin and adiponectin inhibit protein synthesis and cancer cell proliferation R. Dowling1, M. Zakikhani2, M. Pollak2 and N. Sonenberg1 1Biochemistry, Rosalind and Morris Goodman Cancer Centre McGill University, Montre´al. Que´bec, CANADA, 2Oncology, Can- cer Prevention Centre Jewish General Hospital McGill University, Montre´al. Que´bec, CANADA

Maturation, turnover and quality control of RNA is performed by many different classes of ribonucleases. E. coli RNase II, together with RNase R, are the prototypes of the RNase II fam- ily of ribonucleases, a ubiquitous family of enzymes that are cru- cial in RNA metabolism. The ability of RNase R to act on structured RNAs suggests a specific role for this enzyme in RNA metabolism. The fact that RNase R is involved in virulence and is modulated in response to stress made this protein the focus of this work. RNase II/RNase R from different pathogenic organ- ismswere characterized. Salmonella typhimuriumand Streptococcus pneumoniae were chosen as examples of Gram-negative and Gram-positive pathogens. In S. pneumoniae only one member of the RNase II family was identified by sequence analysis, while both RNase II and RNase Rwere found in S. typhimurium. These hydrolytic enzymes were cloned, expressed and purified, and their biochemical and structural characteristics studied. Predicted S. typhimurium RNase II and RNase R behaved essentially as their respective E. coli counterparts. Our results further show that the only hydrolytic RNase found in Streptococcus pneumoniae is an RNase R-like enzyme. Interestingly, different activities observed between the enzymes belonging to virulent and non-virulent strains may account for their role in virulence and stress response.

AMPK is an enzyme involved in regulating energy metabolism and is activated by decreases in cellular ATP levels. Upon activa- tion, AMPK phosphorylates a number of effectors leading to the activation of ATP generating pathways, such as glycolysis, and the inhibition of ATP consuming pathways, such as protein syn- thesis. The anti-diabetic drug metformin has been implicated in the suppression of tumorigenesis, with some of the beneficial effects of metformin being linked to the activation of AMPK. Adiponectin, another AMPK activator, is produced by adipo- cytes and has insulin-sensitizing and anti-cancer effects. To evalu- ate the anti-cancer properties of metformin and adiponectin, we investigated the effects of these agents on cancer cell prolifera- tion, AMPK activation, mTOR signalling and mRNA transla- tion. Metformin and adiponectin activated AMPK and inhibited the proliferation of a variety of cancer cells including those of the breast, prostate, and colon. In breast cancer cells, metformin treatment led to a 30% reduction in global protein synthesis, and more specifically, a dose-dependent decrease in cap-dependent translation. This was associated with a suppression of mTOR sig- nalling and a decrease in the phosphorylation of its substrates: 4E-BP1 and S6K1. Adiponectin exhibited similar effects in pros- tate and colon cancer cells, reducing protein synthesis by 20% and 10% respectively. Adiponectin also reduced mTOR signal- ling and caused the dephosphorylation of S6K1. These results demonstrate that metformin and adiponectin inhibit prolifera- tion, mTOR signalling, and protein synthesis in a variety of can- cer cells, identifying metformin as a potential candidate for anti- cancer therapy and highlighting the importance of adiponectin in neoplasia.

P4–35 Analysis of the pH-dependent interactions between annexin A6 and interfacial lipid monolayers M. Domon1, G. Matar2, F. Besson2 and S. Pikula1 1Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, POLAND, 2Universite Lyon 1, Institut de Chi- mie et Biochimie Mole´culaires et Supramole´culaires, Villeurbanne, FRANCE

binding cholesterol

P4–37 Contribution to metabolic studies of cis-zeatin – cytokinin with so far unknown function S. Gajdosova, M. Kaminek, K. Hoyerova, P. I. Dobrev, A. Gaudinova, P. Klima, E. Zizkova and V. Motyka Laboratory of Hormonal Regulations in Plants, Institute of Experimental Botany, Prague 6, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Cytokinins (CKs) are plant hormones involved in control of numerous physiological processes throughout plant lifetime such as cell division, senescence, nutrient signaling, plant-host interac- tions and many others. The simplest representatives of CKs are N6 isopentenylated derivatives of adenine and their hydroxylated trans- and cis-zeatin. Whereas trans-zeatin (tZ) was analogs, repeatedly reported to be highly active in classical cytokinin bio- assays, cis-zeatin (cZ), although rather abundant in plant king- dom, exhibited less convincing activity. We proved the ability of Annexin A6 (AnxA6) is the largest member of the annexin family of membrane-binding proteins playing an important role in cal- cium homeostasis, trafficking between the endocytic compartment and lysosomes and membrane organization of late endosomes. Previous works suggest that AnxA6 binds to anionic phospholip- ids in a calcium-dependent manner and displays calcium-indepen- dent properties. Thus, AnxA6 may participate in the formation and stabilization of the plasma mem- brane lipid rafts which are enriched in cholesterol and specific proteins. In the present study, we addressed the following ques- tions: does AnxA6 bind preferentially to cholesterol-containing biomimetic membranes? If so, what is the molecular mechanism of the binding? To answer these questions, human recombinant AnxA6 was prepared and used with Langmuir monolayers con- taining various lipids (phosphatidylcholine, cholesterol and/or cholesteryl acetate). The interactions between AnxA6 and the lipid monolayers were examined by kinetic measurements of the interfacial adsorption and Brewster angle microscopy. We

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P4–39 Neurotrophin 3 – Adaptation way to brain hypoxia induced by chronic obstructive bronchopneumopathy M. Gheorghiu, D. Pasarica, B. Mahler, T. Trandafir and C. Bara Pathophysiology and Immunology, Carol Davila University of Medicine and Pharmacy, Bucharest, ROMANIA

cZ and especially its riboside to successfully induce and maintain cell division and to delay dark-induced leaf senescence in plants thus challenging contemporary notion of cZ isomers as unimpor- tant adjuncts. Metabolism of [3H] labeled cZ and tZ in tobacco cells and oat leaves revealed distinct metabolism of both isomers within each material as well as between the two species. In oat leaves, cZ was rapidly degraded to adenine (Ade), subsequently converted to adenosine (Ado) and efficaciously O-glucosylated. On the contrary, tZ was degraded to Ado and Ade and small amount of substrate was N-glucosylated in N7 and N9 positions. In tobacco BY-2 cells, cZ was not metabolized as effectively as in oat leaf segments. It was predominantly ribophosphorylated although less efficiently when comparing to tZ and degraded into Ade at slightly higher rate than trans-isomer. No [3H] cZ M tZ interconversion was observed during feeding experiments in either oat or tobacco. The presented data imply relevance of cZ-type CKs in plant metabolism and their potential role in control of plant development. Acknowledgement: This work was supported by GA AS CR (IAA600380701).

Background: Chronic obstructive bronchopneumopathy is a major cause of hypoxia. In the brain, secondary ischemia devel- ops a complex signaling cascade, leading to fast necrotic cell death or apoptosis. While brain cells are challenged by this dele- terious mechanisms, they activate innate protective programs, including synthesis of inflammatory cytokines and neuronal growth factors, members of neurotrophins family. Neurotrophins are peptides that act as growth or survival factors for specific neuronal populations. Experimental animal models showed that neurotrophin-3 (NT-3) was produced by glial cells as an adapta- tion response to hypoxia. The aim of this work was to begin the NT-3 study in patients with COBP. Patients and Methods: Forty patients with confirmed COBP, treated in ‘Marius Nasta’ Hospital of Pulmonary Diseases Bucharest, were investigated using blood samples. NT-3 was measured using ELISA kits. Results: Significant increase of NT-3 serum levels was obtained. Conclusion: NT-3 up-regulation might represent an activating mechanism for neuronal survival in the injuried brain.

P4–38 Induction of mitochondrial permeability transition (MPT) by iron in isolated rat liver mitochondria requires both NAD(P)H oxidation and iron import J. Gall, J. Skrha Jr, R. Buchal, E. Sedlackova, K. Verebova and J. Platenik Institute of Medical Biochemistry, First Faculty of Medicine Charles University in Prague, Prague, CZECH REPUBLIC

P4–40 Morphogenetic cell migration in 3D collagen matrices varies with growth factor signaling and matrix density S. Rhee1, C. Ho2 and F. Grinnell2 1Department of Life Science, Chung-Ang University, Seoul, SOUTH KOREA, 2Cell Biology, UT Southwestern Medical Center, Dallas, USA

Mitochondrial permeability transition (MPT) plays an important role in necrotic and apoptotic cell death. MPT is induced by cal- cium and promoted by oxidative stress. In vivo the oxidative stress is often catalyzed by iron. In this study we investigated ability of micromolar iron to induce MPT in isolated mitochon- dria. According to literary data, Fe(II) over-loads antioxidant defence that shifts NAD(P)H/NAD(P) to oxidation, and the loss of reduced NAD(P)H promotes MPT opening. Iron can also be imported to mitochondrial matrix by calcium uniporter. Our iso- lated rat liver mitochondria were initially stabilized with EDTA and bovine serum albumine. For MPT induction they were ener- gized by succinate or malate/pyruvate, and stimulated by addi- tion of Ca or Fe(II). We measured mitochondrial swelling (light scatter), the inner membrane potential (fluorescent probe JC-1) and NAD(P)H oxidation (autofluorescence 340/465 nm). Both Ca and Fe(II) could induce cyclosporin A-inhibitable depolariza- tion and swelling (MPT). Fe(II) induced MPT only in the pres- ence of some residual EDTA which formed complex with iron catalyzing rapid oxidation of NAD(P)H. Effect of iron also required membrane potential and could be prevented by post- addition of membrane permeant, but not impermeant iron chela- tors. Iron was apparently needed only for induction of MPT, while its propagation continued through calcium cycling. We conclude that both iron import and NAD(P)H oxidation must occur simultaneously for MPT to occur. This observation can help to elucidate mechanism of iron toxicity, which may be involved in pathogenesis of several liver diseases where cytosolic iron sequestration fails, e.g. hemochromatosis and alcoholic liver disease.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Most research on cell migration has been carried out in serum- containing medium using rigid surfaces. Under these conditions, cells migrate; the surface remains stationary. When fibroblasts interact with collagen matrices, cell tractional force can couple either to matrix translocation or to cell migration, and the bal- ance determines if the matrix moves or the cells move. Platelet- (PDGF) and fetal-bovine-serum (FBS) derived-growth-factor stimulate movement of human fibroblasts interacting with 1.5 mg/ml collagen matrices but by different mechanisms. With PDGF, cells move through the matrix. With FBS, cells contract the matrix and pull themselves closer together. As a consequence, fibroblasts remain individual in PDGF-containing medium but tend to form clusters in the presence of FBS. Clustering is fully reversible. Switching from FBS to PDGF results in cell migration and rapid cluster dispersion. Consistent with the importance of matrix contraction in clustering, blocking myosin-II inhibits FBS – dependent clustering but not PDGF-stimulated migration. On 1.5 mg/ml collagen matrices (stiffness 39 pascals), initial spread- ing of fibroblasts takes place without formation of focal adhe- sions. If collagen matrix density is increased to 4 mg/ml (stiffness 362 pascals), then focal adhesions form between fibroblasts and the matrix during cell spreading. With 4 mg/ml collagen matrices, in the presence of PDGF or FBS, cell tractional force couples to cell migration rather than matrix translocation. Consequently, cell clustering does not occur. These findings demonstrate differ- ential morphogenetic cell migration controlled by the combina- tion of growth factor stimulation and cell matrix density.

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P4–41 Context-dependent enzymatic and nonenzymatic deubiquitination of the ubiquitin-Pex5p thiolester conjugate C. Grou1, A. Carvalho1, M. Pinto1, S. Huybrechts2, C. Sa-Miranda3, M. Fransen2 and J. Azevedo1 1IBMC, OBF, Porto, PORTUGAL, 2Departement Moleculaire Celbiologie, Faculteit Geneeskunde, Leuven, BELGIUM, 3IBMC, Unilipe, Porto, PORTUGAL

cells were first preincubated with resveratrol (10, 50 and 100 mM for 1, 12 and 24 h), followed by incubation with 750 mM H2O2 for 1 h. At the end of the incubation period, supernatants were removed and the cell cytotoxicity was evaluated by lactate dehy- drogenase enzyme assay. Results: In the experiments performed; incubation with 750 mM H2O2 for 1 h resulted in 30–40% cytotoxicity in HCAE cells. When the cells were preincubated with 10 and 50 mM resveratrol for 1, 12 and 24 h, hydrogen peroxide-induced cytotoxicity was significantly decreased. Conclusions: These results display the protective effect of resve- ratrol under the above-mentioned conditions against oxidative stress- induced cell death in in vitro HCAE cells.

P4–43 Resveratrol decreases hypoxia/reoxygenation induced MMP-2 secretion in human brain microvascular endothelial cells Z. Cavdar1, O. Sayin2, N. Arslan3, M. Yuksel-Egrilmez1, Z. Altun4, N. Yener5, S. Genc6, K. Genc7, H. Islekel5, G. Oktay5 and G. Guner8 1Research Laboratory-Health Sciences Institute, Dokuz Eylul Uni- versity School of Medicine, Izmir, TURKEY, 2Research Labora- tory-Health Sciences Institute, Dokuz Eylul University-School of Medicine, Izmir, TURKEY, 3Health Sciences Institute, Dokuz Ey- lul University-School of Medicine, Izmir, TURKEY, 4Department of Oncology, Dokuz Eylul University School of Medicine, Izmir, TURKEY, 5Department of Biochemistry, Izmir, Dokuz Eylul Uni- versity School of Medicine, TURKEY, 6Dokuz Eylul University School of Medicine, Research Laboratory, Izmir, TURKEY, 7Health Sciences Institute, Dokuz Eylul University School of Medi- cine, Izmir, TURKEY, 8Research Laboratory-Health Sciences- Department of Biochemistry, Dokuz Eylul University School of Medicine, Izmir, TURKEY

the Flemish Government

Pex5p is one of the so-called peroxisomal cycling shuttle recep- tors, playing a key role in the import of matrix proteins. Newly synthesized proteins are recognized by Pex5p in the cytosol, and are then targeted to the organelle membrane. There, strong pro- tein-protein interactions between Pex5p and components of the docking/translocation machinery are established resulting in the insertion of Pex5p into the docking/translocation machinery with the concomitant translocation of cargo proteins across the orga- nelle membrane. Next, Pex5p is ubiquitinated at a conserved cys- teine residue, a reaction involving UbcH5 family members and absolutely required for Pex5p dislocation back into the cytosol. The data presented here focus on the last step of the Pex5p-medi- ated import pathway: its deubiquitination. Our results indicate that two mechanisms act on the removal of thiolester-attached ubiquitin: one mediated by deubiquitinating enzymes and the other by nucleophilic compounds, such as GSH. It was previ- ously shown that two populations of Ub-Pex5p can be detected: a membrane-associated pool and a soluble one. We demonstrate that the action of deubiquitinating enzymes and GSH is context- dependent, as soluble Ub-Pex5p is more susceptible to deubiquiti- nation than the membrane-associated one. Acknowledgements: Supported by FCT (PTDC program) and FEDER funds, Portugal, European Union VI Framework program (Grant LSHG-CT-2004-512018), Peroxisomes in Health and Disease, (Geconcerteerde Ond- erzoeksacties GOA2004/08) and the Fonds voor Wetenschappelijk Onderzoek – Vlaanderen (Onderzoeksproject G.0237.04).

P4–42 Protective effect of resveratrol on in vitro human coronary artery endothelial cell injury O. Sayin1, N. Arslan2 and G. Guner3 1Dokuz Eylul University School of Medicine Research Laboratory, Research Laboratory-Health Sciences Institute, Izmir, TURKEY, 2Dokuz Eylul University School of Medicine Research Laboratory, Health Sciences Institute, Izmir, TURKEY, 3Deparment of Bio- chemistry-Research Laboratory-Health Sciences Institute, Dokuz Eylul University School of Medicine Research Laboratory, Izmir, TURKEY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Introduction: There is a balance between formation and elimi- nation of reactive oxygen species which has harmful effects on cell molecules such as lipids, proteins, and DNA; as well as on the physiological functions in the cell. Induced activity of oxida- tive enzymes or reduced activity of antioxidative enzymes disturb this balance and cause oxidative stress. Resveratrol is a polyphe- nolic compound belonging to the stilbene family, found in grapes and red wine. Resveratrol has anti-inflammatory, anti-oxidative, anticarcinogenic, cytoprotective and cardioprotective effects. In this study; the protective effects of resveratrol against hydrogen peroxide-induced oxidative injury in human coronary artery endothelial cells (HCAEC) were investigated. Materials and Methods: Oxidative damage was produced by 100–1000 mM hydrogen peroxide (H2O2) in HCAE cells. The Introduction: Inflammation, oxidative stress and apoptotic or necrotic cell death participate in the pathogenesis of ischemia/ reperfusion-induced injury developed in acute ischemic events. Matrix metalloproteinases (MMPs) play major roles in remodel- ing of extracellular matrix in many pathological conditions including ischemia-reperfusion (I/R) injury. Resveratrol (RSV)a polyphenolic phytoalexin, has been reported to possess anti- inflammatory, anticarcinogenic and antioxidant activities. In this study, we investigated the effect of hypoxia/reoxygenation (H/R), in vitro model of I/R, on MMP-2 and MMP-9 in human cerebral microvascular endothelial (HCMVE) cells and then whether RSV reduce H/R induced MMP-2 and MMP-9 secretion in this model. Method: HCMVE cells were pretreated with RSV for one hour and the cells were exposed to oxygen glucose deprivation (OGD) to mimic H/R (6 h OGD/24 h reoxygenation). Intracellular ROS production was measured fluorometrically by using hydroxyphe- nyl fluorescein probe. MMP-2 and MMP-9 protein activity levels were studied by gelatin zymography. Results: The addition of RSV to the OGD group significantly decreased the ROS level. ProMMP-2 activity was significantly higher in OGD group than normoxia group, and this increased proMMP-2 activity was significantly decreased with RSV in OGD group. MMP-9 was not detected in any group. Conclusion: Our results show the regulation of endothelial MMP-2 secretion after exposure to OGD and suggest, for the first time, that RSV modulates MMP-2 activity in HCMVE cells. Further studies are needed to explore the mechanism of RSV on the H/R induced MMP-2 secretion by HCMVE cells. Acknowledgement: This work was supported by TUBITAK- Health Research Fund (Project No: SBAG 104S513).

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P4–44 Peroxisome proliferation: is the function of Pex11 proteins conserved throughout evolution? J. Koch, A. Huber, A. Hartig, K. Pranjic, F. Kragler and C. Brocard Department of Biochemistry and Cell Biology, Center of Molecular Biology University of Vienna, Vienna, AUSTRIA

monella typhimurium, which lead to increase of the ADA2 expression. After washing the Salmonella typhimurium away, cells were incubated at 37(cid:2)C. The time-dependences of ADA2 activity of human blood cells, incubated separately and mixed, were analyzed. Only the monocyte suspension had shown signifi- cant increase of ADA2 activity. However, more pronounced (2–3 times) effect for monocytes was observed in mixed suspension after addition of lymphocytes or neutrophils to monocytes. After 3 h of incubation the mixed (monocytes and lymphocytes) cell suspension demonstrated drastic increase of ADA2 activity, sup- posedly ‘switch’ effect. These results demonstrate that ADA2 activity produced by monocytes required the interaction of cells with each other.

P4–46 Compensatory changes of respiratory chain complexes in isolated deficiency of ATP synthase V. Havlickova1, J. Paul1, A. Cizkova2, S. Kmoch2 and J. Houstek1 1Institute of Physiology AS CR v.v.i., Bioenergetics, Prague 4, CZECH REPUBLIC, 2Institute of Inherited Metabolic Disorders, Prague 2, CZECH REPUBLIC

Peroxisomes are essential single membrane-bound organelles present in all eukaryotic cells. Their function is mainly associated with lipid metabolism and they enclose hydrogen peroxide-gener- ating and degrading enzymes. These highly versatile organelles quickly adapt their shape, size, number and protein content to the cellular environment. In humans, defects in peroxisome pro- tein transport or biogenesis lead to devastating diseases such as Zellweger syndrome. Over 30 proteins, the peroxins, participate in the formation of mature peroxisomes. In the yeast Saccharo- myces cerevisiae, peroxins of the PEX11-family, namely PEX11, PEX25 and PEX27, participate in peroxisome proliferation. Indeed, yeasts lacking PEX11 display fewer and larger peroxi- somes than wild type cells. Orthologues of the yeast PEX11-pro- teins, PEX11a, b, c and PEX11-1 to -5 have been identified in human and plants, respectively. We sought to analyse the regula- tion of peroxisome proliferation through the PEX11-protein fam- ily in yeast, plant, and human cells. Here we show co-expression studies in human and plant cells using mCherry extended by a peroxisomal targeting signal (mCherry-SKL) as peroxisomal pro- tein marker. We analysed the effect of ectopic heterologous expression of human, yeast and plant PEX11 proteins on peroxi- some size, number and shape. We also performed functional tests in yeast pex11D-mutants. Our analyses suggest that although the peroxisome proliferation mechanism is conserved through all kingdoms some species-specificity does exist regarding the func- tion of PEX11-proteins. To pinpoint the molecular differences between PEX11 orthologues we performed further studies in cell culture with PEX11-proteins harboring point mutations and using siRNA.

P4–45 Stimulation of adenosine deaminase 2 isoenzyme expression in human blood cells in vitro by Salmonella typhimurium H. Harutyunyan, Y. Sargisova, N. Andreasyan and H. Hayrapetyan H.Buniatian Inst. of Biochemistry, Laboratory of Adenylic Compounds Metabolism, Yerevan, ARMENIA

Mitochondrial encephalo-cardiomyophaty due to isolated defi- ciency of ATP synthase is frequently caused by mutations in TMEM70 gene encoding ancillary factor specific for higher eukaryotes (1). In patient cells the content of fully assembled ATP synthase and enzyme activity are reduced to <30% of con- trol. Dysfunction of ATP synthase leads to mitochondrial mem- brane hyperpolarization, lack of ATP and increased ROS production (2) which might affect nucleo-mitochondrial signaling and biogenesis of other complexes of mitochondrial respiratory chain (RC). To analyze possible compensatory changes in patient mitochondria we determined the protein content of the RC com- plexes in fibroblasts with TMEM70 mutation by SDS–PAGE and Western blotting using monoclonal antibodies against subun- its of RC complexes and quantitative detection by infrared imag- ing (Odyssey). Analysis of whole cell lysates of 10 fibroblast cell lines from patient with TMEM70 gene 317-2A>G mutation showed pronounced increase in respiratory chain complex IV and frequent, although variable increase in complex II. Correlation with whole genome expression profiling determined in investi- gated fibroblasts did not show parallel consistent changes in OX- PHOS mRNA levels of subunits or specific assembly factors of RC complexes (3). The results indicate that ATP synthase defi- ciency leads to compensatory changes that are mainly due to posttranscriptional regulation of biogenesis of mitochondrial RC complexes. References: 1. Cizkova A. et al. Nature Genetics 2008; 40: 1288–1290. 2. Houstek J. et al. Biochim. Biophys. Acta. 2006; 1757: 1400– 1405. 3. Cizkova A. et al. BMC Genomics 2008; 9: 38.

P4–47 The role of neuroglobin in the prevention of cell death K. Henty, Y. Yosaatmadja, S. Bonding, J. Skommer, N. Birch and T. Brittain School of Biological Sciences, The University of Auckland, Auckland, NEW ZEALAND

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

ADA2 isoform of Adenosine deaminase (ADA, EC 3.5.4.4) is mainly present in the serum of normal subjects where it is found in negligible quantities. In contrast to ADA1, there is limited information about ADA2, mainly, due to difficulties of purifying this enzyme. ADA2 activity is significantly increased in serum at various diseases, in which monocyte/macrophage cells, as well as strong infections, AIDS and others are activated, indicating that enzyme may be involved in immune system responses to different pathogens. One of the actual questions is the cellular source of ADA2 isoenzyme. We investigated the ADA2 isoenzymes pro- duced by human peripheral blood cells in vitro. Cells were iso- lated according to Boyum and suspensed in Hanks balanced saline solution, HBSS. The ADA isoenzymes activity in isolated cells had shown low ADA1 activity and ADA2 activity reaching zero. Cells then were treated with strong pathogen such as Sal- The mitochondrial pathway of apoptosis is a major mediator of cell death. A key event in this pathway is permeabilization of the

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cells, its inhibition results in an increment of CNT2 activity, spe- cially at low glucose concentration. Moreover, there is an inverse that correlation between CNT2 and GRP58 protein levels depends on glucose availability. Up to date, both aldolase B and GRP58 are the first interacting partners identified for CNT2 and both might link energy metabolism to adenosine transport.

P4–50 Transcriptional regulation of the preimplantation embryo- and testis-specific Rnf33 gene by NFkB subunits p65 and p50 M. C. Hsu1 and C. J. Huang2 1Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, TAIWAN, 2Department of Animal Science, Chinese Culture University, Taipei, TAIWAN

outer mitochondrial membrane and subsequent release of cyto- chrome c from mitochondria into the cytosol. Thereafter, cyto- solic cytochrome c is recruited to the apoptosome complex, which activates procaspase nine and hence the caspase signaling cascade. It has been shown that apoptosis is induced by oxidized but not reduced cytochrome c. Neuroglobin, a recently discov- ered globin, protects various cell lines and tissues from apoptosis. We have shown that neuroglobin efficiently reduces cytochrome c in vitro. We hypothesise that neuroglobin prevents apoptosis by reducing cytochrome c. Our research aims to investigate the properties of the neuroglobin-cytochrome c complex and identify its role in regulation of the mitochondrial pathway of apoptosis in cell-based systems. Surface Plasmon Resonance experiments have been used to explore the nature of the complex and have revealed that the two proteins bind with moderate affinity (Kd 45 lM), forming a transient protein complex that is mediated lar- gely by electrostatic interactions. The likely structure of the com- plex has been determined by computer modeling. We have recently been successful in producing crystals of the complex and X-ray diffraction will be used to determine the detailed structure of the complex. Cell-free lystates and permeabilized cells have been successfully utilized to measure apoptosis activation by exogenous oxidized cytochrome c. We are currently studying the effect of reduced neuroglobin on this system.

P4–48 Abstract withdrawn

P4–49 GRP58 and aldolase B modulate CNT2 activity and link adenosine metabolism to cell energy status I. Huber-Ruano, C. Javier F and M. Pastor-Anglada University of Barcelona-CIBERehd-IBUB, Biochemistry and Molecular Biology, Barcelona, SPAIN

Rnf33 is expressed specifically in the pre-implantation embryo and the testis. RNF33 is a putative TRIM/RBCC protein that has been shown to associate with the cytoplasmic motor proteins, KIF3A and KIF3B, possibly contributing to the KIF3A-KIF3B- dependent cargo mobilization process along the microtubule. The Rnf33 retrogene is structurally composed of a noncoding exon 1, a solitary intron and an undisrupted coding exon. Luciferase assays were used to dissect cis-acting transcriptional elements of the gene. The core promoter elements of Rnf33 are shown to include an atypical TATA box and a transcription initiator. Fur- thermore, removal of a 305-bp sequence, designated as R4, located in the solitary intron resulted in 80% loss of transcription activity indicating the presence of a crucial transcriptional cis ele- ment. Further deletion and mutagenesis experiments indicated that a putative NFkB binding site is essential for Rnf33 tran- scription. EST-based analysis showed that expression of NFkB and Rnf33 is coincidental. The NFkB binding site was demon- strated to be targeted by the NFkB subunits p65 and p50 in EMSA and supershift assays; in vivo binding was demonstrated in chromatin immunoprecipitation experiments in the testis and the TM3 and TM4 testicular cell lines. Our results show that NFjB regulates Rnf33 expression in the preimplantation embryo and the testis.

P4–51 DNA repair and ubiquitination: role of DNA- damage-binding protein-1 (DDB1) B. Iovine, M. L. Iannella and M. A. Bevilacqua Department of Biochemistry and Medical Biotechnology, Universita’ degli Studi di Napoli Federico II, Naples, ITALY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Adenosine is a purinergic agonist implicated in various aspects of cell physiology. It is transported into the cell by CNT2, a high- affinity Na+ dependent purine-preferring nucleoside transporter protein. Moreover, CNT2 itself is known to be up-regulated by A1 receptors by a mechanism involving Katp channels (Duflot et al., Mol. Cell. Biol. 2004) and appears to mediate the adenosine- dependent activation of AMPK (Aymerich et al., J. Cell. Sci. 2006). To better understand how CNT2 function can link to energy status, we aimed at identifying putative CNT2 interacting proteins. To this end, we have realized a two-hybrid screening and GST-pull down assays followed by 2D electrophoresis against the cytosolic N-terminus of the transporter, identifying aldolase B and grp58 as new interacting proteins for CNT2 that colocalize in the plasma membrane and modulate its activity depending on the energy status of the cell. For the former, whose deficiency causes the hereditary fructose intolerance syndrome, we characterized the ability of certain glycolytic precursors, and especially fructose, to transiently up-regulate CNT2 activity in hepatoma Fao cells but without detecting any effect in non-glu- coneogenic cells. The effect on CNT2 activity seems to be the result of an increase in transporter affinity rather than capacity. For GRP58, it is shown that it specifically down-regulates CNT2 activity in co-transfected Hela cells without affecting other nucle- oside transporters of the SLC28 family, while in IEC6 intestinal DNA damage-binding protein (DDB) is a heterodimer made up of the DDB1 and DDB2 subunits. The DDB complex recognizes and binds some UV-damaged DNA lesions and is implicated in nucleotide excision repair (NER) (Wittschieben BO, Wood RD (2003). DNA Repair. 2, 1065–1069). Our results, moreover, show that DDB1 coordinately contributes to the assembly of DNA repair mechanisms and, at the same time, to the regulation of the transcription of UV-induced genes (Bevilacqua MA, Iovine B, Zambrano N, D’Ambrosio C, Scaloni A, Russo T, Cimino F (2005). J. Biol. Chem. 9, 31809–17; Iovine B, Nino M, Irace C, Bevilacqua MA, Monfrecola G (2009). Biochimie. 91,364–72). Recent studies indicate that this protein associated with Skp2

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P4–53 Impairment of adult brain neurogenesis does not promote depressive-like behavior in mice P. Jedynak1, T. Kos2, P. Jaholkowski1, A. Kiryk1, N. B. Abdallah3, H. P. Lipp3, P. Popik2, R. Filipkowski1 and L. Kaczmarek1 1Nencki Institute of Experimental Biology Polish Academy of Sci- ences, Molecular and Cellular Neurobiology, Warsaw, POLAND, 2Institute of Pharmacology Polish Academy of Sciences, Behavioral Neuroscience, Krakow, POLAND, 3Institute of Anatomy University of Zurich, Neuroanatomy and Behavior, Zurich, SWITZERLAND

(i)

and Cul4A is part of the E3 ubiquitin ligase complex and induces proteolysis of the cyclin-dependent kinase inhibitory protein p27Kip1. We investigated the relationship between p27Kip1 turn- over and DDB1 expression following UV irradiation and DNA- damage. We found that low, but not high doses of UVC irradia- tion lead to reduction of p27Kip1. In fact, at 6 h after irradiation we observed the decrease in p27Kip1 nuclear protein and the DDB1 translocation into the nucleus; conversely, high doses of UV induced p27Kip1 accumulation and unchanged level of DDB1. We carried out similar experiments in synchronized BJ5ta cells and in HeLa cells and we showed that this p27Kip1 reduc- tion is a mechanism cell cycle and cell type-indipendent. More- incubated with the proteasoma inhibitor MG132 over, BJ5ta, and exposed to UVC showed the increase of p27Kip1, suggesting that the p27Kip1 degradation may be proteasoma-dependent. Immunoprecipitation and western blot analysis using specific antibodies showed that DDB1 could mediate p27Kip1 degrada- tion following UV-induced DNA damage. These results demon- strated that: low, but not high doses of UV irradiation induced p27Kip1 degradation (ii) decrease in nuclear level of p27Kip1 are related with the DDB1 translocation into nucleus (iii) p27Kip1 degradation is proteasoma-mediated and (iv) p27Kip1 degradation is correlated to a mild DNA damage. The role of DDB1 in ubiquitination and proteasomal degradation of p27Kip1 remains to be investigated, even if, our results provide a significant contribution to address this point.

P4–52 Regulation of organic cation/carnitine transporters in astrocytes by peroxisome proliferator activator receptor agonist E. Januszewicz and K. Nalecz Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology, Warsaw, POLAND

(4-trimethylammonio-3-hydroxybutyrate)

Adult neurogenesis (ANGE) is the process of generation of new neurons in the brain of adult animals. Neuronal stem/precursor cells located in the subgranular zone of dentate gyrus (DG) and subventricular zone of the lateral ventricles proliferate and differ- entiate into mature neurons of the DG and olfactory bulb, respectively. Functional role of ANGE remains elusive, although it has been suggested to play an important role in the etiology of depression and its treatment with antidepressants. In our study we employed cyclin D2 gene knock-out (KO) mice showing approximately 10 times lower level of adult neurogenesis when compared to WT animals (Kowalczyk et al., J. Cell Biol., 2004). We have further characterized this deficit by demonstrating that KO mice show twice less astrocytes (GFAP-positive) when com- pared to WTs in the area of granular cell layer and hilus of the DG in the hippocampus formation. Furthermore, KO mice dis- played a reduction of the apoptotic cells number (expressing active caspase-3) in analogous brain regions. Next, we have investigated whether the suppression of ANGE promotes the depressive-like behavior in KO mice when compared to WTs. We examined the initial depression level using a set of behavioral tests: tail suspension (TST), forced swimming (FST), nest build- ing, novelty suppressed feeding, and anhedonic behavior test (using IntelliCage automated system). There was no significant effect of ANGE repression on KO mice behavior. We have also investigated whether the impairment of the adult brain neurogen- esis alters the behavioral response of KO mice to antidepressants. Single injection of fluoxetine or imipramine (15 mg/kg) produced no difference in immobility between KO and WT animals in TST and FST. These result may suggest that ANGE suppression is rather a consequence than a cause of depressive behavior previ- ously showed in animal models and human.

P4–54 Structural and functional characterization of MGRN1 isoforms expressed in human melanoma cells C. Jimenez-Cervantes, A. B. Pe´ rez-Oliva, M. Abrisqueta, C. Herraiz, C. Olivares and J. C. Garcı´ a-Borro´ n Biochemistry and Molecular Biology, Universidad de Murcia, Murcia, SPAIN

L-Carnitine controls level of free CoASH in the cell by formation of acyl derivatives, a process important in catabolism of fatty acids. Although in the brain ß-oxidation is not a major process in energy delivery, it can account for 20% of oxidative energy and takes place in astro- cytes. Shortening of very long-chain fatty acids and synthesis of docosahexaenoic acid is known to take place in peroxisomes of these cells. The present study was focused on a possible influence of peroxisome proliferators receptor a (PPARa) on expression, presence and localization of two high affinity carnitine transport- ers OCTN2 and OCTN3 belonging to the family of organic cat- ion transporters. Experiments with real time-PCR, Western blot and immunocytochemistry analysis demonstrated expression of both genes in rat astrocytes and out of two rat sequences ascribed as similar to mouse OCTN3, XM_001073573 was found. On the contrary to observations in peripheral tissues PPARa activator (WY-14,643) did not change expression of OCTN2 whilst stimulated by 50% that of OCTN3, an observation corre- lated with an increased Na-independent activity of carnitine transport. An augmented intracellular localization of OCTN3 upon PPARa stimulation confirms its peroxisomal localization, indicating an important physiological role of OCTN3 in glial cells and different from peripheral tissues regulation of carnitine transporters.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

MGRN1 is a RING finger domain protein with hallmark charac- teristics of ubiquitin ligases that is mutated in mahoganoid, a mouse mutation causing coat colour darkening, congenital heart defects and spongiform neurodegeneration. Mammalian pigmenta- tion and body weight are regulated by signalling from MC1R and MC4R, two highly similar G protein-coupled receptors activating the cAMP pathway. Genetic analysis suggested that MGRN1 may act to downregulate MC1R and MC4R. We show that human mel-

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P4–56 Interactions of acyclic nucleoside phosphonates with granzyme A and caspases: implications for apoptotic cell death H. Kaiserova, M. Matousova, M. Hajek, J. Gunterova, A. Holy and I. Votruba Nucleic acid chemistry, Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences, Prague 6, CZECH REPUBLIC

anoma cells express 4 MGRN1 alternative splicing isoforms. Two of them contain canonical nuclear localization signals in the alter- natively spliced exon 12, but they were located in the cytosol when expressed in heterologous cells. However they accumulated in nuclei upon co-expression with either MC1R or MC4R. All MGRN1 isoforms decreased MC1R and MC4R signalling, with- out effect on b2-adrenoreceptor. Inhibition was independent on receptor ubiquitylation or downregulation, occurred upstream of Gas binding to/activation of adenylyl cyclase, and was abolished by overexpression of Gs. Co-immunoprecipitation of MGRN1 and the MCRs suggested their physical interaction. Inhibition of signalling likely resulted from competition with Gs for binding to MCRs. On the other hand, MGRN1 expression levels in mela- noma cell lines transfected with various MGRN1 expression constructs consistently resulted in much higher accumulation of MGRN1 in melanoma cells harbouring N-RAS or BRAF activa- tory mutations. These mutations are the more frequent genetic alteration in melanoma. Therefore, MGRN1 inhibits MCR signal- ling by a Gs displacement mechanism. Simultaneously, its level of expression appears regulated by signalling from N-RAS and B-RAF. among decreasing different ANPs in

P4–55 Collagenolytic potential of rat liver myofibroblasts A. Jiroutova1, P. Cevelova1, L. Majdiakova1, R. Slavkovsky2 and J. Kanta1 1Department of Biochemistry, Faculty of Medicine in Hradec Kra- love, Hradec Kralove, CZECH REPUBLIC, 2CPN, Laboratory of Wound Healing, Dolni Dobrouc, CZECH REPUBLIC

This study aimed to evaluate the molecular mechanisms by which acyclic nucleoside phosphonates (ANPs) execute apoptosis in CCRF-CEM leukemic cells. 9-[2-(phosphonomethoxy)ethyl]guan- ine (PMEG) (5 lM, 72 h) induced apoptosis that was accompa- nied by an elevation of caspase 3, 8 and 9 activity to 630%, 160% and 330%, respectively, compared to untreated control. This suggests preferential involvement of mitochondrial apoptotic pathway. We have also observed a 2.5-fold increase in the activ- ity of a T-cell specific serine protease – granzyme A. This effect was likely due to the up-regulation of granzyme A mRNA (>6- fold). We have found that the effect on granzyme A expression differs order PMEG > PMEDAP > PMEA > (R)-PMPA, consistently with their cytotoxicity. However, other widely used cytotoxic drugs e.g. doxorubicin or etoposide did not influence granzyme A expression in CCRF-CEM cells suggesting that this effect is not a general phenomenon. Finally we have used caspase and gran- zyme broad spectrum inhibitors (Z-VAD-FMK and FUT-175, respectively) to elucidate the role of these proteases in PMEG- induced apoptosis. As none of them managed to diminish the extent of apoptosis, we hypothesize that neither caspase nor granzyme A activation is absolutely necessary for PMEG to exert its apoptotic effects. Acknowledgements: This work was supported by the Research project of the Institute OZ40550506, the Program of targeted projects of Academy of Sciences of the Czech Republic No.1QS400550501 and the Project No.1M0508 by the Ministry of Education, Youth and Sports of the Czech Republic.

P4–57 Ferredoxin-dependent nitrate reductase from a chemoautotrophic bacterium, Hydrogenobacter thermophilus M. Kameya, H. Arai, M. Ishii and Y. Igarashi Department of Biotechnology, The University of Tokyo, Tokyo, JAPAN

synthase

Background/Aims: Myofibroblasts (MFB) are one of the cell types involved in the excessive extracellular matrix (ECM) pro- duction in fibrotic liver. Expression of genes involved in connec- tive tissue metabolism was studied in MFB cultured on plastic and in fibrin or collagen gels. Methods: MFB were obtained by 4–5 passages of hepatic stel- late cell fraction of liver cells and plated on plastic or embedded in fibrin or collagen type I gels. Total cellular mRNA was iso- lated and studied by DNA arrays and by real time RT-PCR. Expression of some genes on protein level was studied by immu- nofluorescence. Results: Embedding the cells in collagen gel resulted in pro- nounced morphological changes of MFB and in increased expres- sion of metalloproteinase genes (collagenase MMP-13, gelatinases MMP-2 and 9, stromelysin MMP-3 and membrane type metallo- proteinase MMP-14), while collagen I expression was not affected. Expression of plasminogen activator inhibitor (PAI-1) decreased. Protein expression paralleled changes in MMPs mRNA. MFB avidly digested collagen gels. The expression of metalloproteinase inhibitors TIMP-1 and -2 did not change. Changes in the expression of cytokines TGF-b1, -b3 and CTGF were found. The effects of fibrin gels were mild. Conclusion: MFB assumed to deposit collagen and other ECM components in fibrotic liver possess great collagenolytic potential that is revealed when MFB are grown in vitro in collagen gel. Acknowledgements: This work was supported by Ministry of Education, grant No. MSM 0021620820, and by Charles Univer- sity Grant Agency, grant No. 86/2006/C.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Hydrogenobacter thermophilus is a hydrogen-oxidizing chemoau- totrophic bacterium, which originated from the deepest branch in the domain Bacteria, along with other Aquificales species. Reflecting such a unique phylogenetic position, a number of novel features have been found in this bacterium, such as car- bon fixation through the reductive tricarboxylic acid (RTCA) cycle. It was recently revealed that H. thermophilus also has unique characteristics in its nitrogen metabolism; ferredoxin- (GOGAT), which had been dependent glutamate believed to be distributed only among photoautotrophs, is pres- ent and contributes to ammonium assimilation in this chemoau- totrophic bacterium. In this study, assimilatory nitrate reductase (NAS), which is one of the key enzymes for nitrate assimilation thermophilus and in many organisms, was purified from H. characterized. It is known that cyanobacterial NASs use ferre- doxin as an electron donor, while many other bacterial NASs use NAD(P)H instead. Interestingly, the H. thermophilus NAS

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in a dose dependent manner. The mRNA expression levels of SREBP1c, C/EBPa, PPARc and Leptin were markedly down- regulated by Glc-6-P. Moreover, Glc-6-P inhibited the protein expression of C/EBPa and PPARc, the key adipogenic transcrip- tion factors. Tumor necrosis factor-a (TNFa) is reported to impair pre-adipocyte differentiation and induce lipolysis. Glc-6-P markedly reduced mRNA expression level of TNFa. These results suggest that inhibitory effect of Glc-6-P on adipocyte dif- ferentiation might be mediated through the down regulated expression of adipogenic transcription factors.

reacted with ferredoxin, like GOGAT in this organism and the cyanobacterial NASs. In contrast to the cyanobacterial enzymes, this NAS reacts not with [2Fe-2S] ferredoxin but with [4Fe-4S] ferredoxin. Disruption of this NAS gene resulted in ammonium auxotrophy, providing further evidence that the ferredoxin- dependent NAS functions as the nitrate assimilatory pathway in H. thermophilus. This study showed that not only photosyn- thetic but also non-photosynthetic bacteria possess ferredoxin- dependent enzymes for ammonium and nitrate assimilation. Since it is known that H. thermophilus uses ferredoxin also in the RTCA cycle, ferredoxin was revealed to play a crucial role as the electron donor in both nitrogen and carbon anabolisms of this organism.

P4–58 Exosomes derived from LPS-treated mice induced in vitro and in vivo immune response against inert allergen J. H. Kim, T. Shin, O. Y. Kim, Y. Kim and Y. S. Gho Department of Life science, Pohang University of Science and Technology, Pohang, SOUTH KOREA

P4–60 Effects of carboxymethyl-chitosan and -chitin on MMP inhibition related with ROS scavenging in HT1080 human fibrosarcoma cells C. S. Kong1, J. A. Kim2, B. N. Ahn2, T. K. Eom2, H. G. Byun3 and S. K. Kim2 1Marine Bioprocess Research Center, Pukyong National University, Busan, SOUTH KOREA, 2Chemistry, Pukyong National University, Busan, SOUTH KOREA, 3Faculty of Science and Biotechnology, Kangnung National University, Kangnung, SOUTH KOREA

Antioxidative and matrix metalloproteinase-2 and -9 (MMP-2 and -9) inhibitory effects of carboxymethyl-chitosan and -chitin (CM-chitosan and -chitin) were investigated in HT1080 cells. CM-chitosan and CM-chitin suppressed intracellular ROS forma- tion by DCFH-DA and radical simulated oxidations of mem- brane protein and lipid in a concentration-dependent manner. In addition, a protective effect against oxidative damage of purified genomic DNA was observed in presence of CM-chitosan and CM-chitin. Moreover, CM-chitosan and CM-chitin reduced the expression levels of MMP-2 and -9 in gelatin zymography, RT- PCR and western blot analysis without any cytotoxic influence. CM-chitin exhibited a higher MMP inhibitory effect than CM- chitosan through transcriptional down-regulations of AP-1 and NF-jB. Findings from the present study should underscore the nutraceutical value of CM-chitosan and CM-chitin as a potent anti-oxidant and MMP inhibitor via alleviations of radical- induced oxidative damage.

Exosomes are nano-sized membrane vesicles (30–100 nm in size) which are released into extracellular milieu after fusion of mul- tivescular body with the cell membrane. Growing evidence shows that various cell types such as hematopoietic, epithelial, and immune cells release exosomes, and these vesicles play multiple roles in intercellular communication including immune modula- tion and signal transduction. Although recent study showed that exosomes are found in the bronchoalveolar lavage (BAL) fluid, the exact roles of exosomes present in BAL fluid have not been investigated. Here, we showed that the intranasal treatment of lipopolysaccharide (LPS) into mice caused significant increase of exosomes release into BAL fluid when compared with PBS-treat- ment. Both exosomes derived from PBS- and LPS-treated mice harbored several exosomal marker proteins, and more intercellu- lar adhesion molecule-1, a marker protein of inflammation, was present in LPS-induced exosomes than control PBS-treated ones. Furthermore, exosomes derived from LPS-treated mice stimu- line and induced immune lated RAW264.7 macrophage cell response against inert allergen in mouse airway inflammation model while those from control PBS-treated mice have not any apparent in vitro and in vivo activities. These observations suggest that exosomes are insoluble communicasomes which transmit environmental information to other cells in vivo.

P4–61 Effect of glucosamine derivatives on induction of apoptosis in MCF-7 human breast cancer cells C. S. Kong1, J. A. Kim2, T. K. Eom2 and S. K. Kim2 1Marine Bioprocess Research Center, Pukyong National University, Busan, SOUTH KOREA, 2Chemistry, Pukyong National University, Busan, SOUTH KOREA

P4–59 Anti-adipogenic effect of glucosamine-6- phosphate in 3T3-L1 adipocytes J. A. Kim1, T. K. Eom1, C. S. Kong2 and S. K. Kim1 1Chemistry, Pukyong National University, Busan, SOUTH KOREA, 2Marine Bioprocess Research Center, Pukyong National University, Busan, SOUTH KOREA

proliferator-activated peroxisome

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Effects of glucosamine-6-phosphate (Glc-6-P) on adipocyte differ- entiation of 3T3-L1 cells were investigated by measuring triglyc- eride level and leptin and glycerol secretions as indicators of lipid accumulation and gene expressions of CCAAT/enhancer-binding protein-a (C/EBPa), sterol regulatory element-binding protein 1c (SREBP1c), receptor-c (PPARc) and Leptin in cultured 3T3-L1 adipocytes. Glc-6-P sig- nificantly reduced lipid accumulation, a marker of adipogenesis, Abilities of glucosamine and its derivatives (glucosamine-6-sul- fate: SGlc-6, glucosamine-2-sulfate: SGlc-2 and glucosamine-6- phsphate: PGlc-6) to inhibit proliferation of MCF-7 human breast cancer cells were evaluated by measuring cell death via induction of apoptosis. Among them, glucosamine-2-sulfate (SGlc-2) exerted the highest anti-proliferative activity in the human breast cancer. SGlc-2 induced significant proliferative inhibition and apoptosis in a dose- and time-dependent manner in MCF-7 human cancer cells. Treatment with SGlc-2 induced the increase in caspase-3, -8 and -9 activities, DNA repair enzyme

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poly-(ADP-ribose) polymerase (PARP) cleavage and pro-apopto- tic gene and the decrease in anti-apoptotic genes. Besides, NF-jB family and -dependent activated genes were down-regulated by SGlc-2. These results indicated that SGlc-2 had a potential against inhibition of growth of MCF-7 human breast cancer cells, which might be associated with induction of apoptosis through NF-jB or -dependent pathway.

as its and identified sequence was

P4–62 1-(3’,5’-dihydroxyphenoxy)-7-(2’’,4’’, 6-trihydroxyphenoxy)-2,4,9-trihydroxydibenzo- 1,4-dioxin inhibits adipocyte differentiation of 3T3-L1 fibroblasts C. S. Kong1, J. A. Kim2, N. Y. Yoon1 and S. K. Kim2 1Marine Bioprocess Research Center, Pukyong National University, Busan, SOUTH KOREA, 2Chemistry, Pukyong National University, Busan, SOUTH KOREA

aspects. However, it’s not clear on arthritis. In the present work, we have purified and charaterized a bioactive peptide from sea- horse hydrolysis using various enzymes. Among hydrolysates, pronase E derived hydrolysate exhibited the higest alkaline phos- phatase (ALP) activity which phenotype marker of differentiation on osteoblast and chondrocyte, than those of other enzyme hy- drolysates. Pronase E hydrolysate was separated in diverse purifi- cation steps. Finally, the peptide having the ALP activity was isolated LED- PFDKDDWDNWK (1821 Da). We have shown that isolated peptide exhibits a significant induction of differentiation in osteo- blastic MG-63 and chondrocytic SW-1353 cells for ALP activity, minerlaization and collagen synthesis. Our result indicates that peptide affects from early to later stages of differentiation in MG-63, SW-1353 cells. Moreover, we assessed peptide inhibition on 12-O-Tetradecanoylphorbol-13-acetate (TPA) which is a com- mon form of phorbol ester, induced expression of MMPs (1, 3, and 13), iNOS, COX-2 in a concentration-dependent manner and inhibits the NO production on MG-63 and SW-1353 cells. To elucidate the mechanism responsible for the inhibitory effect of peptide, we examined the effect of peptide on TPA-induced MAPKs and NF-jB activation and determined that the isolated peptide treatment significantly reduced p38 kinase/NF-jB in MG-63 cells and MAP kinase/NF-jB in SW-1353 cells.

peroxisome proliferator-activated element-binding of sterol regulatory protein

P4–64 Dioxinodehydroeckol suppresses adipocyte differentiation of 3T3-L1 preadipocytes by MAPK pathway C. S. Kong1, J. A. Kim2, N. Y. Yoon1 and S. K. Kim2 1Marine Bioprocess Research Center, Pukyong National University, Busan, SOUTH KOREA, 2Chemistry, Pukyong National University, Busan, SOUTH KOREA

In present study, we isolated phloroglucinol derivative, 1-(3’,5’- dihydroxyphenoxy)-7-(2’’,4’’,6-trihydroxyphenoxy)-2,4,9-tri- hydroxydibenzo-1,4-dioxin (1), from Ecklonia Cava and its potential inhibitory effect on adipocyte differentiation in 3T3-L1 cells. Lipid accumulation and expression of several genes associ- ated with adipogenesis and lipolysis were examined at the end of differentiation period. Lipid accumulation was examined by mea- suring triglyceride contents and Oil-Red O staining. The expres- sion levels of several genes and proteins were examined using reverse transcription-polymerase chain reaction (RT-PCR), real- time PCR and western blot analysis. Presence of compound 1 induced significant reduction of lipid accumulation and down- receptor-c regulation (PPARc), 1c (SREBP1c) and CCAAT/enhancer-binding proteins (C/EBPa) in a dose-dependent manner. Moreover, presence of compound 1 induced down-regulation of adipogenic target genes such as adi- pocyte fatty acid binding protein (aP2), fatty acid transport pro- tein (FATP), fatty acid synthase (FAS), acyl-CoA synthetase 1 (ACS1), lipoprotein lipase (LPL) and leptin genes. According to the lipolytic response, compound 1 down-regulated perilipin and hormone-sensitive lipase (HSL) and up-regulated tumor necrosis factor a (TNFa). Therefore, these results suggest that compound 1 might decrease lipid accumulation during adipocyte differentia- tion by modulation of adipogenesis and lipogenesis and com- pound 1 could be developed as a good functional agent to improve antiobesity.

Dioxinodehydroeckol was isolated from E. Cava and its inhibi- tory effect on adipocyte differentiation of 3T3-L1 preadipocyte was investigated by measuring levels of lipid accumulation and changes in genes and proteins associated with adipogenesis and lipolysis. Treatment with dioxinodehydroeckol significantly reduced lipid accumulation during adipocyte differentiation and induced down-regulation of SREBP1, PPARc and C/EBPa in a dose-dependent manner. Moreover, dioxinodehydroeckol sup- pressed regulation of the adipocyte specific gene promoters such as FABP4, FATP1, FAS, LPL, ACS1 and leptin. As the lipolytic response, dioxinodehydroeckol down-regulated expression levels of perilipin and HSL genes and up-regulated the expression levels of TNFa mRNA compared to fully differentiated adipose tissue. Specific mechanism of dioxinodehydroeckol was examined through transcriptional down-regulations of extracellular signal- regulated kinase (ERK). Therefore, these results suggest that di- oxinodehydroeckol induced antiadipogenic effect on adipocytes via ERK-dependent signaling pathway.

P4–63 Purification of a peptide from seahorse, that inhibits arthritis-related cytokines through MAPK/NF-kB activation, and induces human osteoblastic and chondrocytic differentiation B. M. Ryu1, Z. J. Qian2 and S. K. Kim1 1Chemistry, Pukyong National University, Busan, SOUTH KOREA, 2Marine Bioprocess Research Center, Pukyong National University, Busan, SOUTH KOREA

P4–65 Phosphoryled glucosamine inhibits inflammatory response in PMA-induced THP-1 macrophages J. A. Kim1, C. S. Kong2 and S. K. Kim1 1Chemistry, Pukyong National University, Busan, SOUTH KOREA, 2Marine Bioprocess Research Center, Pukyong National University, Busan, SOUTH KOREA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Inflammation is pathological influences of diseases as diverse as atherosclerosis, cancer, diabetes and Alzheimer’s disease. In these Currently, there have been considerable efforts to search for nat- urally occurring bioactive substances for the amelioration of arthritis. Many substances derived from natural products have been found to possess substantial bioactive properties. Seahorse, a telelost fish, has been exhibited positive effects on multiple

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in this

found at the apical plasma membrane of human epithelial cells. In cancer cells apical proteins are often mislocalized. Since the GPI-anchor and N-glycans in many proteins act as apical target- ing signals we have examined their role in apical targeting of CEA. By utilizing glycosylation inhibitors and targeted mutagen- esis, we show that neither immature N-glycans nor the removal of the GPI-anchor disrupt the apical targeting of CEA in stably transfected MDCK cells. We also mutagenised N-domain, a known interaction domain of CEA, but found no interference with the targeting of CEA. In contrast we found that apical tar- geting was sensitive to organellar pH gradient. Treatment of cells with either ammonium chloride or concanamycin A (a vacuolar H+-ATPase inhibitor) resulted in increased basolateral CEA. Consistent with the potential involvement of lipid raft association and complex formation in apical targeting, drug treatment also increased the TX-100 solubility of CEA and also inhibited com- plex formation as assessed by sucrose density gradient centrifuga- tion. These results suggest that apical targeting of CEA is not directly dependent on its GPI-anchor or N-glycans, but rather relies on pH-sensitive complex formation with other lipid raft constituents. inflammatory-related diseases, macrophages produce lots of pro- inflammatory cytokines, pro-inflammatory enzymes and inflam- matory mediators. Inducible nitric oxide synthase (iNOS) and cy- clooxygenase (COX)-2 are important enzymes that induced by various inflammatory stimuli such as bacterial endotoxic lipo- polysaccharide (LPS), and inflammatory cytokines in macrophag- es. Therefore, study, we synthesized glucosamine-6- phosphate (Glc6-P) through phosphorylation of glucosamine with phosphorous pentoxide (P2O5). Anti-inflammatory effect of Glc- 6-P was investigated in PMA-induced macrophages, THP-1 cell line. Briefly, N-phtalamide glucosamine was dissolved in me- thanesulfonic acid with adding phosphorous pentoxide (P2O5) followed by stirring at 5ordm;C for 4 h. First of all, we measured changes in inflammatory cytokines by using enzyme-linked immunosorbent assay, ELISA. Productions of tumor necrosis factor-a (TNF- a), interleukin-1b (IL-1b) and interleukin-6 (IL-6) were inhibited in presence of Glc-6-P. Furthermore, pretreatment with Glc-6-P dose-dependently inhibited both the mRNA and protein levels of TNF-a, IL-1b, IL-6, iNOS and COX-2 in lipo- polysaccharide (LPS)-stimulated cells. Therefore, these results may provide new insight into the molecular basis for the anti- inflammatory property of Glc-6-P.

P4–66 Histological analysis of thymus of acomys cilicicus S. Kiralp, H. Mutlu, N. Ozsoy and E. Kivanc Biology, Ankara University Faculty of Science, Ankara, TURKEY

P4–68 Nuclear intermolecular interactions of neurofibromin during the cell cycle X. Koliou, G. Leondaritis and D. Mangoura Biomedical Research Foundation Academy of Athens, Center of Preventive Medicine Neurosciences and Social Psychiatry, Athens, GREECE

including its

Spiny Mouse (Acomys cilicicus Spitzenberger, 1978) is an endemic species in Turkey, spread over Silifke. Although there are studies about the taxonomy of this specie, there is no study about its his- tology. In this study, thymus of this specie was analyzed in detail histologically. Thymus is a two-loop lymphoepithelial organ, which provides an area for T lymphocyte maturation. Thymus of 3 adult, 2 male and a female spiny mice were collected from the area and were grown up in the laboratory. Tissue samples were fixed in 10% formalin and embedded in paraffin. Haematoxylin- Eosin, Masson’s trichrome, Gomori’s silver impregnation and Periodic Acid Schiff (PAS) were used for staining the obtained paraffin sections. Histological analysis was done using light microscopy. A thin capsule and thymic septa were observed in sections stained with Haematoxylin-Eosin. Darkly stained cortex in the outer region and lightly stained medulla in the interior region were observed. There were more lymphocytes in the cortex compared to the medulla. Hassal corpuscle was encountered in the medulla. Fibrous connective tissue, capsule and septa and vein walls were stained using Masson’s trichrome. In silver stain- ing, it is found that medulla had a dense network of coarse retic- ular fibers while the cortex contained a loose network of fine fibers. PAS positive cells and strongly PAS positive Hassal’s cor- puscles were also observed.

Neurofibromin is a tumour suppressor gene product and, as a RasGAP, plays an important role in the Ras-ERK pathway. A number of observations, established mobility throughout the cell and the nucleus have, however, suggested that it may have additional tasks. To begin to address the specific function of neurofibromin in the nucleus we first examined its expression in the SF268 glioblastoma cell line, after establishing the duration of the cell cycle phases. Thus, we synchronized cells at G1/S, S, G2, or G2/M cell cycle phases using double thymi- dine and nocodazole blocks and found that the levels of neurofi- bromin transcripts (GRDI and GRDII), retaining their 2 : 1 ratio, increased during the S phase, peaked at S/G2 (2-fold gain), and declined by mid G2. We recorded corresponding increases in neurofibromin expression by imunoprecipitations, with a lag of several hours. Because neurofibromin via its NLS domain may be actively transported into the nucleus, we verified by immuno- fluorescence analysis, subcellular and subnuclear fractionation studies, that a pool of neurofibromin remains associated with the nuclear matrix throughout the cell cycle. More importantly, we found that neurofibromin co-immunoprecipitated with the nuclear intermediate filament lamin A/C particularly during the G2 phase and this interaction was decreased and finally undetect- able as the nuclear lamina dissolved and as neurofibromin was hyperphosphorylated on C-terminus, PKC specific sites. Our results indicate that neurofibromin is actively transported and specifically retained in the nucleus via intermolecular interactions and postranslational modifications for functions possibly related to mitosis. (PENED grant 03ED778)

P4–67 Acidic Golgi pH is necessary for the apical targeting of carcinoembryonic antigen N. Kokkonen, A. Kauppila and S. Kellokumpu Department of Biochemistry, University of Oulu, Oulu, FINLAND

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Carcinoembryonic antigen (CEA, ceacam5) is a highly N-glycosy- anchored protein lated, glycosyl-phosphatidylinositol (GPI)

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increase significant

P4–69 Uncontrolled protein overexpression leads to plant cell autophagy T. Komarova, E. Sheval and Y. Dorokhov A.N.Belozersky Institute, Lomonosov Moscow State University, Moscow, RUSSIA

statistically in GST-CDNB activity (P < 0.05), 1.51 fold, compared to susceptible population. Whereas, GST-DCNB activity was increased statistically signifi- cantly (P < 0.05) in both Mardin and C¸ anakkale (n = 30) field populations, 4.81 and 1.98 folds, respectively, compared to sus- ceptible population. However GST gene expressions (GST and GST Sigma) did not exhibit statistically significant (P > 0.05) change in C¸ anakkale and Mardin populations compared to sus- ceptible population. According to these results, relative expres- sion levels of examined genes were inconsistent with activity of CDNB and DCNB-GSTs. Nevertheless, the cytosolic GSTs may contribute to the pyrethroid resistance of H. armigera. Acknowledgement: This work is supported by Project No. BAP-DPT2002K120510.

P4–71 Location of cell cycle promoters PCNA, Cyclin D1, Cyclin D3, and inhibitors p21, p27 and p57 in rodent placental development E. T. Korgun1, D. Kipmen-Korgun2 and R. Demir1 1Department of Histology and Embryology, Akdeniz University, Antalya, TURKEY, 2Department of Biochemistry, Akdeniz University, Antalya, TURKEY

Autophagy is a major system for the bulk degradation of intra- cellular proteins and organelles in plants playing an important role in nutrient recycling during senescence, stress and hypersen- sitive response to pathogen infection. Normally plant cell is adapted for producing large amounts of protein which is targeted and stored in special cellular compartments. But intensive uncon- trolled and untargeted protein production is likely to result in programmed cell death via autophagy. To study the autophagy of protein overexpressing plant cell we created specific vector sys- tem allowing GFP production in agroinjection experiments. TMV infectious copy delivered by Agrobacterium tumefaciens into plant leaves replicated efficiently providing GFP production in huge quantities (more than 5 g/kg) and resulted in cells death 3 days after agroinjection. Such GFP super-expressing cell was called Pheidippides cell. Using microscopy (light, confocal, elec- tronic) and biochemical approaches we described the following features of the Pheidippides cell: localization of viral factories and GFP production sites; stages of cell and tissue death and autophagy; GFP behavior in aggregates and its proper folding; correlation between BiP and GFP expression level. Moreover we just analyzed the pattern of mRNA expression in plant cell before its death using subtractive hybridization. Several candidate genes including specific biotic cell death-associated protein were found to be upregulated. The dynamics of corresponding mRNAs accumulation proved the role of these proteins in plant cell death via autophagy.

P4–70 Glutathione S-transferases in pyrethroid resistance of Helicoverpa armigera from Turkey M. Konus1, S. Ugurlu2 and M. Iscan1 1Biochemistry Department, Middle East Technical University, Ankara, TURKEY, 2Plant Protection Central Research Institute, Agriculture Protection, Ankara, TURKEY

Objectives: Although placental development and implantation depend on the coordination of trophoblast proliferation, differen- tiation and invasion, little is known about the cell cycle regula- tors. Methods: PCNA, Cyclin D1, Cyclin D3 p21, p27 and p57 were immunolocalised in paraffin embedded tissue sections. Results: p27 and p57 were localized in distinct cell populations of the Junctional zone (JZ) while p21 was not detectable except for a small population in the decidual cells. Giant cells expressed both p27 and p57, with more number of cells being stained with p57 when compared with p27. Except for glycogenic cells, all the other cell types within the JZ were labeled with PCNA. While cy- clin D1 did not show any immunoreactivity within the JZ. p21 labeled mesenchymal cells and labyrinthine trophoblast cells in the labyrinth zone (LZ). p27 and p57 were found with mesenchy- mal cells, labyrinthine trophoblast cells, labyrinthine giant cells and glycogenic cells of LZ. PCNA was visualized in mesenchymal cells, labyrinthine trophoblast cells and labyrinthine giant cells, but not in glycogenic cells in the LZ. Cyclin D1 immunoreaction was apparent in mesenchymal cells, labyrinthine trophoblast cells and especially in endothelial cells but was not detected in either labyrinthine giant cells or visceral endodermal cells in the LZ. The localization of cyclin D3 was similar to that of cyclin D1, with the exception of some cells of visceral and parietal endo- derm being noticeably immunoreactive with anti-cyclin D3. Conclusion: According to our findings all these cell cycle pro- moters and inhibitors have different function in placental devel- opment.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Helicoverpa armigera is a major pest of cotton. It has developed resistance against most of the insecticides, applied in the fields. Pyrethroid insecticides are known to be less harmful on human health, but, excessive usage has resulted in resistance develop- ment in H. armigera. It has been proposed that one of the main factors that causes resistance development may be induction of glutathione S-transferases (GSTs). Although, H. armigera GSTs are not well defined at protein and gene level, 1-chloro-2,4-dini- trobenzene (CDNB) and 3,4-dichloronitrobenzene (DCNB) are accepted as model substrates for H. armigera. In this study, GST activities against CDNB and DCNB were examined in the sam- ples obtained from fields at C¸ anakkale and Mardin provinces in Turkey. In addition, relative variations in GSTs gene expressions were investigated using real-time PCR. The results were com- pared to that of susceptible population obtained from Germany. It was found that only Mardin (n = 30) field population showed

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P4–72 Plasma membrane calcium pump isoforms in calcium regulated catecholamine secretion from PC12 cells M. Kosiorek1, P. Podszywalow-Bartnicka1, L. Zylinska2 and S. Pikula1 1Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, POLAND, 2Department of Molecular Neuro- chemistry, Medical University of Lodz, Lodz, POLAND

derived from AM patients tended to reduce mitochondrial mass, in contrary, in FD where we observed increased mitochondrial content compared to control cell lines. The activity of respiratory chain complex IV (COX) and the ratio between COX and citrate synthase (CS) serving as the control enzyme were increased (P < 0.05) in AM in comparision to controls. Decreased mito- chondrial Ca2+ buffering efficiency, similarly to observed in some of mitochondrial disorders, was detected in all patients with GD. We revealed swelled and damaged mitochondria as well as dilated endoplasmatic reticulum in AM, increased production of mitochondrial superoxide and disturbed calcium homeostasis in GD. Based on our results, we hypothesize, that more profound effect of mitochondrial disbalance may be found in tissues with higher energy demand than in fibroblasts. Acknowledgement: Supported by HUEMAN LSHM-CT- 2006-018692.

P4–74 Influence of potassium ions on calcium- induced redistribution of phosphatidylcholine at the plasma membrane of human erythrocytes D. Kostsin Institute of Biophysics and Cell Engineering, National Academy of Science of Belarus, Minsk, BELARUS

PC12 cell line, derived from rat tumor pheochromocytoma with high level of catecholamines, is frequently used to study mecha- nisms of exocytosis and Ca2+ homeostasis. We have chosen this line to study the impact of plasma membrane calcium pumps (PMCA) on catecholamine secretion. Among four isoforms of PMCA; PMCA1 and PMCA4 are common, while PMCA2 and PMCA3 are restricted to neuronal and neuroendocrine tissues. To test the influence of PMCA2 and PMCA3 on Ca2+-regulated exocytosis of catecholamines PC12 cells were transfected with plasmids expressing antisense sequences to the respective isoforms of PMCA. General aim of this project was to determinate cal- cium homeostasis and catecholamine secretion in our model. PC12 cells stably transfected with plasmids expressing antisense sequences against PMCA2 or PMCA3 were established at the Medical University of Lodz. PMCA expression was determined with Western blot and RT-PCR. Changes in [Ca2+]c were mea- sured with FURA-2AM, while catecholamine secretion with RP- [Ca2+]c and higher Ca2+ HPLC. We observed elevated basal influx upon stimulation of cells treated with the antisense sequences to PMCA2 and PMCA3 in comparison to control cells. This was accompanied by decrease in dopamine secretion. In conclusion decreased PMCA2 and PMCA3 expression in PC12 cells significantly affected Ca2+ homeostasis and catechol- amine secretion. The observed effects were stronger in cells with decresed PMCA2 expression.

P4–73 Mitochondrial ultrastructure and function in fibroblasts from patientswith alpha- mannosidosis, Fabry and Gaucher disease O. Kostkova1, B. Asfaw2, J. Sladkova1, H. Poupetova2, J. Zivny3, J. Krusek4, H. Hansikova1, M. Magner1, M. Tesarova´ 1, J. Ledvinova2 and J. Zeman1 1Mitochondrial laboratory, General University Hospital and First School of Medicine Charles University, Prague 2, CZECH REPUBLIC, 2Institute of Inherited Metabolic Disorders, General University Hospital and First School of Medicine Charles Univer- sity, Prague 2, CZECH REPUBLIC, 3Department of Pathophysi- ology, General University Hospital and First School of Medicine Charles University, Prague 2, CZECH REPUBLIC, 4Academy of Sciences, Institute of Physiology, Prague 4, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Lysosomal storage diseases (LSDs) present class of serious inborn metabolic disorders with well recognised enzymatic defect, however pathophysiology most of them is largely unknown. Growing amount of evidence for mitochondrial contribution in LSDs pathology has appeared during recent years. Our study is focused on ultrastructure of mitochondria and endoplasmatic reticulum (ER) and mitochondrial biochemistry in fibroblasts derived from three patients suffering with LSDs a-mannosidosis (AM), five patients with Fabry disease (FD) and three patients with Gaucher disease (GD). Higher amount of aberrant mito- chondria with rudimental cristae, enlarge volume and dilated en- doplasmatic reticulum was dominant in AM, elevated production of mitochondrial superoxide was detected in GD. Fibroblasts Objective: The aim of investigation was to examine how cal- cium and potassium ions at different concentration in extracellu- lar medium influence on redistribution of phosphatidylcholine (PC) at the plasma membrane of human erythrocytes. Methods: Experiments were performed on donor erythrocytes. In this study 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexa- noyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (16:0/C6-NBD- PC) from «Molecular Probes» (USA) was used. The amount of internalized 16:0/C6-NBD-PC was determined by comparing the probe fluorescence intensity associated with the cells before and after back exchange procedure with 2% bovine serum albumin. Results: A difference in 16:0/C6-NBD-PC distribution between outer and inner membrane layers of control cells and erythrocytes loaded with different calcium concentration (0, 25; 0, 5; 0, 75 and 1 mM) in 10 mM Hepes buffer was received. Content of 16:0/C6- NBD-PC in inner leaflet of erythrocyte membranes under influence of 0, 5 and 0, 75 mM CaCl2 (1 lM ionophore A23187) was in range of 30–45% and 40–50% respectively. Increase in extracellu- lar calcium concentration up to 1 mM lead to more marked accu- mulation of 16:0/C6-NBD-PC (50–60%) in inner leaflet of human erythrocyte membranes. However, decreased calcium concentra- tion in 10 mM Hepes buffer up to 0,25 mM tend to broad distribu- tion 16:0/C6-NBD-PC in inner half of membrane during time course of experiment (about 25% at time interval 20 min to 50% after 6, 5 h incubation). Because no influence on total fluorescence of PC analog incorporated at the plasma membrane of erythro- cytes under action of 1 mM CaCl2 was registered further we stud- ied effect of 3, 90 and 140 mM KCl on Ca2+-induced redistribution of 16:0/C6-NBD-PC only at this extracellular cal- in this condition strongly pro- cium concentration. Moreover, nounced redistribution of 16:0/C6-NBD-PC was obtained. Amount of 16:0/C6-NBD-PC relocated between membrane leaflets was higher under action of 1 mM CaCl2 in presence of 90 and 140 mM KCl (50–60% of probe redistributed in the inner leaflet). At the same time when 3 mM KCl was added in 10 mM Hepes buffer only 25–40% of 16:0/C6-NBD-PC was presented in inner leaflet of erythrocyte plasma membranes. Conclusion: Obtained results testify that concentration of potas- sium ions in extracellular medium determine degree of Ca2+-

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induced redistribution of PC at the plasma membrane of human erythrocytes.

References: 1. Nekipelaya VV, Semenov DV, Potapenko MO, Kuligina EV, Kit YY, Romanova IV & Richter VA. Lactaptin is a human milk protein inducing apoptosis of MCF-7 adenocarcinoma cells. //Dokl Biochem Biophys. 2008; 419: 58–61.

2. Vlassov VV, Richter VA, Semenov DV, Nekipelaya VV, Kuli- gina EV & Potapenko MO. Peptide inducing apoptotic death of human cancer cells. 2008. Patent # 2317304 (February 20, 2008).

P4–75 The optimization of the protocol for immunofluorescence on fish spermatozoa P. Koubek1, A. Kralova1, M. Psenicka2 and J. Peknicova1 1Laboratory of Diagnostics for Reproductive Medicine, Institute of Biotechnology, Prague, CZECH REPUBLIC, 2Laboratory of Molecular Cellular and Quantitative Genetic, Research Institute of Fish Culture and Hydrobiology, Vodnany, CZECH REPUBLIC

P4–77 Role of heme oxygenase-1 in differentiation of muscle satellite cells into mature myocytes M. Kozakowska1, A. Stefanska1, J. Kotlinowski1, A. Grochot-Przeczek1, A. Sierpniowska1, R. Derlacz2, J. Dulak1 and A. Jozkowicz1 1Medical Biotechnology, Jagiellonian University Faculty of Biochemistry Biophysics and Biotechnology, Krakow, POLAND, 2Department of Metabolic Regulation, Institute of Biochemistry Faculty of Biology University of Warsaw, Warsaw, POLAND

In the research of fish spermatozoa, our activity concentrated mainly on the study of proteins that participate in the motility of chondrostean and teleostean fish and on the usefulness of the acrosome for the spermatozoa of chondrostean fish. Immunofluo- rescence experiments with newly prepared monoclonal antibodies (MoAbs) can be a useful tool for the study of these proteins, as we found in our experiments on mammalian models. However, the commonly used protocols for immunocytochemical experi- ments on mammalian spermatozoa failed to meet our require- ments for both undamaged sperm flagellum and intact head. Therefore, our study was focused on the optimization of those steps that are crucial for the preservation of unaffected fish sper- matozoa and on the improvement of the protocol for handling fish sperm during immunofluorescence experiments. Based on our results, it is obvious that several preconditions must be respected in order to get objective results of immunofluorescence labeling on fish sperm. First, the composition of the solutions should cor- respond to the requirements of individual species. Second, the temperature during the experiment should be close to the natural conditions of the samples. Finally, it should be respected that the cells attached to the microscopic slides must not desiccate prior to the fixation and also should dot desiccate even after the fixa- tion. The desiccation prior to the fixation is common when work- ing with mammalian spermatozoa and the structure is not affected at all. The fixation of fish spermatozoa is necessary, especially when the structure of the flagellum should be pre- served.

P4–76 Recombinant analogues of lactaptin – apoptosis induced protein from human milk O. Koval, A. Fomin, V. Matveeva, D. Semenov and V. Richter Biotechnology, Institute of Chemical Biology & Fundamental Medicine, Novosibirsk, RUSSIA

Muscle satellite cells are progenitor cells located beneath basal lamina of muscle fiber, which act as precursors for muscle repair. Upon muscle damage they may differentiate into myocytes, so they are considered as candidates for cell-based regenerative ther- apies. Our aim was to explore the role of heme oxygenase-1 (HO-1), a heme degrading enzyme which displays anti-inflamma- tory, antiapoptotic and antioxidant acivities, in differentiation of satellite cells. Experiments were performed using two models: (i) line C2C12, stably transduced with immortalized myoblast cell retroviral vectors to overexpress HO-1 and (ii) primary satellite cells isolated from hindlimb muscle of HO-1+/+, HO-1± and HO-1-/- mice. In both models the differentiation was induced by incubating the confluent cell cultures in the presence of 2% horse serum. We demonstrated that overexpression of HO-1 in C2C12 cells protected them against oxidative stress induced by the high concentrations of H2O2 or hemin, and upregulated production of stromal cell derived growth factor (SDF-1), whereas synthesis of major proinflammatory cytokines remained unaffected. Impor- tantly, HO-1 overexpression inhibited differentiation of C2C12 line, as indicated by reduced fusion of cells into myotubes, decreased expression of myogenic regulatory factors (such as MyoD or myogenin), and attenuated activity of creatine phos- phokinase. Results of first experiments performed on primary satellite cells, isolated from one month old mice and purified by pre-plate technique, seem to confirm that HO-1 expression may reduce maturation, whereas HO-1 deficiency may cause spontane- ous differentiation of even non-confluent cells. Thus, HO-1 might be an important inhibitor of formation of mature myotubes from satellite cells.

P4–78 Changes in chlorophyll content of cyanobacterial thylakoid membranes in the presence of cadmium ions T. Kucera1, A. Fendrychova2, T. Emmerova1 and A. Sonska1 1Biochemistry, Charles University Faculty of Science, Prague 2, CZECH REPUBLIC, 2Teaching and didactics of chemistry, Charles University Faculty of Science, Prague 2, CZECH REPUBLIC

sub-cellular preparations,

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

chlorophyll Working with plant amount is often used as a marker of sample quantity. Investigat- ing the effect of any factor on the given preparation, therefore, it is important to know whether and how that factor influences the Investigation of natural and synthetic agents induced selectively apoptosis of tumor cells is way for anticancer drugs design. We demonstrated that a protein from human milk – the lactaptin, induces apoptosis in MCF-7 epitheliocytes (1, 2). Here the attempt of amplification apoptotic effects of lactaptine was made. Several recombinants coding lactaptin’s analogues were con- structed. After the rounds of selection the clones that were posi- tive for expression of recombinant analogues of lactaptin were revealed and they were destined as systems for expression and secretion. Recombinant lactaptin analogues from these eukary- otic cells were isolated and tested for they apoptosis activity on human tumor cells: breast adenocarcinoma MCF-7, lung carci- noma derived A549 cells, larynx carcinoma derived HEp-2 cells and on human primary steam cells. The data obtained provide us the opportunity to see deeper inside anticancer therapy. Acknowlegments: This work was supported by the Russian Federal Agency for Science and Innovations (# 02.512.11.2257); Integration SB RAS Project # 18 (2009–2011).

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data emphasize the participation of adenosine deaminases in cap- turing and intracellular trafficking of internalized RNAs.

leading to lowered concentration estimates.

P4–80 The cell surface expressed nucleolin is a glycoprotein that triggers calcium entry into mammalian cells M. E. Losfeld1, D. El Khoury2, P. Mariot3, M. Carpentier1, B. Krust2, J. P. Briand4, J. Mazurier1, A. G. Hovanessian2 and D. Legrand1 1Biology, Unite´ de glycobiologie structurale et fonctionnelle UMR 8576 CNRS, villeneuve d’ascq, FRANCE, 2Biology, Unite´ Propre de Recherche n(cid:2)2228 du CNRS Universite´ Paris Descartes, Paris, FRANCE, 3Biology, 3Laboratoire de Physiologie cellulaireIN- SERM U800, villeneuve d’ascq, FRANCE, 4Biology, Unite´ Propre de Recherche n(cid:2)9021 du CNRS, Strasbourg, FRANCE

chlorophyll content of the studied material. Studying the Cd2+ effect on photosystem 2 in spinach, we found that chlorophyll isolated thylakoid membranes exposed to Cd2+ content of changes with varying Cd2+ concentration. We try to find the relationship between Cd2+ncentration and chlorophyll content. In order to achieve this, we exposed isolated thylakoid mem- branes to various Cd2+ concentrations and studied the pigment composition by means of absorption spectra and reversed-phase liquid chromatography. Similar experiments were carried out also with methanolic extracts of thylakoid membranes to evaluate the effect of a direct interaction of Cd2+ with chlorophylls e.g. dur- ing the routine concentration assays. Cadmium seems to affect the apparent chlorophyll concentration probably in three main in pigment-protein ways: (i) Pheophytinization of chlorophyll complexes, (ii) Decreasing the chlorophyll loss when washing the aqueous sam- ples during their preparation, probably strengthening the pig- interaction in the pigment- ment-protein (or protein–protein) protein complexes. This leads to higher concentration estimates. These two changes show clear dependence on Cd2+ concentra- tion. (iii) Pheophytinization of chlorophyll in the methanolic extract. This reaction seems to be almost complete already at the lowest Cd2+ concentration used, leading to considerably lowered concentration estimates, if Cd2+ is still present at the time of chlorophyll assay.

P4–79 Modification of extracellular RNAs in course of spontaneous internalisation and transport in human cells E. Kuligina, D. Semenov, O. Vratskih and V. Richter Laboratory of Biotechnology, Institute of chemical biology and fundamental medicine, Novosibirsk, RUSSIA

Nucleolin is an ubiquitous nucleolar phosphoprotein involved in fundamental aspects of transcription regulation, cell proliferation and growth. Nucleolin has been described as a shuttling molecule between nucleus, cytosol and the cell surface. Cell surface expressed nucleolin serves also as a receptor for various extracellu- lar ligands implicated in cell proliferation, differentiation, adhe- sion, mitogenesis and angiogenesis. Previously, we reported that nucleolin in the extranuclear cell compartment is a glycoprotein containing N- and O-glycans. In the present study, we show that glycosylation is an essential requirement for surface nucleolin expression, since it is prevented when cells are cultured in the pres- ence of tunicamycin, an inhibitor of N-glycosylation. Accordingly, surface but not nuclear nucleolin is radioactively labeled upon met- abolic labeling of cells with [(3)H] glucosamine. In addition, involvement of the glycosylation on the interaction properties of nucleolin with its ligands was studied by surface plasmon reso- nance thanks to glycosylated or non-glycosylated forms of nucleo- lin produced in a baculovirus/insect cell system. Moreover, we show that ligand binding to surface nucleolin could also induce calcium entry into cells. Indeed, by flow cytometry, microscopy and patch-clamp experiments, we show that the HB-19 pseudopep- tide, which binds specifically surface nucleolin, triggers rapid and intense membrane calcium fluxes. The use of several drugs then indicated that Store-Operated Calcium Entry (SOCE)-like chan- nels are involved in the generation of these fluxes. Taken together, our findings suggest that binding of an extracellular ligand to sur- face nucleolin could be involved in the activation of signaling path- ways by promoting calcium entry into cells.

P4–81 Itch interacts with the pre-initiation complex for gene transcription W. Y. Lui and C. Y. Tam School of Biological Sciences, The University of Hong Kong, Hong Kong, HONG KONG

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

than promiscuous. Fragments of Itch, an E3 ubiquitin ligase, is highly expressed in the testis. Studies have shown that cytoplasmic Itch interacts with a tight junction protein, occludin and is involved in the regulation of occludin deg- radation. However, the interacting partners of Itch in the nucleus remain enigmatic. We expressed and purified the GST-Itch fusion protein. GST-Itch fusion protein was incubated with nuclear extract prepared from Sertoli cells followed by affinity chromatog- raphy and eluted proteins were subjected to mass spectrometry. Their identities are RNA pol II, nuclear actin and myosin I beta (NMIb) and they are the components of the pre-initiation complex (PIC) for gene transcription. To confirm an in vitro interaction of Itch with PIC components in mammalian cells, co-immunoprecipi- Now it is well known that nucleic acids (NAs) are normally pres- ent in mammalian extracellular fluids such as plasma of either blood or milk, urine and the medium condensed by cultured cells. Early studies showed that circulating, as well as exogenously injected NAs, are readily captured, spontaneously internalized and accumulated in the cytoplasm or even in the nucleus of mammalian cells. NAs after internalization are rapidly hydro- lyzed to oligo- and mono- nucleotides, but a part of accepted NAs survive and can be involved in induction and regulation of numerous cellular processes as a signal or guide molecules. The internalization of NAs, in addition to hydrolysis, may be accom- panied by a wide range of covalent modifications. Modifications of purine or pyrimidine residues can influence either stability and functionality of accepted NAs. In order to analyse nucleotide modifications introduced by human cells in internalized extracel- lular RNAs we used synthetic analogs of extracellular RNAs. It was shown that these RNAs and their fragments are readily cap- tured by human adenocarcinoma MCF-7 cells from culture med- ium and delivered to cytoplasm and nuclei. Most of RNAs internalized by human cells were hydrolyzed to mono- and oligo- nucleotides. Accepted full-length RNAs were detected by RT- PCR in cytoplasm since 1 h and in nuclei – 2 h after the pulse addition of RNAs to the culture medium. 5’-Inosine was the only detectable modified nucleotide among canonical ones in P1 hydrolysate of internalized RNAs. Consequently, extracellular RNAs that penetrated into human cells were subjected to partial adenosine deamination. Sequencing of cDNA confirms that full- lenth extracellular RNAs accumulated in nucleus, but not in cytoplasm, partially edited by adenosine deaminases. Deamina- tion revealed in nuclear stored full-length RNAs was site-specific rather internalized RNAs, apparently, were promiscuously modified. Taking together our

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(17b-E2)

tation and immunostaining were performed in both TM4 cells (Sertoli cell cell line) and HeLa cells and confirmed that Itch indeed physically associates with these PIC components. By expression and purification of truncated Itch coupled with co- immunoprecipitation, we confirmed that the WW domain in Itch is responsible for the interaction with RNA pol II and NMIb. To examine whether Itch is involved in the RNA pol II-mediated gene transcription, in vitro gene transcription and in vivo BrUTP incor- poration assays were performed. It was found that in vitro tran- scription is significantly reduced in the presence of anti-Itch antibody, but not serum. Knockdown of Itch by siRNA in cul- tured cells reduced the BrUTP incorporation in the nascent tran- scripts. Taken together, Itch is a PIC component and is crucial for RNA pol II-dependent transcription. Acknowledgements: This work was supported by Hong Kong Research Grant Council (HKU7609/06M) and CRCG Seed Funding for Basic Research.

P4–82 Stimulation-dependent nuclear translocation of myosin VI in PC12 cells L. Majewski1, M. Sobczak1, A. Wasik2, K. Skowronek1 and M. J. Redowicz1 1Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, POLAND, 2Department of Cell Biology, Nencki Institute of Experimental Biology, Warsaw, POLAND

women, 17b-estradiol is a steroid involved in bone metabolism but also in the regulation of cartilaginous matrix homeostasis. The aim of present study was, therefore, to investi- gate the molecular mechanisms regulating type II collagen gene expression under the effect of 17b-E2, and to characterize the genomic pathway via estrogen receptors (ER). Results: We showed that 17b-E2 could stimulate the expression of its own receptor and also type II collagen neosynthesis, in dif- ferentiated and in vitro dedifferentiated rabbit articular chondro- cytes. The effect on type II collagen is accompanied by an increase in the corresponding steady-state levels of COL2A1 mRNAs, suggesting that 17b-E2 acts at the transcriptional level through a genomic effect of ERa. The data showed that ERa induced an activating effect on COL2A1 gene transcription, med- iated by the proximal promoter covering the -266/+121 bp region. This sequence contains eight GC boxes, known to be potential DNA binding sites for the transcription factors Sp1 and/or Sp3. Experiments performed with a binding inhibitor to GC-rich sequences, mithramycin A, allowed us to confirm the these cis sequences in the transactivation of involvement of COL2A1 by ERa. In order to finely delineate the ERa protein sequences involved in transcriptional activation of human COL2A1 gene, transient transfections were performed with estro- gen receptors constructs containing serial deletions of functional domains of ERa: this effect requires the ERa transactivation domain, AF-1, as demonstrated by using a mutant ERalacking this domain. Moreover, physical interactions between ERa/Sp1 transcription factors have been demonstrated by immunoprecipi- tation experiments and validated in vivo by chromatin immuno- precipitation (ChIP) on COL2A1 promoter in primary and dedifferentiated chondrocytes. Conclusion: Understanding the molecular basis for 17b-E2 induction of COL2A1 transcription provides new insights into molecular mechanisms of OA and may facilitate identification of novel approaches for the treatment against degenerative diseases affecting articular cartilage.

Myosin VI (MVI) is the only known myosin walking towards minus end of actin filaments. In secretory adrenal medulla cells, MVI (but not myosins IB and IIB) was detected in isolated chro- maffin granules preparations and was tightly associated with the granule apical surface. MVI was not only found in secretory granules, Golgi, endoplasmic reticulum and clathrin-coated pits but also in nucleus. Nuclear localization was even more pro- nounced upon cell stimulation and/or incubation with leptomycin B, inhibitor of nuclear export. Bioinformatic analysis revealed one possible nuclear export (around leucine 1014) and several nuclear localization signal sequences within MVI tail domain. In contrast, myosin V upon stimulation translocated together with chromaffin granules to the peripheral area. Quantification of MVI distribution before and after stimulation confirmed its shift from cytoplasm to nucleus, and correlated with its nuclear colo- calization with transcription factor Sp1. When MVI expression was reduced up to 70%, loss of nuclear localization, destabiliza- tion of Golgi complexes as well as a slight increase of catechol- amine secretion were observed. These data indicate that in adrenal medulla cells MVI may be engaged in stimulation-depen- dent nucleo-cytoplasmic translocation, Golgi integrity as well as actin cortex organization.

P4–84 Phenotypic, genotypic and proteomic characterization of J774 macrophages upon chronic exposure to fluoroquinolone antibiotics B. Marquez1, N. E. Caceres1, M. Aerts2, C. Vallet1, M. P. Mingeot-Leclercq1, P. M. Tulkens1, B. Devreese2 and F. Van Bambeke1 1Universite´ catholique de Louvain, Unite´ de Pharmacologie cellu- laire et mole´culaire, Brussels, BELGIUM, 2Laboratory of Protein Biochemistry and Biomolecular Engineering, Ghent University, Ghent, BELGIUM

P4–83 Up-regulation of type II collagen gene expression by 17beta-estradiol in differentiated and dedifferentiated chondrocytes L. Maneix1, A. Servent1, B. Pore´ e1, N. Boujrad2, G. Flouriot2, K. Boumediene1, S. Moslemi1 and P. Gale´ ra1 1Laboratory Extracellular Matrix and Pathology EA 3214, Faculty of Medicine, Caen, FRANCE, 2Laboratory of Molecular Endocri- nology of Reproduction CNRS UMR 6026, University of Rennes I, Rennes, FRANCE

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Introduction: Type II collagen, encoded by a unique gene called COL2A1, is a phenotypic marker of articular cartilage whose expression is strongly decreased during osteoarthritis (OA). As reflected by the elevated risk of OA among post-menopausal Multidrug transporter overexpression can be selected through exposure to sublethal concentrations of drug substrates. The flu- oroquinolone ciprofloxacin is substrate of Mrp4 (a multidrug ABC transporter) in J774 macrophages, while a closely-related but more lipophilic molecule, moxifloxacin, is not affected. We have exposed J774 macrophages for several months to increasing concentrations of ciprofloxacin or moxifloxacin to compare the changes in phenotype, gene expression and membrane proteome, in comparison with wild-type cells. Fluoroquinolones were assayed by fluorimetry. Expression levels of Mrps were evaluated by real-time PCR and Western-blot. Relative abundance of mem- brane proteins in wild-type and ciprofloxacin-exposed macro- phages was assessed by ‘Stable Isotope Labelling with Amino acids in Cell culture’ technique (SILAC). Compared to wild-type

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cambialistic nature of SmSOD and prove its regulation by differ- ent metal contents. Therefore, the adaptative response of S. mu- tans during the aerobic exposure in the oral cavity could involve a different metal uptake of SmSOD.

P4–86 Generation and characterization of an MDCK cell line with reduced expression of b4-galactosyltransferase IV (b4GalT-IV) D. Maszczak, P. Drapala and M. Olczak Faculty of Biotechnology, University of Wroclaw, Wroclaw, POLAND

macrophages, cells exposed to ciprofloxacin showed a 5-fold decreased accumulation of this antibiotic, related to faster efflux. In contrast, cells exposed to moxifloxacin showed a 2-fold larger accumulation of ciprofloxacin together with a slower efflux. Moxifloxacin accumulation was high in all cell types. Real-time PCR and Western-blot showed an overexpression of Mrp2 and Mrp4 in ciprofloxacin-exposed macrophages and a reduction of Mrp4 expression in moxifloxacin-exposed macrophages. SILAC analysis revealed a change in expression level for 8% of mem- brane proteins upon exposure to ciprofloxacin, with proteins involved in transport, metabolic and communication pathways being the most affected. Exposure of J774 macrophages to the Mrp4 substrate ciprofloxacin selects for resistant cells overex- pressing Mrp4, and showing complex modifications in their pro- teome. Exposure to a non-substrate (moxifloxacin) selects for an opposite phenotype, with a decreased Mrp4 expression.

forming poly-N-acetyllactosamine structures present

P4–85 Superoxide dismutase from the dental pathogenic microorganism Streptococcus mutans M. Masullo1, A. De Vendittis2, M. Amato3, C. Cappelletti3, R. Cotugno2, M. R. Ruocco2, S. Rengo3 and E. De Vendittis2 1Dipartimento di Scienze Farmacobiologiche, Universita’ degli Studi ‘Magna Graecia’ di Catanzaro, Roccelletta di Borgia (CZ), ITALY, 2Dipartimento di Biochimica e Biotecnologie Mediche, Universita’ degli Studi di Napoli Federico II, Napoli, ITALY, 3Dipartimento di Scienze Odontostomatologiche e Maxillo Facciali, Universita’ degli Studi di Napoli Federico II, Napoli, ITALY

the phenotype of

b4-galactosyltransferases are Golgi-resident membrane-bound enzymes which transfer galactose from the uridine diphosphoga- lactose (UDP-Gal) to the terminal b-N-acetylglucosamine resi- dues in certain glycoproteins and glycolipids. Poly-N-acetyllactosamine chains are also present in keratan sulfate, which is the only galac- tose-rich glycosaminoglycan. Among seven known human b4-ga- lactosyltransferases only b4GalT-IV is linked to the keratan sulfate biosynthesis. MDCK cells produce large amounts of the keratan sulfate containing proteoglycans in contrast to the UDP- galactose transporter-deficient mutant cell line (MDCK-RCAr). Using PCR-based strategy (RACE) we generated and sequenced a cDNA clone containing the complete coding region for b4GalT-IV from MDCK cells. In order to inhibit b4GalT-IV expression in these cells via RNA interference we performed a stable transfection with an appropriate shRNA-generating plas- mid. We also transfected cells with the control vector. We obtained stable transfectants with a markedly reduced b4GalT- IV expression comparing with the control, as confirmed by the RT-PCR technique. We performed a detailed, comparative analy- sis of the glycoconjugates produced by the b4GalT-IV-deficient and control MDCK cell lines revealing that the former exhibited a dramatic decrease in the keratan sulfate content without signifi- cant effect on the structure of the galactose-containing complex type N-glycans. These results suggest that b4GalT-IV is specifi- cally responsible for the keratan sulfate biosynthesis in MDCK cells and not for the galactosylation of the complex type N-gly- cans. Interestingly, the b4GalT-IV-deficient MDCK cells resembled that of the MDCK-RCAr mutant cell line, implying some functional connection between b4GalT-IV and the UDP-galactose transporter.

P4–87 Estimation of heavy metals salts mutagenic activity with application of test-systems of Tradescantia (Clone 02) M. Matevosyan Chair of Genetics and Cytology, Yerevan State University, Yerevan, ARMENIA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

For the estimation of genotoxic effects of separate chemical mat- ters, exactly of heavy metals (HM) it is reasonable to use plant test-systems. The most sensitive among them are: computation of Tradescantia flower stamen hair (Trad-SHM) and Tradescantia micronucleus (Trad-MCN) assay. For the modeling of heavy metals mutagenic activity we have made an experiments with the Streptococcus mutans is an eubacterial pathogenic microorgan- ism involved in the development of dental caries, growing either in aerobic or anaerobic conditions. The bacterium lacks cyto- chromes and catalase, but possesses other anti-oxidant enzymes involved in the control of the intracellular redox potential, such as superoxide dismutase (SOD), the key enzyme scavenging the toxic superoxide anions. Previous researches suggested that SOD from S. mutans (SmSOD) belongs to the cambialistic group, functioning with Fe or Mn in the active site; this feature is prob- ably related to the adaptation of this microorganism to different growth conditions. Recently, the genome of S. mutans has been sequenced, and one SOD gene has been identified. SmSOD was heterologously produced in Escherichia coli, with its C-terminal extremity fused to a His-tag. This recombinant form of the enzyme (rSmSOD) had a specific activity of 1150 U/mg and con- tained both Fe and Mn (0.45 and 0.07 atoms/subunit, respec- tively). To improve its metal content, rSmSOD was incubated in the presence of Mn or Fe ions and, after extensive dialysis, metal content and specific activity were redetermined. The amount of Mn in the sample treated with Mn (SmSOD–Mn) raised to 0.21 atoms/subunit, whereas the Fe content remained unchanged. the specific activity of SmSOD–Mn improved to Moreover, 3500 U/mg. Vice versa, in the sample treated with Fe (SmSOD– Fe), the Fe content reached 0.52 atoms/subunit at the expense of Mn; the specific activity of SmSOD-Fe slightly decreased to 1050 U/mg. rSmSOD was half-inactivated after 10 min exposure at 68(cid:2)C. The effect of typical inhibitors/inactivators of Fe- and Mn-SODs was investigated, in terms of a possible regulation of the sensitivity by samples with different metal contents. Sodium azide functioned as a weak inhibitor of SmSOD, the lowest resis- tance being displayed by SmSOD-Fe. Hydrogen peroxide caused a modest inactivation on either untreated rSmSOD or SmSOD- Fe, whereas it was almost ineffective on SmSOD–Mn. Sodium peroxynitrite provoked a significant inactivation of SmSOD–Mn; the untreated rSmSOD and even more SmSOD–Fe were more resistant to peroxynitrite inactivation. These results confirm the

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application of Tradescantia flower stamen hair and micronucleus tests for the estimation of the following salts of heavy metals mutagenicity CrCl3, Pb (NO3)2, ZnSO4, NiCl2, CuCl2 and chrome oxide. Identified the frequency of induction of somatic recessive mutation and micronucleus under the influence of salts of heavy metals solution CrCl3, Pb (NO3)2, ZnSO4, NiCl2, CuCl2 and chrome oxide with the application Tradescantia stamen hair (Trad-SHM) mutation assay and the Tradescantia micronucleus (Trad-MCN) assay. The ions of the same metals which have the different valence are different by their mutagenic activity. Besides the mentioned mutations with the strengthening of heavy metals salts there are also another types of deviations: branching stamen hair, development of dwarf stamen hair, accretion of stamen hair, development of denuded stamen hair and another without pullen, the change of number of stamen, petal and sepals in flower. It is shown that the ions of investigated heavy metals are genotoxic. They induce somatic point mutations and clastogen effects in saprogenic cells of Tradescantia.

P4–88 Effects of inducible Hsp70 on staurosporine induced apoptosis M. Matokanovic1, R. Novak2 and K. Barisic1 1Department of Medical Biochemistry and Haematology, Univer- sity of Zagreb – Faculty of Pharmacy and Biochemistry, Zagreb, CROATIA, 2Department of Biochemistry and Molecular Biology, University of Zagreb – Faculty of Pharmacy and Biochemistry, Zagreb, CROATIA

Italian MIUR grants genome. We used a rat cardiomyoblast cell line (H9c2) to investi- gate mitochondrial biogenesis at morphological and biochemical level in relation with the cardiomyocyte-like differentiation pro- cess, consistent with the high energy requirement of the heart cells, Differentiation was induced in H9c2 (all-trans-retinoic acid and serum deprivation) and monitored with time by reduction of cell proliferation and expression of specific markers (troponin 1, myosin heavy chain, alfa-sarcomeric actin). The mitochondrial mass increased during differentiation, as documented by confocal microscopy and cytofluorimetry (mitochondrial probes, immu- nolabelling for cytochrome c and beta subunit of F0F1ATP syn- thase) as well as by western blotting analyses (cytochrome c and beta subunit). The mitochondrial morphology, analysed by con- focal high magnification techniques, showed an increase in per- centage of cells exhibiting a reticular network organization in H9c2 induced to differentiation (50% vs. 19%), conversely a fragmented morphology well characterised the parental cardio- myoblasts. We further evaluated functional changes, by compar- ing mitochondrial respiration and F0F1ATP synthase activity in mitochondria isolated from parental and differentiating cardio- myoblats. Mitochondria from differentiating cells showed a higher basal respiration and an improved mitochondrial cou- pling, measured as ratio between ADP-dependent and oligomy- cin-sensitive respiration. This suggests a better bioenergetic efficiency upon cardiomyogenesis, also confirmed by a higher mitochondrial transmembrane potential (JC-1 green and red fluo- rescence flow cytometry analyses). A significant increase of ATP synthesis capacity was also observed in mitochondria from differ- entiating cells with respect to those from control cells. This is in line with the increased expression of beta-subunit and suggests that the F0F1ATP synthase biogenesis and functionality are improved by in vitro cardiomyogenesis. Together these results suggest that mitochondrial number, morphology and function are up-regulated during cardiomyocyte-like differentiation, in accor- dance with the higher energy demand of cardiomyocytes. Acknowledgements: Supported by (PRIN 2007 and Italian Human ProteomeNet Project 2007).

P4–90 Novel RNAse activities of proteasome alpha- type subunits are found to be affected by apoptosis induction T. Moiseeva, O. Fedorova, A. Mittenberg and N. Barlev Institute of Cytology Russian Academy of Science, Structural organisation of genome, Saint-Petersburg, RUSSIA

Apoptosis, the process of programmed cell death, is characterized by a sequence of precisely regulated events that culminate in the self destruction of a cell. Expression of heat shock proteins (Hsp) is known to correlate with increased resistance to apoptosis induced by a range of diverse cytotoxic agents and has been implicated in chemotherapeutic resistance of tumors and carcino- genesis. Staurosporine, often used for apoptosis induction, is an alkaloid with ability to inhibit protein kinases through the pre- vention of ATP binding. Hsp70 is one of the major heat shock inducible proteins of the Hsp family. Jurkat cells (acute T cell leukemia), if exposed to heat stress (30 min, 44(cid:2)C),express higher concentration of inducible Hsp70 (9–12.5 times elevation com- pared to the control cells), depending on the time left for the recovery in custom cell culture conditions. Induction of apoptosis with staurosporine was assessed at morphological level, by Flow Cytometry analysis, and at the level of terminal event of cell death, DNA fragmentation, by immune detection of single- and double-stranded DNA breaks. It is shown that increased concen- tration of inducible Hsp70 shifts the percentage of live cells from 37 to 55% (1, 5·), predicting the role of inducible Hsp70 in pro- tection against staurosporine induced apoptosis with involvement of 36%.

P4–89 Morphological and functional mitochondrial changes upon in vitro cardiomyogenesis M. Comelli, L. Tomasetig and I. Mavelli Biomedical Sciences and Technoogies, University of Udine, Udine, ITALY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

As the energy demand of a cell can change dramatically during development and differentiation, the mitochondrial content and function can be adjusted to suit the current state of the cell by a well-orchestrated regulation of both nuclear and mitochondrial Proteasome is a multisubunit protein complex that is essential in most cell protein degradation. Lately some new roles of protea- somes have been discovered – the participation in transcription regulation and the ability to degrade RNA. The ability of protea- somes to cleave RNA was found to be specific and targeting mostly AU-reach regions. Then two proteasome subunits of alpha-type possessing RNAse activity were identified – alpha5 and alpha1. In order to identify all alpha-type proteasome subun- its, that might be able to degrade RNA, we have performed the 2D-electrophoresis of proteasomes, extracted from nuclei and cytoplasm of K562 cells. Then the spots, corresponding to alpha- type proteasome subunits, were cut out of the gels, the proteins were extracted and tested for their ability to cleave the radioac- tive-labeled RNA-substrate – p53 mRNA, synthesized in vitro. The reaction was performed using three types of conditions in each case – in the absence of bivalent cations and in the presence of Ca2+ or Mg2+. 5 alpha-type proteasome subunits from cyto-

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cells and both DG subunits were analyzed by Western blot exper- iments as well as immunofluorescence. Our results show that c- myc prevents the proteolytic cleavage, when it is located a few amino acids upstream the DG precursor breakdown site. Further studies will be needed to verify whether the uncleaved DG is properly targeted to the plasma membrane; however, preliminary immunofluorescence data seem to corroborate this hypothesis. On the contrary, when the c-myc epitope is inserted beside the DG signal peptide, at the very N-terminal of the a-DG subunit, we detected a possible proteolytic degradation process that pro- duces a shedding into the culture media of the N-terminal por- tion of a-DG; this peptide needs to be further characterized by proteomic analysis. The N-terminal degradation of a-DG can be important in the pathogenesis of muscular dystrophies as well as of other diseases, including cancer progression. Therefore, this novel construct is likely to represent a useful tool for the study of the DG function. References: 1. Sciandra et al. Trends Biotechnol. 2007; 25: 262–268. 2. Bozzi et al. FEBS J. 2006; 273: 4929–4943.

plasm appeared to be able to degrade RNA. Their activities increased in the presence of calcium ions. As for nuclear protea- somes, there were just two subunits, possessing RNAse activity – alpha5 and alpha7. Moreover, nuclear alpha7 subunit was the most active in the presence of magnesium ions. On order to study the regulation of RNAse activity we extracted proteasomes from the nuclei and cytoplasm of the cells, induced to apoptosis by DNA-damaging agent – doxorubicin. The proteasome subunits were separated by 2D-electrophoresis, and proteins, extracted from the spots, corresponding to alpha-type proteasome subunits, were tested for the ability to cleave the same RNA-substrate in the same three types of conditions to compare their activity with that of the proteasome subunits form the control cells. All tested spots were also studied by mass-spectrometry in order to identify the proteins in the spot and to find some changes and posttrans- lational modifications that can modulate the RNAse activity. The same subunits were found to degrade the given substrate, except for alpha6, which had lost its RNAse activity under the action of doxorubicin. Some changes in the conditions preferences were detected for almost all active alpha-type proteasome subunits. Acknowledgements: This work was supported by Molecular and Cell Biology Program of Russian Academy of Sciences (MCB RAS) and Russian Foundation for Basic Research (pro- ject No.08-04-00834).

P4–92 The study of succinate dehydrogenase and bc1 complex interaction in intact liver mitochondria oxidizing artificial substrate duroquinol K. Motovilov1, G. Kolesova1, N. Sumbatyan2 and L. Yaguzhinsky1 1A.N. Belozersky Intitute for Physico-Chemical Biology, Bioener- getics, Moscow, RUSSIA, 2Lomonosov Moscow State University, Chemistry of Natural Compounds, Moscow, RUSSIA

P4–91 Alpha- and beta-dystroglycan: evaluation of processing and targeting through multiple fluorescent taggin S. Morlacchi1, M. Bozzi2, A. Galtieri3, B. Giardina2, F. Sciandra4 and A. Brancaccio4 1Biologia Animale ed Ecologia Marina, Universita degli Studi di Messina, Messina, ITALY, 2Biochimica e Biochimica Clinica, Universita Cattolica del Sacro Cuore, Roma, ITALY, 3Chimica Organica e Biologica, Universita degli Studi di Messina, Messina, ITALY, 4CNR, Chimica del Riconoscimento Molecolare, Roma, ITALY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Inhibitory analysis was applied to study the functional interac- tions of succinatedehydrogenase (SDH) and bc1-complex of liver mitochondria oxidizing artificial substrate (duroquinol). It was found out that highly purified duroquinol is able to interact with the SDH succinate binding site. In presence of dicumarol and rotenone duroquinol induced respiration of mitochondria is partly inhibited (40–60%) by TTFA or malonate and completely inhibited by normal concentrations of myxothiazol, antimycin and cyanide. Semiquinone formation induced by the 10 : 1 mix- ture of duroquinol:duroquinone icreases the rate of duroquinol induced respiration by 20–30% compared to the rate with pure duroquinol, while the effect of SDH inhibitors disappears. Inhibi- tors of bc1-complex and cytochrome c oxidase still suppress respi- ration completely. The results obtained led us to presume that complex III – complex II system brings about three different states formed fully by initial conditions. The first state corre- sponds to oxidation of highly-purified duroquinol when respira- tion rate is limited by N-center of Q-cycle. The second state takes place if respiration rate is limited by P-center of Q-cycle. The precise type of SDH-bc1 system state depends on malonate (1 mM) addition to the medium given before duroquinol. Malo- nate addition has no inhibiting effect on duroqinol induced respi- ration in this case. Third state occures when duroquinone and duroquinol are added to the medium with mitochondria simulta- neously. SDH doesn’t participate in oxidation of duroquinol and mitochondrial respiration rate is not controlled by bc1-complex and in this state (low concentrations of antimycin and my- xothiazol have no effect on DQH2 oxidation rate). Cynide in nor- respiration completely while this mal concentration inhibits myxothiazol and antimycin require enlarged concentrations. Acti- vation of the precise state, one of three, strictly depends on initial conditions. This means that SDH-bc1-complex system is com- pletely controlled by powerful feedbacks. Dystroglycan (DG) is a pivotal member of the dystrophin-glyco- protein complex. DG is composed of two subunits, a and b. a-DG is a highly glycosylated extracellular protein that interacts noncovalently with b-DG and other extracellular proteins; b-DG crosses the plasma membrane and interacts extracellularly with a-DG, whereas its cytosolic domain is connected to dystrophin. In muscular dystrophies, the membrane localization of DG can be largely altered, contributing to the destabilization of sarco- lemma. DG is encoded by the dag1 gene and expressed as a sin- gle polypeptide precursor (1). Two post-translational events are extremely important for DG maturation: the proteolytic cleavage that liberates the two subunits and the decoration with O- and N-glycosylation groups. In order to better understand the molec- ular aspects underlying the DG precursor maturation, we pro- duced a series of knockin DNA constructs inserting the c-myc epitope within our intrinsically fluorescent DG-GFP chimaera (2). By multiple fluorescent tagging, we aimed at obtaining a bet- ter evaluation of the expression, processing and targeting patterns of the two DG subunits in eukaryotic cells. A series of constructs were prepared in which c-myc was inserted into different sites within the portion of the DG gene encoding for the a-DG sub- unit. All the novel constructs were used to transfect HEK 293

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P4–93 Histological Analysis of Spleen of Acomys cilicicus H. Mutlu, S. Kiralp, E. Kivanc and N. Ozsoy Biology, Ankara University Faculty of Science, Ankara, TURKEY

P4–95 bFGF induces changes in hyaluronan synthase and hyaluronidase isoform expression and modulates the migration capacity of fibrosarcoma cells D. Nikitovic, A. Berdiaki, A. Kouvidi, H. Syrogianni and G. Tzanakakis Department of Histology, University of Crete, Heraklion, GREECE

2 (HYAL2) hyalurodinase

Acomis cilicicus from Muridae family of rodentia order is an endemic specie in Turkey, spread over Silifke. Although there are studies about the taxonomy of this specie in the literature, there is no study about its histology. In this study, spleen of this specie was analyzed in detail histologically. Spleen is enveloped by dense connective tissue and it is the largest lymphoid tissue in the body. In this study three adult samples two male and one female spiny mice were collected from the area and were grown up in the laboratory. Spleen tissues obtained from these samples were fixed in 10% formalin and embedded in paraffin. Haematoxylin- Eosin, Masson’s trichrome, Gomori’s silver impregnation and Periodic Acid Schiff (PAS) were used for staining the obtained paraffin sections. White pulpa, red pulpa and trabeculae were observed clearly under light microscope using sections stained with Haematoxylin-Eosin. Connective tissue and vein walls were stained using trichrome. In silver staining, it is found that reticu- lar fibers were stained intensely around red and white pulpa. PAS positive cells were evaluated.

P4–94 Heparin-induced proliferation on human colon cancer cell lines is mediated through p38 mitogen-activated protein kinase D. Nikitovic, G. Chatzinikolaou, A. Berdiaki, A. Zafiropoulos and G. Tzanakakis Department of Histology, University of Crete, Heraklion, GREECE

Hyaluronan (HA), a high molecular weight glycosaminoglycan (GAG), has been correlated with malignant progression in several types of human neoplasia. Fibrosarcoma is a rare malignant tumor originating from fibroblasts whose extracellular matrix is rich in GAGs. In this study, we investigated whether changes in the HA metabolism of two fibrosarcoma cell lines, HT1080 and B6FS, induced by basic fibroblast growth factor (bFGF) associ- ate to their migratory abilities. bFGF inhibited HT1080 cell but did not modulate B6FS fibrosarcoma cell motility. Moreover, bFGF decreased expression (P = 0.0028) inhibiting the degradation of HA in HT1080 cells. The reduction in HYAL2 expression together with hyaluronan synthase 1 and 2 (HAS1 and HAS2) expression stimulation, as previously reported by us, enhanced high molecular weight HA deposition in the pericellular matrix of HT1080 cells. The increased content of endogenous HA correlated with a significant decrease of HT1080 cell migration (P = 0.0022). Treatment of these cells with exogenous high molecular weight HA showed a similar reduction in cell motility (P = 0.0268). Furthermore, induced degradation of the HA content by hyaluronidase treat- ment, resulted in a significant stimulation of HT1080 cells’ motil- ity (P = 0.0098) thus, corroborating the participation of HA- metabolism in their migration. Moreover, HT1080 cells migratory capaqcity was not affected by the inhibition in HAS2 expression, demonstrating that mainly HAS1 synthesized high molecular weight HA regulates their motility. In conclusion, bFGF regu- lates, in a cell-specific manner the migration capability of fibro- sarcoma cells by modulating their HA metabolism.

P4–96 Inhibition of Plasmodium falciparum proliferation in vitro by double-strandedRNA directed against malaria histone deacetylase and topoisomeraseII W. Noonpakdee1, S. Boonma1, N. Sriwilaijaroen2 and S. Panyim3 1Biochemistry, Faculty of Science Mahidol University, Bangkok, THAILAND, 2Graduate studies, Faculty of medicine Thammasat University, pathumtani, THAILAND, 3Biochemistry, Faculty of Science Mahidol University, Bangkok, THAILAND

(1.8-fold, P < 0.001)

Cancer progression requires a continually evolving network of interactions between neoplastic cells and the surrounding micro- environment. Heparin acts as an extracellular stimulus capable of activating major cellular signaling pathways. In the current study the possible involvement of mitogen activated protein kinase (MAPK) signaling pathways on heparin-induced HT29, SW1116 and HCT116 colon cancer cell growth was evaluated by utilizing specific JNK (SP600125), MEK (U0126) and p38 (SB203580) inhibitors. Treatment with the JNK or MEK inhibitor had no significant effect on heparin-stimulated colon cancer cell prolifer- ation. In contrast, the inhibition of p38 kinase caused a statisti- cally significant decrease in heparin-stimulated HT29 (2.7-fold, P < 0.001), SW1116 (2-fold, P < 0.001) and HCT116 (3.3-fold, P < 0.001) cell proliferation. The activation of p38 MAPK path- way by heparin was verified by western blot analysis. Heparin induced an increase in phosphorylation of p38 kinase in HT29 (2.6-fold, P < 0.001), SW1116 and HCT116 (1.9-fold, P < 0.001) cells. Moreover, the effect of hep- arin, p38 inhibitor or their combination on p21WAF1/cip1, p53 and cyclin D1 gene expression was analyzed using real-time quantitative PCR. In the presence of heparin, p21WAF1/cip1 and p53 transcripts levels were strongly reduced (up to 2-fold or 1.8 fold, P < 0.001), whereas cyclin D1 expression was signifi- cantly increased (up to 1.8-fold, P < 0.001) in all three cell lines through a p38-mediated mechanism. In conclusion, heparin, an extracellular glycosaminoglycan, may modulate the expression of cell-cycle genes through the activation of p38 MAPK to stimulate colon cancer cell proliferation.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

The development of new effective antimalarial agents is urgently needed due to the ineffectiveness of current drug regimes on the most virulent human malaria parasite Plasmodium falciparum. Histone deacetylase and topoisomerase II of the malarial para- sites have been proposed as potential new targets for antimalarial compounds. In this study, we report the result of using two long double-stranded RNA encoding a segment of the parasite histone deacetylase (pfHDAC) and topoisomerase II to interfere with the cognate messenger expression. Chloroquine- and pyrimethamine- resistant P. falciparum K1 strain was exposed to pfHDAC or to- poisomerase II dsRNA for 48 h and growth was determined by hypoxanthine incorporation assay. Exogeneously delivery of pfH- DAC dsRNA between 1 and 200 nM significantly inhibited para- site growth up to 45% as compared with either untreated cultures or cultures treated with unrelated dsRNA (gfp) which

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had some inhibitory effect (314%). In addition, the decrease in parasite growth correlated with the decrease in levels of pfHDAC mRNA. Similar result was also observed with topoisomerase dsRNA.

P4–97 Reversal of cyanide inhibition of cytochrome c oxidase by pyruvate H. Nuskova, A. Pecinova, P. Pecina, M. Vrbacky, Z. Drahota and J. Houstek Bioenergetics, Institute of Physiology AS CR v.v.i., Prague 4, CZECH REPUBLIC

MSSP complex bound to the myc (H-P) sequence. Here we fur- ther examined the involvement of Myc in the autonomous repli- cation of pmyc (H-P) by use of PCR. After pmyc (H-P) or the similar plasmid lacking the core 21 bp sequence for c-Myc/ MSSP-binding was introduced into human HeLa cells, low- molecular-weight DNA molecules were recovered in Hirt super- natant, digested by DpnI to exclude the input DNA and sub- jected to PCR. The results indicated that pmyc (H-P) replicated in the transfected cells dependent on the 21 bp sequence. The replicated pmyc (H-P) molecules were increased when the expres- sion vector for c-Myc was co-transfected to the cells, or by induc- tion of Myc-ER fusion protein in Rat-1-derived Myc-ER cells. Myc was thus suggested to beproactively involved in the autono- mous replication of pmyc (H-P). Several cell lines harbouring pmyc (H-P) extrachromosomally have been obtained and there- plication during cell cycle are being examined.

P4–99 Ouabain-induced apoptosis and Rho kinase A. O¨ zdemir1, B. Polat2 and M. Ark1 1Pharmacology, Gazi University, Ankara, TURKEY, 2Perinatolo- gy, Zekai Tahir Burak Maternity Hospital, Ankara, TURKEY

the rate of succinate-dependent

The activity of cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, is inhibited by cyanide. This inhibition can be reversed by a-ketoacids due to their reac- tion with cyanide that yields nontoxic cyanohydrines. We investi- gated the process how pyruvate recovers both electron-transport and H+-pumping activity of COX completely inhibited by 0.25 mM KCN. In isolated rat liver mitochondria, we measured the rate of succinate- and ascorbate + TMPD-dependent respira- tion using the OROBOROS oxygraph. In frozen/thawed mito- chondria, respiration was restored to 90%, whereas ascorbate + TMPD-dependent respira- tion only to 45% of original values. In freshly isolated mitochon- dria (state 3, ADP), the rate of ascorbate + TMPD-dependent respiration was recovered up to 70% of original values. To mea- sure changes of the mitochondrial membrane potential we employed the TPP+-selective electrode. Pyruvate recovered over 60% of proton pumping capacity of COX, which is in agreement with the electron flux data. We also determined free KCN con- centration in the medium. KCN was completely depleted already by 5 mM pyruvate, but neither respiration nor membrane poten- tial became completely restored, although there was no free cya- nide available for COX inhibition. Maximal recovery of COX activity was observed at higher pyruvate concentration (40 mM), which implies that the recovery proceeds further due to the release of KCN bound to COX. Nevertheless, the recovery of electron flux as well as proton pumping capacity is never com- plete, which indicates that KCN binding might induce irreversible modification of COX.

Ouabain is a specific inhibitor of Na+, K+-ATPase but also oua- bain-Na+, K+-ATPase complex influence various cytosolic sig- naling events. Recent studies have showed that the cytotoxic doses of ouabain induced cell death. The Rho GTPase effectors, Rho kinases (ROCK I and ROCK II), which play central roles in the organization of the actin cytoskeleton, contributes to plasma membrane blebbing in several models of apoptosis. In this study, we investigate the possible role of Rho kinase in oua- bain-induced apoptosis. Human umbilical vein endothelial cells (HUVECs) were exposed to ouabain (0, 1–1–10 lM, 36 h), Y27632 (10 lM, 30 min), z-VAD-fmk (50 lM, 2 h). Ouabain- dependent changes were evaluated with phase-contrast and fluo- rescence microscopy, TUNEL assay, Western-blot. Phase con- trast and fluorescence microscopy observations showed that ouabain treatment resulted in a dose dependent increase in apop- totic blebbing. Pretreatment of cells with a specific ROCK inhibi- tor, Y27632, In addition, reduced the formation of blebs. ouabain increased cell detachment from substratum. However, Y-27632 significantly inhibited but did not prevented detachment of the cells. TUNEL reaction demonstrated that ouabain induced apoptosis but Y27632 did not abrogated this effect. Ouabain induced cleavage of ROCK I and ROCK II, and this activation was also prevented by the caspase inhibitor z-VAD-fmk. Our data indicate for the first time in this study that ouabain induces both ROCK I and ROCK II cleavage via caspase dependent mechanisms. Our data also provide evidence that ROCKs involve in ouabain-induced cell detachment. These results may be impor- tant for the treatment of several diseases such as hypertension and malignancies.

P4–98 Myc-dependent autonomous DNA replication of the sequence upstream of human c-myc gene S. Okumura1, T. Niki2, S. Ishikawa3, H. Ariga2 and S. Iguchi-Ariga1 1Graduate School of Agriculture, Hokkaido University, Sapporo, JAPAN, 2Graduate School of Pharmaceutical Science, Hokkaido University, Sapporo, JAPAN, 3Graduate School of Life Science, Hokkaido University, Sapporo, JAPAN

P4–100 The establishment of Green Fluorescent Protein expressing CHO cells by stable transfection using activated dendrimers and G418 selection B. Celtikci1, N. Purali2 and H. A. Ozkara1 1Department of Biochemistry, Hacettepe University Faculty of Medicine, Ankara, TURKEY, 2Department of Biophysics, Hacettepe University Faculty of Medicine, Ankara, TURKEY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Stable transfection of Green Fluorescent Protein (GFP) has been widely used as a marker for both monitoring protein expression Myc has been implicated for key roles in cell proliferation, differ- entiation, and apoptosis, but major function(s) have remained obscure. In addition to the transcriptional activities, Myc func- tions in DNA replication have recently been highlighted again, at global level via histones and at local sites of replication origins. We have previously identified the 210 bp sequence between the HindIII site and the PstI site in the region 2 kb upstream of human c-myc gene as an initiation site of DNA replication and the plasmid pmyc (H-P), bearing the HindIII-PstI sequence (myc (H-P) sequence) in pUC19, showed extrachromosomal, autono- mous replication, not only in human and mouse culture cells, but also in transgenic mice. Moreover, we have shown that c-Myc/

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Poster Presentations

P4–102 Prevalent cardiac mitochondrial toxicity in a sub-chronic in vivo model of doxorubicin-induced toxicity G. C. Pereira1, S. S. Pereira1, M. S. Santos1, A. J. Moreno2 and P. J. Oliveira2 1Department of Zoology, Centre for Neuroscience and Cell Biol- ogy, Coimbra, PORTUGAL, 2Department of Zoology, Institute for Marine Research (IMAR), Coimbra, PORTUGAL

and direct observations of cellular dynamics. Despite this proven utility of GFP as a marker, the appropriate conditions of stable transfection of kanamycine-resistant pIRES-EGFP in CHO cells by activated dendrimers, required concentration of G418 selec- tion and the effect of passage number on fluorescence radiation time has not been fully described. To address these issues and the establishment of GFP expressing CHO cells for further experi- ments, we transfected these cells with pIRES2-EGFP activated dendrimers. We selected these transfected cells by using G418 (500 lg/ml for 3 weeks). Selected transfected cells were visualised by confocal microscopy and were seperated using FACS method. After 48 h following transfection, 5% of cells were transiently transfected and their fluorescence faded within 4 days. Following G418 treatment for 3 weeks, 10% of the stably transfected cells survived, as we observed by their fluorescence. GFP expressing cells had complex morphology which is different from the usual fusiform shape of CHO cells. Even some stably transfected sur- viving cells showed change in their morphology, no fluorescence was detected. Unfortunately, transfected cells could not be sorted by FACS method, because of the low yield in the efficiency of stable transfection. This study demonstrates that fluorescent pro- teins can be succesfully expressed in mammalian cells for multiple purposes. Our future goal is to obtain homogeneous stable trans- fection of GFP in different cell lines.

P4–101 Correlation between neurotrphin 3 and interleukin 10 in the prognosis of viral and non viral neuroinflammation to the central nervous system P. Daniela1, G. Mihaela1, T. Traian1, B. Constantin1, S. Mihnea1, I. Loretta2, B. Coralia2 and S. Mihai2 1Pathophisiology and Immunology Department, Carol Davila University of Medicine and Pharmacy, Bucharest, Romania, 2Stefan S Nicolau, Institute of Virology, Bucharest, ROMANIA

Nowadays, doxorubicin (DOX) is used to treat several types of human tumors. However, treatment is accompanied by a cumula- tive cardiotoxicity with a strong mitochondrial dysfunction com- ponent but which full mechanism remains to be elucidated. The aim of this work was to evaluate and characterize the DOX- induced toxicity in a sub-chronic animal model at the mitochon- drial level. Eight weeks old male Wistar rats were weekly injected with saline solution or 2 mg/kg DOX, during 7 weeks. Heart mitochondria appear to be mostly affected, presenting lower membrane potential together with a decrease in both state 3 and state 4 respirations without differences in ADP/O ratio or RCR regardless of the substrate used. Hepatic mitochondria present only differences at the complex I level (membrane potential, lag phase and state 3) contrarily to renal mitochondria which have increased lag phase and decreased ADP/O ratio, however only when complex II substrates were used. Mitochondrial calcium loading capacity results indicate that cardiac and renal mitochon- dria from DOX-treated rats have increased calcium-release rate and decreased retention time, respectively. Hydrogen peroxide production by the MRC shows that heart and liver mitochondria from DOX-treated rats produce more hydrogen peroxide in the presence of complex I substrates. The present work is the first to show a direct comparison of in vivo DOX mitochondrial effects in three distinct organs from the same animal. In conclusion, our data supports the notion that DOX in vivo treatment causes mitochondrial alterations, which are more evident in the heart and that contribute for DOX-induced cardiomyopathy.

P4–103 The HspA2 chaperone protein accumulates at centrosomes and nucleoli of heat shocked cancer cells W. Piglowski1, P. Filipczak1, E. Malusecka2, Z. Krawczyk1 and D. Scieglinska1 1Department of Tumor Biology, Maria Sklodowska-Curie Memo- rial Cancer Center and Institute of Oncology, Gliwice, POLAND, 2Department of Radiotherapy, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, POLAND

The viral neuroinflammation of the CNS triggers a very complex signalization cascade. The cerebral tissue has naturally tendency to repair the lesion produced if this is achived by its components mediated by anti-inflammatory cytokines, neurotophic factors and gliosis, and the neuronal loos is prevented. In the present work we investigated the possible correlation of NT-3 with IL-10 in the serum and cerebrospinal fluid (CFS) from patients with ischemic stroke (IS) and acute viral meningitides (VIR_M) and encephalitis (VIR_E). 50 patients aged between 45–57 years (58.92 ± 8.76 years) were investigated. According to the clinical cours, these patients were divided into three categories. (1) Patients non-survivors 6.4 ± 3.4 days after the onset of the disease;

and from non-survivors

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Human HspA2 protein belongs to Hsp70 chaperone protein fam- ily. The HspA2 protein is necessary for progression of spermato- genesis, its reduced synthesis was found to be related to male infertility. However, the HSPA2 expression is not specific for sper- matogenic cells. The HSPA2 transcripts were reported to be pres- ent in human somatic tissues. Depletion of HspA2 might be involved in diminished growth and survival of cancer cells. Recently it was shown that HSPA2 promoter is activated by inter- action with HIF-1alpha during hypoxia. Here we present data on the HSPA2 gene expression and intracellular localization of HspA2 protein in cancer cell lines. Transcription level ofthe HSPA2 was determined by RealTime qRT-PCR, the protein level by Western blot using produced and purified by us monospecific the anti-HspA2 polyclonal serum. The highest expression of HspA2 protein was detected in A549 and NCI-H1299 lung cancer cells. Significant expression of the HspA2 was also detected by immunohistochemistry in primary NSCLC specimens. Intracellu- (2) Patients with medium course 10 ± 5.8 days; (3) Patients survivors. Serum and CFS sample were analysed using EuroClone ELISA kits. The Pearson correlation coefficient (r) was calculated. There was a direct correlation between the NT-3 and IL-10 in serum from patients with a medium course, IS r = 0.54 P = 0.025, M_VIR r = 0.59, P = 0.05 IS r = 0.63, P = 0.025, VIR_E r = 0.90, P = 0.05. On the con- trary an inverse CFS and serum correlation between NT-3 and IL-10 may be observed in patients survivors IS r = -0.51, P < 0.005, VIR_M r = -0.69, P < 0.005. An inverse CFS and serum correlation between NT-3 and IL-10 is associated with favorable prognosis of the disease and suggested that NT-3 could play an immunomodulatory role in patients with improve out- come after neuroinflammation. The absence of this correlation corresponded to imminence of death.

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lar localization of the HspA2 was analyzed using cell lines express- ing HspA2-GFP and RFP-HspA2 fusion proteins and confirmed by immunofluorescence using cells naturally expressing HspA2 and the specific anti-HspA2 antibody. In cells growing at normal conditions the HspA2 protein was localized in cytoplasm. During heat shock the HspA2 shifted into nucleus and nucleoli. We observed the HspA2 accumulation at interphase and mitotic cen- trosomes in heat-shocked cells. Our results suggest, that the HspA2 protein can be involved in protecting nucleoli and centrosomes integrity in cells subjected to heat shock, and possibly to other cel- lular stressors.

P4–104 Deubiquitination of Pex5p, the peroxisomal protein cycling receptor, is not a mandatory step in the Pex5p-mediated import pathway M. Pinto1, C. Grou1, A. Carvalho1, S. Huybrechts2, C. Sa-Miranda3, M. Fransen2 and J. Azevedo1 1IBMC, OBF, Porto, PORTUGAL, 2Departement Moleculaire Celbiologie, Faculty of Medicine, Leuven, BELGIUM, 3IBMC, Unilipe, Porto, PORTUGAL

but also is related to a massive deposition of connective tissue in the lamina propria. An important complication of hyperglycemia is the accumulation of advanced glycation end products of pro- teins in tissues, including periodontium. Vessels growth and pro- liferation, endothelial cells function and even collagen turnover could be affected in this condition. The aim of this study is to point out the influence of matrix metalloproteinase-3 (MMP-3) and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) on vascular endothelial growth factor (VEGF) expression, a key mediator of angiogenesis. Detection of MMP-3, TIMP-2 and VEGF was performed on gingiva samples, paraffin embedded, (n = 8 from type 2-diabetes patients and n = 4 from healthy subjects) processed through the usual sequence of the immunohis- tochemical technique. In the diabetic gingiva, pro-inflammatory and endothelial cells expressed both MMP-3 and TIMP-2; MMP-3 was also expressed in keratinocytes. Some pro-inflamma- tory and endothelial cells show intense positive reaction for VEGF. A faint immunopositive reaction for VEGF was also noted in keratinocytes from the basal and spinous layer. These results suggest that the interaction between keratinocytes and pro-inflammatory cells could regulate extracellular matrix turn- over and moreover that the activation of MMP-3 promotes angiogenesis by enhancing extracellular matrix degradation in gingival overgrowth related to diabetes mellitus.

P4–106 Prognostic value of circulating tumor cells in breast cancer V. Prokopova1, I. Janatkova1, T. Zima1, M. Cabinakova2, P. Tesarova2, J. Valchar3, J. Pavlasek4, D. Pinterova4 and K. Kolostova4 1General University Hospital and First Faculty of Medicine Charles University in Prague, Institute of Clinical Biochemistry and Laboratory Diagnostics, Prague 2, CZECH REPUBLIC, 2General University Hospital and First Faculty of Medicine Charles University in Prague, Clinic of Oncology and Radiology, Prague 2, CZECH REPUBLIC, 3General Hospital Kralovske´ Vinohrady and Third Faculty of Medicine Charles University Prague, Radiotherapy and Oncology Clinic, Prague 10, CZECH REPUBLIC, 4Third Faculty of Medicine Charles University Prague, Deptartment of Tumor biology, Prague 10, CZECH REPUBLIC

Newly synthesized peroxisome matrix proteins are transported into the organelle by the shuttling receptor Pex5p. After binding cargo proteins in the cytosol, Pex5p interacts with the docking/transloca- tion machinery (DTM) present at the peroxisomal membrane. The interaction of the Pex5p-cargo complex with this multisubunit machinery ultimately results in Pex5p insertion into the peroxi- somal membrane and release of the cargo protein into the organelle matrix. During its passage through the DTM, Pex5p is monoubiq- uitinated at a conserved cysteine residue, yielding a Pex5p-ubiqu- itin thiolester conjugate. This modification is required for the ATP- dependent dislocation of Pex5p back into the cytosol, by Pex1p and Pex6p, two members of the AAA family. In the cytosol, recy- cled Pex5p is then available to promote further cycles of protein import. Using an established in vitro system, we show that soluble monoubiquitinated Pex5p can still re-enter the peroxisomal DTM in a cargo-dependent way. Accordingly, we found that monoubiq- uitinated Pex5p retains the cargo-binding properties of the uncon- jugated protein. Furthermore, soluble monoubiquitinated Pex5p is a monomeric protein indicating that ATP-dependent dislocation of membrane-bound ubiquitin-Pex5p and deubiquitination of soluble ubiquitin-Pex5p are completely independent steps. Finally, steady- state analyses of rat liver Pex5p revealed the presence of mono- ubiquitinated Pex5p only in the organelle pellets. Thus, although deubiquitination of Pex5p in vivo seems to be a fast event, it is not a mandatory step of the Pex5p-mediated protein import pathway. Acknowledgements: Supported by FCT (PTDC program) and FEDER funds, Portugal, European Union VI Framework pro- gram, the Flemish Government and the Fonds voor Wet- enschappelijk Onderzoek – Vlaanderen.

P4–105 Influence of some mediators of extracellular matrix remodeling on angiogenesis in diabetic gingival overgrowth C. Pisoschi1, M. Banita2, C. Stanciulescu1, M. Fusaru3 and M. Gheorghita4 1Biochemistry, University of Medicine and Pharmacy, Craiova, ROMANIA, 2Histology, University of Medicine and Pharmacy, Craiova, ROMANIA, 3Pharmacy, University of Medicine and Pharmacy, Craiova, ROMANIA, 4Maxillo-Phacial Surgery, University of Medicine and Pharmacy, Craiova, ROMANIA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Introduction: The tumor cell dissemination from the primary tumor may increase the risk of relapse in breast cancer patient. The presence of circulating tumor cells (CTCs) in peripheral blood of breast cancer patients is associated with worse prognosis because of increased metastasis risk. The CTCs are indicating poor clinical outcome if detected after adjuvant therapy. Sequen- tial CTCs monitoring allows identifying patients at increased risk of disease progression and could help to describe the therapeutic efficacy and patient’s response to systematic treatment. Materials and Methods: 48 patients with diagnosed breast can- cer (stage I–III and metastatic disease) were enrolled into a pro- spective study (66 tests have been done in total). Primary tumors of tested patients have been characterized by 44, 83% ER and HER2 negativity. In 31% cases the histology grade was G3 and 49% patients had infiltrated axillary lymph nodes. Patient’s peripheral blood has been drawn before and/or after surgery, chemotherapy or radiotherapy. Immunomagnetic enrichment of CTCs from 5 ml whole blood has been processed using AdnaTest BreastCancerSelect. The isolated CTC cells were characterized by gene expression analysis of tumor-associated genes Her-2, MUC- 1 and GA 733-2 using AdnaTest BreastCancerDetect. A semi- quantitative analysis of PCR fragments has been performed on Agilent Bioanalyzer 2100. Gingival overgrowth is frequently found in subjects with uncon- trolled diabetes mellitus and has not only an inflammatory cause

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Poster Presentations

P4–108 Flotillins are regulating the retrograde transport of Shiga toxin and ricin in mammalian cells S. Pust, A. B. Dyve, M. L. Torgersen and K. Sandvig Biochemistry, Centre for Cancer Biomedicine Norwegian Radium Hospital, Oslo, NORWAY

Results: CTC positivity has been described in 35% from pro- cessed samples. It has been shown, that if patient did not respond to the therapy, the CTC count has increased in comparison to the first blood testing results or has been stable for several weeks. Interestingly 23% patients with HER2 negative primary tumor showed Her-2 positive CTCs. Further molecular characterization should help to improve and individualize treatment strategies in breast cancer patients. There is a big potential to prevent metas- tizing process by regular CTC examinations in diagnosed patients within adjuvant treatment. Conclusion: Detection of CTCs may provide a tool for moni- toring the efficacy of systematic therapy and could aid in appro- priate stratification of breast cancer patients and design of tailored therapy.

P4–107 Noradrenaline influence on the development of 6-hydroxydopamine-induced hyperprolactinemia in rats T. Pronina1, L. Dilmuhametova1, V. Kudrin2 and M. Ugrumov1 1Laboratory of hormonal regulations, Institute of developmental biology RAS, Moscow, RUSSIA, 2Laboratory of neurochemical pharmacology, State foundation Institute of pharmacology RAMS, Moscow, RUSSIA

The ubiquitously expressed flotillin-1 and flotillin-2 proteins are known to be localized to the cell membrane and intracellular compartments and serve as stable scaffolds for multiprotein com- plexes. The flotillins are reported to be involved in a variety of cellular processes, including cell adhesion, signaling, endocytosis, and membrane trafficking. Here, we analyzed the role of the flo- tillins in the uptake and retrograde transport of the bacterial Shi- ga toxin (Stx) and the plant toxin ricin. Our data showed that knockdown of flotillin-1 or flotillin-2 did not affect the endocytic uptake of Stx and ricin. However, we obtained a significant reduction in the endosome-to-Golgi transport of both toxins after flotillin-1 knockdown, measured by determining the activity of a Golgi-specific sulfation of the engineered toxins. In contrast, the knockdown of flotillin-2 did not affect the transport to the Golgi. Surprisingly, the trafficking of ricin to the ER was increased both after depletion of flotillin-1 or flotillin-2, determined by quantifi- cation of ER-specific N-glycosylation of ricin. The overall protein sulfation or glycosylation was not affected by knockdown of flo- tillins. Finally, we demonstrated that the knockdown of flotillin-1 or flotillin-2 resulted in increased toxicity compared to control cells. The retrograde trafficking of Stx and ricin is regulated by flotillin-1 and -2 by partially different means. In conclusion, knockdown of flotillin-1 or -2 seems to increase toxin transport to the ER and the cytosol.

respectively. Control and intraperitoneally,

P4–109 The exosome contains domains with specific endoribonuclease, exoribonuclease and cytoplasmic mRNA decay activities D. Schaeffer1, B. Tsanova1, A. Barbas2, F. Pereira Reis2, E. Ghosh Dastidar1, M. Sanchez-Rotunno1, C. M. Arraiano2 and A. van Hoof1 1Department of Microbiology and Molecular Genetics, University of Texas Health Science Center-Houston, Houston, USA, 2ITQB, Control of Gene Expression Laboratory, Oeiras, PORTUGAL

Introduction: Dopamine synthesizing in the arcuate nucleus neurons inhibits pituitary prolactin secretion. As follows from our earlier studies (Ugrumov, 2008), the 6-hydroxydopamine (6- OHDA)-induced degeneration of the neurons results in the hy- perprolactinemia development, which is compensated with time. In addition to DA-producing neurons, noradrenergic axons are also degenerated under the 6-OHDA action. The goal of this study was to evaluate what is the functional significance of nor- adrenergic afferents in the development of hyperprolactinemia. Therefore, desmethylimipramine (DMI), an inhibitor of nor- adrenaline uptake, was co-administrated with 6-OHDA in order to protect the noradrenergic afferents from degeneration. Methods: 6-OHDA and DMI were injected to rats, to cerebral ventricles rats received saline instead of drugs. The concentrations of dopamine, noradrenaline and metabolites was measured in the arcuate nucleus on the 14th and 45th day after injections. The prolactin concentration was estimated in the pituitary and serum. Results: When comparing with our earlier data, the co-adminis- tration of DMI in addition to 6-OHDA resulted on the 14th day in: (i) a lower decrease of the noradrenaline concentration in the arcuate nucleus; (ii) a stronger decrease of the dopamine concen- tration in the arcuate nucleus; (iii) and the lower increase of the prolactin concentration in serum. The same treatment resulted on the 45th day in: (i) a normalization of the noradrenaline concen- tration in the arcuate nucleus; (ii) a stronger decrease of the dopamine concentration in the arcuate nucleus; (iii) and the increase of the prolactin concentration in serum. Conclusion: Thus, noradrenaline appears to inhibit the dopa- mine secretion in the survived dopamine-producing neurons thereby inducing the irreversible development of hyperprolactin- emia.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

The exosome is essential for the processing, quality control and turnover of RNAs of the cell. The yeast core exosome is com- posed by nine catalytically inactive subunits constituting a ring and the active nuclease Rrp44. The 3¢–5¢ exoribonuclease activity of the exosome is conferred by the RNB catalytic domain of the Rrp44p subunit. We have shown for the first time that a trun- cated protein that contains the PIN domain but lacks the RNB domain has robust nuclease activity. This activity was lost when the PIN domain was mutated. So, we demonstrated that Rrp44 has a second nuclease domain apart from the RNB catalytic domain. We also noted that the degradation profiles of 5¢ and 3¢- labelled substrates were similar and the truncated protein was able to cut a circular substrate in vitro. These results reveal that this protein has endoribonuclease activity in addition to its known exoribonuclease activity making Rrp44 the unique RNase, known so far, that has the two catalytic sites. These results have serious implications on the mode of action of the exosome, namely the mechanism of recruitment and degradation of RNA, so the model of the exosome should be revisited. Furthermore,

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these results underline the conservation of the RNA processing machinery since it is related with a bacterial complex (degrado- some) and archaeal exosome.

P4–110 Hexadecylphosphocholine alters cholesterol transport from the plasma membrane to the endoplasmic reticulum in HepG2 cells P. Rı´ os-Marco1, J. M. Jime´ nez-Lo´ pez1, M. Garcı´ a-Gonza´ lez2, J. L. Segovia1, C. Marco1 and M. P. Carrasco1 1Biochemistry and Molecular Biology I, University of Granada, Granada, SPAIN, 2University School ‘La Inmaculada Concepcio´n’, University of Granada, Granada, SPAIN

Results: CM analysis revealed that cells expressing HCV proteins exhibited: (i) marked mitochondrial membrane potential depolar- ization; (ii) increase of mitochondrial ROS production; (iii) higher intramitochondrial calcium levels. Moreover, endogenous respira- tion and complex I activity were significantly reduced whereas complexes III and IV activities were unaffected. The ATP total amount was significantly higher in HCV positive cells. All the alterations observed were rescued upon treatment of the infected cells with the Ca++ uniporter inhibitor ruthenium red (RR). Note- worthy, in spite of the oxidative phosphorylation impairement, survival of HCV proteins-expressing cells was assured by an adap- tive up-regulation of glycolitic enzymes. This was linked to norm- oxic stabilization of the hypoxia-inducible factor (HIF1a). Conclusions: The modifications observed in infected cells were completely reversed by RR, thus indicating a causative link between the detrimental effect of HCV proteins on the oxidative metabolism and the de-regulation of the intracellular recycling of calcium. The bioenergetic unbalance is compensated by a HIF- dependent transcriptional mechanism these observations provide new insights into the pathogenesis of hepatitis C and may offer new opportunities for therapeutic intervention.

P4–112 Examining the function of the Arabidopsis formin AtFH1: effects of actin disruption on root development E. A. Rosero Alpala, F. Cvrckova and V. Zarsky Plant Physiology, Charles University Faculty of Science, Prague, CZECH REPUBLIC

In previous works, we have established that hexadecylphosphocho- line (HePC) alters intracellular cholesterol metabolism leading to an increased uptake, synthesis and accumulation of cholesterol in HepG2 cells. Our data demonstrate that a main mechanism by which HePC impairs cholesterol homeostasis is by altering the intracellular transport of cholesterol from the plasma membrane to the endoplasmic reticulum (ER). Sterols are critical for eukary- otic cell membrane due to their key role in raft domain-related functions. Membrane lipid rafts not only participate in distributing proteins to the cell surface and to other organelles, but they also play a significant role in many signalling cascades and in the immune response. It has also been reported that HePC strongly interacts with cholesterol and other sterols. Therefore, we postu- late that HePC, due to its amphiphilic nature, acts on cell mem- branes, decreasing the influx of sterol into the ER. We investigated this possibility in three different ways. Firstly, we used cyclodext- rins to remove cholesterol from the plasma membrane to disrupt lipid raft microdomains. Secondly, HepG2 cell membranes were enriched in cholesterol. And finally, we incubated the cells with HePC and cholesterol in the medium. In all cases, we analyzed the effect of HePC on cholesterol traffic from the plasma membrane to the ER. Ours results provide novel information about the mecha- nism by which HePC interacts with the plasma membrane decreas- ing the influx of the sterol into the ER. Acknowledgement: This work was aided by the Carlos III Institute of the Spanish Ministry of Health (PI061268).

P4–111 Hepatitis C virus proteins expression promotes mitochondrial bioenergetic dysfunction M. Ripoli1, G. Quarato1, A. D’Aprile1, R. Scrima1, O. Cela1, D. Boffoli1, D. Moradpour2, N. Capitanio1 and C. Piccoli1 1Biomedical Sciences, University of Foggia Medical School, Foggia, ITALY, 2Gastroenterology and Hepatology, University of Lausanne, Lausanne, SWITZERLAND

The dynamic events in the plant cell require extensive remodeling of the actin and microtubule cytoskeletons. Formins promote rapid assembly of actin filaments, involved in the control of cell polarity, cell and tissue morphogenesis, and cytokinesis. AtFH1, the main housekeeping Arabidopsis formin, belongs to the type-I plant formins, which contain an N-terminal signal peptide or membrane anchor followed by a transmembrane domain, sug- gesting a membrane localization. To understand the function of AtFH1 and actin filaments during root elongation, polarity and morphogenesis, we observed the effects of Latrunculin B (LatB) treatment that disrupts actin microfilaments in AtFH1 mutants upon root growth (by measuring root length and diameter) and on development of root hairs. Hypersensitivity to LatB treatment was observed in the AtFH1 mutant, as LatB caused significant reduction in root elongation with respect to wild type. Changes in root diameter wereobserved, however the differences between mutant and wild type were not significant. Severe abnormalities were found in the trichoblast and atrichoblast morphology in AtFH1 mutant, where bulbous structures at the base of the root hair and branched root hairs were observed. These data support the expected interaction between AtFH1 and cortical actin cyto- skeleton, as disruption of actin filaments in the AtFH1 mutant affects growth, polarity, morphogenesis and cell architecture more than in the wild type. Acknowledgements: This work was supported by the MSM 0021620858 and LC06004 projects.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Introduction: Recent evidence has shown that a proportion of hepatitis C virus (HCV) core protein localizes to mitochondria, suggesting their possible involvement in HCV infection. Hence we investigated the mitochondrial oxidative metabolism in U2- OS cell line inducibly expressing the entire HCV genome. Methods: Human osteosarcoma cell line (U2-OS) stably trans- fected with tetracycline-regulated HCV genomic construct expressing viral proteins were analysed by: high resolution respi- rometry, spectrophotometry-based enzymatic assay, morpho- functional analysis by Confocal Microscopy (CM), qRT-PCR, Immunoblotting.

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P4–113 Comparative NDRG1 gene regulation analysis in an alternating hypoxic microenviroment in different grades of human brain tumors H. M. Said1, S. Stein2, C. Hagemann3, B. Polat4, A. Staab4 and M. Flentje4 1Department of Radiation Oncology, Medical Faculty, Wuerzburg, GERMANY, 2Department of Hematology and Oncology, Johannes Gutenberg University III. Medical School, Mainz, GERMANY, 3Tumor Biology Laboratory Department of Neurosurgery, Univer- sity of Wu¨rzburg Medical Faculty, Wuerzburg, GERMANY, 4Department of Radiation Oncology, University of Wu¨rzburg Medical Faculty, Wuerzburg, GERMANY

detected by Northern-blotting and immunoblotting. Iodoacetate (IAA; 50 lM) was used as glycolysis inhibitor. Materials and Methods: Human glioblastoma cell lines U373, U251, U87-MG and GaMG were grown up to 50% confluence. pSUPER-NDRG1 vectors were with two differerent NDRG1 siRNA sequences that were confirmed via sequencing. Transient transfection of siRNA constructs into tumor cells exposed to extreme hypoxic aeration condition was via Fugene6 solution (Roche, Germany). Also, cells were transfected with empty pSU- PER and pSUPER-NDRG1. Results: NDRG1 was inhibited with one of the two siRNA con- structs, each separately or also when 50 lM glycolysis inhibitor IAA was used for 24 h with 0.1% O2, on protein level. On mRNA level was a complete NDRG1 mRNA expression inhibi- tion when 50 lM glycolysis inhibitor IAA was applied, in vitro, for 24 h with 0.1% O2 was applied to cells exposed to 0.1% O2. Conclusions: NDRG1 inhibition expression in glioblastoma in vitro by either siRNA technology or tumor cell glycolysis inter- ference is a potential therapeutic tool for this gene expression regulation in glioblastoma. Successful tumor cell growth inhibi- tion by siRNA aimed at oncogenes in vitro and in vivo may rep- resent alternative therapeutic applications against such disease regulation together with other transcription factors. NDRg1 is regulated by HIF-1 under hypoxia and not Egr-1.

Aim: NDRG1 (N-myc downregulated gene 1) a gene regulated during cell proliferation, differentiation & response to stress(s) ex. Hypoxia. HIF-1a together with other factors is involved in NDRG1 regulation. NDRG1 expression analysis in gliobastoma in different hypoxic environments highlights some mechanism(s) involved in the regulation of this gene. Materials and Methods: Human U251, U373, GaMG & U87- lines were exposed to (5–0.1%) O2, for MG glioblastoma cell 1 h, 6 h, 24 h in addition to 24 h with reoxygenation over 24 and 48 h. Treatment with desferrioxamin (COCl2, 300 lM, 24 h) served as a positive control, Incubation at (20% O2, 5% CO2) as a normoxic negative control. HIF-1a expression were detected via western blots & mRNA (sq RT-PCR) while, NDRG1 was also detected via Northern blots. b-actin, b-Tubulin and 18 S RNA served as loading controls, respectively. HIF-1a nuclear extracts bound HRE element in a hypoxia dependent manner. Results: With exemption of U87-MG (were NDRG1 was strongly expressed, independent of O2 level, no NDRG1(protein) expression was displayed in a normoxic environment in all other cell lines and at 5% O2. A substancial degree of NDRG1 expres- sion, with a maximum at 0.1% and relative stability during reoxygenation emerged at (1–0.1%) O2. HIF-1a was moderately – strongly expressed after 1 h 0.1% O2, and stable up to 48 h reoxygenation after 24 h at 0.1% O2. Conclusions: NDRG1 protein levels in human cancers aid in disease diagnosis. Glioblastoma cells with minimal O2 concentra- tions display a substancial NDRG1 expression with a relatively long stability. NDRG1 inhibition opens anti tumor therapeutic approaches potential.

P4–115 Comparative expression analysis of the hypoxia marker gene Carbonic anhydrase 9 (CA9) in different grades of human malignant glioblastoma H. M. Said1, C. Hagemann2, B. Scho¨ mig2, G. H. Vince2, M. Flentje1 and D. Vordermark3 1Departement of Radiation Oncology, University of Wu¨rzburg Medical Faculty, Wuerzburg, GERMANY, 2Tumor Biology Laboratory Departement of Neurosurgery, University of Wu¨rzburg Medical Faculty, Wuerzburg, GERMANY, 3Departement of Radiation Oncology, Martin-Luther-University-Halle-Wittenberg Medical Faculty, Halle, GERMANY

P4–114 Targeted Inhibition of Hypoxia induced NDRG1 gene expression in human glioblastoma cells in vitro H. M. Said1, B. Polat1, S. K. Arif2, A. Staab3, C. Hagemann4, M. Flentje1 and D. Vordermark5 1Department of Radiation Oncology, University of Wu¨rzburg Medical Faculty, Wuerzburg, GERMANY, 2Biology Department, Sulaimani Unviversity College of Science, Sulaimani-Kurdistan- Region, IRAQ, 3Department of Radiation Oncology, University of Zuerich, Wuerzburg, SWITZERLAND, 4Tumor Biology Laboratory Department of Neurosurgery, University of Wu¨rzburg Medical Faculty, Wu¨rzburg, GERMANY, 5Department of Radiation Oncology, University-Halle Medical Faculty, Halle, GERMANY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Introduction: siRNA is a powerful tool for drug target discov- ery, human therapeutics validation and for artificially modulating gene expression by interfering RNAs introduction into eukaryotic tumor cells HIF1a is involved under hypoxia, in NDRG1 gene transcriptional Reduced NDRG1 mRNA and protein were Introduction: Hypoxia-inducible enzyme carbonic anhydrase IX (CA IX) a tumor hypoxia marker representing an prognosis indi- cator and a potential malignant glioma therapeutic target. Material and Methods: To characterize CA9 expression pat- terns in human malignant glioma cells, we studied CA IX pro- tein, CA9 mRNA and hypoxia-inducible factor-1a (HIF-1a) protein levels in U87-MG, U251, U373 and GaMG cells exposed to in vitro hypoxia (1, 6 or 24 h at 5%, 1% or 0.1% O2). CA9 mRNA and protein expression were examined in two tumor patient specimens groups, low-grade astrocytoma (LGA; n = 15) & glioblastoma (GBM; n = 15) & compared to a negative nor- mal brain samples (n = 3) control group. CA9 & HIF-1a protein expression were detected via Western blot, CA9 & HIF-1a mRNA expression detection via Northern blot or RT-PCR. Results: In the in vitro experiments, all cell lines displayed a strong hypoxic CA9 mRNA induction in response to severe hypoxia with cell-line specific patterns. U87-MG exhibited a strong constitutive, normoxic CA IX protein expression without a detectable change under hypoxia. In U251 and GaMG, a marked CA IX protein induction in response to severe hypoxia was seen. CA IX changes under severe hypoxia and glycolysis inhibitor iodoacetate (IAA, 50 lM) inhibitory effect on hypoxic

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CA IX overexpression were paralleled by HIF-1a protein results. In patient tumor specimens, CA IX protein was only overexpres- sed in (GBM). CA9 mRNA was predominantly overexpressed in GBM (12/15) compared to LGA patients (3/15). Conclusions: CA9 expression in (GBM) tumors occurs at a higher frequency, both on protein or mRNA levels, rendering CA9 as a suitable hypoxia-related diagnostic marker or target for therapeutic approaches.

P4–117 Immunological Response of Frog (Rana ridibunda) Skin Exposed to Continuous Dark M. Sengezer-Inceli1, S. Sancar-Bas1, A. Kimiran-Erdem1, E. Kaptan1, E. O. Arslan-Aydogdu1 and A. L. Inceli2 1Department of Biology, Istanbul University Faculty of Science, Istanbul, TURKEY, 2Faculty of Art and Sciences, Isik University, Istanbul, TURKEY

P4–116 Validation analysis of GAPDH as a tumor expression reference gene under alternating oxygenation conditions including hypoxic conditions in different tumor cells H. M. Said1, B. Polat1, S. K. Arif 2, C. Hagemann3, J. Anacker4, M. Flentje5 and D. Vordermark6 1Departement of Radiation Oncology, University of Wu¨rzburg Medi- cal Faculty, Wuerzburg, GERMANY, 2Biology Department, Sulai- mani Unviversity College of Science, Sulaimani-Kurdistan-Region, IRAQ, 3Tumor Biology Laboratory Departement of Neurosurgery, University of Wu¨rzburg Medical Faculty, Wuerzburg, GERMANY, 4Tumor biology Laboratory Departement of Neurosurgery, Univer- sity of Wu¨rzburg Medical Faculty, Wu¨rzburg, GERMANY, 5Tumor Biology Laboratory Departement of Neurosurgery, University of Wu¨rzburg Medical Faculty, Wuerzburg, GERMANY, 6Departement of Radiation Oncology, Martin-Luther-University-Halle-Wittenberg Medical Faculty, Wuerzburg, GERMANY

The skin secretions of ranid frogs exhibited antibacterial activity. There is no information about their synthesis and releasing mech- anisms. The antimicrobial activity of secretion shows variations related to physiological conditions. The aim of this study is to determine the effect of aphotic environmental circumstance on antimicrobial activity of the frog skin secretion. The skin secre- tions of animals obtained from nature were emptied (N). They were separated into two groups. First group was kept continu- ously in the dark condition (D), while the second group was kept in the laboratory under the daily light conditions (L). Thereafter, skin secretions of D and L groups were collected after 1 month. The protein profiles of lyophilized skin secretion in all groups (N, D, L) were obtained electrophoretically (SDS–PAGE) as well as the amount and composition of secretion peaks were compared by using HPLC technique. The antimicrobial activity of three groups was studied on different bacteria with diffusion and dilu- tion techniques. In group D, the amount of protein in almost each band increased and almost each peak in HPLC analysis of secretion rose as well. Group D exhibited to the strongest antimi- crobial activity when compared to the other groups. Dark period play an important role in releasing of hormones such as melato- nin related to immunological events. Consequently, it has been claimed that hormone and/or hormones may influence skin secre- tion profile and antibacterial activity. This study is interesting because of displaying factors affecting synthesis mechanism and secretion pattern of antimicrobial substances which had a phar- macological importance in the frog skin.

P4–118 Expression pattern of human HSPA2 protein in normal and cancerous tissues D. Scieglinska1, W. Piglowski1, M. Chekan2 and Z. Krawczyk1 1Department of Tumor Biology, Maria Sklodowska-Curie Memo- rial Cancer Center and Institute of Oncology Gliwice, Gliwice, POLAND, 2Nuclear Medicine and Endocrine Oncology Department, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology Gliwice, Gliwice, POLAND

Intoduction: Cancer diagnosis and treatment related gene expression studies are important. Absolutely reliable housekeep- ing genes are essential to normalize cancer related gene expres- sion. Such genes are present in all cells and their expression levels remain relatively constant under different conditions. Incorrect housekeeping genes choice lead to interpretation errors of experi- mental results including evaluation and quantification of patho- logical gene expression. Materials and Methods: We examined (i) GAPDH expression regulation degree in Hep-1-6 hepatoma and Hep-3-B and HepG2 human hepatocellular carcinoma cells, human lung adenocarci- noma epithelial cell line (A-549) and HT-29, HCT-116 colon can- cer cells, under hypoxic conditions in vitro in comparison to other housekeeping genes like b-actin, serving as experimental loading controls, (ii) potential GAPDH use as a tumor therapeu- tic approaches target was comparatively examined in vitro on both protein and mRNA level, by western blot and semi quanti- tative RT-PCR. Results: No hypoxia-induced GAPDH expression regulatory effect was observed in the cell lines under the different oxygena- tion conditions examined, in vitro both on protein and mRNA level. These results poses a similar tendency to those obtained when in a previous series of experiments glioblastoma cells where analyzed under similar and comparable conditions. Conclusions: In human hepatocellular carcinoma, mouse hepa- toma, human colon cancer, and human lung adenocarcinoma, GAPDH represents an optimal housekeeping gene choice and loading control since this gene is not an attractive target for tumor therapeutic approaches because of the GAPDH regulation under hypoxia abscence.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Heat-shock proteins are a group of evolutionary highly conserved chaperone proteins. Experimental data indicate that Hsp70 pro- teins are cytoprotective and antiapoptotic agents involved in enhancing survival of normal and tumor cells. The HSPA2 gene, the member of Hsp70 family, is highly expressed in spermato- genic cells and is essential for spermatogenesis. We and others have recently reported that HspA2 is expressed in various human cancer cell lines and in some primary human carcinomas. The transcripts of human HSPA2 gene have been also detected in various normal somatic tissues, however the translation of the messenger RNA into corresponding protein has not been unam- biguously demonstrated, so far. Here, we present immunohisto- chemical analysis of HspA2 protein expression in normal somatic tissues and in most frequent human primary cancers. We used commercially available tissue microarrays and monospecific poly- clonal anty-HspA2 antiserum produced by us. Our data indicate that in normal tissues (e.g. breast, thyroid, ovary, bladder) the HspA2 protein expression is either not detectable or is restricted to selected cell populations (e.g. striatum basale and striatum

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lucidum of epidermis, Goblet cells of colon, renal tubules cells). In breast cancers, contrary to normal breast tissue, the HspA2 expression is frequently activated. The HspA2 is also frequently and strongly overexpressed in melanomas and other skin cancers. In cancer cells this protein localizes in nucleus and/or cytoplasm. Interestingly, in melanoma HspA2 can be observed also on cell membrane. The data on HspA2 expression were compared with expression of inducible HspA1 (hsp70) protein and proliferation marker Ki67 protein.

grative vector modified with a cat gene at BamHI and SphI restriction sites) at EcoRI restriction site. In spite of the fact that surfactin producing strains posses an extremely low level of natu- ral competence, the resulting plasmid pK19-sfp was successfully used to transform B. subtilis ATCC 21332 competent cells. The clones with an abolished ability to produce surfactin (impairment of sfp) were identified by examination on blood agar according to the absence of erythrocyte lyses. It is the first time, a sfp knock-out derivative of B. subtilis ATCC 21332 was constructed and will be used to verify the physiological relevance of the adap- tive membrane reconstruction induced in the surfactin producer. Recently, the analyses of both the membrane lipid and proteome components of the cytoplasmic membrane are in progress, sug- gesting a complex defensive cell response.

P4–119 Extracellular Alu-transcripts as intercellular regulators of translation D. Semenov, D. Baryakin, T. Kamynina, E. Kuligina and V. Richter Institute of Chemical Biology & Fundamental Medicine, LBT, Novosibirsk, RUSSIA

P4–121 Type-2 ribosome-inactivating protein SNA-I from elderberry induces apoptosis in insect midgut cells S. Shahidi-Noghabi1, E. J. Van Damme2, M. Iga3 and G. Smagghe3 1Laboratory of Agrozoology Department of Crop Protection, Lab- oratory of Biochemistry and Glycobiology Department of Molecu- lar Biotechnology, Ghent University/Faculty of Bioscience Engineering, Gent, BELGIUM, 2Laboratory of Biochemistry and Glycobiology Department of Molecular Biotechnology,Ghent University/Faculty of Bioscience Engineering, Gent, BELGIUM, 3Laboratory of Agrozoology Department of Crop Proection, Laboratory of Agrozoology Department of Crop Proection, Ghent University/Faculty of Bioscience Engineering, Gent, BELGIUM

in elevated level

It is well-known that mammalian extracellular fluids, such as plasma of blood or plasma of milk, contain cell-free RNAs. Fragments of ubiquitously expressed human rRNA, mRNAs, as well as tumor-specific and viral RNAs have been previously revealed in normal or diseased human plasmas. In the present work we used three independent approaches to construct cDNA libraries coding human blood plasma RNAs. In generated cDNA libraries we have identified copies of snRNAs, siRNAs, Alu con- taining RNAs, and fragments of L1 RNAs. We analyzed the rel- ative content of identified RNAs in human plasma and in culture media condensed by human cells. It was determined that among other RNAs, Alu-transcripts have elevated stability and were present in human extracellular fluids. We designed and synthesized single- and double-stranded analogs of human extracellular RNAs in order to analyze influence of RNAs on proliferation and viability of human adenocarcinoma cells MCF-7. We show that passive transfection with entire Alu- transcripts or with their fragments significantly reduces the viabil- ity of MCF-7, possibly by general and reversible inhibition of translation. These data emphasize the role of extracellular Alu RNAs as stress inducible intercellular RNA messengers.

P4–120 Construction of a surfactin non-producing mutant of Bacillus subtilis as a tool for membrane resistance study G. Seydlova1, M. Patek2 and J. Svobodova1 1Departement of Genetics and Microbiology, Faculty of Science Charles University in Prague, Prague 2, CZECH REPUBLIC, 2Institute of Microbiology, Laboratory of Molecular Genetics of Bacteria, Prague 4, CZECH REPUBLIC

In this study we investigated the cellular uptake of SNA-I, a type-2 RIP from elderberry (Sambucus nigra) bark with specificity for sialic acid. Our previous results have shown the toxicity of SNA-I for several pest insect species such as the beet armyworm Spodoptera exigua (Lepidoptera) and the aphids Myzus nicotianae and Acyrthosiphon pisum (Homoptera) (Shahidi-Noghabi et al., 2008; 2009). For further investigation of the mode of action of SNA-I, a more in-depth study was performed at the cellular and molecular level. To determine the relative potency of SNA-I on cell proliferation, FPMI-CF-203 cells from a continuous midgut cell line of the spruce budworm (Choristoneura fumiferana) were treated with SNA-I at different concentrations. Analysis of cell proliferation and toxicity revealed cell growth inhibition. To investigate the effect of SNA-I uptake in more detail at the DNA level, cells were incubated for different time periods with SNA-I. Analysis of DNA samples from the treated cells showed an oligo- meric ladder beginning at 24 h after treatment. In addition, microscopic analysis of the cells revealed the classical apoptotic morphological characteristics such as plasma membrane blebbing, nuclear fragmentation, and cell shrinkage. Our results clearly indicate for the first time that SNA-I induces apoptosis in FPMI- CF-203 insect midgut cells. Finally, the results are discussed in relation to potential mechanisms of action and uptake of RIPs in insects, and a role in plant resistance against pest species. References: 1. Shahidi-Noghabi S, Van Damme EJM & Smagghe G. Carbo- hydrate-binding activity of the type-2 ribosome-inactivating protein SNA-I from elderberry (Sambucus nigra) is a deter- mining factor for its insecticidal activity. Phytochemistry 2008; 69: 2972–2978

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

2. Shahidi-Noghabi S, Van Damme EJM & Smagghe G. (2009) Expression of Sambucus nigra agglutinin (SNA-I¢) from elder- berry bark in transgenic tobacco plants results in enhanced resistance to different insect species. Transgenic Research DOI 10.1007/s11248-008-9215-2 Surfactin is a lipopeptide surfactant and membrane antibiotic produced by Bacillus subtilis. Its wide commercial application still restricts the unknown mechanism of surfactin resistance of the producing cells and, in turn, the low yields. The aim of this study was to construct a surfactin non-producing mutant strain. Its characterization will reveal the membrane determinants that increase the cell resistance in response to the antibiotic synthesis. The chromosomal region responsible for surfactin synthesis com- prises the entire srfA operon and essential sfp gene coding for phosphopantetheinyl transferase. B. subtilis sfp0 strain comple- mentary to the wild type B. subtilis ATCC 21332 sfp+ was pre- pared by PCR amplification of the non-functional sfp region using B. subtilis 168 chromosomal DNA as a template. The gene bears a frame-shift mutation that makes the gene inactive (sfp0). The amplified fragment was cloned into pK19 (cloned locus inte-

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P4–122 Modulation of TNF-related apoptosis inducing ligand apoptosis in colon cancer cells by omega-3 docosahexaenoic fatty acid B. Skender, M. Hyzdalova, A. Vaculova, A. Kozubik and J. Hofmanova Department of Cytokinetics, Institute of Biophysics AS CR v.v.i., Brno, CZECH REPUBLIC

IAA cytes differentiation and visfatin expression in various tissues of laboratory rat. In addition, we demonstrate a loss of the NamPRTase activity and NAD biosynthetic ability of the cell ly- sates after decrease of visfatin expression. New method for the determination of visfatin enzymatic activity has been designed based on the HPLC analysis in the presence of visfatin prepara- tions from cell cultures. MALDI-TOF analysis of selected protein spots from two-dimensional gels revealed absence of selected pro- teins under RNAi knockdown of visfatin. Two-dimensional gels were also compared by Image Master 2D Platinum program. Acknowledgement: Funding: MSM 6046137305, 500110805, NS9696-4/2008.

(HT-29)

P4–124 Lipopolysaccharide delays apoptosis of bovine lymphocytes P. Slama1, Z. Sladek1 and D. Rysanek2 1Department of Animal Morphology Physiology and Genetics, Mendel University of Agriculture and Forestry in Brno, Brno, CZECH REPUBLIC, 2Department of Immunology, Veterinary Research Institute, Brno, CZECH REPUBLIC

Polyunsaturated omega-3 docosahexaenoic fatty acid (DHA, 22 : 6, n–3) present especially in fish oil, is effectively incorporated into cellular membranes. DHA may alter membrane properties, influence receptor activities, modulate intracellular signalling, oxi- dative metabolism and gene expression. Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) is known to selectively induce apoptosis in cancer cells, while sparing normal cells. Despite it, some cancer cell type including colon cancer cells are resistant to its effect. Supposing modulation of TRAIL apopto- tic signalling by DHA, we investigated the effects of DHA pre- treatment and subsequent TRAIL treatment on cell cycle, viability lines sensitive and apoptosis of two human colon cancer cell (HCT-116) or relatively resistant to TRAIL-induced apoptosis. In addition to cytokinetics we compared the response of these two cell lines with regard to mitochondrial changes in early stage of apoptosis including mitochondrial membrane potential (MMP), reactive oxygen species (ROS) production, expression of caspases and Bcl-2 family proteins. Compared to agents used alone, cell growth was more inhibited and percentage of floating and apoptotic cell increased after DHA pre-treatment followed by TRAIL treatment. These effects were more apparent in resistant HT-29 cell line then in HCT-116 cells. Our results show that DHA is able to potentiate colon cancer cell sensitivity to the apoptotic effect of TRAIL partially through intrinsic mitochondrial apopto- tic pathway. Acknowledgements: This work was supported by grants Nos. 524/07/1178 and 301/07/1557 GA CR and No. 1QS500040507 ASCR.

Bacterial lipopolysaccharide (LPS) is a toxin released from the cell wall of Gram-negative bacteria. Infusion of LPS into the bovine mammary gland induces an inflammatory response. LPS affects lifespan of mammary gland leukocytes. The effect of LPS on bovine neutrophil apoptosis is known, but there is no infor- mation about apoptosis of bovine lymphocytes after LPS stimu- lation. The aim of this study was to determine whether apoptosis of lymphocytes is modulated by LPS. To this end, apoptosis of lymphocytes was studied in in vitro environment (RPMI medium, 37(cid:2)C, 5% CO2) with LPS (0.2, 2 and 20 lg/ml). Heifers were used as mammary gland cell donors for in vitro studies. Intact lymphocytes from the mammary glands were harvested following the phosphate buffered saline intramammary injection. Apoptosis of lymphocytes were analysed by flow cytometry following simul- taneous staining with Annexin-V and propidium iodide. The results of this study demonstrate that apoptosis of lymphocytes was delayed during in vitro incubation with LPS with no signifi- cant differences among concentrations. The question remains, however, as to why LPS delays bovine lymphocyte apoptosis. Acknowledgement: This study was supported by the Ministry of Agriculture of the Czech Republic (MZE0002716202).

P4–123 Selective functional analysis of visfatin in murine cell cultures using rna interference V. Skop1, J. Zidkova1, K. Kontrova1, J. Sajdok1, L. Kazdova2, V. Zidek3 and M. Pravenec3 1Biochemistry and microbiology, Institute of Chemical Technology Prague, Prague 6, CZECH REPUBLIC, 2Department Diabetes Metabolism, Institute for Clinical and Experimental Medicine, Prague, CZECH REPUBLIC, 3Genetics of Diseases models, Institute of Physiology Czech Academy of Sciences, Prague, CZECH REPUBLIC

P4–125 Melatonin as a modulator of chronic microwave radiation effects on rat thymocyte proliferation, apoptosis and oxidative stress D. Sokolovic1, J. Nikolic1, D. Pavlovic1, G. Kocic1, T. Jevtovic-Stoimenov1, B. Djindjic2 and V. Pavlovic3 1Medical Faculty, Institute of Biochemistry, Nis, SERBIA, 2Medical Faculty, Institute of Pathophysiology, Nis, SERBIA, 3Medical Faculty, Institute of Physiology, Nis, SERBIA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Exposition to microwave radiation (MW), from mobile phones and other sources induce disturbances in thymocytes. The pineal secretory product, melatonin (Mel), exerts a variety of effects on the immune system. Melatonin inhibits oxidative stress, DNA fragmentation and apoptosis of T lymphocytes. Wistar rats (40 animals) were divided in 4 experimental groups: I (control-sham exposed), II (Mel group)-rats treated with Mel every day (2 mg/ kg b.w., i.p), III (MW group)-rats exposed to MW (4 h/day dur- ing 60 days), IV (MW + Mel)-rats treated with Mel every day (2 mg/kg b.w., i.p) and exposed to MW radiation (4 h/day during test phone 60 days). MW was produced by a mobile Visfatin, one of relatively newly described protein, belongs to the candidates for associated factors of metabolic syndrome. Visfatin is one of adipose tissue secreted factors so-called adipokines. Visfatin was originally identified as a factor preferentially pro- duced by visceral fat. It is also produced by lymphocytes to sup- port maturation of pre-B cells (pre-B cell colony-enhancing factor, PBEF). Visfatin has several biological functions: regula- tion of NAD level, accumulation of triacylglycerols (TAG) in preadipocytes and enhances TAG synthesis from glucose. To investigate biological/physiological function, of visfatin, we used the RNAi mechanism to the sequence-specific knock-down of visfatin expression in murine 3T3-L1 adipocytes and Fao hepato- cytes. Results of our experiments demonstrate that changes in visfatin levels regulate glucose uptake in Fao hepatocytes this results contribute to a better understanding of the role of visfa- tin. We also studied changes of visfatin expression during adipo-

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Poster Presentations

P4–127 Attenuation of Nrf2 expression and antioxidant response by ochratoxin A – the impact on the profibrotic effect A. Stachurska1, A. Loboda1, M. Kozakowska1, C. Boesch-Saadatmandi2, G. Rimbach2, S. Yla-Herttuala3, A. L. Levonen3, A. Jozkowicz1 and J. Dulak1 1Department of Medical Biotechnology, Jagiellonian University Faculty of Biochemistry Biophysics and Biotechnology, Krako´w, POLAND, 2Institute of Human Nutrition and Food Science, Uni- versity of Kiel Faculty of Agricultural and Nutritional Science, Kiel, GERMANY, 3Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute, University of Kuopio, Kuopio, FINLAND

(SAR = 0.043–0.135 W/kg). MW significantly decreased thymo- cytes’ proliferation, induced by ConA (P < 0.01) and increased apoptosis, detected using the Annexin V-FITC/PI detection kit (P < 0.001). DNA fragmentation in exposed thymocytes is prob- ably triggered by the increase activation of alkaline-DNase I (cas- pase 3-activated) and acid-DNase II (P < 0.05). A significant increase in the thymus malondialdehyde (MDA) and carbonyl group concentration (P < 0.001), and decreased activity of cata- lase (P < 0.001) was registered during exposure. Melatonin was found to be effective on MW induced injury of thymocyte: (i) increase thymocyte proliferation (P < 0.05), (ii) decreased apop- totic rate of thymocytes (P < 0.001), (iii) decreased DNase I and DNase II activity (P < 0.01), (iv) decreased MDA and carbonyl group levels (P < 0.01) and (v) increased activity of catalase (P < 0.05). Melatonin exerts protective effects on rat thymocyte by reducing proliferation, apoptosis and oxidative stress in rats’ thymocytes under exposure of microwave radiation.

P4–126 Metformin reverses hexokinase and 6-phosphofructo-1-kinase inhibition in skeletal muscle, liver and adipose tissues from streptozotocin-induced diabetic mouse M. Sola-Penna1, D. Da Silva1, L. Gomez1, W. Coelho1 and P. Zancan2 1Pharmacy School – Federal University of Rio de Janeiro, Labora- torio de Enzimologia e Controle do Metabolismo – LabECoM Fac- uldade de Farmacia, Rio de Janeiro, BRAZIL, 2Pharmacy School – Federal University of Rio de Janeiro, Laboratorio de Oncobiolo- gia Molecular – LabOMol Faculdade de Farmacia, Rio de Janeiro, BRAZIL

Ochratoxin A (OTA), a nephrotoxic mycotoxin, is considered as a causal agent of Balkan endemic nephropathy (BEN), a chronic kidney disease. Generation of oxidative stress by OTA has been suggested as one of the mechanisms implicated in its genotoxicity and cytotoxicity. Oxidative stress in cells activates expression of superoxide dismutase (SOD), glutathione-S-transferase (GST) and heme oxygenase (HO), the protective enzymes, expression of which is regulated by nuclear factor – erythroid 2 related factor (Nrf2) transcription factor. In cultured kidney tubulus cells (LLC-PK1) OTA diminished antioxidant response via attenua- tion of Nrf2, SOD, GST and HO expression. Moreover, this my- cotoxin potently elevated expression of profibrotic agents: transforming growth factors-b (TGFb1 and TGFb2) with con- comitant decrease in expression of proangiogenic genes, like vas- cular endothelial growth factor (VEGF) and erythropoietin (Epo). Adenoviral vectors were used to investigate the role of Nrf2 and HO-1 in OTA-induced toxicity. Overexpression of Nrf2 as well as application of pharmacological inducer of Nrf2 (PGJ2) led to attenuation of OTA-elevated TGFb expression and caused increase of OTA-diminished HO-1 and Epo mRNA level. How- ever, genetic overexpression of HO1 did not prevent OTA- induced alterations. The phosphorylation of MAP kinases – JNK and ERK42/44 after OTA treatment was observed, and pharma- cological inhibition of ERK42/44 compensated the effect of OTA on TGFb2 expression. Present data indicate the attenuation of antioxidant response as one of the mechanisms of OTA action. Nrf2 overexpression can compensate some but not all of OTA- evoked alterations. Nevertheless, Nrf2 may be considered as one of the important factors in antifibrotic therapy.

P4–128 Opposing roles of annexin A1 and A2 in endocytosis and trafficking of shiga toxin L. Tcatchoff1, S. Andersson1, A. Utskarpen1,2, V. Gerke3 and K. Sandvig1,2 1Centre for Cancer Biomedicine – Department of Biochemistry, Institute for Cancer Research – The Norwegian Radium Hospital, Oslo, NORWAY, 2Department of Molecular Biosciences, Univer- sity of Oslo, Oslo, NORWAY, 3Institute for Medical Chemistry, Center for Molecular Biology of Inflammation, University of Mu¨nster, GERMANY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Annexins form a family of calcium and membrane binding pro- teins with diverse cellular functions, many of them related to membrane trafficking. This study was initiated to investigate the role of annexin A1 and A2 in the uptake and intracellular trans- port of the bacterial Shiga toxin (Stx) and the plant toxin ricin. Upon binding to cell surface receptors, the toxins are endocyto- sed and transported retrogradely to the endoplasmic reticulum. The enzymatically active part is then translocated to the cytosol where it inactivates protein synthesis. Toxin endocytosis was first Diabetes mellitus (DM) is a chronic disease characterized by per- sistent elevated glycemia due to disruption of insulin secretion (type 1 DM) and/or insulin cellular response (type 2 DM). Gly- colysis is the major glucose-consuming metabolic pathway, pre- senting as regulatory hallmarks the enzymes hexokinase (HK), 6- phosphofructo-1-kinase (PFK) and pyruvate kinase. Activation of glycolysis may contribute to decrease glycemia and thus to ameliorate diabetic symptoms. Metformin is an oral hypoglyce- mic drug worldwide used for the treatment of type 2 DM. How- ever, it has been shown that this drug decreases glycemia and augments glucose consumption rate in type 1 DM models, sug- gesting an insulin-independent mode of action. The present work describes the effects of metformin on HK and PFK activities and localization in different tissues from streptozotocin-induced dia- betic mice. Diabetic animals present lower HK and PFK activi- ties (50% in average) in skeletal muscle, liver and adipose tissue, when compared to control (P < 0.05, Student’s t-test). Treat- ment with 250 mg/kg metformin (daily dose for three consecutive days) reverses this pattern of enzyme inhibition with concomitant reversal of hyperglycemia and hipolactacidemia and without sig- nificant effects on the reduced body weight. Furthermore, the treatment of diabetic mice with metformin increases the cytoskel- eton-associated PFK activity in skeletal muscle, which has been described as an important mechanism for the enzyme activation. This effect might be due to the increased phosphorylation of the enzyme in serine residues that has been described to increase the interaction of PFK to f-actin. These effects are not observed in the liver or in the adipose tissue. In the end this work support evidences that metformin hypoglycemic effects involve the activa- tion of glycolysis through its regulatory enzymes, which may function as a potential targets for the development of new hypo- glycemic drugs. Acknowledgement: Supported by FAPERJ, CNPq, FAF/ FECD.

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signal

enzymatic activities, studied in HeLa cells after inhibition of annexin A2 function by siRNA treatment or expression of a dominant negative annexin 2 mutant. Under these conditions, Stx uptake was somewhat reduced, but some toxin molecules were still transported to the Golgi apparatus after 30 min incubation. Treating cells with siR- NA against annexin A1 did not result in any alteration of Stx binding and endocytosis. To study the role of annexins in endo- some to Golgi transport, HeLa and HEp2 cells were incubated with a modified toxin containing available sulfation sites. Sup- pression of annexin A1 expression resulted in an elevated sulf- ation level, whereas knockdown of annexin A2 reduced the amount of Stx reaching the Golgi apparatus. Ricin internaliza- tion and trafficking remained unaltered after depletion of annexin A1 or A2. Our findings demonstrate that both annexin A1 and A2 are implicated in the retrograde transport of Stx but with dif- ferent functions.

P4–129 Oxidative stress response and histopathological changes in the gills of chronic copper intoxicated Carassius auratus gibelio M. C. Munteanu, A. C. Staicu, O. Zarnescu and A. Dinischiotu Molecular Biology Center, University of Bucharest, Bucharest, ROMANIA

in eukaryotic cells. The proteasomes realize programmed proteol- ysis and processing of various regulatory proteins and thereby, play an important role in major cellular processes, including tran- scription, cell cycle progress, apoptosis, transduction, immune response and oncogenesis. According to current views, the composition and specific proteolytic activity of the cell pro- teasome population is heterogeneous. Moreover, cells have been shown to excreted proteasomes into the culture medium or extra- cellular space, and the number of exported proteosomes changed upon malignant the properties of transformation. However, excreted proteasomes, their enzymatic activity, and specificity remain unknown. The data of the present study testifies to the specificity of proteasomes excreted by cells into the culture med- ium. The excreted proteasomes were found to retain their charac- teristic although they differed from intracellular proteasomes in the specificity of proteolytic activity and in characteristics of endoribonuclease activity. Furthermore, the activity of extracellular particles depended on the cell func- tional state. It should be emphasized that changes in intracellular and excreted proteasomes activities were different, which suggests that the excretion of the specific proteasome population from cells is part of a regulatory mechanism that control the intracellu- lar activity of these particles. Furthermore, these extracellular proteasomes may transfer various regulatory molecules (tran- scription factors and proteasome-associated small RNAs) to pro- vide intercellular communication. Acknowledgements: Foundation of the President of the Rus- sian Federation (SS-774.2008.4 and YD-779.2008.4).

P4–131 Thiol oxidoreductases involved in soybean storage protein biogenesis R. Urade Graduate School of Agriculture, Kyoto University, Uji, JAPAN

Among pollutants, heavy metals are of great interest, because they could trigger oxidative stress in fish. Histological and bio- chemical analysis revealed that the gill is the primary target organ for Cu2+. The specific activities of superoxide dismutase (SOD) and catalase (CAT) and the histopathological changes for two sublethal concentrations (100 and 250 lg/l) of Cu2+ on Carassius auratus gibelio were assayed during 7, 14, 21 days. The sections were stained with hematoxylin-eozin as well as silver- enhanced for microscopic analysis following the autometallo- graphic method. In the case of 100 lg Cu2+/l treatment, the SOD activity decreased after 7, 14 and 21 days by 11%, 15% and 46% respectively, compared to the control, while CAT one decreased by 82.7%, 91% and 93% after 7, 14 and 21 days. At 250 lg Cu2+/l, the SOD activity decreased by 6%, 23% and 79% after 7, 14 and 21 days compared to the control, while CAT one decreased by 11.5%, 30% and 63% after 7, 14, 21 days. Hyperplasia, fusion of secondary lamellae, blood congestion and aneurysm were noticed. The presence of Cu2+ was revealed by autometallography in the epithelial cells from the distal tips of gill filaments, in the interlamellar space and hyperplasied epithe- lial cells. Our experiments demonstrated that the histological modifications and impairment of the antioxidant defense was tox- icant dose and exposure time dependent. Carassius auratus gibelio could serve as a valuable bioindicator and useful alternative model for evaluating environmental issues from both human health and ecological perspective.

In eukaryotes, secretory, organelle and membrane proteins are synthesized and folded with the assistance of molecular chaper- ones and other folding factors in the endoplasmic reticulum (ER). In many cases, protein folding in the ER is accompanied by the formation of disulfide bonds. Disulfide bond formation is catalyzed by thiol oxidoreductases localized in the ER lumen. In plants, most nutritional proteins such as the seed storage proteins in food crops are produced in the ER. However, the effect of the plant thiol oxidoreductase family on storage protein folding has not been explored. Seven genes encoding soybean thiol oxidore- ductase family proteins (GmPDIL-1, GmPDIL-2, GmPDIM, GmPDIS-1, GmPDIS-2, GmPDIL-3a and GmPDIL-3b) were cloned. Among them, five recombinant proteins expressed from their cDNAs in Escherichia coli displayed oxidative refolding activity on denatured RNase A. Expression of GmPDIL-1, GmP- DIM and GmPDIS-1 were upregulated under ER stress condi- tions. All thiol oxidoreductases were expressed ubiquitously in soybean tissues such cotyledon, leaf, root, stem and flower, and were localized to the ER lumen. Immunoprecipitation experi- ments suggested that in cotyledon cells GmPDIL-1, GmPDIL-2, GmPDIM and GmPDIS-1 associated non-covalently with pro- glycinin and b-conglycinin. In addition, some thiol oxidoreducta- ses were found to make complexes with other proteins. These results suggest that thiol oxidoreductases play a role in folding and/or quality control of storage proteins.

P4–130 Exogenous 26S proteasomes differ from cellular ones in their structure and activities A. Tsimokha1, J. Zaykova1, L. Gause2 and V. Kulichkova1 1Laboratory of Gene Expression Regulation, Institute of Cytology Russian Academy of Science, Saint-Petersburg, RUSSIA, 2Laboratory of Genetics, Koltsov Institute of Developmental Biology Russian Academy of Science, Moscow, RUSSIA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

The 26S proteasome, composed of a 20S catalytic core and two associated 19S regulatory complexes, is a multisubunit protease complex which executes the non-lysosomal proteolytic pathway

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Poster Presentations

P4–132 Staining procedure for peroxidase activity- educational approach A. Urbanska Department of Biochemistry & Molecular Biology, University of Podlasie, Podlasie, POLAND

tor-15 (GDF-15) secreted by prostate cancer cells on the osteo- clastic differentiation pathway. GDF-15 is a new member of the TGFb superfamily of proteins participating in control of tissue development and repair. Recently, GDF-15 has been associated with many cancers, including prostate adenocarcinoma. However, its role in the metastasis-induced bone remodeling remains unclear. This study documents that both purified GDF-15 and unpurified product secreted by prostate cancer cells strongly interferes with differentiation of osteoclasts by inhibiting expres- sion and activity of NFjB and c-Fos transcription factors. Down-regulation of NFjB and c-Fos is associated with up-regu- lation of p38 kinase phosphorylation and affects the SMAD sig- naling pathways. Moreover, GDF-15 inhibits expression of enzymatic markers carbonic anhydrase II and cathepsin K. The fact that the GDF-15 produced by prostate cancer cells can affect bone homeostasis provides new insight into mechanism of bone metastasis formation and can help to establish a new strategy for therapy of advanced prostate cancer. Acknowledgements: This work was supported by grants 301/ 09/1115, 204/07/0834, 310/07/0961 of the Czech Science Founda- tion, MSM0021622415 of Ministry of Education, Youth and Sports, IGA9875-4 of Ministry of Health of the Czech Republic and AV0Z50040507 of the Grant Agency of Academy of Sciences of the Czech Republic.

P4–134 The effect of varying the 4’ position substituents for the same chalcone structures and reversely on biological activity in mitochondria J. Kubalkova1, L. Vasko1, J. Guzy1 and P. Perjesi2 1Medical Chemistry Biochemistry and Clinical Biochemistry, Faculty of Medicine, Kosice, SLOVAK REPUBLIC, 2Faculty of Medicine, Institute of Pharmaceutical Chemistry, Pecs, HUNGARY

The term peroxidases includes two groups of oxidoreductases act- ing on hydrogen peroxide as hydrogen acceptor, namely specific for the hydrogen donor that oxidize NADH, NADPH, fatty acid, cytochrome, glutathione and L-ascorbate, and non-specific known to biochemists as POD or EC 1.11.1.7 donor: hydrogen in the peroxide oxidoreductases. The POD reactions consist transfer of hydrogen from a variety of compounds and in partic- ular phenols, polyphenols and aromatic amines. In consequence of scientific interest in POD, initiated in 1903 multi-aspect knowl- edge is collected in regard to occurrence in various cell types, subcellular localization and biochemical functions/implications in animal/plant tissues and bacteria. POD assay is applied not only in basic biochemistry but also in medical science (e.g. clinical diagnostics), agriculture (e.g. phytopathology, chemical resistance to pests), nutrition/food chemistry and environmental science. Then, there is logical that POD assay is becoming significant practice for courses i.e. introductory biochemistry, biochemical diagnostics/toxicology and environmental biochemistry/biotech- nology. In my opinion, due to wide distribution in nature, chro- mogenic substrates and fast rate of reaction POD appears to be ‘model enzyme’ for biochemical education. Yet, students practise POD reaction usually with carcinogenic benzidine or harmful pyrogallol/quaiacol, and experimental materials are still horserad- ish roots or potatoes. In this paper experimental technique with 3,3¢,5,5¢-tetramethyl benzidine (TMB) for POD activity in Aphidi- dae, other Homoptera or Heteroptera is propounded. For POD detection in salivary secretions aphids ‘closed’ in plastic ring are allowed to puncture the agarose (1.25% w/v) – sucrose (30% w/ v) gel through parafilm membrane, for 1–2 h. Next the gel is immersed in TMB (6.0 mM) – H2O2 (30 mM) mixture, freshly in dark for prepared in 0.1 M phosphate buffer, pH 6.5–7.0, 30 min to1 h, blue staining demonstrates POD reaction. For POD reaction in digestive system, this is freshly dissected within the TMB-H2O2 incubation medium, and the blue colouring of the midgut appears in 30 s to 3 min and is visible at the light microscope. The TMB-H2O2procedure is fast, non-toxic and sen- sitive for microscopic visualization. Moreover, it is useful for understanding of exogenous and endogenous occurrence of POD activity in phytophagous insects and other animals.

P4–133 Growth differentiation factor-15 secreted by prostate cancer cells inhibits differentiation of osteoclasts P. Vanhara1, E. Lincova2, K. Soucek2 and J. Smarda1 1Department of Experimental Biology, Masaryk University Faculty of Science, Brno, CZECH REPUBLIC, 2Academy of Science of Czech Republic, Institute of Biophysics Laboratory of Cytokinetics, Brno, CZECH REPUBLIC

Methyl-, methoxy-, hydroxy- and dimethylamino- cyclic chalcone analogues with the substituent at the same position were selected to observe affection the mitochondria activities in case of reactive oxygen species production and connected endogenous antioxi- dants. Methyl- substituent appeared in both selected structures E-2-arylmethylene-1-tetralone and E-2-arylmethylene-1-benzosub- erone protective efficacy. Unsufficiency of antioxidant defensive system (reduced glutathione and glutathione related enzymes as glutathione peroxidase and glutahione reductase) were induced predominantly by E-2-arylmethylene-1-benzosuberone structures irrespectlively on the character of the substituent. For the com- plete comparison various chalcone analogue structures of two groups with assessed toxic effect of hydroxy- and dimethylamino- substituents were collected. Evaluated parameters of chalcone, E- 2-arylidene-1-indanone and two previously mentioned structures revealed toxic influence the mitochondria of the E-2-arylidene-1- indanones, additionally. On the contrary, E-2-arylmethylene-1- tetralone pronounced clearly protective action regardless on the structure or the substituent. Acknowledgement: The study was supported by VEGA 1/ 0624/08.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Prostate cancer is a common disease in industrial countries. Pros- tate tumors preferentially disseminate to bones and induce signifi- rearrangement of bone architecture. The mechanisms cant responsible for selective homing and activity of cancer cells in bone microenvironment have not been clarified yet. The aim of this study is to characterize effects of growth differentiation fac-

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Abstracts

P4–135 Evidence for the presence of gamma-tubulin in the nucleolus S. Vinopal1, B. Horejsi1, V. Markova1, V. Richterova1, E. Draberova1, V. Sulimenko1, A. Philimonenko2, P. Hozak2, C. D. Katsetos3 and P. Draber1 1Laboratory of Biology of Cytoskeleton, Institute of Molecular Genetics, Prague 4, CZECH REPUBLIC, 2Laboratory of Biology of the Cell Nucleus, Institute of Molecular Genetics, Prague 4, CZECH REPUBLIC, 3Department of Pathology and Laboratory Medicine, St Christopher’s Hospital for Children, Philadelphia, PA, USA

carnitine/acylcarnitine translocase and CPT2, responsible for the import of long-chain acyl units into the mitochondria for mFAO. This system could reverse its mechanism and export acyl groups from the mitochondria as acylcarnitines. Herein we aimed to extend our previous studies towards CPT2 substrate specificity in order to understand its role in the intramitochondrial synthesis of acylcarnitines as part of a putative mitochondrial detoxifica- tion mechanism. S. cerevisiae homogenates expressing human CPT2 were incubated with saturated and unsaturated C2–C26 acyl-CoAs and acyl-CoAs from the BCAAO. The produced acyl- carnitines were quantified by ESI-MS/MS. CPT2 was found to be active towards medium (C8–C12) and long-chain acyl-CoA esters (C14–C18), whereas virtually no activity was observed with short- and very long-chain acyl-CoAs or with BCAAO intermedi- ates. The trans-2,3-enoyl-CoA intermediates were also found to be poor substrates for CPT2 and preliminary inhibition studies performed with trans-2,3-C16:1-CoA suggest that it may act as a CPT2 competitive inhibitor. These results indicate that CPT2 may be implicated in mitochondrial clearance of C8–C18 acyl- CoAs. Formation of short, very long-chain or BCAAO carnitine derivatives may result from the activity of other enzyme(s). Poor enoyl-CoA intermediates detoxification can explain the absence of trans-2,3-enoyl-carnitines in mitochondrial trifunctional pro- tein deficiency and clarify the severity and specific clinical mani- festations of this pathology when compared with other mFAOD.

expression as well

P4–137 Cryptic promoter activity in firefly luciferase gene can affect experimental results V. Vopalensky1, T. Masek1, O. Horvath2 and M. Pospisek1 1Genetics and Microbiology, Faculty of Science Charles University in Prague, Prague 2, CZECH REPUBLIC, 2Laboratory of Leukocyte Antigens, Institute of Molecular Genetics, Prague 4, CZECH REPUBLIC

Galectin-3, a b-galactoside binding lectin, is spread among differ- ent types of tissues and cells found intracellularly in nucleus and cytoplasm or secreted via non-classical pathway outside of cell, thus being found on the cell surface or in the extracellular space. Through specific interactions with a variety of intra- and extra- cellular proteins galectin-3 affects numerous biological processes. Of special interest is its involvement in modulation of innate and adaptive immune response by participation in e.g. neutrophil adhesion to laminin and endothelial cells, stimulation of oxida- tive burst in neutrophils and macrophages and promotion of leu- kocyte recruitment. Galectin-3 expression is influenced by various stimuli but the precise regulatory mechanisms are not yet eluci- dated. The aim of this study was to ascertain the level of galec- tin-3 expression in resting and activated monocytic cells (taking in vitro and ex vivo approach) and investigate the involvement of JNK, p38 and ERK signaling pathways in the regulation of galectin-3 expression. Additionally, the influence of exogenously added recombinant galectin-3 on transcriptional level of galectin- 3 gene (‘autocrine effect’) and on the activation of aforemen- tioned pathways was studied. Our data revealed a part of the reg- ulatory mechanisms of as galectin-3 mechanisms of its action contributing to the better understanding of physiological role of that lectin in the immune cells physio- logy.

P4–136 Involvement of the carnitine cycle in a potential mitochondrial detoxification pathway: the role of carnitine palmitoyltransferase 2 S. Violante1, L. IJlst2, I. Tavares de Almeida1, R. J. Wanders2 and F. V. Ventura1 1Metabolism and Genetics, iMed.UL – Research Institute for Medicines and Pharmaceutical Sciences, Lisboa, PORTUGAL, 2Laboratory of Genetic Metabolic Diseases, AMC University of Amsterdam, Amsterdam, THE NETHERLANDS

Firefly luciferase (FLuc) from the common North American fire- fly Photinus pyralis is very popular nowadays as a very sensitive reporter with an extraordinarily broad dynamic range of the measurable activity. The sensitivity, versatility and relative sim- plicity of the luciferase-based assays are the reasons why FLuc ranks among the most frequently used reporter genes and as such has been used in thousands of variously designed experiments since the time of its first discovery for application in molecular and cellular biology. We report here a cryptic promoter activity in the luc+ gene, which is the most frequently used version of the firefly luciferase. The firefly luciferase coding region displays cryptic promoter activity in human CCL13 and Huh7 cells, in which cryptic transcription from the luc+ gene is 10–16 times weaker in comparison to the strong immediate-early cytomegalo- virus promoter. Additionally, we will discuss a possible impact of the FLuc gene cryptic promoter on experimental results especially in in some fields of the RNA-oriented research, for example, analysis of translation initiation or analysis of miRNA/siRNA function. Our findings should appeal to the researchers to be more careful when designing firefly luciferase based assays as well as open the possibility of performing some experiments with the hepatitis C virus internal ribosome entry site, which could not be considered until now.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Mitochondrial fatty acid ß-oxidation (mFAO) is a crucial meta- bolic pathway especially in response to fasting and prolonged exercise. Various inborn errors of metabolism have been described to affect mFAO. The pathogenic mechanisms associ- ated with these disorders have been correlated with the intrami- tochondrial and cellular accumulation of toxic metabolites, namely acyl-CoA intermediates. Although described as severe dis- orders many of these patients do survive which suggests the exis- tence of mechanisms protecting the mitochondria from acyl- CoAs. The characteristic acylcarnitines profiles described for most of mFAOD and branched-chain aminoacid oxidation defects (BCAAOD) suggests the export out of the mitochondria of acyl-CoAs as carnitine derivatives. We propose that mitochon- drial synthesis and export of acylcarnitines likely involves the car- nitine-cycle, composed by carnitine palmitoyltransferase (CPT) 1,

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Poster Presentations

P4–140 Functional characterization of interferon induced transmembrane protein 1 (IFITM1) in colorectal carcinogenesis F. Yu, S. S. M. Ng, M. C. T. Lum, W. K. C. Cheung, X. M. An and M. C. M. Lin Chemistry, The University of Hong Kong, Hong Kong, CHINA

P4–138 Glycerol-3-phosphate induced ROS formation and oxidative damage in mammalian mitochondria M. Vrbacky, Z. Drahota, T. Mracek, A. Pecinova, P. Jesina and J. Houstek Bioenergetics, Institute of Physiology Academy of Sciences of the Czech Republic, Prague, CZECH REPUBLIC

(BAT) mitochondria that

IFITM1, a member of interferon induced transmembrane protein family, was firstly cloned from a human lymphoid cell cDNA library. The role of IFITM1 in colorectal carcinogenesis is poorly understood. We found in 15 out of 19 colon cancer patient tis- sues the IFITM1 mRNA expression levels are more than 2-fold higher than that of the corresponding adjacent normal tissues. Knocking down IFITM1 by a small interfering RNA (siIFITM1) could inhibit cell migration and invasion in Lovo and HT-29, two of the high IFITM1-expressing colon cancer cell lines. In contrast, overexpression of IFITM1 by pcDNA3.1(+)-IFITM1 transfection could promote cell migration and invasion in DLD1, a low IFITM1-expressing colon cancer cell line. By DNA micro- array analysis coupled with semi-quantitative reverse transcrip- tion PCR, we identified that several cell migration related genes, such as Cav-1, MDK, Serpina1 and CTTN, are differentially expressed after knocking down IFITM1 in HT-29 cells. In con- clusion, we have demonstrated that IFITM1 can enhance cell migration and invasion in colon cancer cells, and this effect may be modulated by the up-regulation of Caveolin-1 gene expres- sion.

Reactive oxygen species (ROS) are produced by several mito- chondrial enzymes and contribute to mitochondrial damage in a range of pathologies. Here we studied the involvement of mammalian mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH, EC 1.1.99.5) in ROS generation in brown adipose tissue contain high amount of mGPDH as well as in mitochondria from tissues, whose mGPDH content is very low (liver, heart and brain). ROS-sen- sitive spectrofluorometric probes in the presence of specific respiratory chain inhibitors antimycin A and myxothiazol allowed us to analyze the contribution of dehydrogenase and coenzyme Q pool in ROS production as compared to complex III. In all mitochondria, glycerol-3-phosphate induced a high amount of ROS production that was localized to the dehydro- genase + CoQ and complex III, the former being the highest in BAT. When this myxothiazol-insensitive ROS production was related to the activity of mGPDH, 10-fold higher ROS production was found in liver, 14-fold in heart, and 3-fold in brain mitochondria as compared to BAT suggesting its endog- enous inhibition in BAT. Analysis of several oxidative damage markers (lipofuscin-like pigments, 4-hydroxy-2-nonenal; inhibi- tion of respiration) revealed a marked difference in susceptibil- ity of BAT (resistant) and liver mitochondria to ROS. Our results thus demonstrate high efficiency of ROS production by mGPDH and tissue-specific characteristics and consequences of mitochondrial ROS production.

P4–141 Glycolysis in human breast cancer cells: a potential target for therapy with clotrimazole P. Zancan Molecular Oncobiology Laboratory (LabOMol), Pharmacy School – Federal University of Rio de Janeiro, Rio de Janeiro, BRAZIL

P4–139 Molecular dynamics of focal adhesion components: a role for a juxtamembrane cytoplasmic domain H. Wolfenson1, A. Lubelski2, T. Regev1, J. Klafter2, H. Yoav1 and B. Geiger3 1The Department of Neurobiology George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, ISRAEL, 2School of Chemistry Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv, ISRAEL, 3Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, ISRAEL

Cancer cells are characterized by a high rate of glycolysis, which is their primary energy source. Glycolysis is known to be con- trolled by allosteric regulators, as well as by reversible binding of glycolytic enzymes to the cytoskeleton. Clotrimazole (CTZ) is an anti-fungal azole derivative recognized as a calmodulin antago- nist with promising anti-cancer effect. This effect is partially attributed to the ability to alter the intracellular localization of glycolytic enzymes. Here, we show that CTZ induces functional alterations on human breast cancer derived cell lines, MCF-7 and MDA-MB-231. The drug promotes a dose-dependent decrease on cell viability, which is more pronounced on MDA-MB-231 when compared to MCF-7. Moreover, MCF-7 resistance to CTZ treat- ment significantly decreases when insulin is present in growth medium. In addition, CTZ directly alters the activity of the gly- colytic enzymes, 6-phosphofructo-1-kinase and hexokinase, espe- cially upon 3 h of treatment and in doses above 50 lM. Altogether, our results support evidences for the antineoplasic effects of CTZ that might start by inhibiting glycolytic flux, as revealed by the inhibition of the key glycolytic enzymes. Hence, energy metabolism may be an alternative therapeutic target for glycolytic tumors. Financial support: CNPq, FAPERJ, FAF/ FECD and PRONEX.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Focal adhesions (FAs) are specialized membrane-associated multi-protein complexes that link the cell to the extracellular matrix and play crucial roles in cell-matrix sensing. Considerable information is available on the complex molecular composition of these sites, yet the regulation of FA dynamics is largely unknown. Based on a combination of FRAP studies in live cells, with in silico experiments and mathematical modeling, we show that the FA plaque proteins paxillin and vinculin exist in four dynamic states: an immobile FA-bound fraction, an FA-associ- ated fraction undergoing exchange, a juxtamembrane fraction experiencing attenuated diffusion, and a fast-diffusing cytoplas- mic pool. The juxtamembrane region surrounding FAs displays a gradient of FA plaque proteins with respect to both concentra- tion and dynamics. Based on these findings, we propose a new model for the regulation of FA dynamics in which this juxta- membrane domain can act as an intermediary layer, enabling an efficient regulation of FA formation and reorganization.

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Poster Presentations

Abstracts

P4–143 Pathways for release of apoptotic factors from yeast mitochondria R. Zvyagilskaya, E. Sukhanova, M. Kovaleva and T. Trendeleva Laboratory of biological oxidation, A. N. Bach Institute of Biochemistry, Moscow, RUSSIA

P4–142 Plant phospholipase D isoforms regulate cell polarity in tobacco pollen tubes M. Potocky1, R. Bezvoda2, R. Pleskot1 and V. Zarsky2 1Laboratory of Cell Biology, Institute of Experimental Botany Academy of Sciences of the Czech Republic Rozvojova 263, Prague, CZECH REPUBLIC, 2Departement of Plant Physiology, Charles University Faculty of Science Vinicna 5, Prague, CZECH REPUBLIC

intracellular ATP content Introduction: During the last years, several reports described an apoptosis-liked programmed cell death in yeasts in response to different environmental stimuli. Several apoptotic factors were identified in yeast mitochondria, including endonuclease G, AMID, cytochrome c, and Omi-protease, normally confined to the intermembrane space. However, the mechanisms underlying release from mitochondria into the cytosol are still remain unclear. Of note, obvious orthologues of Bcl-2 proteins, key mammalian apoptotic regulators are lacking in yeast cells. A role of fission/division proteins in promoting apoptosis in yeast cells is also uncertain. This prompts us to assess induction of the non- specific permeability transition (pore) as the leading mechanism leading to release of apoptotic factors from yeast mitochondria. Methods: Dipodascus (Endomyces) magnusii and Yarrowia li- polytica cells, aerobes, were used in this study. Large amplitude mitochondrial swelling and collapse of the mitochondrial mem- brane potential were applied in demonstrating pore induction. Results: D. magnusii and Y. lipolytica mitochondria were resis- tant to Ca2+ overload and did not undergo the classical Ca2+/ Pi-dependent cyclosporine A-sensitive pore, or a pore induced by prooxidants, Ca2+ and saturated fatty acids, anaerobiosis, deple- tion of intramitochondrial adenine nucleotide pools, deenergiza- tion of mitochondria, The only pore found in yeast mitochondria under study was a highly regulated ATP-dependent K+-channel of ‘animal type’ closed in response to ATP. Conclusions: We suggested that commitment of D. magnusii and Y. lipolytica cells to a programmed cell death process would be dependent on opening of the ATP-dependent K+-channel fol- lowing substantial depletion of in response to different aggressing stimuli.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

from GAAV-IAA601110916, Phospholipase D (PLD) cleaves structural phospholipids produc- ing phosphatidic acid (PA). PLD and PA play crucial role in many signalling pathways across eukaryotic kingdom. In animal and yeast cells, PLD was implicated in the control of vesicular trafficking, while in plants, PLD research is focused on its role in stress responses. Here we present bioinformatic data describing complex family of PLD isoforms in eukaryotes together with experimental data showing that multiple PLDs are required for tobacco pollen tubes. Phylogenomic analysis of PLD homologs revealed great diversity of this family in eukaryotic lineages and identified distinct PLD subclasses with variable distribution and domain composition. PLD phylogeny indicates that there were tremendous lineage-specific duplications and diversifications of PLD in eukaryotes and especially in land plants. The outcomes from this comparative analysis may facilitate study of this gene family across eukaryotes. In vivo inhibition of PLD-mediated PA production by primary alcohols rapidly disturbed pollen tube cyt- oarchitecture visualized by video-enhanced contrast and electron microscopy. Conversely, exogenous PA was able to stimulate pol- len tube growth and trafficking dynamics. We cloned four PLD cDNAs from tobacco pollen tubes and vegetative cells. Anti- sense-mediated knock-down of NtPLDb1 and NtPLDd lead to inhibition of pollen tube time- and concentration-dependent growth. NtPLDb1 is implicated in the regulation of actin and microtubular cytoskeleton, while NtPLDd signaling operates downstream of reactive oxygen species. PLD-derived PA thus participates in multiple processes controlling polar expansion of plant cells. Acknowledgements: Support MSM-LC06034 and MSM0021620858 grants.

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Poster Presentations

P5 Signal Transduction

Conclusion: These data show that NO stabilizes endothelial barrier by activation of MLCP, leading to an inactivation of con- tractile machinery, and enhancement of cell-cell and cell-matrix adhesions. Co-immunoprecipitation of PKG and MYPT1 indi- cate that PKG may mediate its effect on MLCP by a direct inter- action with MYPT1.

P5–1 Phosphorus NMR of model membrane interaction with nitric oxide Y. Adiguzel1, S. Hippe1, H. J. Hauswald2, R. Stoll2 and R. Heumann1 1Neurobiochemistry, Ruhr University Bochum, Bochum, GER- MANY, 2Biomolecular NMR, Ruhr University Bochum, Bochum, GERMANY

P5–3 Regulation of an inhibitor of the MAP Kinase ERK; the Phosphatase DUSP6 O. Bermudez1, P. Jouandin1, J. Rottier2, G. Pages1 and C. Gimond1 1Institute of Developmental Biology and Cancer, Alpes Maritimes, Nice, FRANCE, 2University of Lyon, Rhoˆne, Lyon, FRANCE

Neuronal rhythmic activity, synaptic transmission and propaga- tion of excitation are processes that involve nitric oxide (NO) and plasma membrane at different cellular levels. Hence, it is aimed in this study to reveal the details of relationship between these two agents, NO and membrane, by the aid of phosphorus (31P) NMR. It was found that NO was leading to structural transitions of the membrane, and affecting the membrane’s physi- co-chemical properties. We believe that these characteristic phe- nomena could be the triggering stimuli of forthcoming structural changes in the cytoplasm, and events such as vesicle movement, budding, fusion, and reorganization of cytoskeleton.

P5–2 Nitric Oxide protects endothelial barrier function via inactivation of the contractile machinery and stabilisation of cell-adhesions M. Aslam, F. Haertel, D. Guenduez and T. Noll Institute of Physiology, Justus Liebig University, Giessen, GERMANY

DUSP6/MKP-3 is a cytoplasmic phosphatase that specifically dephosphorylates and inactivates the MAP Kinases ERK ½. We had previously shown that DUSP6 was phosphorylated and degraded by the proteasome upon growth factor stimulation, in a MEK-dependent manner (Marchetti et al., 2005 Mol. Cell. Biol.). The PI3K/mTOR signaling pathway also plays a role in the phosphorylation and degradation of DUSP6 induced by serum growth factors (Bermudez et al., 2008, Oncogene). We now inves- tigate the molecular basis of DUSP6 regulation at the mRNA level. Others have shown a role for the MEK/ERK pathway in transcriptional activation of dusp6, and we confirmed that their inhibition strongly diminished the amount of dusp6 mRNA. To determine whether the stability of dusp6 mRNA could be sub- jected to regulation, a luciferase reporter was cloned upstream of the non coding 3¢UTR of dusp6, which contains consensus sequences for various stabilization/destabilization factors. The MEK/ERK pathway was found to stabilize dusp6 mRNA, although pharmacological inhibitors also suggest a potential role for other pathways in this regulation. Hypoxic conditions, a hall- mark of many tumors in vivo, induce a modest but reproducible increase in dusp6 mRNA levels, which is HIF1-alpha dependent. Consistent with increased dusp6 mRNA levels in hypoxia, we found that pERK levels are diminished under hypoxia in several albeit not all cancer cell lines tested. Finally, preliminary data point for a role of mRNA-destabilizing factors, such as triste- traprolin (TTP), in the regulation of dusp6 mRNA. Altogether, these results indicate that the regulation of DUSP6 involves the MEK/ERK pathway at different levels, at the mRNA level as well as at the post-translation level. They also show that other major signalling pathways may participate in this regulation.

210 ± 12%, by

P5–4 Lentiviral delived siRNA against ICER protects primary cortical neurons from apoptosis caused by serum depriviation K. Bieganska, A. Klejman, D. Gierej and L. Kaczmarek Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology, Warsaw, POLAND

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Failure of endothelial barrier function induced by inflammatory mediators causes severe edema that impedes functional recovery of the organ. NO by activating cGMP/PKG pathway protects against imminent barrier failure. Here the hypothesis was tested whether NO protects endothelial barrier by inhibiting the con- tractile machinery and stabilizing cell-cell adhesions, two major determinants of endothelial barrier function. Methods and Results: Exposure of cultured monolayers of HUVEC to Spermine-NONOate [NO] (10 lM, 20 min) reduced permeability (P, albumin flux) by 35 ± 7%, isometric tension (IT, cells cultured on collagen gels) by 80 ± 9%, contractile acti- vation, (myosin light chain [MLC] phosphorylation, Western blot) by 31 ± 6% (P < 0.05, n = 5 for all following parame- ters). During the same time period NO caused a 2-fold increase in phosphorylation of vasodilator-stimulated phosphoprotein (VASP). All these NO effects could be blunted by KT5823 (1 lM), a specific PKG inhibitor. Thrombin (0.2 U/ml) increased IT (130 ± 8%), MLC3P permeability (90 ± 8%). These effects were attenuated significantly when cells were preincubated with NO. NO induced assembly and activation of the myosin light chain phosphatase (MLCP) holoenzyme com- plex (3-fold increase in translocation of PP1 catalytic subunit to myosin phosphatase targeting subunit [MYPT1], immunoprecipi- tation, IP), and 1.5-fold increase in PP1 activity. It also induced a 3-fold increase in translocation of PKG to MYPT1 (IP). NO induced translocation of VE-cadherin, b-catenin, and c-catenin to cell-cell junctions. Thrombin induced loss of VE-cadherin, b-cate- nin, and c-catenin from cell-cell junctions. These thrombin effects were attenuated significantly by preincubation with NO. More- over, NO induced phosphorylation of the focal adhesion kinase (FAK) and increased formation of focal adhesions (cell fraction- ation, confocal microscopy). CREB activation and CREB-dependent signaling pathways are crucial for neuronal survival. The term ICER (inducible cAMP early repressor) refers to four protein isoforms that are all endog- enous, inducible antagonists of CREB. It was previously shown, that all 4 ICER isoforms are induced upon proapoptotic treat- ment, and also that each of them separately evokes neuronal cell death in cortical culture transfected with these genes. The ICER proteins are believed to be strong repressors of Immediate Early

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Abstracts

siRNA against

Genes, genes which are involved in cell response to inter and/or intra cellular signals. For our research we applied the siRNA technique to achieve silencing of ICER’s expression. Because ICER’s are members of CREM proteins family and shares with them gene sequence, only the small unique region for ICER was the base for siRNA designing. Despite such little creating space, we obtained functional ICER which block ICER’s but not other CREM proteins. We have investigated if the ICER’s silencing could protect neurons from apoptosis caused by unfavorable environmental conditions such as serum depriviation or excytotoxic effect of glutamate. Using the lentivi- ral vector, as a tool for delivery of siRNA (shRNA) we have shown, that silencing of ICER protect primary cortical neurons from apoptosis caused by serum depriviation.

P5–5 Identifcation of candidate protein tyrosine phosphatases involved in PrPc signalling B. Pantera, P. Paoli, P. Cirri, G. Camici, G. Manao and A. Caselli Scienze biochimiche, Universita di Firenze, Firenze, ITALY

function(s)

grants A600040701, regulating plant development are being intensely researched. To get an insight into CK regulated molecular events at the prote- ome level, we employed 2-DE followed by image analysis and MALDI-TOF-TOF MS to analyze early changes in steady-state protein levels and phosphorylation status of the proteome in CK treated Arabidopsis thaliana seedlings. Effects of four principal CKs, t-zeatin, 6-(g-g)dimethylallylaminopurine, thidiazuron and isopente- 6-benzylaminopurine representing hydroxyisopentenyl, nyl, urea-derived and aromatic CKs, respectively, were compared. For analysis of polypeptide steady state levels, total protein was extracted using acetone/TCA procedure. For phosphoproteome analysis, an isolation procedure was established based on Phos- phoProtein Purification Kit (Qiagen). We observed over 160 and 90 differently expressed proteins in proteome and phosphoprote- ome maps, respectively, which represent about 20% of detected protein spots. Out of them, 102 proteins were identified. They represent a snapshot of early links involved in CK regulated sig- naling circuits and cellular processes including light signaling and photosynthesis, nitrogen metabolism, ethylene biosynthesis, CLAVATA pathway, and protein and gene expression regulation which are in line with previously described CK functions. Fur- thermore, our results point to a link between temperature and CK signaling. The data represent the first comparative study of action of the four distinct CK types on proteomic/phosphoprote- omic level that is expected to contribute to the elucidation of their common and individual in regulating plant development. Acknowledgements: Supported by LC06034, 1M06030 and AV0Z40310501.

P5–7 Stuy on dynamic interplay of STAT3- associated proteins in response to multiple activation pathways R. Cocchiola1, D. Muscolino1, C. Grillo1, S. Chichiarelli1, E. Gaucci1, A. Ferraro1, F. Altieri1, C. Turano2 and M. Eufemi1 1Biochemical Sciences ‘A. Rossi Fanelli’, Sapienza University of Rome, roma, ITALY, 2Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza University of Rome, roma, ITALY

Cellular prion protein (PrPc) is a ubiquitous glycoprotein which physiological role is poorly characterized. It has been suggested that PrPc participates in synaptic structure, neurite formation, cop- per metabolism, and signal transduction (1, 2). PrPc antibody cross-linking stimulates the caveolin-dependent activation of Fyn leading to a rapid and transient phosphorylation of Erk1/2 (2, 3). The activity of Src family tyrosine kinases (SFK) is regulated by tyrosine phosphorylation at two sites, but with opposing effects. Phosphorylation of a tyrosine residue in the activation loop of the kinase domain upregulates enzyme activity, whereas phosphoryla- tion by Csk of a tyrosine residue in the carboxy-terminal tail ren- ders the enzyme less active stabilizing a noncatalytic conformation. An increasing number of protein-tyrosine phosphatases (PTPs) are being implicated in controlling SFK phosphorylation and activity. Such SFK-activating PTPs include both non-receptor (e.g. PTP1B, SHP1, and SHP2) and receptor PTPs (RPTPs, such as CD45, RPTPa, RPTPb, and LAR) (4). Following this lead, we investi- gated the possibility that one of these PTPs is involved in PrPc- dependent activation of Fyn. We peformed co-immunprecipitation experiments, PTPs in gel assay on isolated caveolae-like membrane domains and signalling inhibition tests by using selected synthetic compounds. Our preliminary results suggest RPTPb and/or RPTPa as major phosphatases involved in PrPc signalling. References: 1. Linden R. et al. Physiol. Rev. 2008; 88: 673. 2. Mouillet-Richard et al. Ann N Y Acad Sci. 2007; 1096: 106. 3. Schneider B. et al. Proc. Natl. Acad. Sci. U.S.A 2003; 100: 13326. 4. Vacaresse N. et al. J Biol Chem. 2008; 283: 35815.

P5–6 Early cytokinin response proteins and phosphoproteins of Arabidopsis thaliana identified by proteome and phosphoproteome profiling M. Cerny1, F. Dycka2, J. Bobalova2 and B. Brzobohaty1 1Laboratory of Plant Molecular Biology, Mendel University of Agriculture and Forestry in Brno & Institute of Biophysics AS CR, Brno, CZECH REPUBLIC, 2Department of Proteomics and Glycomics, Institute of Analytical Chemistry AS CR, Brno, CZECH REPUBLIC

In the ChIP assay,

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

The transcriptional processes regulated by nucleoproteins com- plexes, called enhanceosome, in higher eukaryotes play a critical role in the integration of cellular signalling information. In this context the STAT3-dependent enhanceosome is particularly inter- esting. The signal transducer and activator of transcription-3, a cytoplasmatic latent transcription factor, is a point of conver- gence for numerous oncogenic signalling pathways. STAT3, con- stitutively activated in several cancer cells, up-regulates the expression of numerous genes, involved in promoting tumour cell proliferation, angiogenesis, metastasis and cell survival. This research is focused on the influence of diverse extracellular sig- nals on the composition of nuclear multi-protein STAT3-specific complexs. Melanoma cell (M14) is our cellular model of choice, because STAT3 is constitutively activated by SRC but is also responsive to stimulation by cytokine and growth factors. In order to identify the proteins of STAT3-dependent enhanceo- some, we performed experiments in vitro, such as DNA affinity assay, and in vivo such as Chromatin Immunoprecipitation (ChIP) and Co-Immunoprecipitation (CoIP). The proteins so purified, were analyzed by SDS–PAGE then subjected to Mass- Spectrometry analysis and Western Blotting. The results led the presence of proteins PARP-1 or CBP/P300, as components of en- hanceosome, depending on the specific pathways of STAT3 acti- vation. the immunoprecipitated DNA, analyzed by PCR to enrichment of the three sequences containing the putative site for STAT3 protein. The same experiments per- Cytokinins (CKs) are known to regulate diverse developmental processes in plants. The molecular mechanisms of CK action in

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Poster Presentations

formed after pretreatment of the cell with specific inhibitors for PARP-1 and for CBP/P300, showed that interactions STAT3- PARP-1 and STAT3-CBP/P300 were abolished. Therefore, we looked for the effect of these inhibitors on the transcription of the p21, mmp9 and a2- macroglobulin genes, and in both cases the transcription of the three genes was significantly repressed. The results obtained showed that the interactions between STAT3 and its co-activators is strongly dependent on the signal- ling pathways. Particularly for two nuclear proteins, PARP-1 and CBP/P300, involved in the process of chromatin remodelling. When STAT3 is activated by cytokine (IL-6), its enhanceosome contains the P300 protein, while PARP-1 is present following the activation by the SRC kinase.

determining cell fate. Stable overexpression of SRPK1a induces erythroid differentiation of K562 cells. The induction of globin synthesis was accompanied by a marked decrease in proliferation and a significantly reduced clonogenic potential. In addition, small interfering RNA-mediated down-regulation of SRPK1 in K562 cells results similarly in a decrease in proliferative capacity and induction of globin synthesis. An increased SRPK1a/SRPK1 ratio is also observed upon hemin/DMSO-induced differentiation of K562 cells. Aiming to identify proteins specifically associated with SRPK1a, we immunopurified FLAG-tagged SRPK1a from extracts of stably transfected K562 cells and characterized the co- purified proteins by MS–MS based mass spectroscopy. Several RNA-binding proteins involved in the regulation of mRNA sta- bility were identified. These findings suggest that SRPK1a may play an important role in linking signaling to erythroid differenti- ation in human leukaemic cells.

P5–8 Galectin-3/signaling cascade interplay in immune cells S. Dabelic, R. Novak, A. Lepur and J. Dumic Biochemistry and Molecular Biology, Faculty of Pharmacy and Biochemistry University of Zagreb, Zagreb, CROATIA

P5–10 Transcriptional regulation of H-Ferritin gene after TSH stimulation: NF-Y/p300 complex M. Di Sanzo, F. Romeo, G. Epifanio, E. Arcuri, B. M. D’Alessandro, L. Falbo, R. Sottile, B. Quaresima, M. C. Faniello and F. S. Costanzo Experimental and Clinical Medicine, Magna Graecia University, Catanzaro, ITALY

Galectin-3, a b-galactoside binding lectin, is spread among differ- ent types of tissues and cells found intracellularly in nucleus and cytoplasm or secreted via non-classical pathway outside of cell, thus being found on the cell surface or in the extracellular space. Through specific interactions with a variety of intra- and extra- cellular proteins galectin-3 affects numerous biological processes. Of special interest is its involvement in modulation of innate and adaptive immune response by participation in e.g. neutrophil adhesion to laminin and endothelial cells, stimulation of oxida- tive burst in neutrophils and macrophages and promotion of leu- kocyte recruitment. Galectin-3 expression is influenced by various stimuli but the precise regulatory mechanisms are not yet eluci- dated. The aim of this study was to ascertain the level of galec- tin-3 expression in resting and activated monocytic cells (taking in vitro and ex vivo approach) and investigate the involvement of JNK, p38 and ERK signaling pathways in the regulation of galectin-3 expression. Additionally, the influence of exogenously added recombinant galectin-3 on transcriptional level of galectin- 3 gene (‘autocrine effect’) and on the activation of aforemen- tioned pathways was studied. Our data revealed a part of the regulatory mechanisms of galectin-3 expression as well as mecha- nisms of its action contributing to the better understanding of physiological role of that lectin in the immune cells physiology.

TSH modulates survival, proliferation and differentiation in thy- reocyte by binding membrane receptors specific (TSH-R) via the cAMP/PKA pathway. cAMP activates any transcription factors, such as CREB and NF-Y. NF-Y on the H ferritin promoter binds an inverted CAAT box recognized by a protein complex called the B-box binding factor (Bbf). Bbf is composed of the tri- meric transcription factor NF-Y, that directly binds DNA, of the co-activator p300 and of the histone acetylase pCAF. Moreover, cAMP and its analogs have been shown to promote the binding, thus inducing ferritin gene transcription. The ferritin expression is regulated by many pathological conditions, like inflammation and oxidative stress, is stimolated during development, cell differ- entiation and is modulated by several factor, such as cell growth, hormones, and cytokines. In particular, the H- ferritin synthesis in thyroid cells is stimulated by hormones such as TSH. Aim of the present study is to analyze the expression of the H-ferritin gene after TSH stimulation, with particular attention to tran- scriptional modulation and the role played by the complex NF- Y/p300. To this end, thyroid cells were subjected to TSH by using increasing amounts. H ferritin mRNA levels of treated and untreated cells with TSH were valutated by Northen Blot and RT PCR assays. Moreover, protein levels were estimated by wes- tern blot assays. The analysis of transcription induction efficiency were carried on by transient transfection experiments in cell trea- ted and untreated with TSH. Our results showed that H-Ferritin transcription is increased after TSH induction.

P5–9 The ratio of SRPK1a/SRPK1 regulates erythroid differentiation in K562 leukaemic cells M. Daniilidou1, I. Sanidas1, V. Kotoula2, L. Christof3, V. Cruft3, J. Daans4 and P. Ponsaerts4 1Chemistry, Aristotle University of Thessaloniki, Thessaloniki, GREECE, 2Medical School, Aristotle University of Thessaloniki, Thessaloniki, GREECE, 3Applied Biosystems, Proteomics, Darms- tadt, GERMANY, 4Medical School, Antwerp University, Antwerp, BELGIUM

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

SRPK1, the prototype of the serine/arginine family of kinases, has been implicated in the regulation of multiple cellular pro- cesses such as pre-mRNA splicing, chromatin structure, nuclear import and germ cell development. SRPK1a is a much less stud- ied isoform of SRPK1 that contains an extended N-terminal domain and so far has only been detected in human testis. In the present study we show that SRPK1 is the predominant isoform in K562 cells, with the ratio of the two isoforms being critical in

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vation of EGFR and of TLR2/4 in A431 carcinoma cells. In addition, e-Hsp70 was able to induce TLR2/4 association with EGFR. The neutralizing antibodies against both TLR2 and TLR4 decrease e-Hsp70-induced EGFR activation, which sug- gested the cross-talk between TLR2/4 and EGFR signaling sys- tems. However, the neutralizing antibodies against EGFR have no effect on e-Hsp70-induced EGFR activation, which suggested, that neither Hsp70 nor the other extracellular factor is bound to EGFR.

P5–11 Effect of cultivation medium on activity, phosphorylation level and kinetic parameters of phosphoenolpyruvate carboxylase V. Doubnerova1, K. Muller2, M. Cerny1, P. Spoustova3, H. Synkova3 and H. Ryslava1 1Department of Biochemistry, Faculty of Science Charles Univer- sity in Prague, Prague, CZECH REPUBLIC, 2Laboratory of Plant Reproduction, Institute of Experimental Botany Academy of Sciences of the Czech Republic, Prague, CZECH REPUBLIC, 3Laboratory of Stress Physiology, Institute of Experimental Botany Academy of Sciences of the Czech Republic, Prague, CZECH REPUBLIC

P5–13 Functional characterization of Hebp1 in the context of RasGRF1 signaling in neurons A. Ferna´ ndez-Medarde, T. Ortiz-Rivera and E. Santos Laboratory No.1, Centro de Investigacion del Cancer, Salamanca, SPAIN

Phosphoenolpyruvate carboxylase (PEPC; E. C. 4.1.1.31) cata- lyzes the irreversible b-carboxylation of PEP to yield oxaloacetate - ion and and inorganic phosphate with participation of HCO3 divalent metal ion. PEPC is a highly regulated cytosolic enzyme. The phosphorylation status is one of the most important regula- tory mechanisms of PEPC. We examined the effect of carbon ±, saccharose in in vitro cultivation medium) on source (CO2 activity and phosphorylation status of PEPC in tobacco plants (Nicotiana tabacum L.). Kinetic parameters such as Michaelis constant, maximal reaction rate, effects of D-glucose-6-P and L- malate on enzyme activity for phosphorylated and dephosphoryl- ated forms of PEPC were determined. The dephosphorylated form of tobacco PEPC had lower maximal reaction rate, it was less activated by D-glucose-6-P, especially at subsaturation PEP concentrations, but the sensitivity to L-malate was not influenced by phosphorylation. These characteristics of tobacco leaf PEPC were compared with those of PEPC from maize seeds (Zea mays, C4 plant). The dephosphorylation of PEPC from seeds caused the decrease of maximal reaction rate and it changed the kinetics from hyperbolic to sigmoidal one. Unlike to PEPC from tobacco leaves, dephosphorylated form of PEPC from maize seeds was more sensitive to inhibition by L-malate than phosphorylated one. Our results proved that the PEPC phosphorylation caused changes in enzyme kinetic characteristics, which was dependent on plant species. Acknowledgements: This work was supported by the grants of Ministry of Education of the Czech Republic MSM0021620808 and 1M0505.

P5–12 The cross-talk between TLR and EGFR induced by extracellular Hsp70 A. Evdonin, D. Popova and N. Medvedeva Department of Cell Signaling and Transport, Institute of Cytology Russian Academy of Science, Saint-Petersburg, RUSSIA

tetrapyrrole-binding protein isolated from Hebp1 is a small chicken retina and pineal gland. The functional significance of its potential heme-binding activity is unknown, but recent work has shown that a proteolytic fraction of Hebp1 is implicated in neu- trophil chemo-attraction. Our laboratory has recently shown that Hebp1 is strongly over-expressed in the hippocampus of Ras- GRF1 KO mice and we are interested in ascertaining possible Hebp1 functional roles in RasGRF1 mediated signalling in the neuronal environment. We observed that Hebp1 is expressed in many mouse tissues specially in liver, spleen and some regions of the CNS, like the hippocampus CA3 region. RasGRF1 expres- sion is also highest in this area, and this co-expression suggests the possibility of significant functional interactions between both proteins in this cellular context. Hebp1 protein displays an aver- age half-life of 8 hours in PC12 cells expressing full length Hebp1, and accumulates in early, late endosomes and lysosomes. Interestingly, we also detected Hebp1 accumulation at the nucleo- lus and in cells undergoing mitosis, suggesting a role in sister cell segregation. Thus, Hebp1 over-expression is associated to poly- ploidy. Differentiation studies showed that NGF induced PC12 cells over-expressing Hebp1 develop neurite outgrowths at a fas- ter rate than control cells. In the absence of differentiation induc- ers, Hebp1 transfected PC12 cells already showed clear morphological changes, including higher numbers of cellular pro- trusions than the controls. The Hebp1-linked alterations of cellu- lar shape appear to be independent of Ras-ERK pathway activation. Thus, control and Hebp1 over-expressing PC12 cells reach similar levels of ERK phosphorylation, and the specific U0126 ERK inhibitor is unable to inhibit the observed increased protrusion formation. These changes in morphology do not appear to affect either cellular motility or substrate adhesion. In addition, Hebp1 over-expression does not change the rates of cell proliferation or apoptosis. These data uncover a possible partici- pation of Hebp1 in processes of neuronal differentiation and/or mitosis. Further studies will be needed to clarify the exact cellular interactions between mechanisms involved and the potential Hebp1 and RasGRF1 that may contribute to those processes.

P5–14 Src tyrosine kinases modulate relevant male germ cells function: acrosome-reaction L. J. Garcia-Marin, M. C. Gil, A. D. Moreno and M. J. Bragado SINTREP, Veterinary School, Caceres, SPAIN

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Though initially Hsp70 was thought to be a typical intracellular protein, recent studies demonstrated that Hsp70 is being released into the blood or the conditional medium of cultured cells. Previ- ous studies indicated both immunomodulatory and pro-survival functions of extracellular Hsp70 (e-Hsp70). It has been shown that e-Hsp70 can promote tumor rejection, elicit an autoimmune response, or stimulate both adaptive and innate immunity. In addition, e-Hsp70 enhanced stress tolerance of immune and neu- ronal cells in culture. Nevertheless, the e-Hsp70 function is still poorly understood. e-Hsp70 was shown to induce signal trans- duction that engages Toll-like receptors 2 and 4 (TLR2/4) and lipopolysaccharide receptor CD14 in monocytes and macrophag- es. Previously we have shown that e-Hsp70 induces EGF receptor (EGFR) activation in A431 carcinoma cells. The purpose of this study was to investigate the possible cross-talk between TLR2/4 and EGFR in response to e-Hsp70. e-Hsp70 stimulates both acti- Signalling pathways involving protein tyrosine phosphorylation regulate somatic cell functions. However, few tyrosine kinases have been identified in male germ cells. Src family of tyrosine

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P5–16 Rat brain cells nuclear phospholipids under the cisplatin action N. Hakobyan, A. Hovhannisyan, Z. Yavroyan and E. Gevorgyan Faculty of Biology, Yerevan State University, Yerevan, ARMENIA

extracellular stress-related signals,

kinases (SFK) regulates important functions in spermatozoa although its role is specie specific. Our aims are to identify Src family members in Iberian pig spermatozoa and to study their role in relevant functions of these germ cells: motility, viability and acrosome reaction (AR). Identification of SFK members and their phosphorylated (active) forms was studied by Western blot- ting. Motility parameters were evaluated by computer assisted ISAS(cid:3) program and germ cell viability and AR by flow cytome- try. Our data shows that pig spermatozoa express at least two SFK members: cYes and cLyn. Active form of SFK phosphory- lated in Y416 was identified in control male germ cell lysates. Spermatozoa activated in a capacitating medium (TCM) or the induction of AR with calcium ionophore significantly increases Src active form (pY426). SFK specific inhibitor SU6656 did affect neither male germ cells viability nor basal or TCM/8Br- cAMP-stimulated pig spermatozoa motility. However, germ cells incubation with SU6656 significantly increases both non-stimu- lated (14 ± 1% vs. 20 ± 2%) and TCM-stimulated (17 ± 1% vs. 33 ± 2% at 240 min) acrosome reaction. In summary, our data shows that pig spermatozoa express several members of SFK, which are not involved in the regulation of neither motility nor viability. Data suggest an inhibitory role of SFK in the regu- lation of one of the most relevant functions of male germ cells: acrosome reaction. Acknowledgement: Supported by JUEX PRI07A100.

Cisplatin has been used for many years in chemotherapy. Cis- platin induces a number of signaling pathways. Apoptosis may arise from the modulation of a number of signaling pathways by cisplatin including the mitochondrial pathway, the DNA damage signaling, signal-regulated kinase pathway and others. It is known that DNA is the target of metal complex and that the nitrogen atoms of the bases, mainly N7 of guanine, are bound to the platinum following hydrolysis of the chloride ions inside the cell nucleus. The success of cisplatin in cancer chemotherapy derives from its ability to crosslink DNA and to alter DNA structure. The cytotoxic effect of cisplatin when bound to targets other than DNA may also be important. Cisplatin has been found to bind to phosphatidylser- ine, and this can affect the function of the cell membrane. It is of interest to establish to what extent cisplatin interacts with lipids. The in vivo 24-h effect of cisplatin on rat brain cells nuclear phos- pholipids was investigated. Rat brain nuclear phospholipids were fractionated by microTLC technique. The quantitative valuation of fractionated phospholipids was established by computer soft- ware FUGIFILM Science Lab. 2001 Image Gauge V 4.0. The results of our study confirm that rat brain nuclear phospholipids exhibit diversity in sensitivity to cisplatin action. The changes in content of rat brain cells nuclear phospholipids may be result of cisplatin antitumour action. On the other hand, it is possible that rat brain cells nuclear phospholipids are participated in the molecular mechanisms of cisplatin effects realization.

P5–15 Identification of the enzyme and mechanisms involved in the ADP-ribosylation of CtBP1-S/ BARS G. Grimaldi, A. Colanzi, C. Valente, G. Turacchio, C. Cericola, A. Luini and D. Corda Consorzio Mario Negri Sud, Cell Biology and oncology, Santa Maria Imbaro, ITALY

P5–17 The regulatory subunit PP4R1 of protein phosphatase PP4 is implicated in activation of NF-kappaB by the Epstein-Barr virus oncoprotein LMP1 E. Hatzivassiliou and G. Mosialos Biology, Aristotle University of Thessaloniki, Thessaloniki, GREECE

of CtBP1-S/BARS

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TRAFs constitute a family of proteins that have been implicated in signal transduction by members of the Tumor Necrosis Factor (TNF) receptor superfamily and the Epstein-Barr virus oncopro- tein LMP1. TRAF2 and TRAF6 have an E3-ubiquitin ligase activity, which is dependent on the integrity of their RING finger domain and has been associated with their ability to activate the NF-kappaB and AP1 signaling pathways. In order to investigate further the mechanism of signal transduction by TRAF2 and its regulation, we have performed a yeast two-hybrid screen of a human cDNA library with TRAF2 as bait. Among other pro- teins, we identified the regulatory subunit PP4R1 of protein phosphatase PP4 as a TRAF2-interacting protein. PP4 is involved in the regulation of microtubule growth and the centro- some maturation in mitosis and meiosis as well as T lymphocyte apoptosis. TRAF2 could be coimmunoprecipitated with PP4R1, following the coexpression of the two proteins in HEK 293T coimmunoprecipitation of TRAF2 and PP4R1 cells. The depended on the integrity of the RING finger domain of TRAF2. Furthermore, exogenous expression of PP4R1 inhibited TRAF2, TRAF6 and LMP1 mediated activation of NF-kappaB, but did not affect the activation of NF-kappaB by constitutively active IKKbeta. Finally, preliminary experiments showed that downregulation of PP4R1 by RNA interference enhanced the C-terminal-binding protein-1 (CtBP1) is a dual function protein that is involved in two diverse activities: intracellular membrane trafficking and gene transcription. CtBP1-S (short form)/BARS was initially identified as the 50-kDa substrate of BFA-dependent ADP-ribosylation, and later shown to be an essential component of the machinery controlling Golgi tubule fission (Weigert R et al., Nature 1999). Moreover, it has a fundamental role in the partitioning of the Golgi complex during mitosis (Colanzi A et al., EMBO J. 2007). CtBP1 is regulated by two different post- translational modifications and protein interactions that affect its localisation and functions. Indeed, phosphorylation of CtBP1-L (long form) by PAK1 induces its translocation from the nucleus to the cytoplasm and inhibition of its transcriptional activity; an opposite effect has been reported following CtBP1-L sumoylation (Barnes et al., Nature Structural Biology 2003). BFA-induced ADP-ribosylation represents another post-translational modifica- tion that regulates CtBP1-S/BARS activities. Here, we report that BFA-induced ADP-ribosylation occurs through a novel mechanism that comprises two steps: (i) synthesis of a BFA-ADP-ribose conjugate (BAC); and (ii) covalent binding of the BAC into the NAD+-binding pocket of CtBP1-S/BARS, which abolishes the fission-inducing activity of CtBP1-S/BARS. The first step of this mechanism can be catalysed by ADP-ribosyl cyclases, such as the Aplysia californica cyclase and human CD38. Our data demonstrate that this ADP-ribosylation locks CtBP1-S/BARS into a dimeric conformation that is inactive. This mechanism appears to be involved in the regulation of CtBP1-S/ BARS functions, and it also provides tools to modulate CtBP1-S/ BARS functions pharmacologically at the molecular level.

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induction of IL8 by LMP1. Taken together, our data indicate that PP4R1 plays an important role in signal transduction by LMP1 and specific TRAF molecules possibly through the modu- lation of PP4 activity.

P5–18 Activation of Nrf2 may preserve the coupled state of endothelial NO synthase under redox stress E. Heiss, D. Schachner and V. Dirsch Department of Pharmacognosy, University of Vienna, Vienna, AUSTRIA

VEC). Instead, it dose-dependently decreased HO-1 expression, both at mRNA and protein levels. As expected, in all cell lines studied PTX significantly reduced production of TNF. This effect was independent of HO-1 activity, as demonstrated in cells trea- ted with HO-1 activators and inhibitors or in cells overexpressing HO-1. Finally, inhibition of TNF was the same in HUVEC of different HO-1 genotypes, showing that PTX is similarly efficient in carriers of more and less active HO-1 promoter variants. In mice, PTX applied intraperitoneally (10 mg/kg) for seven days did not influence HO-1 protein expression, as measured in the liver, kidney, spleen, heart, and skin tissues. Accordingly, the response of PTX treated animals to LPS (5 lg/kg, i.p., 1.5 h) was the same in the wild type and HO-1 deficient mice. PTX in similar extent increased influx of leukocyte into peritoneal cavity, decreased production of TNF and reduced expression of VCAM- 1 in vascular intima. In conclusion, PTX inhibits production of TNF and may decrease inflammatory reaction both in vitro and in vivo, but these effects are independent of HO-1.

P5–20 Up-regulation of the immediate-early gene product RhoB by the cytotoxic necrotizing factor 1 from Escherichia coli S. C. Huelsenbeck1, G. Fritz2, G. Schmidt3, I. Just and H. Genth1 1Toxicology, Hannover Medical School, Hannover, GERMANY, 2Toxicology, Universitaetsmedizin Mainz, Mainz, GERMANY, 3Pharmacology and Toxicology, University Freiburg/Brsg, Freiburg, GERMANY

inhibitors as well as

The synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28- oic imidazolide (CDDO-IM) activates the transcription factor NF-E2-related factor (Nrf2). Nrf2 is redox-regulated and triggers an antioxidant cellular defense to oxidative stimuli. We adminis- tered CDDO-IM to primary human umbilical vein endothelial cells (HUVEC) in order to study the impact of Nrf2 activation on endothelial redox homeostasis and the endothelial nitric oxide synthase (eNOS) system. Activation of Nrf2 by CDDO-IM dose- and time-dependently reduced intracellular reactive oxygen spe- cies (ROS) with optimal response after 24 h at a concentration of 100 nM, as evident by DCF-based flow-cytometric analyses. Con- sistently with lowered ROS and decreased formation of peroxyni- trite the amount of bioavailable nitric oxide (NO), a major contributor to vascular homeostasis, was increased. Interestingly and in apparent contrast to elevated NO levels, western blot analyses revealed transiently decreased eNOS protein expression which was Nrf2-dependent as demonstrated by Nrf2 knockdown. Employing pharmacological siRNA approaches we identified de novo protein synthesis of heme oxy- genase 1 (HO-1) and subsequent heme deficiency as cause for the observed reduction of eNOS. To reconcile our contradictory find- ings of elevated NO- but reduced eNOS-levels in a physiological context, we hypothesize that under redox stress when the avail- ability of reduced BH4, a pivotal stoichiometric eNOS cofactor is limited, activation of Nrf2 leads (i) to a transient reduction of eNOS protein levels and (ii) to an antioxidant defense in HU- VEC. Both activities ensure a permanent stoichiometric ratio of eNOS and BH4 and may overcome the risk of eNOS uncoupling in situations of redox stress.

The low molecular weight GTP-binding protein RhoB is a tumor suppressor based on its pro-apoptotic and anti-proliferative prop- erties. RhoB is an immediate-early gene product, which is involved in the cellular responses to toxic stress. The cellular level of RhoB is low in most cell lines, due to RhoA- and (H/K/N) Ras-depen- dent suppression of the rhoB promoter. We recently showed that Rho/Ras-inactivating toxins such as the Clostridium difficile toxins A and B or lethal toxin from Clostridium sordellii cause strong up- regulation of RhoB. Here, we report the up-regulation of RhoB induced by the cytotoxic necrotizing factor 1 (CNF1) from E. coli, which deamidates and thereby activates Rac1, RhoA, and Cdc42. The rhoB promoter was activated by CNF1 in fibroblasts. This activation resulted in an increase in the rhoB mRNA and RhoB protein level. RhoB up-regulation was responsive to inhibition by either actinomycin D or cycloheximide, confirming that it was based on transcriptional activation. RhoB up-regulation in CNF1- treated fibroblasts was Rac1-dependent, as pharmacological inhibi- tion of Rac1 reduced RhoB up-regulation. Accordingly, ectopic expression of constitutively active Rac1 caused activation of the rhoB promoter. RhoA and (H/K/N) Ras are known suppressors of the rhoB promoter. We identified Rac1 as an activator of the rhoB promoter, indicating the existence of a not yet identified pathway. This knowledge might be used to specifically modulate the expres- sion of RhoB in tumors.

P5–19 Modulation of inflammatory response by pentoxifylline is independent of heme oxygenase-1 pathway H. Taha1, A. Grochot-Przeczek1, H. Was1, J. Kotlinowski1, M. Kozakowska1, A. Marek1, B. Lackowska2, A. Balcerczyk3, S. Mustafa4, J. Dulak1 and A. Jozkowicz1 1Medical Biotechnology Department, Jagiellonian University Faculty of Biochemistry Biophysics and Biotechnology, Krako´w, POLAND, 2Department of Pathology, Oncology Center, Krako´w, POLAND, 3Department of Molecular Biophysics, University of Lodz, Lodz, POLAND, 4Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Vienna, Vienna, AUSTRIA

P5–21 Thrombin induces NF-kB activation and IL-8/ CXCL8 expression in lung epithelial cells by Rac1 dependent PI3K/Akt Pathway B. C. Chen1, H. W. Cheng2 and C. H. Lin2 1College of Medicine School of Respiratory, Taipei Medical Uni- versity, Taipei, TAIWAN, 2College of Medicine Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, TAIWAN

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Pentoxifylline (PTX) is commonly used for treatment of microcir- culation diseases. It was claimed that PTX can induce expression of heme oxygenase-1 (HO-1), the cytoprotective, antiinflamma- tory and proangiogenic enzyme, and that some effects of PTX are dependent on HO-1 activation. We demonstrated that PTX (0.1–10 mM) did not induce HO-1 in human and murine mono- cytes (U937 and J774 cells), murine brain endothelial cells (MBEC-1), and human umbilical vein endothelial cells (HU- Previously, we found that thrombin activate the c-Src pathway, which in turn initiates IkB kinases a/ß (IKKa/ß) and nuclear

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P5–23 7Betahydroxycholestrol is incorporated in caveolae from endothelial cells M. Jurado1, S. Miyamoto2, A. I. Moretti1 and H. P. Souza1 1Emergency Medicine, School of Medicine, Sao Paulo, BRAZIL, 2Biochemistry, Chemistry Institute, Sao Paulo, BRAZIL

factor-kB (NF-kB) activation, and ultimately induces IL-8/ CXCL8 expression in lung epithelial cells. In this study, we fur- ther investigated the roles of Rac1, phosphatidylinositol 3-kinase (PI3K), and Akt in thrombin-induced NF-kB activation and IL- 8/CXCL8 expression in lung epithelial cells. Thrombin-induced IL-8/CXCL8 release or IL-8/CXCL8-luciferase activity were attenuated by a Rac1 dominant negative mutant (RacN17), a PI3K inhibitor (LY 294002), an Akt inhibitor (1L-6-hydroxym- ethyl-chiro-inositol2-[(R)-2-O-methyl-3-O-octadecylcarbon- lung epithelial cells with ate]), and AktDN. Treatment of thrombin caused activation of Rac and Akt. In addition, SFLLRN-NH2 (a PAR1 agonist peptide), and GYPGQV-NH2 (a PAR4 agonist peptide), but not TFRGAP-NH2 (a PAR3 ago- nist peptide), induced an increase in Akt activation. Thrombin- induced Akt phosphorylation at Ser473 and kinase activity were inhibited by RacN17 and LY294002. Stimulation of lung epithe- lial cells with thrombin resulted in an increase in IKKa/ß phos- phorylation and kinase activity; these effects were inhibited by RacN17, LY294002, an Akt inhibitor, and AktDN. The throm- bin-induced increases in kB-luciferase activity were also inhibited by RacN17, LY 294002, and AktDN. Treatment of lung epithe- lial cells with thrombin induced the Gßr, p85a and Rac1 complex formation in a time-dependent manner. These results indicate that thrombin acting through PAR1 and PAR4 activate the Rac1/PI3K/Akt pathway through the Gßr and p85a to mediate IKKa/ß activation, which in turn induces NF-kB transactivation, IL-8/CXCL8 expression and release in human lung epithelial cell- s.

Caveolae are invaginations in endothelial cell membrane, where a large number of signaling proteins are located, including CD40, eNOS. Caveolae are characterized by their high cholesterol con- tent and the presence of caveolin-1 (CAV-1), a scaffolding pro- tein for molecules belonging to signaling pathways. Oxysterols are derived from cholesterol oxidation; they may modify gene transcription by binding to the nuclear receptor LXR and are known to be present in atherosclerotic plaque. We hypothesized that oxysterols may act also by changing caveolae structure and, therefore, changing signal transduction and cell function. We exposed human umbilical vein endothelial cells (HUVECs) to 7betahydroxycholesterol (10 lM/ml) for 1 h. Membrane sub-frac- tions containing caveolae were obtained by ultracentrifugation in a sucrose gradient and its composition was analysed by mass spectroscopy. In culture medium, TNFalpha and IL-10 were assessed by ELISA. CD40 and CAV-1 mRNA was analyzed by RT-PCR. The chromatographic profile of HUVECs’ fractions rich in caveolae showed a much higher content of oxystherols in treated cells, compared to controls. There was no difference in mRNA for either CD40 or CAV-1 compared to control. Expo- sure to oxysterols did not affect either TNFalpha or IL-10 secre- tion compared to control (79.7 ± 2.3 vs. 94.2 ± 0.3 pg/ml and 35.5 ± 2.2 vs. 49.6 ± 16.9 pg/ml, respectively). 7betahydroxy- cholesterol is incorporated in endothelial cells caveolae after 1 h treatment. Under these experimental conditions, 7betahydroxy- cholesterol did not affect the expression of genes involved in the inflammatory response, therefore, any effects on the activity of these signaling pathways observed after oxysterol exposure might be related to its impact in caveolae structure. Financial support: CAPES, FAPESP.

P5–22 SHP1 regulates macrophage survival and proliferation by M-CSF-induced reactive oxygen species H. K. Choi, H. Jang, T. H. Kim and S. Y. Lee Division of Life and Pharmaceutical Sciences Center for Cell Signaling and Drug Discovery Research, Ewha Womans Univer- sity, Seoul, SOUTH KOREA

P5–24 Bordetella adenylate cyclase toxin subverts both innate and adaptive cell-mediated immunity J. Kamanova1, I. Adkins1, J. Masin1, O. Kofronova1, H. Genth2, H. Janova1, J. Vojtova1, I. Linhartova1, O. Benada1, I. Just2 and P. Sebo1 1Cellular and Molecular Microbiology Division, Institute of Micro- biology, Prague 4, CZECH REPUBLIC, 2Hannover Medical School, Institute of Toxicology, Hannover, GERMANY

The physiological concentration of reactive oxygen species (ROS) may function as a second messenger to mediate various responses including cell migration, differentiation and growth. However, excess ROS is toxic and can prelude diseases. ROS may be pro- duced by many physiological intracellular reactions in response to receptor activation. Macrophage colony-stimulating factor (M- CSF) plays an important role in monocyte/macrophage survival and proliferation. Signals generated by the binding of M-CSF to the cell-surface receptor c-Fms appear to trigger events leading to phagocyte differentiation. Presently, M-CSF-mediated signaling cascades could be modulated by ROS. M-CSF-induced ROS in BMM cells regulated cell proliferation and survival through oxi- dation of Src homology region 2 domain-containing phosphatase 1 (SHP1). Oxidation of SHP1 abrogated phosphatase activity, which sustained phospho-PI3K and Akt activation. To ascertain the source of ROS, BMM cells were treated with the NADPH inhibitor diphenyleneiodonium (DPI). DPI inhibited M-CSF- induced ROS production, thereby decreasing SHP1 oxidation and Akt phosphorylation. Moreover, over-expression of SHP1 decreased Akt phosphorylation and reduced cell survival, and down-regulated D-type of cyclins and cell proliferation. These results suggest that M-CSF-induced ROS in BMM cells regulates cell survival and proliferation through modification of SHP1.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Adenylate cyclase toxin (CyaA) is a key virulence factor of the whooping cough agent Bordetella pertussis. It penetrates myeloid phagocytes expressing the beta2 integrin CD11b/CD18 and by uncontrolled conversion of ATP into a key signaling molecule, cAMP, it knocks-down the bactericidal functions of host innate immunity. We found that CyaA-catalyzed elevation of cytosolic cAMP provokes a rapid and near-complete block of complement- mediated phagocytosis in murine macrophages. This is paralleled by massive actin cytoskeleton rearrangements, due to transient and selective inactivation of RhoA in the absence of detectable activa- tion of Rac1, Rac2 or RhoG, yielding formation of transient sheet- like cell membrane ruffles. Moreover, despite massive cell ruffling, cAMP signaling of CyaA provoked an arrest of macropinocytosis in macrophages as well as dendritic cells (DCs), here without inhib- iting their receptor-mediated endocytic capacity. In parallel, matu- ration of LPS-stimulated DCs was altered and decreased CD40 and CD54 expression and enhancement of IL-10 production was

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observed, possibly accounting for the reduced capacity of CyaA- treated DCs to specifically prime proliferation of antigen-specific CD4+ as well as CD8+ T cells. Collectively, these observations suggest an important role for CyaA in subverting also the adaptive T cell immune response of the host to B. pertussis infection. Jana Kamanova and Irena Adkins contributed equally to this work.

P5–25 Affinity chromatography as the method for brassinosteroid-binding protein isolation M. Kamlar1, O. Uhlik1, L. Kohout2, M. Sanda3 and T. Macek1 1Departement of Natural Products, Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic, Pra- gue 6, CZECH REPUBLIC, 2Departement of Steroid Chemistry, Institute of Organic Chemistry and Biochemistry Academy of Sci- ences of the Czech Republic, Prague 6, CZECH REPUBLIC, 3Departement of Mass Spectrometry, Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic, Prague 6, CZECH REPUBLIC

thought to depend on the regulation of the activation of the Ras- Raf-ERK pathway. EGFR requires activation of the tyrosine kinase Src, with which it exchanges phosphorylations. Src then recruits adaptor proteins and facilitates propagation of the signal through the subsequent activation of the three kinase-module, namely, cRAF-MEK1-ERK. To study the role of Src kinase in the EGF signaling, we have generated a C62B astrocytoma cell line that stably expresses a dominant negative mutant, kinase- dead Src, DNSrc-C62B. Activation of ERK by EGF was low and sustained in the wild-type C62B cells, and led to induction of a differentiated phenotype. In sharp contrast, in the mutant cells EGF induced a high ERK activation with a much shorter dura- tion and within hours most cells were in mitosis. To dissect the protein-protein interactions that occur upon EGF receptor acti- vation, we investigated the spatiotemporal kinetics of activation of the downstream effector of cRAF, using subcellular fractiona- tions and immunoprecipitations as well as fractionations of the plasma membranes into lipid rafts and non-raft membranes. We found that in the wild type cells EGF treatment acutely activated cRAF, which co-immunoprecipitated MEKK1-MEK1-ERK in the membranes. In contrast, in DNSrc-C62B cells, cRAF activa- tion was modest, the cRaf signalosome was formed in the cytosol and did not include MEKK1. MEKK1 (MAP3K) is considered a scaffold protein that facilitates the activation of the three kinase module. Taken together our results suggest that Src-dependent phosphorylation of Raf and possibly MEKK1 is required for proper formation and targeting of the formed ERK signalosome. (PENED grant 03ED778)

P5–27 Functional selectivity of CCR5 interaction with heterotrimeric G-proteins J. Kerr and A. Mueller School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, UK

Brassinosteroids (BRs) are group of phytohormones, plant endoge- nous signal molecules affecting differentiation of plants, growth, photomorphogenesis, senescence and stress tolerance, too. Their mechanism of action is based on interaction of BRs and system of membrane receptors. Signal transduction is supposed to be medi- ated via at least two different receptor-like protein kinases, but the role of brassinosteroid-binding proteins as the helpers in these cas- cade was not explained enough yet. We prepared a range of bioaf- finity columns with different types of brassinosteroid molecules immobilised on polymeric matrices. Plant materials were 4-weeks- old tobacco (Nicotiana tabacum var. Wisconsin 38) callus, young leaves of New Zealand spinach (Tetragonia tetragonioides) and young shoots of flax (Linum usitatissimum var. Venica). All pro- teins with affinity to brassinosteroids were screened on SDS-PAGE gels and the most abundant proteins were analysed with N-termi- nal sequencing or LC/MS/MS. In this paper, we report on the iso- lation and identification of two plant proteins with affinity to the synthetic analogues of naturally occurring brassinosteroids. The first one (from tobacco callus) was identified as an osmotin-like protein precursor (OLPA), an already known pathogenesis-related protein from tobacco, which has connection with plant adaptation to stress factors as drying, wounding or attack of herbivorous pathogens that means the same factors as they are supressed with brassinosteroids. The second one (from flax and New Zealand spinach, too) was identified as ribulose-1,5-bisphosphate carboxyl- ase/oxygenase (RuBisCO), which is the key photosynthetic enzyme converting inorganic carbon into organic compounds. The oxyster- ol ligands were found to increase the activity of the enzyme. The relationship between BRs and OLPA, or BRs and RuBisCO is now under investigation. Acknowledgement: The work is sponsored by research projects 1M06030 and Z 40550506 of the Czech Ministry of Education.

G-Protein coupled receptors (GPCR) are involved a wide range of physiological processes, as such they amount for roughly 30% of modern pharmaceutical targets. Many permutations of G-pro- tein heterotrimers are possible, but certain permutations are more likely to associate with specific GPCRs. The chemokine receptor 5 (CCR5) has a well characterised association with Gai2 hetero- trimers, but has also been shown to interact with several other Ga subunits. This work aims to further examine the relationship between CCR5 and G-protein heterotrimers, by characterising intracellular calcium and cAMP levels. Treatment of CHO and HeLa cells stably expressing CCR5 with pertussis toxin com- pletely abrogates CCR5 activation of associated G-proteins, sug- gesting CCR5 associates with Gai and Gao. However, expression of constituently active Gai2 increased calcium release following CCR5 activation with CCL3 (80 lM) by 37 ± 5%. siRNA knockdown of individual G protein a subunits, has no effect on CCR5 stimulated calcium release. Disruption of Gb1 signalling with gallein, increases calcium release following CCL3 stimula- tion (100 nM) by 150% ± 19 (n = 4), with no effect on potency. Treatment with gallein, in the absence of CCL3 stimulation, reduces basal cAMP levels significantly.

P5–26 Crucial role of Src kinase in EGF-dependent activation of c-Raf, formation of MEKK1-MEK1- ERK signaling complex and cell differentiation S. Karouzaki1, J. Andal2, G. Leondaritis1 and D. Mangoura1 1Biomedical Research Foundation Academy of Athens, Center of Preventive Medicine Neurosciences and Social Psychiatry, Athens, GREECE, 2Department of Pediatrics, The University of Chicago, Chicago, USA

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The epidermal growth factor receptor, EGFR, regulates prolifer- ation and motility in astrocytes. This dual role of EGFR is Taken together, these data show that CCR5 is capable of acti- vating several permutations of Gai/o, with no loss of function when individual Ga subunits are disrupted. Disruption of Gb1 with Gallein suggests that it plays a role in regulating regulatory calcium release from the cytosolic stores following stimulation and may regulate basal cAMP levels.

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HIF-1a protein level was decreased by treatment with p42/44 mitogen-activated kinase (MAPK) inhibitor, PD98059. Taken together, EP significantly inhibited the pVHL-mediated HIF-1a degradation via MAPK pathway.

P5–28 Involvement of EGF receptor tyrosine kinase and MAP kinase ERK1/2 in regulation of microtubule system remodeling during EGF receptor endocytosis M. Kharchenko, M. Zlobina and E. Kornilova Membrane Dynamics Lab, Institute of Cytology Russian Academy of Science, Saint-Petersburg, RUSSIA

P5–30 mH2A1 suppresses enzymatic activity of VRK1 during interphase W. Kim, T. H. Kang, S. Kim, M. W. Jeong, C. H. Park, Y. H. Choi, K. C. Woo, Y. S. Park, K. H. Lee, D. Y. Kim and K. T. Kim Life Science, Postech, Pohang, SOUTH KOREA

VRK1 (vaccinia-related kinase1) is one of eukaryotic counterpart for vaccinia virus B1R kinase and plays a major role in cell-cycle regulation through direct phosphorylation toward 3rd Threonine and 10th Serine of histone H3. Here, we show that macro histo- neH2A1.2 (mH2A1) functions as a suppressor against VRK1 during interphase. Notably, mH2A1 suppresses the enzymatic activity of VRK1 on the histone H3. However, mH2A1 does not suppress activity of other mitotic kinases and phosphorylation of other substrates of VRK1. This mode of suppression does not exist during late G2 phase to mitotic phase, but is restricted dur- ing interphase. The phosphorylation status of histone H3 was strongly related to the level of mH2A1 on nucleosome. Hence our finding on the novel function of mH2A1 which is involved in the regulation of VRK1 activity will provide valuable informa- tion for understanding regulation of cell cycle.

We have reported earlier (Kharchenko et al., Cell Biol Int. 2007; 31: 349–359) that stimulation of EGF receptor endocytosis in numerous cell lines results in coordinated reorganization of inter- phase microtubules (MTs): MTs remain radiality during EGFR stay in early endosomes (EE), but with starting of EGFR transi- tion from early to late endosomes (LE) and during LE matura- tion MTs’ fragmentation/depolymerization is developed. The further MTs reestablishment correlates with EGFR degradation. EGFR tyrosine kinase (TK) requirement for EGF-stimulated sig- naling and for EGFR transition from EE to LE is well estab- lished. In this study we address the question whether EGFR TK or EGF-stimulated signaling cascades are involved in regulation of MT system remodeling. EGFR TK inhibitor AG1478 was found to stabilize radial MTs and prevent further reorganizations only when added not later that in 5 min upon stimulation of endocytosis. Under these conditions the sorting of internalized EGFR from EE into LE is inhibited. This indicates that for the onset of fragmentation EGFR-containing EE maturation is nec- essary. On the other hand, inhibition of MAP kinase ERK1/2 (lying downstream of EGFR) by UO126 had no effect during early stages of endocytosis, However, UO126 abolished MT frag- mentation when added after 30 min upon EGFR endocytosis stimulation, when internalized EGFR is localized in matured LE. By this time the second wave of ERK1/2 phosphorylation was developed, which disappeared after UO126 treatment. We con- clude that ERK1/2 activation on late endosomes provide the sec- ond signal necessary for maintenance of MT fragmentation at late stage of endocytosis.

P5–31 Identification of Protein Phosphatase 2A as a novel regulator of hypoxia-inducible factor-1 Y. S. Kim Department of Anatomy and Neurobiology Biomedical Science Institute School of Medicine, Kyung Hee University, Seoul, SOUTH KOREA

P5–29 Regulation of Hypoxia-inducible factor 1a´ by Ethyl pyruvate S. Y. Kim Department of Anatomy and Neurobiology Biomedical Science Institute School of Medicine, Kyung Hee University, Seoul, SOUTH KOREA

The hypoxia inducible factor-1 (HIF-1) plays crucial roles in can- cer cell growth and survival, angiogenesis, energy metabolism, and erythropoiesis. The function of HIF-1 is regulated by various post-translational modifications such as hydroxylation, ubiquiti- nation, acetylation and phosphorylation via interaction with sev- eral proteins including PHD, pVHL, ARD-1, and p300/CBP. Specifically, the phosphorylation of HIF-1a plays an important role for the transactivation function of HIF-1. Protein phospha- tase 2A (PP2A) is an abundant serine/threonine phosphatase involved in many cellular events. For example, PP2A plays a role in the critical cellular processes of protein synthesis, DNA repli- cation, transcription, and metabolism by regulating phosphoryla- tion of target proteins. Here, we found that okadaic acid, which is a specific inhibitor of PP2A, strongly increased the activity of HIF-1 and VEGF expression. We also found that okadaic acid potently stimulated the HIF-1a protein level through activation of Akt and mTOR. Moreover, we figured out that overexpression of PP2A decreased HIF-1a protein level by direct interaction. From these results, we could speculate that PP2A modulates the function of HIF-1 and angiogenesis via regulating AKT-mediated signal transduction.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

The hypoxia inducible factor-1 (HIF-1) is the master regulator of oxygen adaptation and implicated in a variety of biological pro- cesses including the progression and metastasis of tumors. Under normal oxygen concentrations, HIF-1a, the active subunit of HIF-1, is hydroxylated on proline residues by specific HIF prol- yl-hydroxylases, leading to ubiquitination and degradation by the proteasome. However, under hypoxia hydroxylation and ubiquiti- nation are blocked. And then HIF-1 translocates to the nucleus where it binds to specific hypoxia responsive elements (HREs) thereby activating the transcription of target genes. Recent stud- ies have shown that pyruvate which is an intermediate metabolite of glucose induced accumulation of HIF-1a and expression of its target genes under normoxia. Until now the regulating molecular mechanism of Hif-1a by pyruvate is unclear. To improve the instability of pyruvate in aqueous solution, ethyl pyruvate has been widely used instead of pyruvate. In this study, we investi- gated the regulation of Hif-1a by EP in cancer cell. We found that EP increased HIF-1a protein level by inhibiting the pVHL- mediated proteasome pathway. Moreover we also found that

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P5–32 Effect of ionizing radiation on large conductance Ca2+-activated K+ current and myofilament Ca2+ sensitivity in rat aorta smooth muscle cells I. Kizub and A. Soloviev Institute of Pharmacology and Toxicology of AMS of Ukraine, Experimental Therapeutics, Kiev, UKRAINE

HT-29 cancer cells are more sensitive to TRAIL-induced apopto- sis than the non-cancer FHC cells during adherent cultivation. We detected the significant decrease of apoptosis in the HT-29 cells during non-adherent cultivation in comparison with adher- ent cultivation. This effect can be caused by changes in the amount of TRAIL receptors. Therefore, we investigated altera- tions in TRAIL receptor expression on the cell surface and in the whole cell lysates during the both type of cultivation. However, we did not demonstrate any significant changes in the TRAIL receptor expressions. Subsequently, we examined PI3/Akt pro- survival pathway activation during the non-adherent cultivation, which could be responsible for the decreased sensitivity of the colon cells to the TRAIL action. The results showed an increased phosphorylation of Akt kinase during non-adherent cultivation. Therefore, we detected apoptotic parameters after treatment with TRAIL in combination with specific PI3/Akt pathway inhibitors, to confirm involvement of PI3/Akt pathway. After inhibition of this pathway we observed increased amount of apoptosis. Taken together, our data suggested that non-adherent type of cultiva- tion induced decrease of the TRAIL-mediated apoptosis of colon epithelial cells compared to adherent cultivation. This effect is connected with the activation of pro-survival PI3/Akt pathway. Acknowledgements: This work was supported by grants 305/ 09/1526 GACR a 524/07/1178 GACR and 1QS500040507 IGA ASCR and Masaryk University Rector’s program for support of students creativ activity.

P5–34 Life or death: rooperol’s role as anticancer agent G. Boukes, T. Koekemoer and M. van de Venter Biochemistry and Microbiology, Nelson Mandela Metropolitan University, Port Elizabeth, SOUTH AFRICA

Ionizing radiation increases vascular responsiveness and leads to arterial hypertension but mechanisms of its development still remain unclear. We have supposed that protein kinase C (PKC)- mediated changes in the large conductance Ca2+-activated K+ channels (BKCa) activity and myofilament Ca2+ sensitivity in vascular smooth muscle cells (VSMCs) may contribute to radia- tion-evoked arterial hypertension. The study was performed on isolated rat aorta VSMCs using patch-clamp techniques and chemically permeabilized aorta VSMCs using vascular tone mea- surement, from healthy and irradiated (source Co60, dose 6 Gy) animals taken on 9th (IR9) and 30th days postirradiation (IR30). Whole-cell outward K+ current in VSMCs from IR9 and IR30 was significantly decreased. Ca2+-activated K+current was sepa- rated pharmacologically using 1 mkM apamin, 1 mkM charybdo- toxin and 500 nM paxilline showing that BKCa current is the main component of K+ current. Selective BKCa inhibitor paxil- line (500 nM) depressed K+ current in IR9 and had not effect on it in IR30 VSMCs suggesting that on this term BKCa current eliminates completely. PKC inhibitor chelerythrine (100 nM) renewed K+ current reduced by ionizing irradiation in IR9 and IR30 aorta VSMCs, suggesting that inhibition of BKCa current is mediated by PKC. In permeabilized aorta preparations from irra- diated animals myofilaments Ca2+sensitivity was significantly increased. The value of pCa50 (-log [Ca2+] 50% effective) which reflects shifting in pCa-tension relationship curve, was signifi- cantly lower for IR9 and IR30 vs. in control. PKC inhibitors chelerythrine (1 mkM) and staurosporine (0.1 mkM) had no effect on Ca2+ sensitivity in healthy tissue, but significantly decreased Ca2+ sensitivity in aorta from IR9 and IR30. PKC activator phorbol dibutyrate (10 mkM) increased Ca2+ sensitivity in control tissue but had no effect in irradiated vascular rings. Our data indicate that ionizing radiation leads to inhibition of outward K+ current in rat aorta VSMCs manly via PKC-medi- ated inhibition of BKCa current as well as PKC contributes to irradiation-evked miofilament Ca2+ sensitization in this tissue. Thus, both PKC-mediated inhibition of BKCa current and increasing of myofilament Ca2+ sensitivity in VSMCs may be involved to vascular tone elevation and subsequence arterial hypertension development under ionising radiation impact.

P5–33 Involvement of PI3K/Akt pathway in TRAIL-induced apoptosis during non-adherent cultivation L. Koci, M. Hyzdalova, J. Hofmanova and A. Kozubik Department of Cytokinetics, Institute of Biophysics AS CR v.v.i., Brno, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Colonic epithelial cells are shed into intestinal lumen at the top of the crypt and die by detachment-induced apoptosis (anoikis). We have studied the effects of TNF-related apoptosis inducing ligand (TRAIL) on cytokinetic parameters and adhesive proper- ties of the cell lines derived from normal human foetal (FHC cells) and human adenocarcinoma (HT-29 cells) colon tissues in association with anoikis induction. To mediate anoikis, we estab- lished the model of non-adherent cultivation using 3D rotator. Introduction: Hypoxoside, isolated from Hypoxis hemerocallidea (African potato), is converted to rooperol phase II metabolites in the human body. Rooperol has been shown to kill cancer cell lines in vitro but the mechanism of action has not been identified. This study investigated the mode of cell death and some of the signaling pathways involved. Methods: The four cell lines HeLa, MCF7, HT29 and U937 were used. IC50 for rooperol was determined with the MTT assay. Flow cytometry was used for the following assays: DNA cell cycle analysis using propidium iodide staining; Annexin V- FITC/PI to distinguish between apoptosis and necrosis; intracel- lular immuno-staining of Akt, pAkt, pBcl-2, p21, and active cas- pase-3 and -7; TdT-mediated FITC-dUTP nick end labeling for DNA fragmentation. Results: IC50 values for rooperol against the three adherent cancer cell lines ranged between 13 and 29 lg/ml. Cell cycle anal- ysis revealed S-phase arrest and endoreduplication in MCF7, HT29 and HeLa cells upon exposure to IC50 concentrations of rooperol. Since endoreduplication can be an apoptotic or cell sur- vival strategy, apoptosis assays were conducted. DNA fragmenta- tion was observed and apoptosis confirmed in U937 cells with Annexin V-FITC/PI. An increase was observed in the levels of p21, pAkt, pBcl-2 and activated caspase-3 and -7. In MCF7, which lacks functional caspase-3, activation of caspase-7 was observed. Conclusion: This study has shown that rooperol induces en- doreduplication which leads to cell cycle arrest and subsequent apoptosis in cancer cells.

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Abstracts

Poster Presentations

P5–35 A role of nucleolin in the pleiotrophin-induced cell migration M. Koutsioumpa and E. Papadimitriou Pharmacy, University of Patras, Patras, GREECE

that methylation of PP2A-C and subsequent PP2A-Ba binding is required for mast cell degranulation. In contrast, shRNA down- regulation of the PP2A-B’d subunit enhanced degranulation and enhanced p38 MAPK phosphorylation, suggesting that PP2A complexes containing PP2A-B’d attenuates mast cell degranula- tion via p38 MAPK dephosphorylation. Taken together these data show that PP2A is both a positive and negative regulator of mast cell degranulation, and this differential role is regulated by carboxymethylation and specific PP2A B subunit binding. Identi- fication of the PP2A regulatory subunits involved in mast cell degranulation now provide potential targets for improved asthma therapy.

P5–37 Thermosensory neurons regulate the longevity response to temperature in C. elegans S. J. Lee1 and C. Kenyon2 1POSTECH, life science/I-Bio/WCU ITCE, Pohang, SOUTH KOREA, 2Biochemistry and Biophysics, UC San Francisco, San Francisco, USA

Pleiotrophin (PTN) is an 18 kDa heparin-binding, secreted poly- peptide growth factor with an established role in angiogenesis in vivo and in vitro. It acts directly on endothelial cells, stimulating their migration through its receptor protein tyrosine phosphatase b/e (RPTPb/e) and integrin amb3 that form a functional complex on the cell surface. Nucleolin, a 100 kDa multifunctional protein, has also been mentioned as a low affinity receptor for PTN. Nu- cleolin acts as a shuffle between cytoplasm and nucleolus and may import PTN into the nuclear fraction. Although the func- tion of cell surface nucleolin is poorly described, it is increased on the surface of angiogenic endothelial cells and binds a variety of ligands that play critical role(s) in tumorigenesis and angiogen- esis. In the present work, we studied whether nucleolin plays a role in PTN-induced cell migration. Knockdown of nucleolin by siRNA or blockage of cell surface nucleolin by its ligand HB-19 in human endothelial cells completely abolished PTN-induced migration. Nucleolin directly interacts not only with PTN, but also with both RPTPb/e and integrin amb3 on the membrane of human endothelial and cancer cells, as evidenced by a combina- tion of immunoprecipitations and Western Blot analyses. Treat- ment of cells with pleiotrophin enhances the internalization of nucleolin and the short isoform of RPTPb/e but not of amb3 inside the cell. Knockdown of integrin amb3 by siRNA decreased the interaction of nucleolin with RPTPb/e, suggesting that amb3 may play a role in the interaction of nucleolin with RPTPb/e on the cell surface. This is further supported by the observation that in human glioma M059K cells that express RPTPb/e but not amb3, nucleolin can be detected only in the nucleolus of the cells. Our data suggest that nucleolin participates in the functional complex on the surface of the cells that is responsible for PTN- induced cell migration.

P5–36 Protein phosphatase 2A carboxymethylation and regulatory B subunits differentially regulate mast cell degranulation G. Kranias, L. F. Cottrell, H. Carpenter, A. T. R. Sim and N. M. Verrills School of Biomedical Sciences, University of Newcastle, Callaghan, AUSTRALIA

including the roundworm C. elegans, have Many ectotherms, shorter lifespans at high temperature than at low temperature. High temperature is generally thought to decrease the lifespan of ectotherms simply through its effects on metabolic rates. We questioned this view and asked whether the temperature-depen- dence of lifespan is subject to active regulation. C. elegans has a pair of thermosensory neurons called AFD, which allow the ani- mal to sense and respond to temperature. We asked whether the AFD thermosensory neurons in C. elegans have regulatory roles in the temperature dependence of lifespan. We found that genetic or laser ablation of the AFD neurons led to even shorter lifespan at high temperature, suggesting that the thermosensory AFD neurons are required for maintaining normal lifespan at high temperature. We then found that the AFD thermosensory neu- rons influenced lifespan through the steroid signaling pathway comprising DAF-9 (cytochrome P450) and DAF-12 (nuclear hor- mone receptor). We showed that the thermosensory AFD neu- rons were required for normal expression of daf-9 at high temperature and that this in turn prevented animals from living even shorter than normal at high temperature. Together, our findings suggest that C. elegans actively transmits signals from thermosensory system to this steroid pathway to regulate lifespan at high temperature. We propose that this thermosensory system allows C. elegans (and possibly other cold-blooded animals) to reduce the effect that warm temperature would otherwise have on processes that affect aging, something that warm-blooded ani- mals do by controlling temperature itself.

P5–38 MicroRNA in epithelial-mesenchymal transition of benign prostate hyperplasia cells E. Lincova, A. Starsichova, Z. Pernicova, A. Kozubik and K. Soucek Departement of Cytokinetics, Institute of Biophysics AS CR v.v.i., Brno, CZECH REPUBLIC

transition (EMT),

PP2A-Ba inhibits

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Asthma is characterised by antigen-mediated mast cell degranula- tion and secretion of inflammatory mediators. PP2A is a serine/ threonine protein phosphatase composed of a structural A sub- unit, regulatory B subunit and a catalytic C subunit. Inhibition of protein phosphatases using pharmacological agents has sug- gested a positive regulatory role in mast cell degranulation. Indeed, we find that a high okadaic acid concentration (1 mM) inhibits mast cell degranulation. However we show for the first time that a low concentration of okadaic acid (0.1 mM) has a strikingly opposite effect, enhancing degranulation. Furthermore, selective inhibition of the PP2A-Ca subunit by short hairpin RNA (shRNA) also enhances degranulation, suggesting that the primary role of PP2A is to negatively regulate degranulation. Carboxymethylation of the PP2A-C subunit alters B subunit binding. We show that carboxymethylation of PP2A-C is dynam- ically altered during degranulation, and inhibition of carboxyme- thylation subunit degranulation. The preferentially binds to methylated PP2A-C, and shRNA downre- gulation of PP2A-Ba also decreased degranulation, suggesting Epithelial-mesenchymal in which epithelial cells lose their polarity and become motile mesenchymal cells, occurs during development and is viewed as an essential early step in tumour metastasis. Transcription factors and EMT regu- lators of the ZEB family are thought to be involved in tumour progression, thus having potential clinical interest. MicroRNAs (miRNAs) are an abundant class of small non-protein-coding regulators of gene expression that play an important role in

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P5–40 Activation of HIF-1 diminishes activity of Nrf2, NF-kB and AP-1 and decreases expression of IL-8 in endothelial cells A. Loboda1, A. Stachurska1, U. Florczyk1, D. Rudnicka1, A. Jazwa1, M. Kozakowska1, K. Stalinska2, L. Poellinger3, A. L. Levonen4, S. Yla-Herttuala4, A. Jozkowicz1 and J. Dulak1 1Department of Medical Biotechnology, Jagiellonian University Faculty of Biochemistry Biophysics and Biotechnology, Krako´w, POLAND, 2Department of Cell Biochemistry, Jagiellonian University Faculty of Biochemistry Biophysics and Biotechnology, Krako´w, POLAND, 3Department of Cell and Molecular Biology, Karolinska Institute, Stockholm, SWEDEN, 4Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute University of Kuopio, Kuopio, FINLAND

tumorigenesis and, depending on their targets, can function as tumour suppressors or oncogenes. miR-200 family and miR-205, which regulate expression of ZEB transcription factors, have been found downregulated during EMT, suggesting an important role in inhibition of EMT. The aim of this study is to describe the role of miRNA in EMT of benign prostate hyperplasia cells. EMT of BPH-1 cell line was induced by TGF-b|1 treatment and assessed by cell morphology and expression of epithelial (E-cadh- erin) and mesenchymal markers (N-cadherin, vimentin) on both protein and mRNA level. Expression of ZEB1, ZEB2, miR-200 family and miR-205 was analyzed by qRT-PCR. The effect of miR-200 on ZEB proteins translation was assessed by luciferase assay. The early changes of ZEB proteins transcripts observed suggest a possible role of miR-200 family and ZEB proteins in early stages of the EMT process of nontumorigenic prostate epi- thelial cell line. Acknowledgements: This work was supported by grants No. 204/07/0834, No. 310/07/0961 of the Czech Science Foundation and by the Academy of Science of the Czech Republic, grants no. AV0Z50040507 and AV0Z50040702 and by the Masaryk University Rector’s Programme for Support of Students’ Creative Activity.

P5–39 Destabilization of LKB1 by SirT1: a novel anti-ageing mechanism through switching lysine acetylation to ubiquitination L. Liu1, Y. Wang2, K. S. Lam1 and A. Xu1 1Department of Medicine, The university of Hong Kong, Hong Kong, CHINA, 2Department of Pharmacology, The university of Hong Kong, Hong Kong, CHINA

The transcription factor hypoxia-inducible factor 1 (HIF-1) is one of the key regulators of oxygen homeostasis. HIF-1-regulated genes are involved in many biological processes, including angio- genesis. The regulation of angiogenic mediators expression by HIF-1 seems to be complex, and the mechanisms of those effects have not been fully elucidated yet. HIF-1 was induced in human microvascular (HMEC-1) and in human umbilical vein endothe- lial cells (HUVEC) by hypoxia or by treatment with dimethylox- alylglycine (DMOG) an inhibitor of prolyl hydroxylases. Moreover, adenoviral overexpression of the stable form of HIF- 1a was also used to activate HIF-1. Interestingly, hypoxia, prolyl hydroxylases inhibitor and AdHIF-1a attenuated IL-8 expres- sion. Down-regulation of IL-8 production was reversed by cheto- min, inhibitor of p300 binding to HIF-1 or by siRNA specifically targeting HIF-1. DMOG-mediated inhibition of IL-8 promoter activity was associated with down-regulation of activity of NF- kB and AP-1 transcription factors. Interestingly HIF-1-mediated decrease of IL-8 level was also associated with lowered NF-E2- related factor 2 (Nrf2) transcription factor expression. Concomi- tantly, Bach1, a repressor of Nrf2 transcriptional activity was induced after HIF-1 activation. Accordingly, adenoviral overex- pression of Nrf2 enhanced both IL-8 transcription and mRNA stability and reversed the inhibitory effect of HIF-1 on IL-8. Importantly, inhibition of HIF-1 activity by chetomin reverses the inhibitory effect of DMOG on Nrf2 activity without influenc- ing AP-1 and NF-kB activity. The data indicate that HIF-1- dependent inhibition of IL-8 expression is dependent on the down-regulation of Nrf2. Cross-talk between HIF-1 and Nrf2 may influence the outcome of anti-angiogenic therapies aimed at targeting HIF-1.

P5–41 The role of sterol-binding in elicitin interaction with tobacco J. Lochman, J. Tieffova, P. Literakova, N. Ptackova and T. Kasparovsky Biochemistry, Masaryk University, Brno, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Cryptogein is a proteinaceous elicitor isolated from Phytophthora cryptogea able to trigger plant defence reaction. We prepared a series of cryptogein mutants which showed no-binding of sterol to study influence of sterol-binding for defence reaction activa- tion. Cryptogein mutants were tested for induction of defence related genes expression covering the pathogenesis related pro- teins (PR1a (acidic), PR1b (basic), PR3Q) together with genes for epi-aristolochene synthase (EAS) and 9-lipooxygenase (LOX). Moreover, the accumulation of signalling compound salicylic acid (SA), phytoalexin capsidiol and malonyldialdehyde (MDA) as marker of cell death was measured. The mutants without sterol- binding activity showed different sensitivity to activate defence Introduction: SirT1, a highly conserved NAD+-dependent pro- tein deacetylase in mammals, has been reported to extend life- span. LKB1, a tumor suppressor protein kinase, is an energy sensor that regulates cellular metabolism. The objectives of this study are to investigate whether LKB1 is a downstream target of SirT1 and the mechanism whereby SirT1 regulates LKB1 and its subsequent cellular functions. Methods: Stable HEK293 cell lines overexpressing LKB1 or SirT1 were established to evaluate how SirT1 regulates LKB1 stability and post-translational modifications. The effect of SirT1 on LKB1-mediated cellular senescence and cell cycle arrest were analyzed by b-gal staining and flow cytometry. Mass spectrome- try and site-directed mutagenesis were employed for determina- tion of precise lysine acetylation sites on LKB1. Results: Western blot analysis showed that SirT1 interacts with LKB1 and causes LKB1 lysine deacetylation, thereby promoting proteasome-mediated protein degradation of LKB1. Flow cytom- etry and b-gal staining results demonstrated that SirT1 antago- nizes the effects of LKB1 on inhibition of cell proliferation and promotion of cellular senescence. Proteomic and mutagenesis studies revealed that mutations of LKB1 on lysine 48, 62, 64 abolished the deacetylation effects of SirT1 on LKB1 stability and subsequent cellular responses. Conclusions: SirT1 regulates LKB1 stability through either switching LKB1 lysine acetylation to ubiquitination or inducing LKB1 translocation from nuclei to cytosol. The inhibition of cel- lular senescence by SirT1 is attributed in part to its ability in pro- moting the proteosome-mediated degradation of LKB. Acknowdgement: Hong Kong RGC Collaborative Research Fund (HKU 2/07C).

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study was

reaction in all tested markers; however mutant L15W/L36F dem- onstrated higher efficiency to trigger defence reaction. In addi- tion, correlation of active oxygen species with EAS expression and capsidiol accumulation was observed. Additional mutated proteins attribute influencing triggering of defence reaction was isoelectric point (pI) when low pI was connected with lower effec- tiveness. Acknowledgements: This supported by grants: MSM0021622413 by Ministry of Education of the Czech Repub- lic, 522/06/0156 and 203/09/P248 by Grant Agency of the Czech Republic.

P5–42 K+-flux measurements across the plasma membrane of Saccharomyces cerevisiae using potassium selective electrodes and Microelectrode Flux Estimation (MIFE) J. Ludwig1, S. Shabala2, A. Mpangara1, S. Shabala2, M. Kahm3, M. Kschischo3 and H. Lichtenberg-Frate´ 1 1Universitaet Bonn – IZMB, AG Molekulare Bioenergetik, Bonn, GERMANY, 2School of Agricultural Sciences, University of Tasmania, Hobart, AUSTRALIA, 3FH Koblenz – Rhein Ahr Campus, FB Mathematik und Technik, Remagen, GERMANY

[Ca2+]i,

loop which contains a conserved CCG motif. There are 33 mem- bers of human tetraspanin family, each capable of interacting with their specific partners or other members of the tetraspanin family creating a complex tetraspanin network, termed tetra- spanin-enriched microdomains (TEMs). CD9 is a well known tet- raspanin member implicated by results of previous studies in gamete fusion, motility and adhesion. However, despite its abun- dant expression on numerous cell types including immune cells, its role in immunoreceptor signaling is still poorly understood. Here we show that treatment of murine bone marrow-derived mast cells (BMMCs) with either our own anti-CD9 (2H9) or commercially available (KMC8.8) monoclonal antibody causes a significant impairment of degranulation in response to antigen challenge, compared to isotype controls. This inhibition is anti- body-concentration dependent and is not caused by steric block- ing of the high-affinity IgE receptor (FceRI), as determined by binding of fluorescently labeled antigen to the cells sensitized with IgE specific for the antigen, and treated or not with anti- CD9. Surprisingly, anti-CD9 antibody treatment alone elicited small but significant BMMCs degranulation. Further investiga- tion of these processes revealed that binding of anti-CD9 anti- body to BMMC surface is accompanied by an increase in the level of free intracellular calcium [Ca2+]i. Pretreatment of the cells with anti-CD9 followed by antigen triggering led to decreased levels of suggesting that CD9 regulates BMMCs responses upstream or at the level of [Ca2+]i. This con- clusion was supported by further data indicating that the RNA interference-mediated decreased expression of CD9 led to an inhi- bition of the anti-CD9 effects. From these results and those obtained with antibodies against other tetraspanins (CD63 and CD81), we conclude that TEMs are integral parts of the mast cell FceRI signalosomes, and act as regulators that are fine-tuning their proximal signaling activity. Acknowledgement: Supported by grant 301/09/1826 from the Grant Agency of the Czech Republic.

P5–44 Different association kinetics between hetero- and homo- dimers of the BMP Receptor family B. T. Marom1, P. Knaus2 and Y. I. Henis1 1Department of Neurobiology, Tel Aviv University, Tel Aviv, ISRAEL, 2FU Berlin, Institute for Chemistry/Biochemistry, Berlin, GERMANY

related processes (epithelial-to-mesenchymal

High affinity K+-uptake in S. cerevisiae is primarily mediated by the K-transporter Trk1p and to a lower degree by its homologue Trk2p. Additionally, a voltage-gated K-channel Tok1p of yet unknown physiological function is present in the yeast plasma membrane. To analyse the activity of transport systems (i.e. to determine the ion fluxes), often uptake assays using radioactive isotopes or analogues are carried out. Intracellular concentrations of elements can be measured using atomic absorption spectros- copy. Both techniques however require cell lysis at given time points and suffer from poor time resolution. Furthermore, it is difficult to simultaneously determine the fluxes of several species simultaneously. As an alternative to measure [K]-changes in the cytosol, we used (i) K+- and H+-selective electrodes to determine the corresponding [K]- and [H]-changes in the surrounding and (ii) the MIFE technique to simultaneously measure the fluxes across the membrane. The activities of K+-translocation systems were examined in a set of isogenic strains (wt, trk1,2, trk1,2,tok1, tok1). Transient K+-uptake accompanied by H+-extrusion was observed in wt andtok1 cells after adding KCl (from 10 lM to 1 mM) to starved cells and energizing the cells with glucose (acti- vating the plasma membrane ATPase Pma1p). H+-extrusion increased with K+-uptake, indicating that H+-efflux is responsi- ble for charge equalisation. Almost no net K+-uptake and strongly decreased H+-efflux was detected with trk1,2 and trk1,2,tok1 cells. At 10 mM external KCl, the differences between the strains vanished, indicating the prevalence of ‘unspecific’ K+- uptake at higher concentrations. Using MIFE, K+-uptake was also detected when only KCl and no glucose were added. Inter- estingly, this was also accompanied by a net proton efflux that is not consistent with a K+/H+ symport.

for

P5–43 Tetraspanin CD9 is modulator of antigen- mediated mast cell degranulation M. Machyna, D. Smrz, L. Dra´ berova´ and P. Dra´ ber Laboratory of Signal Transduction, Institute of Molecular Genetics, Prague, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Bone morphogenetic proteins (BMPs) are crucial in many biolog- ical processes, including embryogenesis, bone formation and can- transition, cer proliferation, etc.). These processes can evolve from functional changes in the ligand, the type I BMP receptors (BRIa and BRIb) or the type II receptor. We have shown in previous work that BRIa, BRIb and BRII form hetero- and homo-dimers to some degree already in the absence of ligand, and ligand (BMP2) binding mostly enhances complex formation. However, the inter- action dynamics of BMP receptor complexes were unknown, in spite of the potential importance of a shift between transient and stable interactions signaling. Here we investigated the dynamic nature of the different BMP receptor complexes (stable vs. transient interactions) at the surface of live cells. To this end, we combined antibody- mediated patching of one receptor type (e.g. HA-BRIa) with FRAP studies on the lateral diffusion of another coexpressed receptor (e.g. myc-BRII). Using this method the hetero- complexes (termed patch-FRAP), we show that (BRIa-BRII and BRIb-BRII) are transient. On the other hand we show that all the homomeric complexes (BRIa-BRIa, BRIb- BRIb, BRII-BRII) as well as BRIa-BRIB, are stable on the FRAP time scale. Since only the signaling complexes are tran- Tetraspanins are proteins spanning plasma membrane four times. They are characterized by their structural features, namely two short cytosolic terminal ends and a small and a large extracellular

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sient we propose that transient nature is required for signaling, perhaps to avoid prolonged signaling that may occur due to sta- ble association. This issue will be further investigated in future studies where we will aim to generate stable heteromeric com- plexes, either by the use of other ligands or by specific mutations.

P5–46 A RCAN- derived peptide that selectively inhibits calcineurin- NFATC signalling without affecting calcineurin phosphatase activity S. Martinez Hoyer1, E. Serrano- Candelas1, M. C. Mulero1, A. Aubareda1, M. Orza´ ez2, J. Messeguer3, A. Messeguer3, E. Pe´ rez- Paya2 and M. Pe´ rez-Riba1 1IDIBELL, Medical and Molecular Genetics Center, Hospitalet de Llobregat Barcelona, SPAIN, 2entro de Investigacio´n Prı´ncipe Fel- ipe, Medicinal Chemistry, Valencia, SPAIN, 3Centro Superior de Investigaciones Cientı´ficas CSIC, Biological Organic Chemistry, Barcelona, SPAIN

P5–45 Genetic and protein changes of AXIN1 and beta-catenin in neuroepithelial brain tumors T. Nikuseva Martic1, N. Pecina-Slaus1, V. Kusec2, T. Kokotovic3, H. Musinovic3, D. Tomas4 and M. Zeljko3 1Medical school University of Zagreb, Department of Medical biol- ogy. Laboratory of Neurooncology Croatian Institute for Brain Research School of Medicine University of Zagreb, Zagreb, CROATIA, 2Clinical Hospital Centre Zagreb, Clinical Institute of Laboratory Diagnosis, Zagreb, CROATIA, 3Medical school Uni- versity of Zagreb, Laboratory of Neurooncology Croatian Institute for Brain Research School of Medicine University of Zagreb, Za- greb, CROATIA, 4University Hospital ‘Sisters of Charity’ Zagreb, 4Ljudevit Jurak Department of Pathology, Zagreb, CROATIA

Calcineurin (Cn) is a serine- threonine phosphatase involved in many physiological functions, among them the activation of the T cell. In the lymphocyte, upon stimulation of the T- Cell Recep- tor, there is an activation of Cn and subsequent dephosphoryla- tion of the nuclear factor of activated T cells (NFATc) family of transcription factors. This dephosphorylation enables NFATc to translocate to the nucleus and start the gene expression pro- gramme driving T cell activation. Current immunosuppressive therapies rely on the inhibition of Cn to inhibit this activation of NFATc. Drugs as Cyclosporin A or FK506 are commonly used in transplanted patients, but its chronic administration produce severe side effects, as a consequence of inhibiting general Cn activity. Therefore, there is a need in the field to discover novel immunosuppressive molecules with fewer side effects. The protein family of Regulator of Calcineurin (RCAN) are endogenous modulators of Cn function. In humans there are three members: RCAN1, RCAN2, RCAN3. These proteins are characterized by three functional motifs: FLISPP, considered the family signature; PxIxxT, which binds Cn; and CIC, involved in the binding to and the inhibition Cn- NFATc signalling. Our group has shown that a 21 aminoacid peptide of the CIC motif is necessary and sufficient to achieve specific inhibition of Cn towards NFATc. Moreover, this peptide does not affect the phosphatase activity of Cn towards other of its substrates. This observation suggests that this peptide possess immunosuppressive potential by itself and would probably show fewer side effects than the current anti- calcineurinic drugs. Finally, using this peptide we have developed a low throughput assay to identify novel immunosup- pressive drugs that mimic this effect.

P5–47 High osmolality causes cell cycle arrest and stimulates a p53-mediated DNA damage response in nucleus pulposus intervertebral disc cells E. Mavrogonatou and D. Kletsas NCSR ‘Demokritos’, Biology, Athens, GREECE

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

We have shown that hyperosmotic treatment reduces nucleus pulposus cells’ proliferation by activating the G2 and G1 cell cycle checkpoints (FEBS J. 2008 Jun; 275: 100, Suppl. 1). The G2 arrest was found to be p38 MAPK-dependent since inhibition of the kinase’s activity released the cells from G2 phase into mitosis, while the activation of the G1 checkpoint was established by the ATM-mediated phosphorylation of p53 on Ser15, the up- regulation of the cyclin-dependent kinase p21WAF1 and the hypo- phosphorylation of the retinoblastoma protein. Single cell gel electrophoresis revealed the existence of comet tails with signifi- cant length evidential of DNA damage in the nuclei of nucleus pulpsous cells. Additionally, H2A. X was phosphorylated on Ser139 and accumulated on the sites of the damage forming foci. In order to investigate the implication of p53 in the G1 arrest Neuroepithelial brain tumors are central nervous system neo- plasms that embody a series of primary brain tumors including astrocytic, ependymal, choroid plexus, pineal parenchymal, and embryonal tumors. Tumors of neuroepithelial origin represent a heterogeneous group of intracranial neoplasms with distinct fea- tures that control their ontogeny, pattern of invasion, clinical outcome, and prognosis. These features may reflect the complex- ity of the molecular and genetic alterations in pathways involved in the maintenance and progression of CNS tumors. It has been well documented that wnt genes, together with other components of wnt signaling pathway, are implicated in tumorigenesis. In the present study changes of components of wnt signaling pathway, axin (AXIN1) and b-catenin (CTNNB1) were investigated in neu- roepithelial brain tumors in order to elucidate the genetic back- ground ot this tumors. Seventy-two neuroepithelial tumors were analyzed in this study regarding genetic changes of two genes: axin and b-catenin. LOH of the AXIN1 gene was detected by PCR amplification of the dinucleotid polymorphism (D16S521). The same samples were also analysed by immunohistochemistry for both axin and b-catenin proteins and evaluated by image analysis as staining density. PCR amplification of polymorphic marker for AXIN1 gene, showed LOH in 11.1% of total infor- mative tumors. LOH was distributed to 6.3% of glioblastomas, one was found in neuroepithelial dysembrioplastic tumor and one in medulloblastoma. Immunostaining and Image analysis revealed the quantity and localization of relevant proteins. Axin was observed in the cytoplasm in 68, 8% of samples, in 28, 1% in both the cytoplasm and nucleus and 3, 1% had no expression. b-catenin was observed mainly in the nucleus and cytoplasm (59,4%). In 34, 4% it was localized in cytoplasm and 6, 3% of our sample had no expression. Comparison of mean values of relative increase of axin and b-catenin showed that they are sig- nificantly reversely proportional (P = 0,014). Relative quantity of b-catenin in patients with gross deletion of AXIN1 was signifi- cantly higher in comparison to patients without LOH (P = 0,040). In conclusion we believe that wnt signaling has important role in neuroepithelial brain tumor development. Our novel findings on changes of the wnt molecular components may contribute to better understanding of human brain tumors genetic profiles and also indicates for the first time the relation of quantities of those proteins.

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and the DNA damage response of nucleus pulposus cells stimu- lated by high osmolality, siRNA-mediated knocking down of the protein was used. p53 depletion decreased the expression of p21WAF1 and inhibited its hyperosmolality-induced up-regulation, abolished the hypophosphorylation of the retinoblastoma protein and abrogated the G1 arrest provoked by hyperosmotic stress in nucleus pulposus cells, as demonstrated by cell cycle analysis. Finally, p53 and the consequent G1 arrest were found to partici- pate in the DNA repair pathway of nucleus pulposus interverte- bral disc cells under conditions of increased osmotic pressure, as their inhibition led to an enhanced and prolonged H2A. X phos- phorylation.

P5–48 PARP10 can inhibit the G-protein beta-gamma- signalling through its mono-ADP-ribosylation E. Mayo, N. Dani, A. Stilla, S. Di Paola, A. Marchegiani and M. Di Girolamo Cell Biology and Oncology, Consorzio Mario Negri Sud, Santa Maria Imbaro, ITALY

polyubiquitinylation. We requires are its

been implicated in various forms of stress response. Adaptation to arsenic stress involves coordinated activation of two members of this family. The key regulator Yap8 plays an essential and well-defined role in arsenic detoxification by regulating the expression of the arsenate reductase and the arsenite efflux-pro- tein whereas Yap1, the major regulator of oxidative stress responses in yeast, controls transcriptional activation of the vacu- olar-pump encoded YCF1 and the antioxidant genes in order to maintain the redox homeostasis disturbed by these compounds. In this work, genome transcriptional profiling of the wild type strain stressed with arsenate reveals that many genes encoding proteins of the ubiquitin/proteasome pathway are induced by arsenate treatment including the transcription factor Rpn4 and the ubiquitin chain elongation factor Ufd2/E4. We also com- pared the transcriptome of wild type cells submitted to arsenate exposure with the one of yap1 mutant cells under the same con- ditions and found that transcriptional activation of RPN4 is par- tially dependent on YAP1. These results support the notion that the ubiquitin/proteasome pathway contributes to full arsenic tol- erance and reveal the importance of Yap1 in this process. The biological significance of Ufd2/E4-mediated polyubiquitinylation to arsenic stress responses was assessed by growth assays showing that it is required to full adaptation of yeast cells to arsenic-con- taminated environments, consistent with the idea that it facili- tates cell survival under stress conditions. Moreover, a yeast two- hybrid cDNA library screening identified Ufd2/E4 as a Yap8- interaction partner. This enzyme, which is necessary for the effi- cient polyubiquitinylation of specific substrates, binds to the ubiquitin moieties of preformed conjugates and catalyzes ubiqu- itin chain assembly in conjunction with the ubiquitin-activating enzyme (E1), the ubiquitin-conjugating enzyme (E2) and the ubiquitin ligase (E3). Thus, our results suggest that post-transla- tional regulation of Yap8 through the ubiquitin/proteasome path- way currently addressing this issue by monitoring the ubiquitinylation status of Yap8 in the wild type and ufd2 mutant strains.

P5–50 The morphogene bolA has a major effect on cellular architectural features R. N. Moreira, P. Freire and C. M. Arraiano ITQB, Control of Gene Expression Lab, Oeiras, PORTUGAL

The heterotrimeric G proteins are the most common transducers for G-protein-coupled receptors (GPCRs), the largest family of cell surface receptors, which switch biological signals from the cell sur- face to the intracellular compartment. When activated, GPCRs catalyse the exchange of GDP for GTP on Galpha, leading to the dissociation of the Gbeta-gamma dimer. Both the Galpha subunit and the Gbeta-gamma dimers regulate effector proteins, such as the adenylyl cyclases, phospholipases, mitogen-activated protein kinase and ion channels. The Gbeta-gamma dimer does not have a catalytic site, and thus it acts as a modulator of G-protein signal- ling through regulated protein-protein interactions. Here we describe a novel interactor of the Gbeta-gamma dimer, and pro- vide evidence that this interaction can lead to a Gbeta-gamma dimer modification that regulates its activity. We have previously demonstrated that the beta subunit is endogenously ADP-ribosy- lated (1). This reversible, post-translational modification occurs on Arg129 of the beta subunit, and it is catalysed by a not-yet-identi- fied mono-ADP-ribosyltransferase (1). Once modified, the beta- gamma dimer is inactive towards its effector enzymes (1, 2); thus, this mono-ADP-ribosylation represents a novel mechanism for the regulation of beta-gamma-dimer activities (1–3). We now show that PARP10, a recently identified member of the poly-ADP-ribose polymerase (PARP)-like proteins (4), interacts directly with the beta subunit and can catalyse its ADP-ribosylation. This finding opens the way to the analysis of the physiological relevance of this post-translational modification for Gbeta-gamma signalling. References: 1. Lupi R, Corda D & Di Girolamo M. J. Biol. Chem. 2000: 275; 9418–9424

2. Lupi R. et al. Biochem. J. 2002: 367; 825–832.367. 3. Corda D, Di Girolamo M. EMBO J. 2003; 22: 1953–1958. 4. Kleine et al. Mol Cell. 32 (1): 57–69

P5–49 The ubiquitin chain assembly factor Ufd2/E4 contributes to full arsenic adaptation in Saccharomyces cerevisiae L. Batista-Nascimento1, L. Batista-Nascimento2, R. Menezes1, D. Thiele2 and C. Rodrigues-Pousada1 1ITQB, Genomics and Stress Laboratory, Lisboa, PORTUGAL, 2Department of Pharmacology and Cancer Biology, Sarah W. Stedman Nutrition and Metabolism Center, Durham, USA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

In natural environments, most bacteria live attached to surfaces in structures known as biofilms. Overexpression of the morpho- gene bolA in Escherichia coli was shown to induce biofilm devel- opment while its deletion presents a consistent reduction in biofilm accumulation. The bolA gene is highly induced in stress conditions and during stationary phase of growth causing a cellu- lar round morphology when overexpressed. bolA was shown to regulate the levels of PBPs 5 and 6 and of AmpC lactamase. Evi- dence shows that induction of bolA results in increased resistance of OM integrity to SDS and a decrease of cell sensitivity to vancomycin. In parallel to outer membrane proteins accessibility changes, the expression ratio of porins OmpC and OmpF was shown to vary according to bolA levels. It has been hypothesized that the bolA morphogene was related to the switch between cell elongation and cell division. bolA overexpression can influence the mechanism by which bacteria elongate and retard cell growth rate. Thus, BolA affects cellular mechanisms in various separate ways. Here we show that increased BolA levels can inhibit cell elongation mechanisms. MreB polymerization is crucial for the bacterial cell cytoskeleton, and this protein is essential for the maintenance of a cellular rod shape. We demonstrate that bolA overexpression affects the architecture of MreB filaments. An increase in BolA leads to a significant reduction in MreB protein In the yeast Saccharomyces cerevisiae a family of AP-1 transcrip- tion factors designated as YAP (Yeast Activator Proteins) has

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lines having a wild type

levels and mreB transcripts. BolA affects the mreBCD operon in vivo at the level of transcription. Furthermore, our results show that BolA is a new transcriptional repressor of MreB. The altera- tions in cell morphology induced by bolA seem to be mediated by a complex pathway that integrates PBP5, PBP6, MreB, and probably other regulators of cell morphology/elongation.

P5–51 Cholesterol depletion increases CCR5 calcium release in monocytic cells and macrophages C. Moyano and M. Anja School of chemical sciences and pharmacy, Molecular pharmacology, University of East Anglia, Norwich, UK

human tumors. The functional capacity of mutated p53 can be abrogated, leading to an abnormal (or absent) response to stress. Thus, we investigated cell death signaling of two neuroblatoma cell (SHSY5Y) or a mutated [SKNBE2(c)] p53 gene, following an oxidative stress (with ROS generation) due to cobalt chloride treatment. Quickly (from 6 h), CoCl2-induced a mitochondrial signalization through up-regula- tion of p53 (with Ser15 and Thr81 phosphorylation), PUMA, and Bax expression in WT SHSY5Y cells and not in [SKNBE(2c)]. In these mutated-p53 cells, no recruitment of pro- teins involved in death signaling was observed. A delay (about 12–15 h) in mt collapse and Caspase-3 and -9 increase activity was observed, suggesting mechanisms of repairs and/or recycling. An autophagic process attested by the punctiform staining of LC3 and a level enhancement of Beclin-1, Atg5-Atg12 and LC3. For later time of exposure, autophagy seemed inadequate as regard to ROS production, the executive phase of cell death became obvious after 18 h of CoCl2 addition. Although, no sig- nificant Caspase-1 (a protease implied in inflammation) activity was detected, the autophagic cell death seems to correspond to necrosis.

P5–53 Arabidopsis LIM domain proteins involved in actin bundling exhibit different modes of regulation J. Papuga, C. Thomas, M. Dieterle, F. Moreau and A. Steinmetz CRP Sante´, Plant Molecular Biology, Luxembourg, LUXEMBOURG

The chemokine receptor CCR5 serves as a co-receptor for HIV-1 entry into cells and is involved in many inflammatory diseases. CCR5 is mainly situated in lipid rafts, which are cholesterol rich is essential for regions of the plasma membrane. Cholesterol CCR5 signal transduction and processes such as chemotaxis or HIV-1 infection (1). It is known that cholesterol depletion impairs HIV-1 infection in macrophages (2). In this work we seek to investigate CCR5 signalling dependence on intact lipids micro- domains further in monocytic cells. We use various cholesterol depleting agents and employ flow cytometry, immunofluorescence microscopy and the measurement of intracellular calcium mobili- sation to analyse the signalling pattern of four different cell types. We have previously shown that MCD treatment of CHO.CCR5 and HEK.CCR5 cells inhibits intracellular calcium mobilization upon CCR5 stimulation and switches CCR5 coupling to a PTX- insensitive G protein (3). However, here we demonstrate that this treatment leads to a high increase in intracellular calcium release in THP-1 and especially in THP-1 derived macrophages. We also observe a MCD-induced association between CCR5 and a PTX- resistant protein. MCD depletes around 70% of membrane cho- lesterol while it has no effect on CCR5 membrane expression. Calcium release increase in THP-1 cells is chemokine dose depen- dent and cholesterol re-loading reduces MCD stimulation of the CCl3 induced signalling, proving that the effect of MCD in THP- 1 and macrophages is due to cholesterol reduction. Moreover, MCD abrogates the inhibitory effect the SERCA inhibitor thapsi- gargin and the IP3R inhibitor caffeine have on calcium release from the ER stores in THP-1 cells. This indicates that cholesterol depletion does not only influence plasma membrane molecules but affects proteins expressed in the ER membrane as well. This research highlights for the first time the importance of cholesterol depletion for signal transduction in THP-1 cells and macrophages and addresses the issue of whether HIV-1 infection and signalling are independent processes or not. References: 1. Nguyen DH & Taub D. Blood 2002; 99: 4298–4306. 2. Carter GC, Bernstone L, Sangani D, Bee JW, Harder T & James W. Virology 2009. 3. Cardaba CM, Kerr JS & Mueller A. Cell Signal 2008; 20: 1687–1694.

Actin cytoskeleton organization and dynamics are regulated by different types of actin-binding proteins. Recently, a novel family of actin filament cross-linkers involved in the formation of paral- lel bundles has been reported in both plants and animals: the two LIM domain-containing (LIM) protein family. In plants it splits into to subgroups differing in their expression patterns: the WLIMs which are widely expressed in vegetative tissues and the PLIMs which are specifically expressed in pollen. The present study aims at comparing the actin-associated activities and mode of regulation of one member of each Arabidopsis LIM family subgroup. Confocal analyses of transgenic Arabidopsis expressing GFP-fused LIM proteins and biochemical investigations confirm that both members possess the ability to bind to, stabilize and bundle actin filaments. However, they exhibit different respon- siveness to [Ca2+] and pH. Whereas modifications in these parameters have no effects on WLIM’s activities, their elevation significantly inhibits PLIM’s activities. Based on these data and the well-known patterns of [Ca2+] and pH in pollen tubes, we propose a model where PLIMs regulate the formation and posi- tioning of actin bundles in pollen tubes. The relatively low values of [Ca2+] and pH in the pollen tube shank would allow PLIMs to generate the thick and stable actin bundles required for vesicle transport. In contrast, the relatively high values of [Ca2+] and pH in the tip region would inhibit PLIM’s activities and there- fore allow the formation of dynamic actin structures required for tip growth.

P5–52 Cobalt chloride induces oxidative stress in neuroblastoma cells, with different cell death responses according to their p53 statut T. Naves, C. Stenger, M. O. Jauberteau, M. H. Ratinaud and M. Verdier EA 3842 Home´ostasie cellulaire & Pathologies, Universite´ de Limoges, Limoges, FRANCE

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

The p53 plays a key role in regulation of cell growth arrest and apoptosis, thus mutations in its gene are implicated in numerous

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P5–56 Yap4 PKA- and GSK3-dependent phosphorylation affects its stability but not its nuclear localization J. Pereira, C. Pimentel, C. Amaral, R. Menezes and C. Rodrigues-Pousada ITQB, Genomics and Stress Laboratory, Lisboa, PORTUGAL

P5–54 Differential role of PKC-epsilon in calcium influx and exocytosis in chromaffin cells C. H. Park, Y. S. Park, S. Baek, W. Kim, M. W. Jeong, S. Kim, Y. H. Choi, K. C. Woo, K. H. Lee, D. Y. Kim, J. S. Kim and K. T. Kim Life Science, Pohang University of Science and Technology, Pohang, SOUTH KOREA

Phorbol 12-myristate 13-acetate (PMA) treatment enhances exo- cytosis via activating Protein kinase C (PKC), while inhibiting Ca2+ influx in chromaffin cells. It has been not fully understood which isozymes of PKC can induce potentiation of exocytosis in spite of the inhibition of Ca2+ influx. Here, we provide evidence that PKC-epsilon is involved in PMA-induced inhibition of Ca2+ influx and potentiation of large dense-core vesicle (LDCV) exocy- tosis. First, treatment with 200 nM PMA inhibits 29.3% of con- trol Ca2+ influx elicited by high potassium (KCl), but expression of dominant negative (DN) PKC-epsilon completely reverses inhibitory effect. Second, electron microscope and amperometry analysis reveal that PMA treatment increases docking of LDCVs to the plasma membrane (PM), suggesting that potentiation of exocytosis is associated with an increase in docked vesicle num- ber. Lastly, DN PKC-epsilon attenuates PMA-induced potentia- tion of LDCV exocytosis. These results suggest that PKC-epsilon regulates PMA-induced potentiation of LDCV exocytosis by increasing the docked vesicle number, even though reducing Ca2+ influx.

YAP4 belongs to the YAP family of bZIP transcription factors and is involved in the stress response in the yeast Saccharomy- ces cerevisiae. Yap4 is phosphorylated and induced upon expo- sure to several stress conditions and its regulation involves the Protein Kinase A. PKA negatively regulates YAP4 expression via Msn2 and Yap4 phosphorylation is abolished in the null PKA mutant. In order to ascertain whether Yap4 is directly or indi- rectly phosphorylated by PKA, we searched for stress and PKA- related kinases that could phosphorylate Yap4. We showed that this modification is independent of the kinases Rim15, Yak1, Sch9, Slt2, Ste20, Ptk2 and Mck1. However, Yap4 phosphoryla- tion is abolished in the double GSK3 mutant rim11 mck1. Yap4 presents several putative phosphorylation sites, but only mutation of the PKA consensus residue S196 and the GSK3 consensus res- idue T192 impairs its phosphorylation. Taking together, these results suggest that Yap4 is sequentially phosphorylated by PKA in residue S196 and by Rim11 in residue T192. The role of Yap4 and its phosphorylation in the yeast stress response remains unclear. We were able to show that lack of phosphorylation does not impair Yap4 nuclear localization or its ability to partially res- cue the hog1 severe sensitivity phenotype. However, phosphoryla- tion seems to be required for the stability of the protein as the non-phosphorylated form has a shorter half-life compared to the phosphorylated one. Additional experiments are necessary to fully understand the role of Yap4 in the stress response and the relevance of the PKA and GSK3-dependent phosphorylation in its regulation.

P5–55 Analysis of cell cycle regulation during vegetative cell growth in Dictyostelium discoideum S. J. Park and S. O. Kang Laboratory of Biophysics School of Biological Sciences and Insti- tute of Microbiology, Seoul National University, Seoul, SOUTH KOREA

P5–57 Induction of neuroendocrine transdifferentiation by androgen ablation is associated with expression of markers of senescence Z. Pernicova, E. Lincova, A. Starsichova, A. Kozubik and K. Soucek Department of Cytokinetics, Institute of Biophysics AS CR v.v.i., Brno, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Prostate cancer belongs to world’s most extended types of cancer in men. At the beginning, patients usually benefit from the con- ventional anti-androgen therapy. But the disease recurs in more severe and aggressive manner and cancer cells become androgen- independent. Neuroendocrine (NE) cells are present in both nor- mal and cancer prostate epithelium. They secrete many types of neuropeptides, through which they can stimulate the process of neuroendocrine transdifferentiation (NED) of surrounded pros- tate cancer cells. Cells that undergo NED acquire NE phenotype; they are able to secrete neuropeptides and are androgen-indepen- dent. So it seems that NE cells may contribute to acquisition of androgen independence of prostate cancer cells. The aim of our study was to find out how we can modulate process of NED in vitro and if this process is associated with senescence. To evoke NED of LNCaP cancer cell line in vitro, we used prolonged culti- vation in media without androgens. This leads to cell cycle arrest and increased expression of NED markers – neuron-specific eno- lase and b-III tubulin. Next we wanted to investigate, if increased cell density can induce transdifferentiation. We found out that Reduced glutathione serves as a primary redox buffer and its depletion causes growth inhibition or apoptosis in many organ- isms. We recently reported that the null mutant (gcsA-) of gcsA encording c-glutamylcysteine synthetase shows G1 phase growth arrest. Methylglyoxal (MG) accumulated by GSH depletion inhibits cell growth in Dictyostelium discoideum. For a better understanding of the cell cycle during vegetative cell growth, we examined cell growth in gcsA- cell. The growth rate of gcsA- cells, which is unable to synthesize GSH, decreases as GSH concentra- tions in media decreases and is completely halted by depletion of GSH. Interestingly, when 1 mM GSH was added to the medium, gcsA- cell growth is recovered. To address what kind of cell cycle factors induces growth arrest of Dictyostelium, we performed a proteomic analysis to see the difference in transcriptional expres- sion patterns of nuclei between parental KAx3 cells and treated with MG. Cyclin (cyc) and cyclin-dependent kinase (cdk) have been known cell cycle regulation factor. To evaluate the regula- tion of cell cycle factor in vegetative cell, we tested cyc mRNA expression pattern using Northern bolt analysis and cdk activity. Northern blot analysis and activity measurement revealed that transcriptional level of cyclin B (cycB) induced and Cdc2 (cdk2) activity was changed by GSH depletion and MG treatment. Using a fluorescently tagged replication factor, proliferating cell nuclear antigen (PCNA), to mark S-phase cells, we examined G1/S phase transition when GSH was depleted. GFP-PCNA has a characteristic dynamic distribution.

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P5–59 17beta-estradiol binding to synaptosomal mitochondria isolated from rat brain nucleus caudatus, hippocampus and brain steam S. Petrovic, M. Milosevic, D. Drakulic, I. Stanojevic, N. Velickovic and A. Horvat Institute for Nuclear Sciences Vinca, Laboratory for molecular biology and endocrinology, Belgrade, SERBIA

conventional marker of

expression of NED markers increased with increased density. This observation was confirmed in mouse cell line TRAMP-C1. NED is associated with cell cycle arrest and downregulation of cyclin D1. We wanted to assess, whether depletion of cyclin D1 can induce NED of LNCaP cell line in presence of androgens. Using RNA interference we showed that level of cyclin D1 does not reflect changes in expression of NED markers. Senescence associated with permanent cell cycle arrest is well known in pri- mary cells, but it was observed also in cancer cells. Using stain- ing for senescence – senescence associated b-galactosidase (SA-b-gal), we showed, that cells trans- differentiated by androgen depletion express this marker, as shown as blue staining in cytosol. Acquisition of senescent phe- notype was confirmed by increased level of another marker of senescence p16INK4A. We can summarize that cyclin D1 is not the key regulator of NED and that NED is associated with induction of senescence in prostate adenocarcinoma. Acknowledgements: This work was supported by MU Rec- tor’s Programme for Student’s Creative Activity Support, grants 204/07/0834, 310/07/0961 of the Czech Science Foundation and AV0Z50040507, AV0Z50040702 of the Grant Agency of Acad- emy of Sciences of the Czech Republic.

P5–58 Histochemical detection of GM1 ganglioside in liver sections using cholera toxin B – subunit after various types of fixation T. Petr1, V. Smid1, J. Smidova2, H. Hulkova3, L. Muchova1, M. Jirkovska2, L. Vitek1 and F. Smid1 1Institute of Clinical Biochemistry and Laboratory Diagnostics, 1st Faculty of Medicine, Charles University, Prague, CZECH REPUBLIC, 2Institute of Histology and Embryology, 1st Faculty of Medicine, Charles University, Prague, CZECH REPUBLIC, 3Institute of Inherited Metabolic Disorders, 1st Faculty of Medicine, Charles University, Prague, CZECH REPUBLIC

17beta-estradiol, gonadal steroid hormone, binds specifically to synaptosomal mitochondria isolated from whole rat brain. The same estradiol concentrations that specifically bound to mito- chondria in parallel, modify the mitochondrial and consequently synaptosomal content of Ca2+, modulating neuronal neurotrans- mitter releasing and nerve impulse passing. In this study 17beta- estradiol binding to synaptosomal mitochondria isolated from different brain regions: nucleus caudatus, hippocampus and brain in order to evaluate diversity between stem were examined, regions indicating differences in Ca2+ sequestration and cytosolic ion content. Mitochondria (0.5 mg/ml) were incubated with increasing concentrations (10-12–10-8 mol/l) of labeled estradiol, (3H)estradiol, for total hormone bound and non-specific bound estradiol was determined by incubating mitochondria with labeled and 100-fold excess of unlabeled estradiol. Specific hor- mone binding was calculated by subtracting non-specific from total bound estradiol. The specific estradiol binding was found in all investigated structures. The binding capacity (Bmax) found: 37.4 ± 3.1 fmol/l; 33.8 ± 2.5 fmol/l and 29.1 ± 2.7 fmol/l for nucleus caudatus, brain steam and hippocampal mitochondria respectively, indicated that capacity of estradiol binding is nearly the same in all investigated structures. Observed Km for estradiol was 0.22 ± 0.04; 0.16 ± 0.07 and 0.53 ± 0.08 nmol/mg for mitochondria isolated from nucleus caudatus, brain steam and hippocampus respectively. These findings indicate that affinity for estradiol is significantly higher in nucleus caudatus and brain steam mitochondria comparing to hippocampus, which could be the cause of differences in estradiol neuromodulatory effect in investigated regions.

study was

extracted from liver gangliosides tissue

P5–60 T-cadherin modulates growth, motility, invasion and cytoskeleton organization of carcinoma cells and their interaction with the vascular endothelium M. Philippova1, D. Pfaff1, K. Maslova1, M. B. Joshi1, E. Kyriakakis1, P. Erne2, S. Buechner3 and T. Resink1 1University Hospital, Department of Biomedicine, Basel, SWITZERLAND, 2Kantonsspital Luzern, Division of Cardiology, Luzern, SWITZERLAND, 3Dermatology Clinic, Dermatology, Basel, SWITZERLAND

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

T-cadherin (T-cad) is an atypical glycosylphosphatidylinositol (GPI)-anchored member of the cadherin superfamily. Data on T- cad expression and function in skin cancer are controversial and show either downregulation or upregulation in squamous and basal cell carcinoma, with varying localization patterns within different tumor zones. In vitro studies also give inconsistent data on T-cad effects on cell motility. Aim: To analyze effects of T-cad overexpression and silencing on growth, migration and invasion of normal keratinocytes and carcinoma cells and their interaction with vascular endothelial cells. Very poor solubility of glycosphingolipids (GSLs) from tissue sections in course of acetone fixation and an extensive solubility from hippocampal cell culture have been described. The aim of our to explain this discrepancy. Moreover, an increase of ganglioside labeling was observed in brain sections after acetone fixation when compared with formaldehyde one. A similar study in liver section has not been performed yet. Cryostat liver sections were fixed with 4% formaldehyde or dry acetone and GM1 ganglioside labeled with cholera toxin B sub- unit was histochemicaly detected. Image analysis of GM1 on dry cryostat sections did not show any significant differences between formaldehyde and dry acetone fixation. Contrary to it, more marked staining after acetone fixation was observed in brain tissue. It could be caused by high cholesterol concentra- tion in brain and its depletion after acetone fixation. TLC ana- lysis of sections supported the microscopic observation. Loss of GM1 ganglio- side was about 2% with dry acetone extraction at -20(cid:2)C and about 4% at room temperature. The loss significantly increased to 30.5% when 10% water was added to acetone. Photometric analysis of lipid sialic acid in extracts from dried liver homoge- nates with dry acetone showed the loss of gangliosides into acetone 3.0 ± 0.3% only. We conclude that sequential diffusion of acetone from zero concentration into aqueous intracellular milieu can be the explanation of high solubility of glycosphingo- lipids from cell culture when compared with that from dry sec- tions. Acknowledgement: Supported by grand IGA-MZ 9366-3.

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cation is also dependent on the presence of the remaining Yap members. We are currently investigating the nature of this post- translational modification and trying to identify the Yap mem- ber(s) involved in Yap1 transcriptional regulation.

P5–62 Akt down regulation by CYP2C9-induced ROS generation mediate dose-dependent cell damage elicited by natural antioxidants in human endothelial cells G. Pintus1, V. Pasciu1, A. M. Posadino1, A. Cossu1, B. Tadolini1, L. Gaspa1, A. Marchisio1, S. Dessole2, G. Capobianco2 and B. Sanna1 1Department of Biomedical Sciences, University of Sassari, Sassari, ITALY, 2Department of Pharmacology Gynecology and Obstetrics, University of Sassari, Sassari, ITALY

Methods: Keratinocyte and carcinoma cell lines HaCat and A431 lentivirally transduced to express either T-cad gene or T- cad shRNA were analyzed with respect to their proliferation, invasion in collagen gels and Matrigel, motility (time-lapse micro- scopy analysis), T-cad expression and cytoskeleton organization (Western blotting, immunocytochemistry). Adhesion of T-cad- overexpressing or silenced A431 labeled with dsRed onto micro- vascular endothelial cells HMEC-1 was measured by flow cyto- metry. Results: HaCat expressed mostly mature 105 kDa T-cad pro- tein; A431 had both mature T-cad and its precursor, their expres- sion level being markedly reduced, and the apparent molecular weights being 10kDa less than in normal keratinocytes. T-cad was present at intercellular borders in monolayers and in fine filopodia extensions formed by migrating cells. T-cad silencing stimulated HaCat and A431 proliferation, promoted random motility, invasion from spheroids into 3D gels, induced cell elon- gation and cortical actin polymerization suggesting effects on Rho GTPase activity. T-cad silencing resulted in increased adhe- sion of A431 cells onto endothelial monolayers. Conclusion: T-cad effects on skin cells are complex and include modulation of growth, migration, invasion and phenotype. Com- bination of expression and localization patterns may differentially regulate tumor expansion. Increased adhesion of T-cad silenced cells onto the endothelium suggests that T-cad acts as a negative guidance cue for tumor cells preventing their spreading at sites with high T-cad levels. Loss of T-cad in tumors may result in local directional promotion of invasion, facilitation of intravasa- tion into blood vessels and metastasis.

involvement

indicating that Akt

P5–61 YAP1 is transcriptionally regulated under oxidative and cadmium stress in a Yap-dependent way C. Pimentel, A. Raposo, C. Amaral, R. Menezes and C. Rodrigues-Pousada ITQB, Genomics and stress, Lisboa, PORTUGAL

High intake of natural antioxidants (NA) from plant-derived foods and beverages is thought to provide cardiovascular benefit. The endothelium plays a pivotal role in cardiovascular homeosta- sis and for this reason the molecular events resulting from NA actions on endothelial cells (ECs) are actively investigated. Here we show the direct impact of two NA, coumaric acid and resver- atrol, on intracellular reactive oxygen species (ROS) generation and cell physiology in human ECs. While at lower doses both NA promoted antioxidant effects, at moderately high doses NA elicited a dose-dependent pro-oxidant effect, which was followed by apoptosis, cell damage and phospho-Akt (p-Akt) down-regu- lation. Treatment of ECs with the mitochondrial permeability transition pore (MPTP) inhibitor cyclosporine A (CsA), comple- tely prevented oxidative cell damage strongly indicating mito- chondrial in NA-induced ECs impairment. NA- induced pro-oxidant effects were counteracted by sulfaphenazole (SPZ), suggesting a role for Cytochrome P450 (CYP) 2C9 in NA-induced toxicity. SPZ also prevented NA-induced p-Akt down-regulation and mitochondrial membrane potential (MMP) impairment can work downstream of CYP2C9 in mediating cellular responses to NA. Stimulation of p-Akt by insulin, dramatically counteracted NA-induced MMP impairment and cell death, an effect abolished by the Akt inhibi- tor wortmannin further suggesting that mechanistically Akt regu- lates cell survival in response to NA-induced stress. Our study is the first to show in a human vascular model that moderately high-doses of NA can induce mitochondrial-dependent cell damage mediated by CYP2C9- and the Akt pathway.

P5–63 Role of diacylglycerol kinase in regulation of plant cell polar growth R. Pleskot1, V. Zarsky2 and M. Potocky1 1Institute of Experimental Botany, Laboratory of Cell Biology, Prague 6, CZECH REPUBLIC, 2Department of Plant Physiology, Charles University in Prague, Prague 2, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Polar cell expansion is one of the most important mechanisms of the cell morphogenesis. Minor membrane phospholipids like phosphatidic acid represent eminent regulators of cell polarity in yeast and animal cells. Phosphatidic acid (PA) is produced by two pathways, either by phospholipase D (PLD) or by cascade of phospholipase C (PLC) and diacylglycerol kinase (DGK). So far in plant cells most studies addressed to the generation of PA via PLD pathway, that is why we focus on study of DGK. No comprehensive study about origin and evolution of DGK gene Yap1 belongs to a family of eight b-ZIP transcription factors of Saccharomyces cerevisae that has been implicated in a variety of stress responses including oxidative, osmotic, arsenic, heat and drug stress, among others. Yap1 is the major regulator of oxida- tive stress and it is also in forefront of cadmium stress response. It was showed that that activity of Yap1 is primary controlled at the level of nuclear export. To definitely understand whether YAP1 is the sole Yap member involved in oxidative and cad- mium stress, we transformed a Yap0 strain (a strain without all the YAPs, our unpublished data) with YAP1 and tested its abil- ity to cope with oxidative (0.4 mM, H202) and cadmium (25 lM) stress. Spot assay analysis clearly demonstrate that the Yap0 complemented with one copy of YAP1 is more sensitive than the wild type strain to both stresses. Cellular localization of YAP1 fused to GFP in the Yap0 strain, revealed that the nuclear localization of this transcription factor is not affected by the absence of the other Yap members. Phenotypic analysis of the single yap mutant strains suggests that indeed YAP1 is the major regulator in both stresses. Therefore, we investigated the tran- scriptional regulation of YAP1 in the Yap0 strain. Northern blot analysis upon cells incubation with 0.4 mM of H2O2 and 1 mM of cadmium reveal that YAP1 expression is induced after 15 min- utes (H2O2) or 30 minutes (Cd), but strikingly this increase is ser- iously compromised in a strain deleted in the remaining Yap family members. These results were further corroborated by Wes- tern blot. Interestingly, protein analysis additionally revealed that (i) Yap1 is posttranslationally modified and that (ii) this modifi-

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different role of NTAL in the signaling pathways in human and mouse mast cells, rather than of the procedure used for silencing. Acknowledgement: Supported in part by KAN200520701 and 301/09/1826.

P5–65 Characterization of the roles of PAK5 in neuronal development H. F. Poon, H. T. Xu and Y. P. Ching Anatomy, The University of Hong Kong, Hong Kong, CHINA

support grants from family has been done yet. Therefore we performed phylogenetic and motif analysis of the DGK family in 32 eukaryotic genomes with special interest to plants. On the basis of these analyses we speculate that ancestral DGK had a simple organization with only catalytic and accessory domain, while in the higher eukar- yotes DGKs have much more complex structure. We also con- structed model of the catalytic core of plant DGK in an effort to uncover similarities in the reaction mechanism and the membrane docking with its bacterial homolog. To obtain initial data about involvement of DGK in regulation of plant cell growth we used tobacco pollen tubes as a model system. We compared PLD and PLC/DGK pathways using inhibitors of PLD and DGK. We found that PA produced by DGK is crucial for pollen tube growth. Our results imply that cellular functions of DGK-pro- duced PA are different from PLD-produced PA. Acknowledgement: Financial IAA601110916 and LC06034 is acknowledged.

P5–64 Is the adaptor protein NTAL a positive or negative regulator of the high-affinity IgE receptor signaling? I. Polakovicova, M. Simicek and P. Draber Institute of Molecular Genetics, Laboratory of Signal Transduc- tion, Prague 4, CZECH REPUBLIC

P21-activated kinase 5 (PAK5) is a serine/threonine kinase down- stream of the small GTPase Cdc42. PAK5 is predominantly detected in the brain and is involved in a number of vital cellular processes including cell survival and microtubules stabilization. It is also known that Pak5 promotes neuronal cell differentiation, but the molecular mechanism(s) by which Pak5 operates in this role is not entirely understood. To understand how PAK5 regu- lates neurite outgrowth, we have performed yeast two hybrid screening using PAK5 as the bait. After screening a yeast fetal brain library, we have identified a positive cDNA clone that matches to the sequence of a motor protein, called dynein cyto- plasmic 1 intermediate chain 1 (DYNC1Il). To confirm the inter- action between PAK5 and DYNC1Il, we have performed an affinity pull down assay. Purified glutathione-S-transferase (GST) tagged PAK5 was shown to interact with a purified his-tagged DYNC1Il, indicating that PAK5 can directly interact with DYN- C1Il. This DYNC1Il isoform was isolated from the human fetal brain library and has 17 amino acids missing in the N-terminal region comparing with the longest DYNC1Il isoform. To charac- terize the binding between PAK5 and DYNC1Il, an in vitro reconstitution kinase assay was performed. A constitutively active form of purified GST-tagged PAK5 (mutation of histidine resi- dues 19 and 22 to leucine) could phosphorylate the purified His- tagged Dync1i1 isoform. It has already been showed that cyto- plasmic dynein participates in neuronal growth cone remodeling and neurite outgrowth. Taken together, the results suggest a pos- sibility that PAK5 exert its functions, particularly neuronal cell differentiation, through phosphorylation of DYNC1Il.

P5–66 Invasive potential of A375 melanoma cells. The role of cofilin A. Popow-Wozniak, D. Nowak, D. Baczynska and M. Malicka-Blaszkiewicz Cell Pathology, University of Wroclaw Faculty of Biotechnology, Wroclaw, POLAND

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Non-T cell activation linker (NTAL) is a transmembrane adaptor which is found in several immune cell types including mast cells. Its function in the high-affinity IgE receptor (FceRI) signaling is not fully understood. Previous studies using bone marrow- derived mast cells (BMMC) from wild-type (WT) and NTAL- deficient mice have shown that NTAL plays a negative regulatory role in FceRI signaling. In contrast, other studies using human mast cells with reduced NTAL levels via silencing RNA oligonu- cleotides showed that NTAL functions as a positive regulator of FceRI signaling. The observed contrast could reflect the different techniques used for NTAL silencing [knock-out (KO) versus knock-down (KD) by RNA interference (RNAi)], or different roles of NTAL in mast cells from different species (human versus mouse). To determine the effect of the techniques currently employed for NTAL silencing, we compared the properties of mouse WT BMMC, BMMC from NTAL-deficient mice (NTAL- KO) or WT BMMC with reduced levels of NTAL after lenti- virus-mediated transfection with vectors possessing NTAL short hairpin (sh)RNA which resulted in reduced levels of RNA by (NTAL-KD). Immunoblotting analyses indicated that RNAi NTAL levels in NTAL-KD BMMC are impaired by more than 90%. Detailed analysis of the properties of these cells activated by antigen revealed that NTAL-KO and NTAL-KD exhibited similar properties. First, secretory and calcium responses in anti- gen-activated NTAL-KO and NTAL-KD BMMC were signifi- cantly enhanced when compared to WT cells. Second, activation- induced tyrosine phophorylations of the transmembrane adaptor LAT (linker for activation of T cells) and ERK were also enhanced in cells with reduced NTAL levels. Third, both acti- vated NTAL-KO and NTAL-KD exhibited reduced spreading on fibronectin, compared to WT cells. Finally, rapid depolymeriza- tion of filamentous actin in activated cells was more profound in NTAL-KO and NTAL-KD BMMC than in WT BMMC. The combined data indicate that NTAL in mouse BMMC functions as a negative regulator of FceRI signaling; an effect that is inde- pendent of the silencing procedure utilized (KO versus KD). Furthermore, our data suggest that the observed differences in the properties of mast cells with silenced NTAL could reflect a Migration ability of every tumor cell depends on the strict coop- eration between actin cytoskeleton and it’s binding proteins that regulate actin dynamics and therefore cell movement. Cofilin, a small actin depolymerizing factor is one of the key players in the process of cell migration. It’s a downstream effector of EGF – initiated signaling preceding the occurrence of directional cell movement. We have selected a highly motile population of human melanoma A375 cells and focused on their invasive potential, morphology, actin polymerization level, distribution and the level of actin cytoplasmic isoforms as well as several actin binding proteins including cofilin. To show the correlation between the level of cofilin phosphorylation, invasive potential of A375 cells and the state of actin polymerization we have stably transfected human melanoma A375 cells with eGFP-hu-S3A-cofi- lin, eGFP-hu-S3D-cofilin and eGFP-hu-wt-cofilin. To assign the

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invasive factor of cells we used Transwell invasion assay on Matrigel coated filters. Real time PCR and quantitative densito- metric analysis of Western blots were used to examine the expres- sion levels of indicated proteins. Subcellular distribution of F-actin and cofilin were examined by means of confocal fluores- cence microscopy. To measure the level of actin polymerization we conducted experiments based on the standard DNa-se I inhi- bition assays. In summary, our data demonstrated a clear corre- lation between metastatic ability of human A375 melanoma cells, the activity of cofilin and the state of actin polymerization.

domains of PKCs were studied (the classical gamma, and the novel delta and epsilon), together with different lipid mixtures containing three types of acidic phospholipids and three types of activating diacylglycerols. The results show that C1Bgamma and C1Bepsilon exhibit a higher affinity to bind to vesicles containing POPA, POPS or POPG, with C1Bepsilon being the most relevant case since its affinity for POPA-containing vesicles increased by almost two orders of magnitude. When the effect of the diacyl- glycerol fatty acid composition on membrane binding was stu- died, the C1Bepsilon domain showed the highest binding afinity to membranes containing SAG or DOG with POPA as the acidit phospholipid. Of the three diacylglycerols used in this study, DOG and SAG showed the highest affinities for each isoenzyme, while DPG showed the lowest affinity. DSC experiments showed this to be a consequence of the non-fluid conditions of DPGcon- taining systems.

P5–67 HGF induces cell migration, ruffling and Rac activation through a novel signalling pathway coupling diacylglycerol kinase alpha to PKCz/i and RhoGDI E. Rainero1, F. Chianale1, P. E. Porporato1, C. Cianflone1, V. Bettio1, N. Filigheddu1, G. Serini2 and A. Graziani1 1University of Piemonte Orientale ‘‘A.Avogadro’’, Clinical and Experimental Medicine, Novara, ITALY, 2Department of Oncological Sciences, Institute for Cancer Research and Treatment, University of Torino Candiolo, Torino, ITALY

P5–69 Association of ion channels and cytoskeleton proteins to lipid rafts from human placenta G. Riquelme, M. Berrios, N. De Gregorio, P. Diaz and C. Vallejos Faculty of Medicine, Physiology and Biophysic, University of Chile, Santiago, CHILE

Diacylglycerol kinases (Dgks) convert diacylglycerol (DG) into phosphatidic acid (PA), acting as molecular switches between DG- and PA-mediated signalling. We previously showed that activation of Dgka is required for growth factor-induced cell migration and proliferation and for Hepatocyte Growth Factor- induced membrane ruffling by regulating Rac membrane target- ing and activation in MDCK epithelial cells. However the mole- cular mechanisms by which Dgka regulates Rac function still remained to be elucidated. Here, we elucidate a still undescribed signalling pathway linking activation of HGF receptor to Rac small GTPase. We show that RhoGDI, which associates with GDP-bound Rac in a cytosolic complex, is required for Rac membrane targeting and that Dgka regulates both Rac-RhoGDI complex formation and RhoGDI localization. Recent evidence indicates that PKCz, an atypical PKC regulated by PA, associ- ates with and phosphorylates RhoGDI, thus allowing its disso- ciation from Rac. Thus we raised the hypothesis that Dgka may control RhoGDI by providing the lipid regulating PKCz/i and we show that PKCz/i activity is necessary for both HGF- and myr-Dgka-induced ruffle formation and both Rac and RhoGDI recruitment to the sites of ruffling. Moreover, we provide evi- dence that PA locally produced by Dgka at the plasma mem- brane is required for PKCz/i function. In conclusion, we have unveiled a novel signalling pathway triggered by HGF and lead- ing to cell migration: at sites of ruffle formation, Dgka-produced PA triggers PKCz/i-mediated regulation of RhoGDI function leading to Rac targeting and activation, with consequent forma- tion of membrane ruffling preliminary to cell migration.

P5–68 Membrane binding properties of C1B domains of PKCs J. C. Gomez-Fernandez, S. Sanchez-Bautista, A. Perez-Lara and S. Corbalan-Garcia Universidad de Murcia, Bioquimica y Biologia Molecular -A, Murcia, SPAIN

by FONDECYT 1070695 Ion channels are known to play important roles in epithelial function (e.g., volume regulation, maintenance of membrane potential, secretion and absorption processes). Human placental syncytiotrophoblast (hSTB) is an epithelium without paracellular route responsible for materno-fetal exchange. It is assumed that ion channels in placental membranes have functions similar to other epithelial channels, and that the activity of these channels and consequently their influence on placental function can be localization on specialized substantially modulated by their domains and microdomains. Previously, using differential sucrose density migration, we have described two isolated fractions of the apical membrane: a classical fraction (MVM) and a light fraction (LMVM) where MVM might correspond to the finger-like regio- nand L-MVM could correspond to the basal region of the micro- villous. Using electrophysiological methods, we have investigated the characteristics of anionic and cationic channels present in these membranes. We also reported the expression of two func- tional microdomains (lipid rafts) in both membranes and the importance of the cytoskeletal participation in their differential composition, using detergent resistant membranes (DRMs) and cholesterol sensitive depletion. Currently our aim is to determine the differential targeting of potassium channel subtypes (BK, TREK, Kir, TASK and Kv) and cytoskeleton protein (Ezrin, actin, Cytokeratin 7) to raft and non raft domains. Our results show that although these proteins are located in both fractions of the apical membranes, some of them have a differential locali- zation within the membrane microdomains. For example we show that BK and Ezrin target to specialized cholesterol rich lipid raft domains on LMVM but not on MVM. Membrane compartmentalization of these channels and cytoskeleton proteins may be critical to understand the molecular mechanism of trans- port processes that occur in the placental hSTB from normal and pathological pregnancies. Acknowledgement: Supported (Chile).

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

The C1 domains of classical and novel PKCs mediate their dia- cylglycerol-dependent translocation. Using FRET, we studied the contribution of different negatively charged phospholipids and to membrane binding. Three different C1B diacylglycerols

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P5–70 Absence of Egr-1 regulation in vitro under dynamic oxygenation including hypoxic conditions in human glioblastoma H. M. Said1, B. Polat1, S. K. Arif2, C. Hagemann3, G. H. Vince3, M. Flentje4 and D. Vordermark5 1Department of Radiation Oncology, University of Wu¨rzburg Med- ical Faculty, Wuerzburg, GERMANY, 2Biology Department, Sulaimani University College of Science, Sulaimani-Kurdistan- Region, IRAQ, 3Department of Neurosurgery, Tumor biology Laboratory, University of Wu¨rzburg Medical Faculty, Wu¨rzburg, GERMANY, 4Department of Radiation Oncology, University of Wu¨rzburg Medical Faculty, Wu¨rzburg, GERMANY, 5Martin- Luther-University-Halle-Wittenberg Medical Faculty, Department of Radiation Oncology, Halle, GERMANY

cell signalling pathways. Expression of HCV core in insect cells can direct the formation of capsid-like particles in the absence of the envelope glycoproteins and the 5¢untranslated region. In this study, we investigated whether non-enveloped HCV nucleocap- sids could be uptaken by cells of the immune system. We showed that the naked capsids could be uptaken not only by human liver cells-the main cell target of HCV-but also by human immune cells such as T and B lymphocytes, as well as human monocytes. Furthermore, we provided evidence that uptake in T cells leads to activation of the MAPKs-p38 pathway in a dose-dependent way, which in turn can affect IL-2 transcriptional activity. This uptake was strongly repressed by preincubation of cells with SB203580, a p38 inhibitor. Following incubation with HCV nonenveloped capsids, IL-2 mRNA was up-regulated, as demon- strated in Jurkat and Hut-78 T cells. Transcriptional activity of IL-2 promoter was confirmed by transient transfection of Jurkat cells with an IL-2 promoter-luciferase reporter plasmid and repressed when cells were pre-treated with p38 inhibitors.

P5–72 Methanol: a new stress regulator in plants A. Shvarts, O. Frolova and Y. Dorokhov Vavilov Institute of General Genetics, Genetic control of resistance to stress, Moscow, RUSSIA

A range of low-molecular organic evaporating compounds known to transfer signals in plants, is being widely studied now. Of these, the most actively emitted but the least studied is the methanol. This alcohol is generally produced in the process of pectin diestherithication by enzyme pectinmethilestherase (PME). Our data show that both, activity of this enzyme and emitted methanol quantity increase at transgene expression, viral infec- tion, and physical damage of the plant. Therefore, we suggested that methanol might take part in viral (and, probably bacterial or fungal) infection signal transfer in order to activate plant defense mechanisms. We found that viral infections development and transgene expression effectivity decreased after treating plant leaves with methanol in low non-toxic concentrations. We can also claim that methanol stimulates post-transcriptional gene silencing. Methanol in non-toxic dosage was also shown to increase expression of genes which are responsible for antivirial, antibacterial, and antifungal plant protection.

Background: It has been shown in several publications (Ellen T et al. 2007, Zhang P et al., 2007) that the transcription factor Egr-1 is regulated via hypoxia, and they hypothesized that Egr-1 is responsible for hypoxia induced NDRG1 gene regulation in human tumors cells. HIF-1a is the main regulator of several hypoxia induced genes. Materials and Methods: Egr-1 regulation level was examined in human glioblastoma cells lines like U373, U251, GaMG and U87-MG under extreme hypoxic oxygenation conditions (0.1 O2), reoxygenation after hypoxia for 24 and 48 hour and oxyge- nated conditions (21% O2 and 5% CO2) in vitro. Protein and mRNA level were detected via western blots and RT-PCR. Cells incubated for 24 hour with 100 lM DFO served positive control for hypoxia and b-tubulin and b-actin served as loading control, respectively. Results: Egr-1 was not up-regulated via hypoxic development in different glioblastoma cells in vitro under extreme hypoxic condi- tions (0.1% O2) or reoxygenation after hypoxia, both on protein and mRNA level. Further, there was no association between Egr- 1 expression and the expression of hypoxia induced, nHIF-1 a regulated genes in human glioblastoma tumor specimes exam- ined, in vitro. Conclusions: Egr-1 regulation as an answer to hypoxic develop- ment in glioblastoma, both on protein and mRNA level is not a general phenomenon. No hypoxic conditions influenced Egr-1 regulation were present. Therefore at least in glioblastoma, HIF- 1 a still can be considered as a major regulator of NDRG1 under hypoxic conditions. In our opinion, Egr-1 regulation under hypoxia is a cell specific post translational event.

P5–73 Estrogen receptor and HIF-1alpha signaling in hypoxia – tolerant breast cancer cells A. Scherbakov1, Y. Lobanova2, V. Shatskaya2 and M. Krasilnikov2 1N.N.Blokhin Cancer Research Center, Laboratory of Clinical Biochemistry, Moscow, RUSSIA, 2N.N.Blokhin Cancer Research Center, Laboratory of Molecular Endocrinology, Moscow, RUSSIA

P5–71 Modulation of p38 signaling pathway by HCV non-enveloped capsid like particles E. Serti1, K. Katsarou1, P. Foka1, G. Thyphronitis2, P. Tsitoura1, P. Mavromara1 and U. Georgopoulou1 1Molecular Virology, Hellenic Pasteur Institute, Athens, GREECE2Biological Applications and Technology, University of Ioannina, Ioannina, GREECE

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Background: The breast cancer cells exposure to acute hypoxia results in estrogen receptor (ER) down-regulation via proteasome degradation, which occurs concurrently with the activation of unoccupied ER, possibly through the direct interaction between ER and HIF-1alpha. HIF-1alpha is a key regulator of the cell response to hypoxia, however its role in the regulation of the hormone responsiveness of breast cancer cells is still unclear. Objectives: To elaborate the role of ER/HIF signaling in the regulation of breast cancer growth under chronic hypoxia. Hepatitis C virus is the major cause of chronic hepatitis liver cir- rhosis and hepatocellular carcinoma. HCV infects not only liver cells but also T and B cells and thus affects their normal func- tions. Although HCV is an enveloped virus, naked nucleocapsids have been reported in the serum of infected patients, and most recently novel HCV subgenomes with deletions of the envelope proteins have been identified. By using the baculovirus expression system we have generated recombinant non-enveloped HCV cap- sid-like particles, and investigated their putative interference with

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P5–75 CHIP ligase plays a central role in p53 regulation during senescence C. Sisoula, V. Trachana, S. Kapeta and E. S. Gonos National Hellenic Research Foundation, Institute of Biological Research & Biotechnology, Athens, GREECE

results demonstrate that

induced senescence. In specific, stress Methods: Using a long-term treatment of MCF-7 breast cancer cobalt chloride, we have developed a-cells with hypoxia mimetic compound hypoxia-tolerant cell subline, designed as MCF-7/H. Primary antibodies to HIF1 and ER were used for Western blot. To determine the activity of ER, the cells were transfected with the plasmid containing luciferase reporter gene controlled by the promoter with estrogen responsive elements (ERE-TK-LUC). Results: The established cell subline MCF-7/H was character- ized with the increased rate of cell growth and survival under hypoxia, the constitutive activation of HIF-1, and restored level of estrogen receptor signaling and hormonal dependency. Study with the plasmid ERE-TK-LUC revealed an increase in the ER activity in MCF-7/H cells. Similarly, under hypoxic conditions the retained ER activity and expression were higher in MCF-7/H cell line if compared with wild-type MCF-7 cells, demonstrating the possible cross-talk between both estrogen and hypoxic signal- ing pathways. Conclusions: Thus our chronic hypoxia/HIF-1alpha signaling may contribute to the maintaining of estrogen receptor machinery and hormonal dependency of breast cancer cells. Acknowledgement: This work was supported in part by Rus- sian Foundation for Basic Research, grant 07-04-00809-a.

P5–74 Correlation between polar auxin transport specificity and auxin signalling S. Simon, M. Kubes, J. Petrasek and E. Zazimalova Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Rozvojova´ 263 16502 Prague 6, CZECH REPUBLIC

p53 is the best characterized tumour suppressor protein and a key player in stress response, apoptosis, cell cycle regulation and senescence. Various posttranslational modifications, includ- ing ubiquitination, are necessary for stability and activity regu- lation of p53. Mdm2-mediated ubiquitination of p53 is central for establishing p53 low levels in normal cells. However, other p53-ligases have been identified, like COP1, Pirh2, Topors and CHIP, but this level of diversity is unclear. It is possible that different ligases are expressed and act optimally in different cell or tissue types or at different developmental stages. In order to elucidate this possibility, and given the role of p53 in cellular senescence, we tested whether specific p53-ligase(s) might be of central importance for this condition. In primary human fibro- blasts we observed altered expression of p53 ligases during repli- cative or there is a decrease in the levels of Mdm2 as well as COP1. In contrast, the levels of Pirh2 and Topors are unaltered. Interestingly, we found that the levels of CHIP ligase are significantly up-regu- lated upon senescence. Localization studies of Mdm2, COP1 and Pirh2 ligases revealed no alteration in their pattern. How- ever, CHIP ligase in young fibroblasts is localized in the cyto- plasm and clearly translocates to the nucleus in senescent cells. Since terminal senescence is also accompanied by a significant down-regulation of p53, as we demonstrated for different cell types, we hypothesize that an inverse co-regulation of CHIP ligase and p53 might be implicated in the manifestation of the senescent phenotype.

P5–76 Life/death decisions in growth factor signaling: role of mitochondrial events J. Smigelskaite, K. Koziel and J. Troppmair Innsbruck Medical University, D. Swarovski Research Laboratory, Innsbruck, AUSTRIA

trigger the auxin-responsive

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Phytohormone auxin plays a major role in various growth and developmental processes in plants. Among all plant hormones, auxin is unique in its polar distribution through plant tissues via active polar transport. This polar distribution provides the neces- sary auxin maxima to trigger various physiological events in plants. Our aim was to determine specificity of auxin transport machinery in relation to auxin signalling; we have used a group of structural analogues of major auxins and analyzed their accu- mulation in cells and their roles in TIR1-mediated auxin signal- ling. Detailed transport kinetics of various auxin analogues has been studied in suspension-grown tobacco BY-2 (Nicotiana taba- cum L., cv. Bright-Yellow 2) and Ath Ler (Arabidopsis thaliana ecotype Landsberg erecta) cells on both influx and efflux levels. Both auxin influx and efflux specificities were determined by a simple displacement of radio-labelled 2,4-D (2,4-dichlorophenoxy acetic acid) and 1-NAA (1-naphthalene acetic acid), respectively, by non-labelled auxin analogues. Further, we examined their involvement in TIR1-mediated auxin signalling pathway by asses- reporter sing their ability to induce DR5rev::GFP in the roots of Arabidopsis seedlings. Our results demonstrated a remarkable difference in auxin transport specifi- city between BY-2 and Ath Ler cells. We have also observed that those compounds, which are transported actively via polar auxin transport machinery, are capable of stimulating the TIR1- mediated auxin signalling pathway. Acknowledgement: E-mail: simon@ueb.cas.cz, zazimalova@ ueb.cas.cz. This work was supported by the Ministry of Educa- tion, Youth and Sports of the Czech Republic, project No.: LC06034. Lack of growth factors is a common physiological stimulus for the induction of apoptosis. Cell death under these conditions can be delayed through survival kinases or antiapoptotic Bcl-2 family proteins. It is currently unclear, which events translate the lack of survival signals into a death stimulus and how sur- vival proteins cope with these changes. We were able to show that following IL-3 withdrawal, reactive oxygen species (ROS)- induced mitochondrial Ca2+ overload functions as an essential apoptosis in IL-3 dependent promyeloid 32D cells, whose action can be annihilated by activated RAF or AKT or the overexpression of Bcl-2. Our data also suggest that in the case of activated RAF this effect is not mediated via altera- tions in the expression of Bcl-2 family proteins or of compo- nents of the antioxidant defense systems. The goal of ongoing experiments is to further dissect mechanisms leading to apopto- tic cell death and to define additional sites, where survival sig- naling interacts with the induction or execution of cell death. To this end our data suggest that RAF fails to prevent BAX translocation in 32D cells but not NIH 3T3 fibroblasts. The underlying mechanisms and functional consequences are cur- rently dissected.

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P5–77 LPS and IFN-gamma modulate DDAH2/iNOS pathway in endothelial cells A. I. Soares Moretti, M. C. Jurado, L. A. Passos and H. Possolo de Souza School of Medicine, Emergency Medicine, Sao Paulo, BRAZIL

started at 6 hours that

transition. TGF-b1 signals through SMADs complex or SMAD- independent pathways such as p38 and ERK. The aim of the study was to describe the crosstalk between IL-6 and TGF-b1 pathways and find possible mechanism of such interaction in prostate epithelial cells. We have established experimental model for this study in the cell line derived from the benign prostatic hyperplasia (BPH-1). Our results demonstrated that IL-6 and TGF-b1 treatment causes chronic activation (phosphorylation) of signaling molecules STAT3 and SMAD2 in BPH-1 cell line. TGF-b1 has antiproliferative effects as we determined by measur- ing the cell numbers and cell cycle, whereas it seems that IL-6 has opposite effects, respectively IL-6 does not decrease the cell numbers and percentage of cells in S-phase. IL-6 treatment did not affect TGF-b1 induced epithelial-mesenchymal transition of BPH-1 cells. However, long-term treatment of cells with TGF-b1 resulted in abolition of IL-6 induced STAT3 phosphorylation. Taken together, our data demonstrate that there is a crosstalk between IL-6 and TGF-b1 pathways and TGF-b1 modulates pro- inflammatory microenvironment in prostate tissue by downregu- lation of IL-6 signaling. Acknowledgements: This work was supported by MU Rector´ s Programme for Students’ Creative Activity Support MUNI/31/ A0004/2008 and grants 204/07/0834, 310/07/0961 of the Czech Sci- ence Foundation and AV0Z50040507 and AV0Z50040702 of the Grant Agency of Academy of Sciences of the Czech Republic.

effective. Untreated HUVEC cells

FAPESP, In endothelial cells, dimethylarginine dimethylaminohydrolase (DDAH) controls the levels of assymetrical dimethylarginine, which by its turn acts as an endogenous competitive inhibitor of nitric oxide generation. It is known that inflammatory stimuli may enhance inducible nitric oxide synthase (NOS2) expression and cytokine secretion, however the role of endogenous NOS2 in- hibiors under these conditions is not yet clarified. Therefore, our objective was to determine whether LPF and IFNg could modu- late NO production through the control of DDAH activity in endothelial cells. Human umbilical vein endothelial cells (HU- VECs), quiescent and under confluence, were stimulated for dif- ferent times (2–48 hour) with LPS (5 mg/ml) and IFNg (25 mg/ ml). Cytokine release (TNF-a, IL-6 and IL-10) was measured by ELISA. DDAH and NOS2 expression were assessed by western blotting in cell lysates. IL-6 levels exhibited time-dependent aug- (494 ± 75 vs. 1594 ± 144, ment p < 0.01). TNF-a levels increased only after 48 hours (17 ± 0.4 vs. 26 ± 1.7, p < 0.05), while IL-10 concentrations did not change. Together these data confirm that the pro-inflammatory stimulus was expressed DDAH-2 constitutively, while no NOS2 protein was detected. After treatment, DDAH-2 protein expression increased after 2 hours (3-fold) and iNOS protein levels raised at 6 hours (3- fold). Together, these findings suggest that, under inflammatory conditions, increased DDAH expression may work as an addi- tional mechanism controling NOS2 function, improving NO pro- duction through elimination of endogenous inhibitors. Acknowledgement: Financial support: CNPq, Direx-LIM.

P5–79 Complexes of membrane-associated gamma- tubulin with Fyn kinase and phosphoinositide 3-kinase in differentiating embryonal carcinoma cells V. Sulimenko1, L. Macurek1, E. Draberova1, V. Richterova1, T. Sulimenko1, L. Draberova2, V. Markova1 and P. Draber1 1Laboratory of Biology of Cytoskeleton, Institute of Molecular Genetics, Prague 4, CZECH REPUBLIC, 2Laboratory of Signal Transduction, Institute of Molecular Genetics, Prague 4, CZECH REPUBLIC

P5–78 Interaction of interleukin-6 and transforming growth factor-beta1 pathways in prostate epithelial cells A. Starsichova, E. Lincova, Z. Pernicova, A. Kozubik and K. Soucek Department of Cytokinetics, Institute of Biophysics AS CR v.v.i., Brno, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

to tumor Molecular mechanisms controlling microtubule formation in cells with non-centrosomal microtubular arrays are not yet fully understood. The key component of microtubule nucleation is gamma-tubulin. Although previous data suggested that kinases might serve as regulators of gamma-tubulin function, their exact roles remain enigmatic. Here we show that in P19 embryonal car- cinoma cells undergoing neuronal differentiation a large pool of gamma-tubulin is associated with membranes in the form of pro- tein complexes of various sizes. A subset of gamma-tubulin is also bound to detergent-resistant membrane domains, called membrane rafts. Microtubule assembly assays demonstrated that membrane-bound gamma-tubulin complexes nucleate microtu- bules. Pretreatment of cells with Src family selective tyrosine kinase inhibitors abolished the nucleation activity of gamma- tubulin. Immunoprecipitation experiments revealed that mem- brane-bound gamma-tubulin forms complexes with Fyn kinase and active phosphoinositide 3-kinase (PI3K), implying that these kinases are involved in the regulation of microtubule nucleation. Direct interaction of gamma-tubulin with C-terminal Src homol- ogy 2 domain of p85alfa regulatory subunit of PI3K was deter- mined by pull-down experiments. These findings suggest that Fyn kinase and PI3K take part in the regulation of nucleation proper- ties of membrane-bound gamma-tubulin. Prostate cancer is the most common non-cutaneous malignancy and the leading cause of death among men in Western countries. Chronic inflammation is probably important factor in initiation and progression of prostatic pathologies including cancer. Inter- leukin-6 (IL-6) is a pleiotropic cytokine that triggers inflamma- tory response and regulates complex of cellular processes such as proliferation, differentiation and apoptosis. IL-6 activates multi- ple prosurvival signaling pathways including JAK/STAT, Ras/ ERK, PI3K/AKT. Therefore, modulation of IL-6 signal trans- duction is potentially appropriate strategy for the prevention and therapy of prostate cancer. Transforming growth factor-beta1 (TGF-b1) is a multifunctional cytokine that can be a negative regulator of inflammation and tumor suppressor. On the other hand, TGF-b1 can also contribute invasiveness in the process of epithelial-mesenchymal and metastasis, e.g.

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Poster Presentations

P5–80 Pleiotrophin increases intracellular ROS levels and cell migration through activation of anb3 integrin S. Tsirmoula and E. Papadimitriou Pharmacy, University of Patras, Patras, GREECE

Also RSK, another ribosomal protein S6 kinase that is down- stream of MAPKs, was inactivated by the suppression of Src kinase activity. Treatment of v-Src-transformed cells either with SU6656 or rapamycin reduced oncogenicity of these cells suggest- ing that mTOR may be a mediator of oncogenic signals issued by the activated Src. Although Akt/PKB is assumed to stimulate mTOR signalling by phosphorylation and thus inactivation of tumor suppressor tuberin, the expression of constitutively active ectopic Akt1 enhanced phosphorylation of tuberin, but did not overcome the inhibitory effects of the Src inactivation or rapamy- cin treatment on the mTOR-dependent signalling downstream of tuberin. The Src-dependent activation of signalling proteins that control translation may thus promote the selective expression of proteins that are involved in malignant process induced by v-Src- transformation. This supports Src as a selective target for cancer therapy with possibly direct clinical applications. Acknowledgement: This study was supported by Research Projects AVOZ50520514 and MZO00064211.

suggesting that amb3

P5–82 Positive regulatory role of NTAL adaptor protein on mast cell spreading and adhesion to fibronectin M. Tumova, A. Koffer, M. Simicek and P. Draber Laboratory of Signal Transduction, Institute of Molecular Genetics, Prague 4, CZECH REPUBLIC

Pleiotrophin (PTN) is an 18 kDa secreted growth factor that dis- plays high affinity for heparin. We have recently shown that PTN induces migration of endothelial cells through binding to both integrin amb3 and receptor protein tyrosine phosphatase b/e (RPTPb/e). In the present work, we studied the effect of exoge- nous administration of PTN on generation of reactive oxygen species (ROS) in human umbilical vein endothelial cells. Human recombinant PTN significantly increased endogenous ROS levels in a concentration and time dependent manner. RPTPb/e appears to be involved in this process, since suppression of the receptor using genetic and pharmacological inhibitors or inhibition of src kinase activity abolished PTN-induced ROS generation. A syn- thetic peptide known to block PTN-amb3 interaction abolished is also PTN-induced ROS generation, involved. The latter was confirmed in CHO cells that do not express amb3 (mock transfected) or CHO cells over-expressing wild-type b3 or mutant b3Y773F/Y785F. Interestingly, PTN increased endogenous ROS levels only in cells expressing wild- type b3, while phosphorylation of tyrosine Y773 seems to be involved in this effect, similarly to what we have previously shown for PTN-induced migration. In the same line, in human glioma M059K cells that do not express amb3 and do not migrate in response to PTN, ROS levels are not affected. The inhibitor of Erk1/2 activation U026 abolished PTN-induced ROS generation, suggesting that ROS production is down-stream of Erk1/2 activa- tion by PTN. Finally, the ROS scavengers catalase, apocynin and allopurinol blocked PTN-induced ROS generation, as well as PTN-induced cell migration, suggesting that ROS production is required for PTN-induced cell migration through the cell mem- brane functional complex of amb3 and RPTPb/e. Further studies are in progress in order to elucidate the intracellular system involved in PTN-induced ROS generation.

P5–81 Differential effects of Src on signalling proteins in v-src-transformed cells; functional interplay of Src, Akt, mTOR-dependent and MAPK-dependent pathways Z. Tuhackova1, M. Vojtechova2, J. Tureckova2, E. Sloncova2 and J. Vachtenheim3 1Laboratory of Molecular Biology, Institute of Molecular Genetics and University Hospital, Prague, CZECH REPUBLIC, 2Laboratory of Cell and Developmental Biology, Institute of Molecular Genetics, Prague, CZECH REPUBLIC, 3Laboratory of Molecular Biology, University Hospital, Prague, CZECH REPUBLIC

cell morphology. Since mast cells and/or

We found previously that non T-cell activation linker (NTAL) serves as a negative regulator of the high-affinity IgE receptor signaling in mast cells. In this study, we analyzed its role in cell adhesion and spreading. NTAL-/- bone marrow-derived mast cells (BMMC) showed less extensive spreading to fibronectin than wild-type (WT) cells in response to antigen (Ag), while spreading induced via c-kit was NTAL independent. Adhesion to fibronec- tin increased after Ag or stem cell factor (SCF) triggering in both NTAL+/+ and NTAL-/- cells, but the Ag-induced increase was smaller in cells lacking NTAL. Moreover, absence of NTAL caused a significant defect in actin polymerization in response to Ag, which could not be ascribed to the (previously reported) higher calcium levels in Ag-activated NTAL-/- cells. In contrast, actin polymerization, as well as calcium signals, were unaffected by the absence of NTAL after stimulation with SCF. However, when both SCF and Ag were present, the defect in filamentous (F)-actin polymerization in NTAL-/- cells became even more pro- nounced. In order to further elucidate the role of NTAL, we transfected NTAL-enhanced green fluorescent protein (EGFP) fusion construct into BMMC and analyzed the properties of the cells. We found that NTAL-EGFP was localized almost exclu- sively in the plasma membrane. Introduction of NTAL into NTAL-/- BMMC completely restored the defect in F-actin poly- merization in response to Ag, and partially restored the defect in spreading on fibronectin. The combined data indicate an impor- tant role of NTAL in signaling to actin cytoskeleton and in regu- lation of their precursors need to migrate to target tissues where they undergo further maturation, operational NTAL may be crucial to mast cells function. Acknowledgement: Supported by 1M0506, 301/09/1826 and KAN200520701.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Activity of Src protein tyrosine kinase is increased in many malignancies, suggesting Src as a promising target for drug ther- apy. Suppression of Src activity either by treatment of v-Src- transformed fibroblasts with Src specific inhibitor SU6656 or by the expression of dominant negative Src kinase-dead mutant in nontransformed fibroblasts displayed the regulatory effect of Src on the endogenous Akt/PKB which was directly phosphorylated by Src. Moreover, disruption of the Src protein kinase activity resulted in a substantial reduction of the phosphorylation and activity of mTOR and its downstream targets S6K1 and 4E-BP1, similar to that induced by mTOR specific inhibitor rapamycin.

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Abstracts

the salivary secretions, whereas CAT is absent. Two izoenzymes are characteristic of the aphid PPO and POD. Yet, these appear to be constitutive enzymes. In contrast to PPO and POD, CAT activity is inducible by dietary DP, PP and H2O2. First chemical products of exogenous POD activity towards plant phenolics are toxic quinones, that spontaneously condense with unoxidized phenols and next are covalently bound to proteins, and further they may be detoxicated/inactivated in consequence of ‘immobili- zation’ within the ‘solid salivary sheath’, typical for Aphididae. Whereas the midgut POD and CAT of aphids appear as a ‘scav- engers’ of toxic H2O2, released in the course of PPO reaction, and finally may reduce oxidative stress, in vivo.

P5–83 Expression of survivin in serrated polyps of the large intestine F. Shimamoto1, H. Tuncel2, G. Qi3, E. Aoki4, T. Takata3 and M. Tatsuka5 1Department of Pathology, Prefecture University of Hiroshima, Hiroshima, JAPAN, 2Department of Biophysics, Istanbul University Cerrahpasa Medical Faculty, Istanbul, TURKEY, 3Department of Oral and Maxillofacial Pathology, Hiroshima University, Hiroshima, JAPAN, 4Department of Cellular and Molecular Biology, Hiroshima University, Hiroshima, JAPAN, 5Department of life science, Prefectural University of Hiroshima, Shoubara, JAPAN

serrated polyps

P5–85 Distinct isoforms of Arabidopsis phospholipase D are involved in defence response against Pseudomonas syringae O. Valentova´ 1, H. Na´ varova´ 1, M. Kalousova´ 1, Z. Novotna´ 1 and L. Burketova´ 2 1Department of Biochemistry and Microbiology, Institute of Chemical Technology Prague, Prague 6, CZECH REPUBLIC, 2Department of Plant Pathophysiology, Institute of Experimental Botany AS CR, Prague 6, CZECH REPUBLIC

Inhibition of apoptosis has been proposed as important pathoge- netic mechanism of colorectal (hyperplastic polyp, serrated adenoma and serrated adenocarcinoma). Overex- pression of survivin, a member of the inhibitor of apoptotic pro- tein (IAP) family, is commonly detected in many cancers but not in adult normal tissue. The IAP function of survivin is believed to be critical for cancer progression. Survivin is also known to be a component of chromosome passenger protein (CPP) complex essential for chromosome segregation and cytokinesis as well as Aurora-B. Our previous study showed that cytoplasmic expres- sion of survivin may be implicated in lymphnode metastasis of colorectal cancer patients. Here we show that by immunohisto- chemical analyses serrated adenocarcinomas reveal high-grade cytoplasmic and nuclear expression of survivin more frequently than serrated adenomas, and Aurora-B expression is more fre- quent in the nucleus of serrated adenocarcinomas than in that of serrated adenomas. These findings indicate that the expression of chromosome passenger proteins may be deeply involved in the pathways of histogensis in serrated polyps, suggesting that cyto- plasmic survivin may have a certain function to survive cancer cells.

P5–84 Oxidoreductases of aphids involved in phenolic metabolizm and oxygen stress A. Urbanska Department of Biochemistry and Molecular Biology, University of Podlasie, Siedlce, POLAND

In plants, the attack of pathogen leads to the accumulation of salicylic acid in the infected tissue and could lead to the develop- ment of systemic acquired resistance (SAR) against pathogen. Salicylic acid triggers a complicated cascade of signalling events resulting in the expression of PR (Pathogenesis-Related) genes enabling the plant to deal with pathogen. The molecular mecha- nism of the early events in signalling pathway leading to SAR development is largely unknown but increasing evidence points to phospholipid signalling as important component of this path- way. Recently, we brought the evidence that phospholipase D and its product phosphatidic acid is involved in a rapid response of Arabidopsis suspension cells to the salicylic acid treatment. In comparison to other eukaryots, plant phospholipase D consti- tutes multiple gene family (12 genes in Arabidopsis thaliana) divided to five groups differing significantly in their biochemical characteristics. This fact brings the question whether and what are their specific roles. In the presented study we show on Ara- bidopsis T-DNA mutant lines the involvement of distinct iso- forms of phospholipase D in the defence response of Arabidopsis plants against virulent pathogen Pseudomonas syringae pv maculi- cola. Acknowledgement: Presented study was funded by Ministry of the Czech Republic, grants no. LC06034 and Education of MSM6046137305.

P5–86 Mac-1-induced clustering of ICAM-1 is required for the transient formation of docking structures J. Van Buul1, E. Bakker2, F. Van Alphen1, K. Houben3, J. Van Marle3 and P. Hordijk1 1Molecular Cell Biology, Univeristy of Amsterdam at Sanquin Research, Amsterdam, THE NETHERLANDS, 2Biophysics, Univeristy of Amsterdam at Sanquin Research, Amsterdam, THE NETHERLANDS, 3Electron Microscopy, Univeristy of Amsterdam at Sanquin Research, Amsterdam, THE NETHERLANDS

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Inflammation has emerged as a crucial force driving the initia- tion and progression of atherosclerotic lesions. Therefore, it is critical to understand the details of leukocyte migration across in inflammation. Upon the endothelial barrier, a key event Phenolic compounds are recognized as fundamental plant metab- olites as regards ‘anti-pathogen/pest efficiency’. There is broadly demonstrated that both diphenols (DP) and polyphenols (PP) participate in constitutive and/or inducible defence/resistance of Poaceae against Aphididae. Yet, biochemical/enzymatic mecha- nisms of detoxication and toxication of DP and PP in aphids, and in particular toxic effects of reactive intermediates and by- products accompanying of oxidative reactions of phenolic allelo- chemicals remain debatable. This paper is concerned with poly- phenol oxidase, PPO (EC 1.10.3.1), peroxidase, POD (EC 1.11.1.7) and catalase, CAT (EC 1.11.1.6) of Rhopalosiphum padi L. and Sitobion avenae F., essentially occurrence and implication for conversion of plant phenols and red-ox state in vivo and in vi- tro. Hydrogen peroxide (H2O2) generation and PPO activity are focused in the salivary secretions and midgut of R. padi and S. avenae. PPO catalyses reaction of oxidation of caffeic acid, (+)- catechin and derivative DP and PP to quinines, in which molecu- lar oxygen (O2) takes part. In the main, this reaction appears as essential step towards H2O2 generation, due to aryloxy radicals -). Haemoprotein enzymes, namely and superoxide anion (O2 POD that catalyses oxidation of DP and PP into quinones and eliminates en route H2O2 and CAT that decomposes H2O2 into O2, are located in the midgut. In vitro, POD activity is present in

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Poster Presentations

P5–88 Identification of the cellular targets and mechanisms of action of the glycerophosphoinositols A. Varone1, S. Mariggio1, B. M. Filippi1, G. Nicolosi2 and D. Corda1 1Consorzio Mario Negri Sud, Cell biology and oncology, Santa Maria Imbaro, ITALY, 2Biomolecular Chemistry, CNR Institute, Catania, ITALY

imaging in time,

binding of the leukocyte integrin LFA-1 and Mac-1 to endo- thelial ICAM-1, ICAM-1 is clustered and induces membrane ruffles that protrude out of the apical plane of the endothe- lium, surrounding the adhering leukocyte. These protrusions are called docking structures (DS). To mimic leukocyte binding to ICAM-1 and to study the dynamics of these DS in detail, we used ICAM-1 antibody-coated beads. 3-D fluorescent confo- imaging revealed that ICAM-1-rich membranes protruded cal around the adhered bead. Scanning electron microscopy showed that first microvilli surround the adhered beads, followed by extensive sheets of membrane. After approximately 30 minutes of adhesion, 60% of the beads were surrounded by membrane ruffles. Using isolated murine arteries we could observe these membrane-ruffles as well. Little is known about the dynamics of DS. Using 3-D fluorescent confocal it appeared that the DS existed for only 5 minutes. Interestingly, the time to induce a DS differed per leukocyte type. Detailed analysis of the expression of the integrins present on the leuko- cytes showed that there was a strong correlation between Mac- 1 and the formation of ICAM-1-positive DS on the endothe- lium. Thus, based on these experiments, we propose a role for Mac-1 to cluster ICAM-1 to induce apical membrane ruffles that form DS to assist leukocytes on their route through the endothelium.

P5–87 Successful monitoring of insulin stimulated signaling events in adherent cell cultures using flow cytometry M. van de Venter and G. Wilson Biochemistry and Microbiology, Nelson Mandela Metropolitan University, Port Elizabeth, SOUTH AFRICA

The glycerophosphoinositols (GPIs) are water-soluble metabolites produced by a phospholipase A2 (PLA2) activity through two sequential deacylation reactions from the membrane phosphoi- nositides. Among these GPIs, glycerophosphoinositol 4-phos- phate promotes actin cytoskeleton reorganisation through the activation of the monomeric GTPases Rac1 and RhoA. Dynamic changes in the actin cytoskeleton are central to a wide variety of cellular events, including cell motility, adhesion and phagocytosis, and tumour-cell invasion. With the aim of defining the mecha- nisms of action of the GPIs, we have used newly synthesized glycerophosphoinositol (GroPIns) derivatives with a biotin sub- stituent bound on different positions of the glycerol backbone these biotinylated- (biotinylated-GroPIns). The localisation of GroPIns in different cell lines was investigated. Using confocal immunofluorescence, we have seen that biotinylated-GroPIns staining is diffuse within the cytosol, with specific localisation in the nucleus also seen. We are also using a proteomic approach to identify and characterise the cellular targets involved in the func- tions of the GPIs. The use of affinity chromatography with mouse-brain cytosol and a Sepharose-based matrix with cova- lently bound GroPIns has led to the identification of several in- teractors. A different approach based on the biotinylated-GPIs and streptavidin-conjugated affinity resins has also been devel- oped. Altogether, we have identified 74 putative interactors, which we are selecting and validating. These can be divided into clusters of proteins involved in cell signalling, cytoskeleton orga- nisation, protein folding and enzymes of metabolic processes. We are presently focussing on the components of signalling cascades that might be directly activated by the GPIs.

P5–89 Platinum(IV) complex LA-12 induces cell cycle phase specific apoptosis in colon carcinoma cell line HCT-116 O. Vondalova Blanarova1, I. Jelinkova1, R. Jendzelovsky2, K. Soucek1, J. Hofmanova1, P. Sova3 and A. Kozubik1 1Department of Cytokinetics, Institute of Biophysics AS CR v.v.i., Brno, CZECH REPUBLIC, 2Faculty of Science, Institute of Biology and Ecology, P. J. Safarik University, Kosice, SLOVAK REPUBLIC, 3Pliva-Lachema a.s., R&D, Brno, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Platinum drugs are employed in a treatment of various tumor types. They are able to create DNA adducts generating DNA damage signals resulting in cellular stress response, which forces the cell to slow down or arrest its cell cycle, repair DNA or undergo cell death. LA-12 is a novel platinum(IV) complex, which expresses cytotoxic effects in many cancer cell lines. It has been shown to overcome intrinsic and acquired resistance to cis- platin and oxaliplatin in ovarian and colon cancer cell lines. In colon carcinoma cell line HCT-116, LA-12 induced accumulation of the cells in G2 phase of the cell cycle, whereas the rate of mitotic cells declined only slightly. In contrast, cells expressing cyclin B1 and active form of CDK1 dephosphorylated on tyrosin 15 were still present, even after 48 hours of incubation with LA- Introduction: Insulin resistance is commonly associated with decreased levels of signaling pathway intermediates and/or decreased activation through phosphorylation. Flow cytometry provides a quick, sensitive method to monitor these processes in cells through immune-labeling. However, adherent cell cultures pose the problem of possible damage to plasma membrane recep- tors (including insulin receptors) during harvesting to obtain a cell suspension for flow cytometry. We used C2C12 mouse myo- cytes to optimize a method yielding insulin responsive cells in suspension that can be successfully used for flow cytometry after immune-labeling of insulin signaling intermediates. Methods: Various protocols were compared to optimize cell harvesting and incubation conditions. After insulin exposure, cells were permeabilised and monoclonal primary – and second- ary FITC labeled-antibodies used to probe intermediates in the PI3-Kinase/Akt, AMP Kinase and the Mitogen Activated Protein Kinase (MAPK) pathways. Flow cytometry was done on a Beck- man-Coulter FC500 flow cytometer. Results: C2C12 myocytes had to be used before fusion into myotubes occurred. The best insulin response was observed when C2C12 myocytes were detached with a gentle detachment med- ium before exposure to insulin whilst in suspension. The three pathways were all shown to be activated as was evident from increases in levels of phospho-Akt (PI3-Kinase/Akt pathway) and phospho-MEK1/2 (MAPK) and a decrease in phospho- AMP Kinase. Conclusions: This method provides a faster, more sensitive and quantitative method than Western blotting which is currently the most commonly used method for monitoring signal transduction pathways in adherent cells. It can be useful in assessing insulin sensitivity and identification of novel compounds that stimulate insulin signaling pathways.

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Abstracts

sign of intracellular signal initiation to the signal initiation in the given cell point) and local signal growth rate. In the cell center we observe longer delay and slower growth rate in comparison to the signal initiation region. The mechanism of the local signal induction is not clear, since both players of cell calcium response: P2Y2 receptors and intracellular calcium stores (endoplasmic reticulum) are evenly distributed. Receptors are present on the whole cell membrane while ER is spread regularly in whole lamellipodium.

12, when apoptosis was massively induced. Bivariate analysis of caspases-cleaved cytokeratin 18 and DNA content revealed an increase in portion of early apoptotic cells with G1 DNA con- tent. On the basis of our results, we hypothesize that in HCT-116 cells, LA-12 activates DNA damage signaling by disturbance of DNA replication. This early event is followed by accumulation of the cells in G2 phase. However, certain part of the cells is somehow able to overcome this arrest and proceed to mitosis and G1 phase, where apoptotic cascade is activated and cells eventually die. Acknowledgement: This work was supported by grants No. 1QS500040507 IGA ASCR and 301/07/1557 GACR.

P5–90 Signal transduction in HepG2 cells infected with a bacterium Klebsiella pneumoniae J. Wu and W. X. Chen Microbiology and Immunology, Chang Gung University, Tao Yuan, TAIWAN

P5–92 Proteomic characterization of cytotoxic mechanism of Tubeimoside-1, a potential anticancer drug Y. XU1, J. F. Chiu2, Y. Zhou2, Q. Y. He3 and F. Chen1 1School of Biological Sciences, The University of Hong Kong, Hong Kong, CHINA, 2Anatomy, The University of Hong Kong, Hong Kong, CHINA, 3Institute of Life and Health Engineering, Jinan University China, Guangzhou, CHINA

Emerging liver abscess caused by a commensal enterobacterium, Klebsiella pneumoniae, illustrates the increased virulence in bacte- ria of human normal flora. The infection is especially severe in diabetic patients. Our aim is to examine the signaling pathways in infected HepG2 cells. Western blotting was used to investigate the live bacteria-infected cells for signaling molecules. The DNA degradation, Annexin V/Propidium Iodide staining and cell cycle changes were also investigated. Our results showed that both Toll-like receptor (TLR)2 and TLR4 were upregulated. MEK1/2- p44/42-p90RSK and MKK3/6-p38 kinase-ATF2 pathways were activated early after infection. Bax/Bcl-2 ratio was increased and Bcl-xL was decreased at late phase of infection. A very small amount of caspase3 and poly(ADP-ribose) polymerase (PARP) cleavage were observed early after infection. Cells were arrested at G1 phase and displayed necrosis at later phase of infection. DNA laddering was apparent during all hours of infection stages. This illustrates that early after infection, balance between the two MAPK pathways causes cell apoptosis, however, at late phase of infection, cells turn to necrosis and arrested cell cycle to yield cell death.

There has been increasing interest in the development of medici- nal plants as anticancer drugs. Tubeimoside-1 (TBMS1), a trit- erpenoid saponin isolated from the tuber of Bolbostemma paniculatum (Maxim.) Franquet (Cucurbitaceae), exhibits cyto- toxicity towards human cancer cells. In this study, the micro- somal fraction proteomic analysis revealed that the expression of mitochondrial and endoplasmic reticulum (ER) associated pro- teins was substantially altered upon TBMS1 treatment. Further biochemical studies showed the depletion of mitochondrial trans- membrane potential and alterations in protein level of ER hall- marks, which indicated that mitochondria and ER play a role in TBMS1-induced cell death. Flow cytometric study indicated that TBMS1 inhibited cell growth through abrogating cell cycle at G2/M phase and induced apoptosis in Hela cells in a time- and dose-dependent manner. Molecular studies showed that G2/M arrest was associated with a decrease in cyclin B1, cyclin-depen- dent kinase (CDK)1 and cell division cycle 25C (Cdc25C), together with an increase in phospho-Chk2 and CDK inhibitor (CDKI) p21. Further, confocal microscopy showed TBMS1 caused cytoplasmic sequestration of cyclin B1 and cdc2 contrib- uting to G2/M arrest. Current study in TBMS1-treated Hela cells suggested that TBMS1 exerts cytotoxicity and growth inhibitory activity through mitochondrial and ER dysfunction and Chk2- Cdc25C-Cdc2/cyclin B cell cycle checkpoint pathways and it would be a potential anticancer drug.

P5–91 Does flatter means faster? Studies on local dynamics of calcium transients in glioma C6 cells D. Wypych and P. Pomorski Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, POLAND

P5–93 SIRT1 promotes cell proliferation and prevents cellular senescence through targeting LKB1 in primary porcine aortic endothelial cells Y. Zu1, L. Liu2, A. Xu2, K. S. L. Lam2, M. Y. K. Lee1, P. M. Vanhoutte1 and Y. Wang1 1Department of Pharmacology and Pharmacy, The University of Hong Kong, Hong Kong, CHINA, 2Department of Medicine, The University of Hong Kong, Hong Kong, CHINA

initiating subsequent

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Here in a single-cell calcium visualization system we use for the first time a fast protocol to see subcellular dynamics of metabo- tropic calcium response in glioma cells. The signal is evoked by activation of P2Y2 nucleotide receptors with UTP in glioma C6, a well-known model of non-excitable cells. We use fura 2-AM, very popular ratiometric dye. The use of ratiometric technique ensures us that calcium-dependent changes in the thickness of very flat parts of the cell will not disturb the results, what may happen during confocal calcium measurements when a slice is thicker than lamellipodium. Sub-second measurement is sufficient to observe that calcium response begins in one region situated on the edge of the cell and is spread to the whole cell body within couple of seconds. However depending on the position of mea- surement point: in lamellipodium area or in the cell center we can see different dynamics of the local response. For every time series two parameters are estimated: signal delay (from the first Normal endothelial function relies on a balance between injury and repair of endothelial cells. But regenerated endothelial cells inflammatory often undergo senescence, responses. Senescent vascular endothelial cells are present in human atherosclerotic lesions. SIRT1 is a conserved deacetylase mediating lifespan extension and anti-aging effects in a variety of species. However, its role in regulating endothelial senescence has not been well elucidated. In present study, we have found that both mRNA and protein levels of SIRT1 decrease during pro-

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Poster Presentations

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

ity as well as protein levels. Over-expression of LKB1 in PAECs induces endothelial senescence and retards the proliferation. These effects of LKB1 can be reversed by over-expression of SIRT1. Similar result is not seen when using a mutant type of SIRT1 that lacks deacetylase activity, suggesting that the effects are deacetylation dependent. In summary, our results demon- strate that SIRT1 plays an important role in antagonizing senes- cence and maintaining normal endothelial functions, which at least in part through targeting the functions of LKB1. longed culture of primary porcine aortic endothelial cells (PAE- Cs), which is a cellular model for endothelial replicative senes- cence established by our group. Exogenously added SIRT1 can prevent cellular senescence and recover the proliferative capacity of PAECs, while enhancing eNOS phosphorylation. When screening the molecules that could interact with SIRT1, we have identified LKB1, a tumor suppressor, which dramatically increases during the process of senescence. Our results have proved that SIRT1 could deacetylate LKB1 and reduce its stabil-

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P6 Organism – The Network of Interactions

P6–1 3T3-L1 adipocyte cell responses to mild mitochondrial uncoupling T. Arnould, A. De Pauw, S. Tejerina, A. Houbion, E. Delaive, M. Dieu, P. Renard and M. Raes Laboratory of Biochemistry and Cell Biology, University of Namur (FUNDP), Namur, BELGIUM

this study, because of its putative connection to T cell activation and cell death; we examined TNF-Related Apoptosis Inducing Ligand (TRAIL) and its receptor expression profile on T cell subsets in RA patients and compared it to healthy controls. Twenty RA patients and 12 healthy individuals were included in the study conducted at the Rheumatology Clinic of Akdeniz Uni- versity Hospitals and Clinics. Analysis of TRAIL and its receptor expression profile on T lymphocyte subsets were performed using flow cytometry. The correlation of TRAIL and its receptor expression profile were compared with clinical parameters includ- ing RA activity scoring (DAS28), ESR and serum CRP levels. Despite no change was detected in the ratio of CD4+ to CD8+ T cells between the two groups, significant up-regulation of TRAIL and its receptors (both death and decoy) was detected on both CD4+ and CD8+ T cells in RA patients compared to con- trols. Furthermore, CD8+ T cell associated DR4 death receptor and decoy receptors (DcR1 and DcR2) but not those present on CD4+ T cells displayed a positive correlation with RA disease activity. Differential alteration in expression levels of TRAIL death and decoy receptors on T cells indicates that these profiles might differently modulate disease activation. Thus, TRAIL/ TRAIL receptor system might play an essential role during the course of RA by way of modulating T cell activation or suppres- sion.

P6–3 The effects of ceftriaxone and diclofenac on viability and proliferation of lymphocytes isolated from diabetic patients A. Causevic, T. Dujic, S. Semiz, T. Bego and M. Malenica Faculty of pharmacy, Department of Biochemistry and Clinical Analysis, Sarajevo, BOSNIA and HERZEGOVINA

As impairment of mitochondrial activity affects lipid-metaboliz- ing tissues, mild-mitochondrial uncoupling has been proposed as a possible strategy to fight obesity and its major associated pathologies. We thus characterized the 3T3-L1-adipocyte ‘dedif- ferentiation’ induced by carbonyl cyanide (p-trifluoromethoxy)- phenylhydrazone (FCCP), a mitochondrial uncoupler. We found a decrease in TG content in adipocytes incubated with this mole- cule comparable to the TG reduction in adipocytes treated with TNFalpha, a pro-inflammatory cytokine known to induce adipo- cyte ‘dedifferentiation’. We next analyzed the expression of genes encoding adipogenic markers and effectors and compared the dif- ferentially expressed genes in adipocytes treated with FCCP or TNFalpha. Furthermore, a significant decrease in the transcrip- tional activity of PPARgamma and CEBP/alpha transcription factors was found in adipocytes with impaired mitochondrial activity. Metabolic assays also evidence that TG reduction could be mediated by a down-regulation of lipid synthesis rather than an up-regulation of free fatty acid beta-oxidation. Stimulation of lipolysis in response to the uncoupler also seems to contribute to the TG reduction, a process associated with perilipin A down- regulation. In these conditions, the proteomic analysis of highly purified mitochondrial fractions isolated from adipocytes incu- bated with FCCP for 6 days reveals a specific mitochondrial response in adipocytes incubated with the uncoupler. This data indicates that the mitochondrial population is qualitatively differ- ent in adipocytes that display a mild mitochondrial uncoupling when compared with mitochondria of adipocytes. In conclusion, our results suggest that molecular mechanisms triggered by either mitochondrial uncoupling or TNFalpha leading to the loss of tri- glycerides in adipocytes are different and are reflected at the mitochondrial protein content.

P6–2 Rheumatoid Arthritis disease activity is correlated with TRAIL death receptor-4 and decoy receptor expresions on CD8+ T cells A. Bisgin1, E. Terzioglu2, C. Aydin3, V. Yazisiz2, N. Balci4, H. Bagci3, R. M. Gorczynski5, C. A. Akdis6 and S. Sanlioglu1 1Akdeniz University Faculty of Medicine, Human Gene Therapy Unit and Department of Medical Biology and Genetics, Antalya, TURKEY, 2Akdeniz University Faculty of Medicine, Department of Rheumatology Allergy and Immunology, Antalya, TURKEY, 3Akdeniz University Faculty of Medicine, Human Gene Therapy Unit and Department of Medical Biology and Genetics, Antalya, TURKEY, 4Akdeniz University Faculty of Medicine, Department of Physical Medicine and Rehabilitation, Antalya, TURKEY, 5Toronto Hospital University Health Network, Division of Cellular and Molecular Biology, Toronto, CANADA, 6Swiss Institute of Allergy and Asthma Research, (SIAF), Davos, SWITZERLAND

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Diclofenac, like various NSAIDs, has been shown to induce apoptosis in various cell lines. Ceftriaxone, known beta-lactam antibiotic, on the other hand, reduces cell death and, according to recent findings, protects neurons against apoptosis. Since minor interactions between the two drugs have been reported in humans, it was of interest to us to examine the effects of combi- nation of these drugs on cell proliferation of peripheral lympho- cytes. Diabetic population, which is especially prone to cell injury and apoptosis, due to a high glucose level, was of a special inter- est to us in this sense. After separation from blood of diabetic and control patients, lymphocytes were suspended in RPMI 1640 medium, transferred to a 96-well plate and then cultured at 37(cid:2)C. Solutions of ceftriaxone were added to cultures after 24 hours, in final concentrations of 80, 160 and 260 mg/l. Two combinations of ceftriaxone and diclofenac (160 mg/l of ceftriaxone plus 3 mg/l of diclofenac and 260 mg/l of ceftriaxone plus 3 mg/l of diclofe- nac) were also applied. After 24 hour, proliferative response of lymphocytes was evaluated by WST-1 assay. Treatment with dic- lofenac and/or ceftriaxone in above concentrations did not result in any significant effect on growth of lymphocytes isolated from diabetic or control subjects. However, although, statistically insignificant, detected absorbances of treated samples of lympho- cytes from diabetic patient showed slight decrease compared to untreated samples from the same patient. Further studies with larger number of samples are needed in order to specify charac- teristic effects of these substances in diabetic population. Rheumatoid Arthritis (RA) is a chronic autoimmune inflamma- tory disorder. Although, the exact origin and pathogenesis of the disease are still unknown, circulating T cells are the primary cells responsible for both the development and initiation of RA. In

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Poster Presentations

P6–4 Dexamethasone effects on rat hippocampal apoptotic molecules expression D. Drakulic, N. Velickovic, M. Milosevic, S. Petrovic, I. Stanojevic and A. Horvat Laboratory for Molecular Biology and Endocrinology, Institute for Nuclear Sciences Vinca, Belgrade, SERBIA

P6–6 Mitochondrial creatine kinase-induced raft-like organization in cardiolipin containing monolayers mimicking the inner mitochondrial membrane T. Granjon1, O. Maniti1, M. Cheniour1, M. F. Lecompte2 and O. Marcillat1 1Universite´ Lyon 1, ICBMS-UMR CNRS 5246, Villeurbanne, FRANCE, 2Universite´ Paul Sabatier, Faculte´ de Me´decine de Rangueil, Toulouse, FRANCE

little is known about

three biophysical

Dexamethasone (DEX), synthetic glucocorticoid (GC) is com- monly used in the treatment of inflammatory disorders, autoim- mune diseases, tissue oedema and also, as co-medication in cancer therapy. Apoptosis, physiological and controlled process of programmed cell death induced by glucocorticoids, in hippo- campal neurons could be caused by glucocorticoid receptor acti- vation, mostly by tumour suppressor p53 signalling pathway activation. The aim of our study was to highlight GC-mediated apoptotic activation mechanism by assessing both protein and gene expression of apoptosis related molecules after chronic treat- ment with DEX. In our model system 3 months old male Wistar rats were treated 8 days with DEX (100 lg/kg/day, i.p.). The lev- els of p53, Bax, Bcl-2 and procaspase-3 in hippocampal whole cell extract were investigated using Western blotting and PCR techniques. In DEX treated animals, the p53 protein level incre- ment and the increment of pro-apoptotic protein Bax, down- stream gene regulated by p53 protein, have been shown. However, expression of anti-apoptotic protein Bcl-2 as well as procaspase-3 protein has not been alternated. In our experiments, glucocorticoid receptor activation has been confirmed by GR protein level decrement after DEX treatment. Our results indicate that DEX application induces p53 protein activation in rat hip- pocampus concomitantly with increment of Bax protein expres- sion. Pro- and anti-apoptotic proteins ratio defines the cell’s destiny in response to apoptotic signals, throughout mitochon- drial membrane’s permeability regulation. In this paper, the aug- mentation of hippocampal whole cell extracts Bax: Bcl-2 protein ratio has been detected, indicating that GR agonist could have potential apoptotic effect. It is well established that the cubic octameric mitochondrial crea- tine kinase (mtCK) binds to the outer face of the inner mitochon- interactions with drial membrane mainly via electrostatic cardiolipin (CL). However, the conse- quences of these interactions at membrane and protein levels. To characterize these consequences, techniques were used: (i) Brewster angle microscopy (BAM) investigations indicates that mtCK binding induced CL cluster formation by selectively recruiting this phospholipid within monolayers mim- icking the inner mitochondrial membrane composition. (ii) Dif- ferential capacity measurements indicate protein insertion into a (iii) Polarization modulation condensed CL-containing film. infrared reflection-absorption spectroscopy (PM-IRRAS) moni- tors modifications in the conformation and the orientation of the protein upon membrane binding and shows that the mean orien- tation of a-helices shifts upon CL binding from 30(cid:2) to 45(cid:2) with respect to the interface plane. The morphology of the monolayer as well as protein domain movement occurring upon membrane binding are however dependent on cardiolipin acyl chain length and saturation. It is therefore tempting to attribute to mtCK a role in organizing membrane lipid distribution and modulating mitochondrial inner membrane morphology into a raft-like orga- nization. Additionaly, in pathologic or physiologic situations such as Barth syndrome, oxidative stress or apoptosis process, known to modify cardiolipin fatty acid composition, modification of mtCK binding may influence the modulation of cardiolipin distribution at the mitochondrial inner membrane level.

P6–5 Inflammatory cytokines in chronic obstructive bronchopneumopathy – preliminary results M. Gheorghiu, D. Pasarica, B. Mahler, T. Trandafir and C. Bara Pathophysiology and Immunology, Carol Davila University of Medicine and Pharmacy, Bucharest, ROMANIA

P6–7 Serine-threonine kinase receptor-associated protein (STRAP) negatively regulates apoptosis signal-regulating kinase 1 activity H. Ha, H. A. Seong, H. Jung and R. Manoharan Biochemistry, Chungbuk National University, Cheongju, SOUTH-KOREA

interleukin 1-beta (IL-1ß),

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Background: Statistically, chronic obstructive bronchopneum- opathy (COBP) will be the third cause of death in 2020. The inflammation. main pathophysiological mechanism is bronchial Inflammatory cytokines tumoral necrosis factor alpha (TNFa), interleukin 6 (IL-6), interleukin 8 (IL-8) and interleukin 10 (IL-10) elicit defensive responses in lung parenchymal cells and bronchial cells, including activation of apoptosis. They could also play an important antiapoptotic role, stimulating organ repair after injury. In addition, IL-8 promotes neutrophilia as well as neutrophil infiltration of bronchial tissue. We were interested in studying the cytokine signals which are involved in COBP development. Patients and methods: 40 patients with confirmed COBP, trea- ted in ‘Marius Nasta’ Hospital of Pulmonary Diseases Bucharest, were investigated using blood samples. IL-1ß, TNFa, IL-6, IL-8 and IL-10 were measured using ELISA kits. Results: Significant increases of IL-6, IL-8 and IL-10 serum lev- els were obtained. Conclusion: Inflammatory cytokines could be used as paraclini- cal markers of evolution in COBP. Serine-threonine kinase receptor-associated protein (STRAP) interacts with TGF-b receptors and inhibits TGF-b signaling. Here, we identify STRAP as an interacting partner of apoptosis signal-regulating kinase 1 (ASK1). The association between ASK1 and STRAP was mediated through the carboxy-terminal domain of ASK1 and the fourth and sixth WD40 repeat of STRAP. Using cysteine to serine amino acid substitution mutants of ASK1 (C1005S, C1351S, and C1360S) and STRAP (C152S, C270S, and C152S/C270S) we demonstrate that the Cys1351 and Cys1360 of ASK1 and Cys152 and Cys270 of STRAP are required for ASK1-STRAP binding and this interaction is decreased by H2O2 treatment. Expression of wild-type STRAP, but not STRAP double mutant (C152S/C270S), inhibits ASK1 activity and down-regulates ASK1-mediated signaling to both JNK and p38 kinases by stabilizing the ASK1-Trx complex or decreasing the complex formation between ASK1 and its sub- strate MKK3. In addition, STRAP directly affected ASK1 activ- ity and suppressed H2O2-mediated apoptosis in a dose-dependent

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manner by inhibiting the ASK1 activity through physical interac- tion. These results suggest that STRAP plays an important role in the negative regulation of ASK1 activity.

P6–8 Influence of HCV infection on serum profile of IGF-1 and proinflammatory cytokines: association with risk for growth hormone resistance development G. F. Helaly1 and N. M. El-Afandy2 1Microbiology Department, Medical Research Institute – Alexan- dria University, Alexandria, EGYPT, 2Applied Medical Chemistry Department, Medical Research Institute – Alexandria University, Alexandria, EGYPT

endocrine, relationships between the

serum levels considerably elevated

fibrosis and cirrhosis worldwide. Despite distinct virologic fea- tures, both are hepatotropic but not directly cytopathic. They slowly wear away person’s liver, harshly damaging liver function with increasing the risk of liver cancer development. Liver fibro- sis is a progressive pathologic process characterized by altered turnover of the extracellular matrix proteins. Matrix metallopro- teinases (MMPs) are central to tissue remodeling. MMP-9 is one of the gelatinases that may be particularly important in liver fibrosis. This study aimed at evaluating whether MMP-9 is simi- larly involved in relation to viral load and hepatic dysfunction in the development of hepatitis B and C liver disease. Twenty chronic HBV, 30 HCV infected and 15 healthy subjects were studied. HBV and HCV viral load was assessed quantitatively by Real-time PCR. ELISA technique was used for quantification of serum levels of MMP-9 and liver biomarkers in the form of am- inotransferases’ activities were measured. HCV-infected patients had significantly higher MMP-9 values than chronic HBV- infected patients (203.32 ± 23.62 ng/ml vs. 106.1 ± 26.56 ng/ml, p = 0.003). Compared to control subjects, HCV infected patients showed of MMP- 9(203.32 ± 23.62 ng/ml vs. 43.2 ± 4.8 ng/ml, p = 0.0001) as well chronic HBV-infected patients (p < 0.05). A highly signifi- cant positive correlation was found between MMP-9 levels and HBV viral load (r = 0.842, p = 0.0001) also with AST /ALT ratio (r = 0.614, p = 0.01). Conversely, MMP-9 levels were not correlated with HCV viral load, however significantly correlated with AST /ALT ratio (r = 0.652, p < 0.001). Based on these results, it is concluded that MMP-9 levels could reflect progres- sive liver damage in HBV and HCV infection. However, a dis- tinction between the pathological mechanism of HCV and HBV could be suggested, as HCV probably promotes hepatocyte dam- age and fibrosis through the release of inflammatory cytokines and the net liver damage will depends on the balance between the host’s antiviral mechanisms and the virus ability to challenge them. While in HBV infection, the integration of HBV genome with continuous expression of HBV genomic RNA through the replication process and secretion of viral antigens may contribute to transcriptional regulation of MMP-9, thus promoting liver damage and fibrosis.

P6–10 The c-jun N-terminal kinase pathways play a distinct role in apoptosis mediated by anti-cancer drugs E. H. Hur, M. J. Kang, D. Y. Kim and J. H. Lee Hematology, University of Ulsan College of Medicine, Seoul, SOUTH-KOREA

infection is an emergent challenge Hepatitis C virus (HCV) the worldwide. Close immune systems and the liver have been postulated. Insulin-like growth factor (IGF) system is a striking target in this respect as IGF-1 synthesis is coupled to growth hormone (GH) via stimu- lating hepatocyte IGF-1 production. Although proinflammatory cytokines are an integral part of inflammation in chronic liver diseases, their involvement in the pathogenesis of GH resistance during HCV infection remains to be elucidated. To address this issue, our study aimed at evaluating the influence of HCV infec- tion on serum profile of IGF-1, TNF-a and IL-6 to asses their possible relation to hepatic dysfunction and GH resistance devel- opment. Twenty-five HCV-infected patients have been studied together with 15 healthy control subjects. Serum concentrations of IGF-1, TNF-a and IL-6 have been determined through ELISA techniques. HCV viral load was assessed by Real-time polymer- ase chain reaction using TaqMan probe technology. Basal serum GH levels were determined by chemiluminescence assay and serum aminotransferases’ activities were also measured. TNF-a and IL-6 demonstrated considerably higher serum levels, while IGF-1 levels were significantly lower in HCV-infected patients compared with healthy controls. A statistically significant positive correlation was observed between GH and IL-6 levels (r = 0.451, p < 0.05), a similar trend was found between GH levels, GH/IGF-1 ratio and AST/ALT ratio (r = 0.526, p < 0.01, r = 0.515, p < 0.01, respectively). A significant nega- tive correlation was observed between HCV viral load and GH levels (r = -0.400, p < 0.05). The progressive increase in HCV viral load matches the decrease in circulating IGF-1 levels but without reaching statistical significance (r = -0.281, p > 0.05). We conclude that GH resistance during HCV infection is assumed to be multifactorial as it could be induced by HCV infection which decrease the liver functional mass with dimin- ished production of IGF-1, also could be mediated by proinflam- matory cytokines through their possible role in blunting the hepatic response to GH. This crosstalk between proinflammatory cytokines and GH-IGF-1 axis could be responsible for triggering impaired glucose metabolism and diabetes later on in chronic HCV infection.

P6–9 Differences in circulating MMP-9 levels with regard to viral load and hepatic dysfunction between HBV and HCV infected patients G. F. Helaly Microbiology Department, Medical Research Institute – Alexandria University, Alexandria, EGYPT

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Hepatitis B virus (HBV) and Hepatitis C virus (HCV) are the two major causes of chronic liver disease including inflammation, c-jun N-terminal kinase (JNK) is activated in response to many stressful stimuli including heat shock, UV irradiation, protein syn- thesis inhibitors, and inflammatory cytokines. Elucidating of apop- tosis and survival pathways implicated in the action of anticancer drugs is important for understanding drug mechanisms and modes of resistance. In this study, we investigated a role of JNK pathways in the cellular response to different anti-cancer drugs. Three anti- cancer drugs including daunorubicin, vinblastine and etoposide were treated to several human leukemic cell lines with various time and dose conditions. All three drugs strongly activated the JNK pathways with various patterns and induced apoptotic cell death in each leukemic cell, with similar kinetic profiles of PARP cleavage, and caspase3 activation. Inhibition of the JNK pathways by SP600125 protected leukemic cell lines from apoptotic cell death by anti-cancer drugs. Our findings suggest that activation of the JNK pathways is an important component of cellular response to anti-cancer drugs. Our understanding of JNK function may pro- vide new insights on anti-cancer drug targets.

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Poster Presentations

P6–11 The role of Hsp90 in folding and stabilization of p53 mutants R. Hrstka, P. Muller, P. Hublarova, R. Nenutil and B. Vojtesek Oncological and Experimental Pathology, Masaryk Memorial Cancer Institute, Brno, CZECH REPUBLIC

P6–13 Endothelial adhesion molecule ESAM provides an adhesion machinery with MAGI-1 regulating the actin polymerization and Rho activity R. Kimura1, Y. Hayashi1, T. Satoh2 and T. Ishida3 1Kobe University Graduate School of Medicine, Division of Molecular Medicine and Medical Genetics International Center for Medical Research and Treatment (ICMRT), Kobe, JAPAN, 2Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe, JAPAN, 3Kobe University Graduate School of Medicine, Division of Cardiovascular Medicine, Kobe, JAPAN

The p53 tumour suppressor protein lies at crossroads of multiple cellular response pathways that control the fate of the cell in response to endogenous or exogenous stresses. Inactivation of the p53 tumour suppressor-signalling pathway is seen in most human cancers. In contrast to other tumour suppressor genes that are mainly altered by truncating mutations, the majority of TP53 mutations are missense substitutions within the core domain, leading to the expression of full-length mutant p53 protein. These mutants could be characterized by a prolonged half-life of the mutant protein and by the inability to recognize wild-type p53 specific DNA-binding sites resulting in loss of their transcrip- tional activity. Several tumour-associated mutants of the p53 tumour suppressor protein may exert oncogenic activities ‘gain of function’ (GOF). Predominant mechanism of mutant p53 GOF is its ability to act as a transcription factor causing the expression of genes that are responsible for malignant transformation of the cells. To explore the involvement of Hsp90 in molecular mecha- nism of mutant p53 GOF, we analysed Hsp90-p53 interactions in relation to cancer therapy. We found that Hsp90 prevents degra- dation, especially of denatured p53 mutants, by stabilising of mutant p53, enabling its novel transcription activities. Interest- ingly, the Hsp90 inhibitor 17AAG reduces oncogenic properties of p53 mutants and initiates their degradation. Thus blocking the function of Hsp90 with 17AAG could be a critical for fighting cancers via the p53 tumour suppressor pathway. Acknowledgement: This work was supported by GACR 301/ 08/1468.

tight

Aims and Methods: Endothelial cell-selective adhesion molecule (ESAM) is an immunoglobulin-like transmembrane protein asso- ciated with endothelial tight junctions. ESAM binds to MAGI-1, in a PDZ a membrane-associated guanylate kinase protein, domain-dependent manner. MAGI-1, as a scaffold protein at cell junctions, interacts with various molecules and functions. To gain more insight into the putative function of ESAM, we constructed stable transfectants expressing wild-type ESAM and MAGI-1 as well as deletion mutant of ESAM which lacks a PDZ domain and wild-type MAGI-1. Results: ESAM mediated a calcium-independent homophilic adhesion and was accumulated at cell–cell contacts, and only an extracellular domain, not transmembrane or intracellular domain, of ESAM was essential for this function. In stable transfectants expressing wild-type ESAM and wild-type MAGI-1, MAGI-1 was recruited into ESAM-based cell contacts and strengthened ESAM-mediated cell adhesion with actin polymerization at cell– cell contacts. In stable transfectants expressing deletion mutant of ESAM which lacks a PDZ domain and wild-type MAGI-1, MAGI-1 showed a cytoplasmic localization and activated Rho, a GTPase implicated in the destabilization of tight junctions. Conclusions: These findings suggest that ESAM together with MAGI-1 provides an adhesion machinery at junctions, which may regulate the actin polymerization and alter Rho activ- ity. When MAGI-1 is localized free from ESAM and diffusely distributed in the cytoplasma, the intercellular adhesions become weak and the activation of Rho is increased. These results might be well corresponding to the physiological function of Rho which increases permeability of endothelial cells and blood vessels.

P6–12 Analysis of HCV IRES quasispecies A. A. Khawaja1, V. Vopalensky1, L. Roznovsky2, I. Orsagova2, J. Mrazek3 and M. Pospisek1 1Charles University in Prague Faculty of Science, Genetics and Microbiology, Prague 2, CZECH REPUBLIC, 2University Hospital Ostrava, Infectious Diseases and AIDS Clinic, Ostrava, CZECH REPUBLIC, 3Institute of Public Health Ostrava, MPI Centre, Ostrava, CZECH REPUBLIC

P6–14 Immune response to vaccinia virus in atopic mice J. Knitlova, G. A. Yendewa and Z. Melkova 1st Faculty of Medicine, Institute of Immunology and Microbiology, Prague 2, CZECH REPUBLIC

post-vaccination encephalitis, vaccinia,

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Diversification of HCV is a dynamic process that gives rise to genetically distinct but closely related variants. The main aim of the study was to analyze the heterogeneity of these variants described as quasispecies originating from HCV-IRES (Internal Ribosomal Entry Site). Three patients were selected suffering from HCV genotype 1. One patient was non-responsive to the medication of IFN-alpha and ribavirin while the other two patients did not receive any treatment. High-efficiency cloning of HCV-IRES from the clinical samples was done in a new bicistron- ic reporter assay system; a reliable means of studying the activities of HCV-IRES diversity individually and on the level of covering the whole population. Further, DGGE was used to analyze the diversity of HCV IRES on molecular basis. Clones from each library, derived from the patients, were run on optimized DGGE conditions and were compared. Variability among clones was suc- cessive and was further sequenced for analysis. The sequenced data and DGGE-derived variation of mutated HCV-IRES was estimated for correlation. Based on the results, the activity of diversified HCV-IRES sequences that persist in an individual or comprising the whole population could be characterized and eval- uated in terms of its structural and functional relevance. Vaccinia virus has been used in the past for a wide-spread vacci- nation against smallpox, leading to eradication of this life-threat- ening disease. In 1980, the World Health Organization Smallpox Eradication Program declared the disease as eradicated and vac- cination of the general population has been stopped. Recently, due to the threats of bioterrorism, certain countries have been re- introducing vaccination programs among selected groups of pop- ulation. However, the old vaccines that are available, and proba- bly also the new vaccines based on the same virus strain but prepared in tissue culture, reveal a substantial risk of post-vacci- nation complications. The most severe complications include pro- gressive eczema vaccinatum, and a recently described myopericarditis. Eczema vaccinatum, a disseminated vaccinia virus infection, is a complex condition occurring primarily in atopic individuals. Our under-

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standing to its pathogenesis at the current level of immunology is limited and it would be greatly advanced by characterizing immune responses towards vaccinia virus in an animal model of atopic dermatitis. Therefore, we have decided to characterize these responses in Nc/Nga mice, a spontaneously mutated mouse strain revealing features of atopic dermatitis. Namely, size of the lesions, spread of the infection, levels of vaccinia-specific antibod- ies and expression of Th1 and Th2 cytokines are being deter- mined. The results of these studies will be presented.

tor of caspase-3. The activity of these particular caspases was then verified in lysates of infected cells using an in vitro cleavage assay involving specific fluorogenic substrates. Again, the specific- ity of the assays was confirmed using specific non-fluorescent inhibitors. Finally, we characterized the cleavage of caspase sub- strates in vaccinia virus-infected cells using a western blot analy- sis. While we found cleavage of cytokeratin-18, we did not observe any cleavage of PARP. In conclusion, we describe activa- tion and activity of caspases-2 and 4 in epithelial cells lytically infected with vaccinia virus. The role of these caspases during vaccinia virus infection will be discussed.

P6–15 Adeno-associated viral (AAV) vectors carrying short-hairpin RNA for the focal deletion of sleep-active adenosine A2A receptors M. Lazarus1, X. F. Yue1, T. Higashi1, T. Enomoto2, C. E. Bass3, H. Yasuda2, Y. Urade1 and O. Hayaishi1 1Osaka Bioscience Institute, Behavioral Biology, Suita, JAPAN, 2Oriental Yeast Co. Ltd, Institute for Biochemical Science, Nagahama, JAPAN, 3Physiology and Pharmacology, Wake Forest University Health Sciences, Winston-Salem, NC, USA

P6–17 The role of Kaposi’s sarcoma-associated herpesvirus-encoded vIRF-3 in activation of viral interleukin-6 B. Lubyova1, J. A. Frisancho2 and P. M. Pitha2 1Institute of Immunology and Microbiology, First Medical Faculty of Charles University, Prague, CZECH REPUBLIC, 2Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA

The sleep drive is believed to lead to the accumulation of endoge- nous sleep substances during wakefulness. Adenosine (AD) is a good candidate of such a sleep substance and its action is believed to be mediated by AD A2A receptors (A2AR). The infu- sion of CGS21680, an agonist for A2AR, into the subarachnoid space induces sleep and A2ARs mediate the arousal effect of caf- feine (Huang et al., Curr Opin Pharmacol 2007; 7: 33). However, the neural entry points of the somnogenic signal of AD acting on A2AR remain unclear. In this study, we established a novel tech- nology for systems somnology, called focal RNA interference (fRNAi), to silence the expression of A2AR in rats by using adeno-associated virus (AAV)-carrying short-hairpin RNAi ele- ments. In AAV transduced neurons, we found that shRNA spe- cific for A2AR suppressed efficiently the expression of A2AR in a pattern dictated by the site of AAV injection. Stereotaxic micro- injections of AAV caused minimal tissue injury and AAV of serotype 10 produced enhanced and widespread infection of neu- rons. We are currently studying the effect of focal gene manipula- tion in the ventral striatum on sleep regulation. The focal knockout approach used in our studies has wide applicability to neuroanatomical problems involving dissection of the effects of different receptors with complex brain distributions (Lazarus et al., Nat Neurosci 2007; 10: 1131).

Kaposi’s sarcoma-associated herpesvirus is the causative agent of Kaposi’s sarcoma, multicentric Castleman’s disease and primary effusion lymphoma (PEL). The virus encodes a nuclear protein, vIRF-3/LANA2, which is constitutively expressed in latently infected PEL cells. Here we report, that vIRF-3 may play a role in stimulating the promoter of viral IL-6 (vIL-6) gene. vIL-6 repre- sents KSHV-encoded homolog of cellular IL-6 and serves as a potent growth factor for PEL cells. The finding of four potential ISRE-like sites in the vIL-6 promoter prompted us to address their role in transcriptional activation of vIL-6. Using transient transfec- tion assays with the vIL-6 reporter, we observed that vIRF-3 together with cellular IRFs, namely IRF-4, IRF-7 and p48, can effectively stimulate vIL-6 expression. Employing co-immunopre- cipitation assay in PEL and transfected HEK293 cells we showed that vIRF-3 binds to IRF-4, IRF7, and p48. DNA pull-down and chromatin immunoprecipitation assays demonstrated the binding of vIRF-3 and IRFs to single ISRE elements within the vIL-6 pro- moter. To further delineate the role of vIRF-3 in the transcrip- tional activation of vIL-6, we employed RNA interference to specifically knock-down vIRF-3 expression in PEL cells. PEL cell lines with specific down-modulation of the vIRF-3 gene expression would allow us to study the vIRF-3 function in the context of the entire KSHV genome in its natural cell host. Out of four potential vIRF-3-specific siRNAs, we selected two with approximately 70% knock-down efficiency. The construction of PEL cell lines with Dox-inducible knock-down of vIRF-3 is currently in progress and the results will be discussed. This work was supported by the Acad- emy of Sciences of the Czech Republic (Grant IAA501050701) and the Grant Agency of the Czech Republic (Grant 204/09/0773).

P6–16 Activation of caspases in cells lytically infected with vaccinia virus J. Liskova and Z. Melkova Institute of Immunology and Microbiology 1st Medical Faculty of Charles University, Molecular Virology, Prague, CZECH REPUBLIC

P6–18 A novel quantitative flow cytometric method for the determination of a diabetes associated adipocytokine, omentin, in human plasma E. Maratou1, E. Boutati2, M. Peppa2, V. Lambadiari2, G. Dimitriadis2 and S. A. Raptis1 1Hellenic National Diabetes Center, 2nd Department of Internal Medicine Research Institute and Diabetes Center, Athens, GREECE, 22nd Department of Internal Medicine Research Institute and Diabetes Center, Athens University ‘‘Attikon’’ University Hospital, Athens, GREECE

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In most cell types, vaccinia virus causes a lytic infection, an equivalent of necrosis. However, we observed caspase activation and/or activity, a hallmark of apoptosis, in epithelial cell lines BSC-40 and HeLa G lytically infected with this virus. The cas- pase activation was first characterized using a fluorescent pan- caspase inhibitor FITC-VAD-FMK and flow cytometry. The specificity of the assay was confirmed using a non-fluorescent individual pan-caspase inhibitor and/or specific inhibitors of caspases (caspase-1, 2, 3, 4, 6, 8, 9, 10, 13) to compete out the binding of the fluorescent inhibitor. The fluorescent signal was competed out by the pan-caspase inhibitor as well as by specific inhibitors of caspases-2 and 4, and to a lesser extent by an inhibi- Background: Adipocytokines such as leptin, adiponectin, resi- stin and visfatin belong to a family of proteins secreted specifi-

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expression. The heat tolerance was evaluated in terms of final Tco, brain activation and inflammation and we assessed the sys- temic stress reaction by the plasma corticosterone concentration. Using the a posteriori k-means clustering method based on Hsp70 mRNA, we obtained three distinct groups of animals. The rats with the lowest Hsp70 mRNA level presented the lowest increase in Tco, the lowest brain activation as shown by low c- fos mRNA level, and the highest IkappaBa mRNA expression: they are well tolerant to heat. Conversely, those having the high- est Hsp70 mRNA level exhibited the highest level of final Tco, the largest c-fos mRNA increase and inflammatory brain activa- tion, as suggested by the highest IL-1b mRNA and the lowest I- they could be considered as kappaBa mRNA expression: intolerant to heat. Moreover, intolerant rats also these heat exhibited lower plasma corticosterone level compared to the well tolerant rats. In conclusion, high cell aggression is associated with inflammatory activation without inhibitory factor activation. These results could be related to poor systemic stress reaction.

correlated with body mass index assessment

P6–20 Low-power laser therapy effects on cell cycle progression, seen in Jurkat T cells M. M. Mocanu1, L. Vlaicu1, B. Andrei1, R. P. Sinha1, E. Radu1, J. Horvath2, E. Tanos2 and E. Katona1 1Biophysics Department, ‘‘Carol Davila’’ University of Medicine and Pharmacy, Bucharest, ROMANIA, 2Lasereuropa, Department of Lasers, Budapest, HUNGARY

cally by mature adipocytes. These adipocytes-derived mediators exert profound metabolic and pro- or anti-inflammatory effects. Adipocytokines are currently investigated as potential future drug targets in diabetes, lipid metabolism, endothelial dysfunction and inflammation in general. Omentin is a recently described adipocy- tokine, selectively expressed and secreted from visceral, but not from subcutaneous adipose tissue. Visceral obesity is strongly associated with insulin resistance, hyperglycemia, dyslipidemia and hypertension. Methods: Blood (10 ml) was withdrawn from 17 healthy women. The levels of plasma omentin were measured by a novel flow cytometric method, which is based on a bead array tech- nique. The bead surface is covered by anti-omentin antibody (Ab), allowing the bead to capture the molecules of circulating omentin. Capture beads and plasma samples are incubated with a biotinylated anti-omentin Ab and streptavidin-phycoerythrin in order to form a sandwich complex. The results were quantified by the usage of a standard curve. Results: The introduced bead array method has allowed the measurement of the plasma omentin in 17 women (average level 106 + 59 ng/ml, range 20–250 ng/ml). The omentin levels are (r = -0.56, negatively (r = -0.55, p = 0.019), homeostasis model of p = 0.019), waist circumference (r = -0.55, p = 0.021) and waist to heap ratio (r = -0.55, p = 0.021). These parameters are indicators of the metabolic status and reflect the distribution of the adipose tissue. Conclusions: The results lead to the conclusion that omentin secretion is diminished by the increased adiposity. This decrease may contribute to the insulin resistance observed in obesity, dia- betes and generally in metabolic syndrome. Additionally, the measurements prove that the introduced flow cytometric tech- nique produces results similar to those produced by the classical method of quantitative western blotting assay. The advantages of the flow cytometric technique are the easiness of handling, the small amount of required blood, and the fact that the results are rapidly produced without compromising specificity and accuracy. This experimental approach permits the determination of this newly discovered adipokine in a large sample size of humans, in order to clarify the physiological role of omentin.

P6–19 Heat intolerance is characterized by high cell aggression and inflammation, associated with low systemic stress activation V. Michel1, J. B. Drouet1, A. Peinnequin2, A. Alonso2, R. Cespuglio3 and C. Frederic1 1Centre de Recherche du Service de Sante´ des Arme´es, Facteurs Humains, La Tronche, FRANCE, 2Centre de Recherche du Service de Sante´ des Arme´es, Radiobiologie et Radiopathologie, La Tron- che, FRANCE, 3Universite´ C. Bernard, EA4170, Lyon, FRANCE

Background: Low-power laser therapy (LPLT) promotes wound healing and reduces pain and inflammation; at the same time LPLT is involved in the prevention and treatment of oral muco- sitis (OM) and oral pain (OP) associated with cancer therapy. Recently, LPLT has been applied to moderate Alzheimer’s dis- ease (AD) hoping to act on the apoptotic processes, but the underlying mechanisms remain unknown. We previously reported metabolic modulation of LPLT and photobiomodulation of flavonoids effects on cell viability, proliferation and mitochon- drial reticulum state. Material and Methods: In this study, we irradiated human T cells with red/ near infra-red light emitted by semiconductor lasers (single doses of 0.5–2 microJ/cell, total doses of 1–15 mi- croJ/cell). Microscopy and flow cytometry techniques were used to investigate cell-cycle progression and apoptosis induction. Results: The cell viability assays showed that LPLT could inhi- bit the apoptosis in radiation wavelength, dose, exposure time and cell state dependent manner. We also revealed that LPLT may promote the survival of Jurkat T cells under serum-free con- ditions that normally lead to the apoptosis. In addition, data indicated that appropiate LPLT stimulates cell cycle entry under serum-free conditions and this LPLT acts synergistically with the addition of serum. Conclusion: Taken together, these data reveal cellular mecha- nisms which might underlie beneficial effects of soft laser therapy used as non-drug alternative for the management of cancer ther- apy associated OM and OP. Acknowledgement: Partial financial support of the Romanian Ministry of Education and Research (grant 42139/2008 ‘REUM- ALAS’) is gratefully acknowledged.

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Body responses to heat exposure involve systemic and cellular level. At systemic level, heat activates hypothalamo-pituitary- adrenal and sympathetic axis. At cellular level, heat favors the free radicals and denatured proteins occurrence, inducing there- fore Hsp70 mRNA expression. For a given heat exposure, heter- ogeneity of heat tolerance exists: the animals having the highest body core temperature (Tco) also exhibit a limited corticosterone production and a metabolic imbalance. We thus hypothesized that heat tolerance as reflected by Tco is the result of cell aggres- sion level and inadequate systemic stress reaction. To test this hypothesis, we exposed 68 rats at an ambient temperature of 40(cid:2)C for 90 minutes. We then classified the animals according to their cell aggression level, which is hippocampal Hsp70 mRNA

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P6–21 Apoptosome pathway activation in non-small cell lung cancer cells and tissues E. Moravcı´ kova1, E. Krepela1, J. Cermak2 and K. Benkova3 1Laboratories of Molecular and Cell Biology, Department of Pneumology and Thoracic Surgery, University Hospital Bulovka, Prague, CZECH REPUBLIC, 2Division of Surgery, Department of Pneumology and Thoracic Surgery, University Hospital Bul- ovka, Prague, CZECH REPUBLIC, 3Department of Pathology, University Hospital Bulovka, Prague, CZECH REPUBLIC

lyzed the systems formed by the chemokine receptors CXCR3, CXCR4, CXCR7, CCR7, and CCR10 and their ligands in human melanoma cell lines, using flow cytometry. We have char- acterized the following cell lines: MeWo, A-375, SK-MEL-28, Malme-3M (obtained from ATTC), Mel-RC08 (Department of Pathology, University of Valencia) and, as control, the cell line of human T cell lymphoma HUT78 (obtained from ECACC). Moreover, we extracted RNA from all cell lines for quantifica- tion of gene expression of the chemokines receptors and ligands, and for quantification of melanoma-associated markers and stem cell markers. Our results show that the melanoma cell lines do not express the chemokine receptors on their cell surface, while the control HUT78 cell line shows surface expression of CXCR3, CXCR4 and CCR7. However, we have found the interesting fact, not described before, that melanoma cell lines show intracellular expression of all the forementioned receptors and most of their respective ligands. Also, the pattern of intracellular expression of chemokines is very similar between the different cell lines. The control HUT78 cell line expresses all the receptors but does not express or expresses in a lower degree ligands for CXCR3, CXCR4 and CCR7. These results suggest that the autocrine pro- duction of chemokines by the melanoma cell lines may block the expression of the receptors on the cell surface. The inhibition of the chemokine ligands production, with interference RNA, and colocalization studies using confocal microscopy, will be done to further clarify this hypothesis. Acknowledgement: Supported by: FIS 07/0805 and FIS 07/ 1203. S. Pinto is supported by the FCT Grant SFRH/BD/40301/ 2007 (Portugal).

P6–23 Recognition of DNA modified with antitumor cisplatin by p53-family proteins H. Pivonkova and M. Fojta Department of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics AS CR v.v.i., Brno, CZECH REPUBLIC

Aim: The apoptosome pathway is a cell death signaling mecha- nism which is initiated by massive release of mitochondrial cyto- chrome-c (cyt-c) into the cytoplasm during a variety of cellular stresses. In order to evaluate the functional status of this path- way in non-small cell lung cancer (NSCLC) we investigated its activatability in cytosol samples from NSCLC cell lines and NSCLC tissues and matched lungs. Methods: Cytosol samples were prepared from six NSCLC cell lines and from NSCLC tissues and matched lung parenchyma obtained from forty-eight surgically treated patients. The activa- tion of apoptosome pathway in cytosols was accomplished by addition of cyt-c and dATP and the generated caspase-3-like activity was measured with a fluorogenic tetrapeptide substrate Ac-DEVD-AFC. Results: The analysis of apoptosome pathway activation showed that this pathway could be activated in the cytosol samples from two of six examined NSCLC cell lines, from 17 of 48 NSCLC tis- sues and from four of 48 lungs. The endogenous as well as the (cyt-c + dATP)-induced caspase-3-like activities were signifi- cantly higher in NSCLC tissues as compared to the lungs. In the cytosols resistant to the (cyt-c + dATP)-mediated apoptosome pathway activation the XIAP-neutralizing peptides AVPIAQK and ATPFQEG were unable to remove this repression. Conclusions: The results of the present work indicate that there is a larger subset of NSCLC cells and tissues and lungs which cy- tosols are resistant to the (cyt-c + dATP)-mediated apoptosome pathway activation. Acknowledgement: This work was supported by a research project (No. NS/9715-4) from the Ministry of Health, Czech Republic.

P6–22 Cytomic analysis of chemokine receptors and their ligands in human melanoma cell lines S. Pinto1, A. Martinez-Romero1, D. Gil1, J. E. O’Connor1, R. Gil-Benso2, A. Pellı´ n Carcele´ n2, C. Monteagudo3 and R. Callaghan1 1Prı´ncipe Felipe Research Centre, Cytomics, Valencia, SPAIN, 2Department of Pathology, Medical School University of Valencia, Valencia, SPAIN, 3Department of Dermatology, Hospital Clinico Universitario, Valencia, SPAIN

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Chemokines represent a large family of polypeptide signalling molecules that are notable for their role in chemotaxis, leukocyte homing, directional migration, and G protein coupled receptor activation. Chemokines have been implicated in tumor progres- sion and metastasis. In melanoma, chemokine receptors have been implicated in organ selective metastasis by regulating pro- cesses such as chemoattraction, adhesion and survival. In particu- lar, CCR7 has been implicated in lymph node metastasis, CXCR4 in pulmonary metastasis, and CCR10 in skin metastasis, using a mouse model of melanoma. In human melanoma, it is known that primary and metastatic cancer cells express several chemokines receptors and that their chemokine ligands are highly expressed in metastasis sites. In the present study, we have ana- The p53-family proteins (1) are checkpoint proteins involved in cell cycle regulation, differentiation, development and apoptosis control. Functions of the tumor suppressor protein p53, known as a stress-induced transcription factor, are closely related to maintaining genetic integrity of the cells and to defense against malignant transformation. The p73 protein shares a remarkable homology with p53 and is able to induce many of the p53-target genes (2). Both proteins bind to similar DNA response elements (p53CON) and this binding can be influenced by many factors (3). Using an immunoprecipitation assay on magnetic beads cov- ered with protein G we studied interactions of wild type (wt) and mutant (mut) p53 proteins, as well as different isoforms of the p73 protein with linearized plasmids pBSK (lacking p53CON) or pPGM1 (containing p53CON) globally modified with a clinically used anticancer drug cisplatin (primarily forming intrastrand cross-links within GG, AG and GNG motifs) in the presence of unmodified competitor DNA. We found that the p73 isoforms alpha and delta differ in their binding to the cis-platinated DNA. First, p73alpha captured at the magnetic beads via its N-terminus bound the cisplatinated pBSK with a remarkable selectivity, while p73delta did not. Second, binding of p73delta to pPGM1 was strongly inhibited due to damage of p53CON by cisplatin (similarly as observed for wt p53); this inhibition was apparently much less pronounced in the p73alpha. The mut p53 proteins (G245S, R248W and R273H), which were unable to bind pPGM1 sequence-specifically, bound both types of platinated DNA with a higher affinity than the same but unmodified DNA.

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Acknowledgement: This work was supported by GA ASCR (IAA500040701), GACR (204/07/P476) and MEYS CR (LC06035). References: 1. Flores ER & Jacks T. In: Science + Business Media, Inc., G.P. Zambetti, (ed), New York: Springer, 2005, pp. 187–198. 2. Levrero M, De Laurenzi V, Costanzo A, Gong J, Wang JY & Melino G. J Cell Sci (2000); 113: 1661–1670. 3. Pivonkova H, Pecinka P, Ceskova P & Fojta M. FEBS J (2006); 273: 4693.

P6–24 The role of LRP380 in follicle and chicken embryonic development D. B. Raich, W. J. Schneider and M. Hermann Medical Biochemistry/Mol. Genetics, Medical University Vienna, Vienna, AUSTRIA

monocytes or polynuclear cells induces a temporary boost of ga- lig transcription. Although mitogaligin is not related to the Bcl-2 family, several points led us to investigate possible functional interactions with proteins of the Bcl-2 family. Members of this protein family control mitochondria homeostasis and are the most well-defined regulators of the apoptotic mitochondrial path- way. More particularly, we were interested to know whether Mcl-1 (Myeloid Cell Leukemia 1), could interfere with galig func- tion. Mcl-1 is one of the main anti-apoptotic proteins of the Bcl- 2 family expressed in the same cell type as galig. Cotransfection experiments of vectors encoding galig and Mcl-1 have been per- formed. Our results indicate that galig-induced cytotoxicity is inhibited by Mcl-1 overexpression. This protecting effect against cell death was not observed with Bcl-2. Confocal microscopy and western blotting studies indicated that production of endogenous Mcl-1 was decreased in cells transfected with a galig encoding vector confirming the antagonism activities of galig and Mcl-1. These results suggest a common pathway between the cytotoxic activity of galig and the anti-apoptotic activity of Mcl-1. Future investigations will aim to characterize the nature of interaction between galig and Mcl-1.

P6–26 Proteinase inhibitor-9 in non-small cell lung carcinoma cells and tissues I. Rousalova1, E. Krepela1, J. Prochazka1, J. Cermak2 and K. Benkova3 1Laboratories of Molecular and Cell Biology, Department of Pneumology and Thoracic Surgery, University Hospital Bulovka, Prague, CZECH REPUBLIC, 2Division of Surgery, Department of Pneumology and Thoracic Surgery, University Hospital Bulovka, Prague, CZECH REPUBLIC, 3Department of Pathology, University Hospital Bulovka, Prague, CZECH REPUBLIC

The development of the domesticated chicken (Gallus gallus) is closely linked to lipid and lipoprotein metabolism. For the devel- oping chicken embryo, yolk is the major source of nutrients, since all required nutrients until hatching have to be provided by the egg. The major components of the yolk are VLDL and vitel- logenin (VTG), and LR8 (which is the chicken analogue of the human VLDLR) is the main receptor for yolk deposition during chicken oocyte growth. In our studies, we analyzed the role of another member of the LDL receptor gene family in the cellular uptake of these two yolk proteins. This recently found family member is a protein with an apparent molecular mass of 380 kDa, termed LRP380. The protein is suggested to fulfill a similar function as LR8 during the growth phase of the oocytes. Northern blot and RT-PCR analysis showed that the expression of this novel component is restricted to oocytes and yolk sac, similar to LR8. Biochemically, the binding properties of LRP380 are highly similar to those of LR8, in that it binds apo-B, VLDL, and VTG. Immunohistochemical studies showed that LRP380 as well as LR8 are localized in close proximity of granulosa cells, possibly in endocytic compartments of the oocyte. Furthermore, we have analyzed LRP380 mRNA transcript and protein levels in different stages of oocyte growth of wildtype and LR8-defi- cient R/O hens, and during embryogenesis.

P6–25 Mcl-1, a protein of the Bcl-2 family, protects cell death induced by galig gene expression P. Robinet, S. Charpentier, M. Dubois, L. Mollet, T. Normand and A. Legrand Centre de Biophysique Moleculaire, cnrs, Orleans cedex, FRANCE

that

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Aim: There is evidence that the proteinase inhibitor-9 (PI-9)/ser- pinB9 can suppress the granzyme B (GrB)-initiated apoptotic pathways in cancer cells and thereby it can protect them from immune-mediated deletion. In this study, we examined the PI-9 mRNA and protein expression status in non-small cell lung carci- noma (NSCLC) cell lines and NSCLC tissues and lungs from surgically treated patients. In NSCLC cell lines, we also evalu- ated the influence of the PI-9 protein expression level on the GrB-induced caspase-3-like activity. Methods: The expression of PI-9 mRNA was quantitated by real time RT-PCR. The PI-9 protein level was analyzed by SDS- PAGE and immunoblotting. The endogenous and GrB-induced caspase-3-like activities in NSCLC cell lines extracts were mea- sured with a kinetic fluorometric assay. Results: All major NSCLC tissue types and lungs showed the expression of PI-9 mRNA. In 24 (16%) of 150 studied NSCLC patients, the tumors had more than 2-fold higher PI-9 mRNA level as compared to matched lungs. PI-9 mRNA and protein expression was strong in six of 10 examined NSCLC cell lines and it was weak in the remaining four ones. In NSCLC cell lines extracts, the GrB-induced caspase-3-like activity showed negative correlation with the level of PI-9 protein. Conclusions: This work indicates that the expression of PI-9 mRNA and protein is upregulated in a subset of NSCLCs. More- over, our results suggest the PI-9 protein expressed in NSCLC cells can inhibit the active GrB. Acknowledgement: This work was supported by a project (MZO 00064211) from the Ministry of Health, Czech Republic. Galig (Galectin-3 Internal Gene) is a human cytotoxic gene that promotes cell death. This gene presents a particular organization in that it contains two different overlapping open reading frames and encodes two unrelated proteins. Mitogaligin, one of these proteins, is a 97 amino acid protein essentially targeted to mito- chondria. Mitogaligin destabilizes mitochondrial membrane by a still unknown process which involves interaction with cardiolipin, a specific phospholipid of mitochondria. Galig expression causes morphological alterations in human cells, such as cell shrinkage, cytoplasm vacuolization, nuclei condensation, and ultimately cell death. These alterations are associated with aggregation of mito- chondria and extramitochondrial release of cytochrome c. In vivo function of galig is still unknown. However, the gene is specifi- cally expressed in leukocytes from peripheral blood but not in bone marrow suggesting a possible regulation during differentia- tion. Indeed, differentiation of HL60 promyelocytic cells toward

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P6–27 Rapid detection of toxigenic Vibrio cholerae via PCR application from water samples collected in Sulaimania-Iraq S. K. Arif1, A. M. Rostam2, R. a. M. Al-Obaidi3, C. Hagemann4 and H. M. Said5 1Biology Department, Unviversity of Sulaimani College of Science, Sulaimani-Kurdistan Region, IRAQ, 2Biology Department, Unviversity of Sulaimani College of Science, Sulaimani-Kurdistan- Region, IRAQ, 3Department of Medical Microbiology, Unviversity of Sulaimani College of Vetrinary Medicine, Sulaimani-Kurdistan- Region, IRAQ, 4University of Wuerzburg Medical Faculty, Tumor Biology Laboratory Department of Neurosurgery, Wuerzburg, GERMANY, 5University of Wuerzburg Medical Faculty, Department of Radiation Oncology, Wuerzburg, GERMANY

Navigator System was used for PFGE, the running condition was; 100 mA/200 Volt/switching time 50 seconds/20 hours using isolates molecular analysis Not I restriction enzyme. Bacterial was by Pulse Field Gel Electrophoresis (PFGE). Parallel bio- chemical and serologic samples identification was done. Results: V. cholerae isolation from stool of nine patients with profuse watery diarrhea, eight were O1 (six ogawa and two in- aba, El Tor Biotype) and one isolate was NAG. Water samples were positive to Vibrio cholerae in Wasit (four isolates) in Bagh- dad (one isolates). PFGE analysis showed two clinical isolates (Inaba) genetically identical therefore epidemiologically related while, NAG isolates one clinical and one environmental isolates were genetically identical therefore epidemiologically unrelated representing a novel local strain and indicated that water is the infection source. Conclusions: Clonal relationship between clinical and environ- mental isolates exist and PFGE is a useful tool for epidemiologic surveillance of V. cholerae in the region. Also a new local ende- mic V. cholerae type was detected.

P6–29 Effects of 17ß-estradiol on oxidative stress and uncoupling proteins in MCF-7 breast cancer cells J. Sastre-Serra1, A. Valle2, J. Oliver1 and P. Roca1 1Fundamental Biology and Health Science, Balearic Islands Uni- versity, Palma, SPAIN, 2Fundamental Biology and Health Science, IUNICS, Palma, SPAIN

Background: Toxigenic Vibrio cholerae, the cause of cholera, is a native flora of the aquatic environment which is transmitted through drinking water and still remains the leading cause of morbidity and mortality in many developing countries. Methods: In the present study, we examined the presence of vir- ulence genes (ctxA, ctxB and tcpA of El Tor) in environmental strains of V. cholerae cultured from three sampling stations of the Sarchnar stream in Sulaimania-Iraq. We also evaluated the effect of different pre-PCR conditions: (i) water filtered and enriched in alkaline peptone water for 6 hours before PCR, (ii) water filtered without enrichment before PCR, and (iii) use of only enrichment in APW for 6 hours before PCR. Results: Unexpectedly, the PCR analysis of two out of 30 iso- lates revealed the presence of cholera toxin genes (ctxA, ctxB) and gene for toxin co-regulated pilus (tcpA) among environmen- tal non-O1 V. cholerae isolates. Conclusions: To our knowledge, this is the first evidence of the presence of toxin genes in non-O1 V. cholerae from the water environment in north Iraq. This study supports the idea that cholera toxin has an environmental derivation and that the intri- cate aquatic environment can give rise to pathogenic Vibrio organisms.

P6–28 Comparative molecular analysis of local Vibrio cholerae strains isolated from human patients and detection of a new V. cholerae type in Iraq S. K. Arif1, R. a. M. Al-Obaidi2, A. Al-Thawaini3, C. Hagemann4 and H. M. Said5 1Biology Department, Unviversity of Sulaimani College of Science, Sulaimani-Kurdistan Region Iraq, GERMANY, 2Unviversity of Sulaimani College of Science, College of Veterinary Medicine- Sulaimani University, Sulaimani-Kurdistan Region Iraq, GERMANY, 3Institute of Genetic Engineering and Biotechnology for higher studies, Baghdad University, Baghdad Iraq, GERMANY, 4University of Wu¨rzburg Medical Faculty, Tumor biology Laboratory Department of Neurosurgery, Wuerzburg, GERMANY, 5University of Wu¨rzburg Medical Faculty, Department of Radiation Oncology, Wuerzburg, GERMANY

Reactive oxygen species (ROS) are products of cell metabolism that not only provoke damage to cell structure, but also play a significant role in numerous chronic pathologies such as cancer. Estrogens are important signals for breast cancer risk since pro- mote cell proliferation and also are involved in the expression of antioxidant enzymes and ROS production. Uncoupling proteins (UCPs), by means of uncoupling electron transport chain from ATP synthesis, may influence ROS production in mitochondria playing a possible role as antioxidants. Our goal was investigate the effects of estrogens on oxidative stress and uncoupling pro- tein levels in breast cancer. Estrogen receptor alpha (ER-a)-posi- tive MCF-7 cells and (ER-a)-negative MDA-MB-231 cells were treated with 1 nM 17b-estradiol. Cellular ROS levels were deter- mined with DCFHDA by flow cytometry. Carbonyl protein con- tent was measured as oxidative damage parameter. Mn-SOD, catalase as well as UCP2, UCP3 and UCP5 protein levels were determined by western blot. Both ROS levels and oxidative dam- age in proteins were higher in 17b-estradiol treated MCF-7 cells whereas protein levels of Mn-SOD, catalase, UCP2, UCP3 and UCP5 were lower compared to vehicle treated cells. No effects were found in MDA-MB-231 cells. The higher ROS levels in 17b-estradiol treated MCF-7 cells may be in relation with a decreased antioxidant defenses as well as an impaired uncoupling capacity in mitochondria. Downregulation of UCP2, UCP3 and UCP5 proteins, through an enhancement of ROS production, may be one of the mechanisms contributing to the estrogen- induced progression and transformation of breast cancer.

P6–30 Interaction of Bordetella adenylate cyclase toxin with the beta 2 integrin CD11b/CD18 R. Osicka, J. Morova, A. Osickova, J. Masin and P. Sebo Cell and Molecular Microbiology Division, Institute of Microbiology, Prague 4, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Introduction: Between 2000–2008, serious cholera outbreaks occurred in Iraq. Here, pulse field gel electrophoresis (PFGE) was used as a DNA fingerprinting tool for first time in Iraq in order to detect possible infection sources and to compare between environmental and clinical isolates associated to cholera outbreaks. Materials and Methods: Two sample groups, namely, 100 acute diarrhea patients stool samples and 100 water samples from nine sites across Iraq were collected 2004–2006. Pathogen isola- tion and identification was according to standard methods. Gene Adenylate cyclase toxin (CyaA) is a member of the RTX (Repeats in ToXin) family and a key virulence factor of Bordetella pertussis,

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layer using immunohistochemistry,

the causative agent of whooping cough. CyaA delivers into cells an enzymatic adenylate cyclase domain, which catalyzes uncontrolled conversion of ATP to cAMP, a key signaling molecule subverting cell functions. Recently, CyaA was shown to use the b2 CD11b/ CD18 integrin as a specific cellular receptor. We show that degly- cosylation of cell surface proteins by glycosidase treatment, or inhi- bition of protein N-glycosylation by tunicamycin, ablates CyaA binding and penetration of CD11b-expressing cells. Competitive inhibition of CyaA binding to the integrin by excess of free sugars showed that CyaA directly recognizes the N-linked oligosaccha- rides of CD11b/CD18. Moreover, after initial interaction of CyaA with oligosaccharide chains, residues 614–682 of the CD11b sub- unit are crucial for specific binding of the toxin to target cells and their subsequent intoxication. Glycosylation of CD11a/CD18, another receptor of the b2 integrin family, was also essential for cytotoxic action of other RTX cytotoxins, the leukotoxin of Aggre- gatibacter actinomycetemcomitans (LtxA) and the Escherichia coli a-hemolysin (HlyA). These results show that binding and killing of target cells by CyaA, LtxA and HlyA depends on recognition of N-linked oligosaccharide chains of b2 integrin receptors and sets a new paradigm for action of RTX cytotoxins.

responsible for the molecular oscillations are also expressed in many peripheral tissues. The rat intestine exhibits daily rhythms in many physiological processes. The aim of the study was to provide an insight into the molecular basis of the circadian rhyth- micity within the rat intestine. The results demonstrate for the first time the rhythmic expression of canonical clock genes Per1, Per2, Cry1, Rev-Erba, Bmal1 and Clock, as well as of ionic exchanger NHE3 and cell cycle checkpoint kinase Wee1, in the in situ intestinal epithelial hybridization and real-time RT-PCR. Restricted feeding schedule shifted the circadian clock within the intestine and the liver, but not the clock within the SCN. A follow-up study focused on pre- cise analysis of circadian phase of the molecular clock along the intestinal tract. Expression of Per1, Per2, Rev-Erba, Bmal1 and Wee1 was measured in the duodenal, ileal, jejunal and colonic epithelial cells by real-time RT-PCR. Significant differences in phasing between the individual intestinal parts were proven, such that the upper part of the gut represented by the duodenum was phase-advanced to the lower part represented by the distal colon. In conclusion, the rat intestinal epithelium contains functional circadian clock, synchronized independently of the central clock and phased gradually along the cranio-caudal axis. The intestinal clock is likely responsible for local rhythms in absorption, motil- ity and cell proliferation.

P6–31 Effect of the ethacrynic acid on immune responses towards vaccinia virus P. Shankaran, J. Knitlova, Z. Cizek and Z. Melkova 1st Faculty of Medicine Charles University in Prague, Institute of Immunology and Microbiology, Praha 2, CZECH REPUBLIC

P6–33 Heme oxygenase in peripheral blood mononuclear cells of HCV infected patients I. Subhanova1, L. Muchova1, M. Lenicek1, L. Vitek1, H.J. Vreman2, O. Luksan3, T. Zima1 and P. Urbanek4 1Institute of Clinical Biochemistry and Laboratory Diagnostics, First Faculty of Medicine, Charles University in Prague, Prague, CZECH REPUBLIC, 2Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA, 3Institute of Clinical and Experimental Medicine, Centre of Experimental Medicine, Prague, CZECH REPUBLIC, 4Department of Internal Medicine, First Faculty of Medicine, Charles University in Prague, Prague, CZECH REPUBLIC

Wild type vaccinia viruses of various strains have been used in the past for a wide-spread vaccination against smallpox, leading to eradication of this life-threatening disease. Recently, certain countries have been re-introducing vaccination programs among selected groups of population and reconsidering a vaccination of the general population. However, the vaccines available reveal a substantial risk of post-vaccination complications, while our cur- rent therapeutic possibilities for poxvirus infections are rather limited. Consequently, there is a need of new agents effective against poxvirus infection. We have recently published an origi- nal observation that the ethacrynic acid, a common diuretic agent, inhibits vaccinia virus growth in vitro. On the other hand, ethacrynic acid has been shown to inhibit activation of NFkap- pa-B and thus affect various immune responses. Since inhibition of antiviral responses by ethacrynic acid would hamper the bene- ficial antiviral effects of this agent, we have decided to character- ize the effect of ethacrynic acid on immune responses towards vaccinia virus in vitro and in vivo. Our preliminary results indi- cate: (i) increasing concentrations of ethacrynic acid did not affect activation of a human T-cell line A3.01 by PMA, as char- acterized by expression of CD69. (ii) In sub-lethally infected mice, 2-day administration of the ethacrynic acid somewhat decreased the levels of vaccinia-specific IgG2a, as determined by ELISA, but the levels of antibodies produced remained consider- ably high. Additional analyses will be presented.

reduced (48.5 ± 17.4 patients was

P6–32 Circadian clock within the rat intestine M. Sladek1, L. Polidarova1, M. Sotak2, Z. Jindrakova1, J. Pacha2 and A. Sumova1 1Neurohumoral regulations, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, CZECH REPUBLIC, 2Epithelial physiology, Institute of Physiology Academy of Sciences of the Czech Republic, Prague, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Introduction: The mechanisms responsible for hepatocellular injury in the chronic hepatitis C are not fully understood. Oxida- tive stress induced by hepatitis C virus (HCV) infection is believed to play an important role. Heme oxygenase (HO) mark- edly participates in a response to oxidative stress. Indeed, reduced HO expression was reported in HCV-infected hepato- cytes. The aim of the present study was to characterize HO activ- ity and expression in peripheral blood mononuclear cells (PBMC) and HO expression in the liver of HCV-infected patients. Methods: The PBMC study was performed on 34 therapeuti- cally naive patients with HCV infection and 29 healthy subjects. Liver tissues were analyzed in 10 HCV-infected patients and 10 subjects with non-inflammatory liver diseases. HO activity was assayed by gas chomatography. HO expression was assayed by qRT-PCR. Results: HO expression in the liver of HCV infected patients was reduced to 55% compared to controls (0.25 ± 0.09 vs. 0.46 ± 0.23, respectively, p = 0.02), whereas no significant change in HO expression between patients and controls was seen in PBMC. However, compared to controls HO activity in PBMC vs. of HCV-infected 21.7 ± 19.1 pmol CO/106 cells/hour, respectively, p = 0.004). Significant correlation between mRNA and HO activity in PBMC was detected in control samples (p = 0.007). However, this relationship was suppressed in HCV-infected patients. While the central circadian pacemaker resides within the suprach- iasmatic nucleus of the hypothalamus (SCN), the clock genes

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IGA MZCR Conclusion: Patients with HCV infection exhibit suppression of HO expression in the liver as well as HO activity in PBMC sug- gesting HCV-related impairment of oxidative stress defense sys- tem. Acknowledgement: Supported by the grant NR9412-3.

P6–34 Radiation-induced HPA axis activation is associated with up-regulation of pro- inflammatory cytokines in rat brain N. Velickovic, D. Drakulic, S. Petrovic, I. Stanojevic, M. Milosevic and A. Horvat Laboratory for Molecular Biology and Endocrinology, Institute for Nuclear Sciences Vinca, Belgrade, SERBIA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

dose of 10 Gy c-rays or were sham-irradiated; animals were sac- rified 1, 2, 4, 6 and 24 hours after irradiation. The expression lev- IL-1b and TNF-a were els of pro-inflammatory mediators analyzed by ELISA both in the serum and hypothalamus. In par- allel, final product of HPA axis activity, corticosterone (CORT), was assessed in serum using ELISA assay. A significant up-regu- lation of TNF-a and IL-1b was observed in hypothalamus iso- lated from irradiated animals, while there were no changes of analyzed cytokines in circulation. Besides, cranial irradiation raised serum CORT level followed by down-regulation of GR protein and mRNA in hippocampus. To elucidate the molecular mechanisms of radiation-induced brain inflammation, expression of pro-inflammatory transcription factor NF-kB protein and mRNA in hippocampus were also examined. Observed NF-kB activation was consistent with the acute increase of pro-inflam- matory cytokines IL-1b and TNF-a in hypothalamus. These data suggest that irradiation induces pro-inflammatory pathway in the brain through overexpression of pro-inflammatory cytokines and activation of transcription factor NF-kB. In addition, our find- ings suggest that central, rather than peripheral, IL-1b and TNF- a production account for up-regulation of HPA axis, manifested finally by increase of circulating glucocorticoids. Exposure of juvenile rats to cranial irradiation adversely affects HPA axis stability, leading to its activation concomitantly with disturbed balance of corticosteroid receptor expression. We tested the hypothesis that radiation-induced rise of serum corticosterone is mediated by pro-inflammatory mechanisms. Male Wistar rats (18 days old) received either whole brain irradiation with a single

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P7 Trends in Biotechnology

P7–1 Production of monoclonal antibody against r-HBeAg and development of serological diagnostic kit I. Sogut, I. Hatipoglu, E. A. Akcael, F. Yucel, A. Manav, H. Kocaaga and A. Basalp Genetic Engineering and Biotechnology Institute, TUBITAK Marmara Research Center, Kocaeli, TURKEY

Methods: Mousehybridoma cells producing MAb against FMD (foot and mouth disease) virus (HIB85, HUKUK, Ankara, Tur- key) were transfected with Bcl-2 and control plasmids by using Lipofectamin reagent. Stable transfectants were selected in 400 mg/ml G418. The protein samples (3 · 106 cells per sample) taken from the cultures of two transfected hybridoma cell lines (HIB85-bcl2: over expressing the Bcl-2 gene and HIB85-PEF: control cell line) and one wild type cell line (HIB85) were ana- lyzed by Western blotting to assess Bcl-2 expression. Their apop- levels in batch cultures were also determined by an totic cell automated flow cytometric analysis and hemacytometric coun- tings. Results and Conclusions: Western blot analysis confirmed the expression of Bcl-2 in the Bcl-2 gene transfected cells (HIB85-bcl- 2) although there was no evidence of Bcl-2 expression in the con- trol and wild type cells. Flow cytometric analysis showed that apoptotic cell densities of control and wild type cells on 7th day were about 95% and 85%, respectively. However, apoptotic cell density of HIB85-bcl-2 transfectant cells remained at about 30% after 7th day. The viable cell number of HIB85-bcl-2 was about 7 · 105 cells/ml on 8th day without feeding or passaging while the all cells in control and wild type cultures were dead. These results indicate that Bcl-2 gene expression in HIB85 hybridoma cells suppresses the apoptosis in sub optimal culture conditions. It might be also deducted that HIB85-bcl-2 cells can survive in serum-free bioreactor cultures. Acknowledgements: This study was supported by Hacettepe University Research Found (project number: 0201602005) and NATO Science Fellowships Program by TUBITAK.

P7–3 Comparison of diagnostic kits developed with two different labeled-antibodies for the detection of HBsAg E. A. Akcael, A. Manav, A. Basalp and F. Yucel Genetic Engineering and Biotechnology Institute, TUBITAK Marmara Research Center, Gebze, Kocaeli, TURKEY

Hepatitis B virus (HBV) is a small DNA virus, causes viral hepa- titis, cirrhosis and liver cancer and is still considered a significant health problem worldwide. Serological and molecular assays are used for diagnosis of HBV and the test involves the measurement of HBV antigens (HBsAg, HBeAg) and antibodies (anti-HBsAg, anti-HBcAg, anti-HBeAg) in sera. HBeAg encoded by c gene indicates high infectivity and active viral replication. In this study, monoclonal antibody against recombinant Hepatitis e anti- gen (r-HBeAg) was produced by hybridoma technology and labeled with horseradish peroxidase (HRP) enzyme for the devel- opment of serological diagnostic kit. BALB/c mice were injected with 10 lg of r-HBeAg intraperitonally and anti-HBeAg response was tested by ELISA. After three subsequent immunization, mouse with the highest antibody titer for r-HBeAg was selected for fusion. Lymphocytes from spleen and lymph nodes were used in order to maximize the probability of generating monoclonal antibodies and fused with F0 myeloma cells (ATCC CRL 1646). After selection of positive clones with ELISA, it was found that out of 390 hybrid clones, 12E5 was producing antibody recogniz- ing both r-HBe and r-HbcAg as shown by immunblot analysis. Subisotype of antibody was determined as IgG2b. Antibody was produced in large scale and purified by ammonium sulfate precip- itation and Protein A column. Purified antibody was labeled with HRP enzyme and tested for the diagnosis of HBVe antigen in patient’s sera. The results have shown that 12E5-HRP conjugate can be effectively used in diagnostic kit for the detection of HBeAg. Acknowledgement: This work has been supported by TUBI- TAK Public Research Grant Committee (KAMAG 1007 – 105G056).

P7–2 Suppression of apoptosis by the Bcl-2 gene expression in HIB85 hybridoma cell cultures E. A. Akcael1, M. Gumusderelioglu2 and M. Al-Rubeai3 1Genetic Engineering and Biotechnology Institute, TUBITAK Marmara Research Center, Gebze Kocaeli, TURKEY, 2Chemical Engineering Department, Hacettepe University, Beytepe, Ankara, TURKEY, 3School of Chemical and Bioprocess Engineering, University College Dublin, Belfield Dublin, IRELAND

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Hepatitis B is a serious global public health problem. Hepatitis B surface antigen (HBsAg) is the most important protein of the envelope of Hepatitis B Virus causing acute and chronic viral hepatitis. HBsAg is also the first serological marker to appear in the serum of HBV-infected patients. In the last years, the ability to detect HBsAg with high sensitive immunoassays has led an understanding of its distribution worldwide and dramatically reduced the risk of infection in transfusion. The aim of this study is to compare two ELISA systems that were developed in insti- tute laboratory. In this study, two different conjugates were developed by labeling 2G3 monoclonal antibody (MAb) with biotin and horseradish peroxidase (HRP). 2G3 MAb with high specificity for Hepatitis B virus surface antigen (HBsAg) had already been produced in our previous project. Activities of the conjugates (2G3-HRP and 2G3-biotin) at different dilutions were tested by direct ELISA in microwells coated with HBsAg. Sand- wich ELISA kit systems were generated by using both 2G3 MAb (as capturing agent) and 2G3-HRP and 2G3-biotin conjugates (as detecting agents). In direct ELISA test, biotin and HRP con- jugates responded high absorbance values until about 1:64000 and 1:16000 dilutions, respectively. For HBsAg detection, ELISA test systems of HRP and biotin were compared by using hepatitis B positive and negative human sera. While showing higher activ- ity, biotin conjugate had some unexpected false positive reac- Introduction: Monoclonal antibodies (MAbs) by hybridoma cell cultures are produced in industrial processes for a range of com- mercial applications including diagnostic assays, drug targeting, antigen purification and direct immunization. However, some of these cells are very fragile both in serum-free media and under conditions of sub-optimal pH, oxygen levels, and shear stresses in reactors. Therefore, they easily undergo apoptosis in bioreac- tor environment. The objective of the present study is to suppress the apoptosis in HIB85 hybridoma cells by Bcl-2 gene transfec- tion. In our previous study, it was shown that HIB85 hybridoma cells are very sensitive to serum-free and stirred reactor condi- tions

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tions. Eventually, it was decided to use 2G3-HRP conjugates in optimization studies of HBsAg ELISA kit. Acknowledgement: This work has been supported by TUBI- TAK Public Research Grant Committee (KAMAG 1007 – 105G056).

P7–4 Fungal chitosan: isolation and biological characterization R. Albulescu1, E. Gheorghiu1, C. Ponta2, D. Barbulescu1, A. Casarica1, C. Marinescu1, D. Ghera1, V. Zaharachescu1 and A. Stan1 1Pharmaceutical Biotechnology, National Institute for Chemical Pharmaceutical R&D, Bucharest, ROMANIA, 2National Horia Hulubei Institute for National Institute of Physics and Nuclear Engineering – IFIN HH, Technical Irradiation Center, Bucharest, ROMANIA

carotenoids of green sulfur bacteria (2) were shown to induce aggregation of BChl c. Different properties of natural chloro- somes can be mimicked by selection of the aggregation inducing molecule. For instance, ability of a redox dependent excitation quenching, which is typical for chlorosomes of green sulfur bacte- ria, is achieved by co-aggregation of BChl c with quinones (3). The aim of this contribution is to find out why only carotenoids possessing at least one aromatic J-ring are found in chlorosomes from green sulfur bacteria. Several carotenoids with different end groups not naturally occurring in chlorosomes were selected, including those containing keto- and/or hydroxy-groups. Their co-aggregation with BChl c was studied. In addition, singlet energy transfer efficiency from carotenoids to BChl c in final assemblies was explored by fluorescence excitation spectroscopy. References: 1. Hirota M, Moriyama T, Shimada K, Miller M, Olson JM & Matsuura K. High degree of organization of bacteriochloro- phyll c in chlorosome-like aggregates spontaneously assembled in aqueous solution. Biochim. Biophys. Acta 1992; 1099: 271– 274.

2. Klinger P, Arellano JB, Vacha F, Hala J & Psencik J. Effect of carotenoids and monogalactosyl diglyceride on bacterio- chlorophyll c aggregates in aqueous buffer: implications for the self-assembly of chlorosomes. Photochem. Photobiol. 2004; 80: 572–578.

3. Alster J, Zupcanova A, Vacha F & Psencik J. Effect of qui- nones on formation and properties of bacteriochlorophyll c aggregates. Photosyn. Res. 2008; 95: 183–189.

P7–6 Phospholipid membrane permeabilization by a Rhodococcus sp. trehalose lipid biosurfactant A. Zaragoza, J. A. Teruel, F. J. Aranda and A. Ortiz Bioquı´mica y Biologı´a Molecular-A, Universidad de Murcia, Murcia, SPAIN

classified ‘weak’

Chitosan from fungal residual biomass was isolated, modified by gamma irradiation and investigated for toxicity and antimicrobial activity. Chitosan is present in significant amounts in cell walls of fungi; extraction of chitosan from residual fermentation biomass may provide an improvement in the use of renewable resources, providing a valuable raw material for pharmaceutical and cos- metics industry. Biosynthesis and isolation studies were carried on a citric acid producing strain of Aspergillus niger (A. niger ICCF 346), using standard biosynthesis conditions for citric acid biosynthesis in a 15 L New Brunswick BioFLo 2000 Fermenter. Biomass was isolated by filtration, alkaline conditions were used to eliminate other organic from biomass, then chitosan was extracted in hydrochloric acid and precipitated at pH 9.5. Purifi- cation by re-precipitation was performed, yielding 1.6 g purified chitosan. The product was characterized by chemical methods and by IR spectrometry. Further structural modifications were achieved by gamma irradiation (at levels of 33.7 and 50.8 kGy). Antimicrobial activity was assayed for irradiation modified chito- san on several fungal and bacterial strains; significant inhibitory lutea ATCC 9341 and C. albicans activity was proved for S. (inhibition zone between 20–35 mm), while moderate effects were demonstrated for S. typhymurium TA 100 and S. salivarius IP 55126 (inhibition zone 12–20 mm). Antimicrobial activity of non- irradiated precursor was (inhibition zone < 12 mm). Processing of residual biomass from industrial fer- mentation may provide a useful and functional derivative of chitosan, suitable for pharmaceutical and cosmetic applications.

trehalose

P7–5 Effect of carotenoid end groups on formation and properties of bacteriochlorophyll-c aggregates J. Alster1, J. Arellano2, T. Polivka3, F. Vacha4 and J. Psencik1 1Department of Chemical Physics and Optics, Faculty of Mathematics and Physics, Charles University, Prague, CZECH REPUBLIC, 2Instituto de Recursos Naturales y Agrobiologı´a (IRNASA-CSIC), Estre´s abio´tico, Salamanca, SPAIN, 3Academy of Sciences of the Czech Republic, Biology Centre, Ceske Budejovice, CZECH REPUBLIC, 4Institute of Physical Biology, University of South Bohemia, Nove Hrady, CZECH REPUBLIC

trehalose

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Surface active amphiphilic compounds of biological origin, known as biosurfactants, are encountering interesting applica- tions as new chemical and biological properties are being described. In general, biosurfactants are preferred over surfac- tants of chemical origin because they are more biodegradable and environmentally friendly. In addition, some biosurfactants display interesting biological activities, for instance antifungical or, in general, antimicrobial, which is an added value to their potential uses. Furthermore, some of these compounds can be produced through biotechnological processes using waste materi- als as carbon sources, which is an added value not only from the economical point of view, but also because of ecological reasons. Thus, a strong research activity is directed toward the identifica- tion of new biosurfactants and characterization of their chemical and biological properties. Here, we report on the interactions of a succinoyl bacterial trehalose lipid biosurfactant produced by Rhodococcus sp. with phospholipid vesicles, leading to membrane permeabilization, as studied by means of calorimetric and fluores- cence and absorption spectroscopical techniques, in search for a molecular model. Binding of lipid to palmi- toyloleoylphosphatidylcholine membranes was studied by means of isothermal titration calorimetry. The partition constant, in conjunction with the CMC, indicated that trehalose lipid behaved as a weak detergent, which preferred membrane incorporation over micellization. Addition of lipid to palmi- toyloleoylphosphatidylcholine large unilamellar vesicles resulted in a size-selective leakage of entrapped solutes to the external medium. Experimental evidence support the requirement of a stage of flip-flop previous to membrane permeabilization, and the rate of flip-flop was measured using fluorescent probes assays. Carotenoids are together with bacteriochlorophyll (BChl) c, d or e aggregates important constituents of chlorosomes, the light-har- vesting antennae of green photosynthetic bacteria. BChl aggre- gates with optical properties similar to those of chlorosomes can also be prepared in vitro, in aqueous environments from a mix- ture of BChl and a suitable non-polar molecule. Lipids (1) and

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lipid incorporates idea that trehalose the

The lipid composition of the target membrane was found to modulate the leakage process to a great extent. It is proposed that trehalose lipid incorporates into phosphatidylcholine mem- branes and segregates within lateral domains which may consti- tute membrane defects or ‘pores’, through which the leakage of small solutes might take place. The results presented here con- tribute to the knowledge of the molecular mechanisms underlying the membrane-related biological actions of this bacterial trehalose lipid biosurfactant.

experiments showed that trehalose lipid increases the fluidity of the phosphatidylglycerol acyl chains and decreases the hydration of the interfacial region of the bilayer. Fluorescence polarization of probes with different location in the bilayer showed that treha- lose lipid modulates the order of the bilayer. These results sup- port into the phosphatidylglycerol bilayers and produces structural perturba- tions which might affect the function of the membrane. Acknowledgements: Supported by Project CTQ2007-662445 from Ministerio de Educacio´ n y Ciencia; and BIO-QMC-06/01- 001 from Comunidad Auto´ noma de la Region de Murcia, Spain.

P7–7 Effect of enzymes on after treatment of acid based dyed nylon fabric G. Arabaci and A. Usluoglu Chemistry, Sakarya University, Sakarya, TURKEY

P7–9 Selenocysteine Se-methyltransferase gene in Astragalus chrysochlorus S. Ari and O. Cakir Molecular Biology and Genetics, Faculty of Science, Istanbul University, Istanbul, TURKEY

Enzymes are very important applications in textile industries. Nowadays, hydrolytic enzymes such as cellulases, amylases, pec- tinases and proteases have been realized as powerful tools in vari- ous textile-processing steps such as removing wool-fibre-scales, removing organic protein-based stains, removing fibrils and fuzz fibres. Ligninolytic enzymes such as laccases and peroxidases have also been shown to decolourise textile dyes. Another enzyme is catalase for the conversion of hydrogen peroxide pres- ent in bleaching to oxygen and water. This work concerns the development of a syntan based after treatment for acid dyed Polyamide/Elasthane (PA/EL) fabric, in improving the fastness after syntan after treatment by using different enzymes. The enzymes used in this work were laccase, amylase, lipase, protease and pectinase. Polyamide/Elasthane (PA/EL) fabric were dyed with acid based dye (Telon Blue M2R: 2% [DyStar]) in acetic acid (pH 4.0–4.5) at 102(cid:2)C and later at 79(cid:2)C. Dyed samples were after treated using commercially available syntan. After the sam- ples were rinsed with tape water, the enzymes were applied to the dyeings at pH 5.5. Then, the samples were analyzed by using Wet Colour Fastness (ISO 105 C06 B1S) method at 50(cid:2)C. The results were evaluated according to Grey Scale. The results showed that after treatment with amylase had no effect the col- our fastness at all. But, the after treatment with pectinase, lipase and protease enzymes were increased the colour fastness of the fabric by means of 0.5 point. Laccase had the best effect the col- our fastness by 1 point. Laccase can be used to improve after- treatment processes in industry.

Selenium (Se) plays an indispensable role in human nutrition and has been implicated to have important health benefits, including being a cancer preventative agent. While different forms of Se vary in their anticarcinogenic efficacy, Se-methylselenocysteine (SeMSC) has been demonstrated to be one of the most effective chemopreventative compounds. However, excess Se may be toxic, and the observation that Se bioaccumulation is toxic to wildlife, has generated a surge of interest in phytoremediation of Se. The largest group of Se-hyperaccumulating plants belongs to the genus Astragalus (Fabaceae). The well known hyperaccumulator Astragalus bisulcatus accumulates up to 0.65 w/w dry weight Se. Selenocysteine methyltransferase (SMT), specifically methylates selenocysteine (SeCys) to produce the nonprotein amino acid Se- methyl selenocysteine (SeMSC) and plays a key role of removing selenium toxic effect at higher levels. Selenocysteine methyltrans- ferase (SMT) has been cloned from A. bisulcatus. Transformation of the A. bisulcatus SMT gene to A. thaliana and Indian mustard were incresed their selenium tolerance. Here we report the sele- nium accumulating ability of Turkish endemic Astragalus chrys- ochlorus by selenium analysis with ICP-MS in plants which were grown in MS medium supplemented with sodium selenate (1– 25 ppm). ICP-MS studies showed that A. chrysochlorus accumu- lates up to 300 mg/kg DW selenium. Additionally, cDNA frag- ment of selenocysteine methyltransferase which obtained by RT- PCR and RACE-PCR, was shown 95% similarity between A. bi- sulcatus SMT gene.

P7–8 Effects of a bacterial biosurfactant trehalose lipid on phosphatidylglycerol membranes F. J. Aranda, A. Ortiz and J. A. Teruel Bioquimica y Biologia Molecular, Universidad de Murcia, Murcia, SPAIN

P7–10 Use of galactomannan edible coatings for minimally processed mango fruits A. M. C. Oliveira1, S. R. S. Arcanjo2, F. J. Beserra3, A. C. O. Monteiro-Moreira4 and R. A. Moreira4 1Nutricao, Universidade Federal do Piauı´ – UFPI, Picos, BRAZIL, 2Nutricao, Universidade Federal do Maranhao – UFMA, Imperatriz, BRAZIL, 3Nutricao, Universidade de Fortaleza – UNIFOR, Fortaleza, BRAZIL, 4Pharmacy, Universidade de Fortaleza – UNIFOR, Fortaleza, BRAZIL

techniques. Differential scanning

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Edible films and coatings are an interesting alternative to increase the shelf-life of food in comparison to packages based on petro- leum derivatives, both due to the non toxicity as well as to eco- logically reasons. Their ability to regulate the migration of moisture, lipids and gases, can be used to improve the quality and exteshelf life of foodstuffs. The minimally processed products are usually more sensible to contamination than the intact fruit, due to severe physical stress occurred during the process, when Trehalose lipids are biosurfactants produced by rhodococci that, in addition to their well known potential industrial and environ- mental uses, are gaining interest in their use as therapeutic agents. The study of the interaction of biosurfactants with mem- branes is important in order to understand the molecular mecha- nism of their biological actions. In this work we look into the interactions of a bacterial trehalose lipid produced by Rhodococ- cus sp. with dimyristoylphosphatidylglycerol membranes by using differential scanning calorimetry, and infrared and fluorescence spectroscopy calorimetry showed that trehalose lipid makes the pretransition to disappear and broadens and shifts the phospholipid gel to liquid crystalline phase transition to lower temperatures, with a decrease of the enthalpy of the transition. Trehalose lipid presents good miscibil- ity both in the gel and the liquid crystalline phases. Infrared

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identified many new potential feruloyl

from these polymeric structures and have received an increas- ing interest in industrial applications such as in the food, pulp and paper and bio-fuel industries. BLAST analysis of 26 fun- gal genomes using characterized feruloyl esterase encoding genes demonstrated that some FAE-types are only found in the fungal species (e.g. FaeA only in Aspergilli), subsets of while others are more commonly found throughout the fungal kingdom. Phylogenetic analysis allowed the classification of putative feruloyl esterases into seven sub-families. The substrate specificity of the esterases does not correlate with the division over the families suggesting that prediction of substrate speci- ficity of novel feruloyl esterases can not be done solely on sim- ilarity to already characterized feruloyl esterases. Interestingly, several fungal species contain multiple candidates per subfam- ily, suggesting gene duplication, possibly as a result of a high need for this enzyme activity in their natural biotope. This study has esterase encoding genes that may be interesting candidates for indus- trial applications.

rapid chemical and biochemical reactions can occur. These reac- tions may cause severe modifications in the sensorial and nutri- tional qualities. Edible films and coatings may protect the food from deterioration, improving their shelf life. The use of Cae- salpinia pulcherrima endospermic galactomannan film for packag- ing minimally processed mango was evaluated. ‘Tommy Atkins’ mango fruits, after been washed with detergent and tap water, sanitized (200 mg/ml), refrigerated (10(cid:2)C, 12 hour), peeled and cut in small slices were coated with a galactomannan (1% to 1.5%) and glycerol (1%) blends and stored at 5(cid:2)C in plastic vials. Every 4 days, until 40 days of treatment, samples were taken to evaluate the humidity, loss of weight, pH, total and soluble solids and total titrated acidity. The results obtained showed that the galactomannan coating application positively influenced the pres- ervation of the mango physico-chemical characteristics, with all parameters showing retardation in the fruit senescence when compared to the uncoated samples. The coated fruits showed the same physicochemical and chemical values when compared with non-cut samples. Acknowledgement: Supported by Capes, CNPq, FUNCAP, RENORBIO.

P7–13 Abstract withdrawn

P7–11 Beta-D-fucosidase activity of beta-D- galactosidase from the bacterial strain Paenibacillus thiaminolyticus E. Benesova, P. Lipovova and B. Kralova Department of Biochemistry and Microbiology, Institute of Chemical Technology Prague, Prague, CZECH REPUBLIC

P7–14 Carbon as a label for the detection of bacterial nucleic acids by immunochromatographic tests M. Blazkova1, Z. Smidova1, L. Fukal1, P. Rauch1, J. Wichers2 and A. V. Amerongen2 1Department of Biochemistry and Microbiology, Institute of Chem- ical Technology, Prague, CZECH REPUBLIC, 2Agrotechnology & Food Innovations B.V. (A&F), Diagnostics & Bioactive Peptides, Wageningen, THE NETHERLANDS

Beta-D-galactosidase (beta-D-galactoside galactohydrolase) is a widespread enzyme very often possessing two different activities – hydrolytic and transglycosylation activity. The former one is used in food processing in the production of low-lactose milk, the latter one is useful in enzymatic synthesis of different types of oligosac- charides or glycoconjugates. The transglycosylation activity in con- nection with not strict substrate specificity of some beta-D- galactosidases opens the possibility to use these enzymes for trans- fer of fucosyl residue to different acceptor molecules and thus for the production of e.g. bifidus-factors or in the synthesis of alkyl fu- cosides as non-ionic surfactants. In this work a gene encoding beta-D-galactosidase with beta-D-fucosidase activity from the bac- terial strain Paenibacillus thiaminolyticus was obtained, sequenced and as a recombinant His-tagged protein produced in the cells E. coli BL21. After the purification by affinity chromatography on Ni-NTA agarose column the enzyme was characterized and its ability to catalyze transfucosylation reactions was tested. It was confirmed that different types of molecules (p-nitrophenyl glyco- sides, alcohols) can serve as acceptors in these reactions. It indi- cates, that this enzyme could find several applications in synthesis of different types of fucosylated molecules. Acknowledgements: This work was supported by the Ministry of Education, Youth and Sports (MSM6046137305).

P7–12 Biotechnological applications and potential of fungal feruloyl esterases based on prevalence, classification and biochemical diversity I. Benoit1, E. G. J. Danchin2, R. Bleichrodt1 and R. P. de Vries1 1Microbiology, Utrecht University, Utrecht, THE NETHERLANDS, 2INRA, IBSV, Nice Sophia-Antipolis, FRANCE

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Feruloyl esterases are part of the enzymatic spectrum employed by fungi and other microorganisms to degrade plant polysac- charides. They release ferulic acid and other aromatic acids The genus Listeria consists of a group of gram-positive, faculta- tive anaerobic rodshaped bacteria. All of the six recognised Liste- ria species can be isolated from a diversity of environmental sources, including soil, water, effluents, a large variety of foods, and from faeces of humans and animals. Although occurrence of Listeria strains in food may indicate errors in good hygienic and manufacturing practice, only L. monocytogenes is a significant human and animal pathogen responsible for the serious illness listeriosis. Lateral flow tests based on the principles of immuno- chromatography are used for the detection of a wide range of target analytes. For the visualisation of the method different nano-particles (gold, carbon, latex) are used. The benefits of these tests include: user-friendly format, short time to get test result, long-term stability, and relatively low price. A new prom- ising application is the detection of genetic material of pathogen microorganisms. Firstly, the DNA is isolated from bacterial cells, and used for PCR with labelled primers. Secondly, a small vol- ume of the final PCR solution is directly added to the assay device and the appearance of a black line is indicative of the presence of the specific amplicon. The method ‘Nucleic acid lat- eral flow immunoassay’ (NALFIA-Listeria) previously developed in our laboratory for the detection of listerial cells is using car- bon particles. The NALFIA was applied on a range of real food samples (e.g. milk, vegetable, delicatessen salads, and sprouted seeds). Acknowledgements: This work was supported by the Grant of Ministry of Education MSM 6046137305 and National Research Programme II 2B08050.

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P7–15 The detection of N-methylcarbamate pesticide methiocarb in baby foods M. Blazkova, P. Plackova, Z. Smidova, P. Rauch and L. Fukal Department of Biochemistry and Microbiology, Institute of Chemical Technology, Prague, CZECH REPUBLIC

revealed that the estimated half-life of recombinant mini TyrRS protein expressed in E. coli was more than 10 hours which classifies the protein as stable. The catalytic activity of recombinant N-mod- ule of mammalian TyrRS has been studied in the aminoacylation reaction of tRNA. It was shown the absence of inhibition of tyro- syl-tRNA synthetase lacking the COOH-terminal module by non- cognate tRNAs in the aminoacylation reaction. In order to test the angiogenic activity of N-terminal module of bovine tyrosyl-tRNA synthetase, in vivo angiogenesis assay on chorioallantoic membrane (CAM) was performed with 10 day chick embryos. It was shown that recombinant N-module of TyrRS strikingly induced blood vessel development at 0.26 nM concentration. These data support the possibility that N-terminal module of mammalian tyrosyl- tRNA synthetase can act as an angiogenic factor.

P7–17 Quantification of secondary structure elements of food proteins by diffuse reflectance FT-IR spectroscopy (DRIFTS): relationship to in vivo utilization M. Carbonaro1, A. Nucara2 and P. Maselli2 1Department of Nutrition, INRAN, Rome, ITALY, 2Department of Physics, University of Rome Sapienza, Rome, ITALY

N-methylcarbamates are widely used for agricultural and residen- tial applications as a bird repellent and as an insecticide. How- ever, carbamate pesticides are toxic to non-targeted wildlife (fish and birds). Their residual concentration in agricultural products must be monitored in order to avoid environmental pollution and to keep our health. The aim of this work was to apply sim- ple, user-friendly and portable competitive lateral flow immuno- assay for rapid detection of methiocarb in juices and baby foods. The lateral flow immunoassay for the detection of carbamate pes- ticide methiocarb using monoclonal antibodies was previously developed in our laboratory. Tests were prepared by spraying im- munoreagents (coating methiocarb-OVA conjugate as test line, RASw as control line) on a nitrocellulose membrane strip using Linomat V. Before the application to the test, analyzed sample was mixed together with reaction mixture containing running buffer SwAM-carbon conjugate, and anti-methiocarb antibody. 10 minutes after application of 100 ll of this solution, intensity of colour in detection zone of the strip was observed and judged by eyes or by grayscale densitometry. During this work, the influ- ence of baby food matrices was studied, the detection limits were estimated and spiked samples of different types of baby foods were analyzed and statistically evaluated. Acknowledgement: This work was supported by the Czech Grant Agency, No. 525/07/0618 and Ministry of Education, grant No. MSM 6046137305.

P7–16 Bacterial expression of N-terminal catalytic module of mammalian tyrosyl-tRNA synthetase and its function as an angiogenic factor I. Bozhko and O. Kornelyuk Protein Engineering and Bioinformatics, Institute of Molecular Biology and Genetics, NAS of Ukraine, Kiev, UKRAINE

FT-IR spectroscopy has been largely applied to detect the sec- ondary structure of proteins through analysis of amide I band (1600–1700 cm-1), whose sensitivity to conformational changes makes possible to study protein folding/unfolding as well as aggregation processes. In the present study, the secondary struc- ture of food proteins of known 3D structure – a-lactalbumin, b- lactoglobulin, BSA, a-casein, b-casein, concanavalin A, 7S globu- lin – was determined by diffuse reflectance FT-IR spectroscopy (DRIFTS). The same experimental procedure was then applied to proteins in whole food powder before and after thermal treat- ment to investigate relationship between structural properties and in vivo protein utilization. In the spectra of amide I band the components at 1638, 1645, 1654 and 1666 cm)1 were assigned to b-sheet, random coil, a-helix and b–turns modes, respectivety, the bands at 1618 cm)1 and 1683 cm)1 to intermolecular and intramolecular aggregates, respectively. In a-lactalbumin and BSA, a-helix was the major component (42% of the total inten- sity), while in b-lactoglobulin the main element was b-sheet. In a- and b-casein, random coil was the dominant contribution (70%). 7S protein and concanavalin A contained a similar amount of b- sheet (35%) and aggregates (40%). Analysis of whole food (pas- teurized milk, raw and autoclaved legumes) showed that proteins in milk were characterized by random coil conformation. Legume flours contained a high content of b-sheet structures (up to 47%). The autoclaving treatment reduced the content of b-sheets and turns, while increasing inter- and intra-molecular beta aggre- gates of high stability. Such modifications may adversely affect protein utilization, as suggested by a significant impairment in protein digestibility and amino acid bioavailability measured in our animal (rat) trials after heating of legumes. Acknowledgement: Research supported by MiPAAF with funds released by C.I.P.E. – Resolution 17/2003.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Mammalian tyrosyl-tRNA synthetase is composed of NH2-termi- nal catalytic module and appended non-catalytic COOH-terminal domain. Recently it was shown that tyrosyl-tRNA synthetase can be split into two fragments having distinct cytokine activities estab- lishing a link between protein synthesis and signal transduction processes. The non-catalytic COOH-terminal domain reveals cyto- kine activities which are similar to endothelial monocyte activating polypeptide II. On other hand, NH2-terminal catalytic TyrRS fragment may be chemotactic for neutrophils and functions as an inductor of angiogenesis in a concentration-dependent manner. In this regard it is important to explore both structural and functional properties of N-terminal module of mammalian tyrosyl-tRNA syn- thetase as a novel biotechnological product which has therapeutic potential across human diseases. In this work we have performed cloning and bacterial expression of the N-terminal module of bovine tyrosyl-tRNA synthetase into pET23d vector with COOH- terminal localization of 6His-tag sequence. The bacterial expres- sion of recombinant N-terminal module of bovine tyrosyl-tRNA synthetase was performed in E. coli BL21 (DE3) cells for subse- quent structural and functional studies. The optimization of bacte- rial expression of mini TyrRS in soluble form was achieved in the conditions when cell growth was performed at 20(cid:2)C and 1 mM IPTG concentration. Bioinformatical analysis with ProtParam tool

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P7–18 Transient expression of E7 and L2 derived peptides from human papillomavirus type 16 using Potato virus X-based vector N. Cerovska1, H. Plchova1, T. Moravec1, H. Hoffmeisterova1, J. Folwarczna1, M. Salanska1, M. Smahel2 and V. Ludvikova2 1Virology, Institute of Experimental Botany v.v.i.CAS, Prague, CZECH REPUBLIC, 2Experimental Virology, Institute of Hema- tology and Blood Transfusion, Prague, CZECH REPUBLIC

ber of dividing cells, dispersed randomly among the three-dimen- sional culture. These results were confirmed by the established sustained steady-state level of ERK1/2 activation and cadherin and cylin D down regulation. In addition, we found that in these in vivo-like conditions, plasma membranes are enriched in raft domains, as judged by the increased amount of cholesterol and sphingomyelin, suggesting different mode of signaling induced by the three-dimensional environment. Our future aim is to develop this 3D culture further as a model system for studies of fibrosis, cancer cell invasion and drug testing.

P7–20 Alpha-amylase production by a newly isolated Bacillus subtilis under solid state fermentation using banana husk as substrate S. Ozdemir1, K. Guven1, Z. Baysal2 and O. Demirci1 1Biology, Dicle University, Diyarbakir, TURKEY, 2Chemistry, Dicle University, Diyarbakir, TURKEY

The production of extracellular alpha-amylase by Bacillus subtilis was studied in solid state fermentation (SSF). In a sequential order, the various process parameters were optimized for maxi- mal alpha-amylase production. The tested process parameters were different solid substrates, different incubation time, particle size, initial moisture content of the substrate, inoculum size, inoc- ulum concentration and screening of substrate mixtures in the ratio of 1:1 and 1:3 for the production of alpha-amylase by Bacil- lus subtilis. The maximum productivity of amylase (3593 U/mg) was achieved by utilizing with banana husk (BH) + wheat bran (WB) in the ratio of 3:1 for 72 hours at 37(cid:2)C, at an inoculum level of 30%, an initial moisture content of 60%, a particle size of 1500 lm. Transient expression of heterologous proteins in plants represents an attractive strategy for vaccines production combining cost- effectiveness and safety. The optimized expression of recombi- nant Potato virus X coat proteins (XCP) carrying three different epitopes from human papillomavirus type 16 (HPV-16) was developed. Two epitopes derived from the L2 minor capsid pro- tein and one from the E7 oncoprotein were joined as N-terminal or C-terminal fusions with XCP of a Potato virus X (PVX)-based vector and these recombinant proteins were initially expressed in E. coli to prove their ability to form virus-like particles (VLPs). Then, the transient expression in plants using Agrobacterium tum- efaciens mediated inoculation was performed. To increase the level of the produced proteins the transgenic Nicotiana benthami- ana plants expressing the Potato virus A HC-Pro gene were tested. The expressed proteins purified either from bacteria or plant material by means of gradient centrifugation were exam- ined for correctness and stability of their sequences. From plants, only recombinant VLPs carrying the L2 (10–120) epitope on the XCP N-terminus were obtained. Immunogenicity of these VLPs was tested after immunization of mice. VLPs were injected subcu- taneously or administered by a tattooing device. In animal sera the antibodies against the viral CP and the L2 epitope were found after both methods of vaccine delivery. Acknowledgement: This research was supported by the grant No. 521/09/1525 of the Czech Science Foundation.

P7–21 Heavy metal bioaccumulation by thermophilic Bacillus thermoantarcticus and Anoxybacillus amylolyticus S. Ozdemir1, K. Guven1, E. Kilic2, O. Demirci1 and B. Nicolaus3 1Biology, Dicle University, Diyarbakir, TURKEY, 2Chemistry, Dicle University, Diyarbakir, TURKEY, 3Istituto di Chimica Biomolecolare, CNR, Napoli, ITALY

P7–19 Biochemical and immunohistochemical characterization of tissue-like three dimensional culture system R. Damianova1, N. Stefanova1, R. Pankov1, G. Staneva2, D. Petkova2 and A. Momchilova2 1Faculty of Biology, Sofia University ‘‘St. Kliment Ohridski’’, Sofia, BULGARIA, 2Bulgarian Academy of Sciences, Institute of Biophysics, Sofia, BULGARIA

In this study, heavy metal resistance and bioaccumulation were studied by thermophilic Bacillus thermoantarcticus and Anoxyba- cillus amylolyticus. It was determined that the highest bioaccumu- lation capacity performed during 24 hours incubation by B. thermoantarcticus for Cd, Co, Cu, Mn was; 774.8 (20 hour), 620.1 (20 hour), 604.05 (8 hour) and 24503.07 (8 hour) lg/g dry bacteria, respectively. According to these results, it was deter- mined that the highest metal capacity which is bioaccumulated by B. thermoantarcticus was Mn, the lowest was Cu. It was found that the highest bioaccumulation capacity performed during 24 hours incubation by A. amylolyticus for Cd, Co, Cu, Mn was; 50 739 (20 hour), 3273 (20 hour), 92 968 (20 hour) and 28566 (20 hour) lg/g dry bacteria respectively. It was determined that the highest bioaccumulation capacity performed was Mn and lowest bioaccumulation capacity performed was Co.

collagen and laminin. Our

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Current model system for investigation of cell signaling mecha- nisms is conventional two-dimensional (2D) cell culture. How- ever, the living organism is a three-dimensional (3D) structure in which each cell is in contact with other cells and/or components of the extracellular matrix. The cell-cell and especially cell-matrix contacts in three-dimensional environment are different from the contacts organized in 2D culture. Since cell contacts affect pro- foundly the signal transduction cascades, our studies are aimed at development and characterization of three dimensional culture system suitable for studies of cell signaling in in vivo-like condi- line that can proliferate tions. We used mouse GD25b1 cell beyond confluent monolayer. After reaching 100% confluency, cells were grown for additional 5 days to generate tissue-like sys- tem of cell embedded in extracellular matrix enriched in fibronec- tin, results demonstrated that confluent GD25b1 culture maintained linear growth up to 16 days after confluency without signs of senescence or increased apoptosis. Immunohistochemistry with cyclin D demonstrated that the growth of the culture was supported by a constant num-

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detected as HbS heterozigous, 10 were HbS mutant and 12 fetuses were wild type. This method was confirmed by invasive techniques. Cell-free fetal DNA circulating in maternal plasma seemed to increase its usage at noninvasive prenatal diagnosis of various hereditary diseases.

P7–22 The effect of Astrazone Red FBL textile dyes on catalase enzyme of Phanerochaete chrysosporium O. Demirci1 and D. Asma2 1Biology, Dicle University, Diyarbakir, TURKEY, 2Biology, Inonu University, Malatya, TURKEY

P7–24 Transfection of spermatozoa and production of transgenic embryos and larvae in Bivalve Molluscs P. Esponda1 and R. Guerra2 1Cell Biology & Development, CIB-CSIC, Madrid, SPAIN, 2Faculty of Sciences, University of Valparaı´so, Valparaı´so, CHILE

We employed the p-Gene Grip gene construction which expresses the Green Fluorescent Protein (GFP) for the transfection of sper- matozoa in two Bivalve Molluscs (Mytilus galloprovincialis and Mytilus chilensis). Both species are an alimentary resource and they have commercial importance. After short periods of incuba- tion with the gene construction the transgene was located in the sperm nucleus using a laser confocal microscopy. The efficience of transfection was of 58.5% in M. galloprovincialis and of 70.0% in M. chilensis. Furthermore, the use of PCR method also demonstrated that the transgene was located into the sperm DNA, and Southern Blot techniques indicated a possible integra- tion of the transgene in the spermatozoon DNA. Transfected spermatozoa of M. chilensis were mixed with eggs for fertiliza- tion, and the fluorescence analysis showed that a number of transgenic embryos and larvae (Veliger type) were developed. These transgenic embryo and larvae show green fluorescence in their cells when observed under the fluorescence microscope. Nevertheless, the number of modified embryo and larvae pro- duced by transfected spermatozoa appeared decreased when com- pared to those produced by non treated spermatozoa. This result seems to be an important and goal for future gene manipulations in these species of commercial importance.

A numerous amount of polluting substances are disposed to natu- ral resources in an uncontrolled manner. Due to this, all livings have been subjected to different ‘xenobiotic’ or toxic substances. And some of these substances are textile dyes. Metabolites formed as a result of biodegradation of textile paints can be carcinogenic, mutagenic or toxic materials. Antioxidant protection system, made up of enzymes of the living such as catalase, has function for pro- tection from these xenobiotic and toxic materials as well. This enzyme is the strong biomarker of oxidative stress formed in cell. The aim of this study is to show effected Antioxidant protection system by Astrazone Red FBL textile dyes. In this study the effect of different concentrational (20 ppm and 50 ppm) of textile dyes on the different seasonal (2, 4, 8, 12, 16 vs. 20 days) Phanerochaete chrysosporium and how antioxidant system is effected have been investigated and changes in Cathalase, enzyme activity and in total level of Glutathione have been determined. The enzyme activity was determined by Luck method. An induction in Cathalase, Glu- tathione s-transferase and Glutathione reductase activity in the early phase of low concentration (20 ppm) of paint application and Glutathione level, and an inhibition in the late phase have been determined. Enzyme activities in the early and late phase of high concentration (50 ppm) of paint application and inhibition in total Glutathione level have been determined. We showed that Astrazone Red FBL textile dye effects cellular defense systems and demonstrated the susceptibility of P. chrysosporium to textile dyes- induced oxidative stress. Given the negative impact on Glutathi- one and defense mechanism (e.g., as catalase) the dye can also decrease the degradation potential of the fungus. In conclusion, in an environment where not only the antioxidant response of P. chrysosporium but also the risks of xenobiotics, caused especially by textile industry and discharged irresponsibly to the environment leading environmental pollution to which all living systems are exposed to, were tried to be found out in this study.

P7–23 High resolution melting analysis for genotyping cell free fetal DNA E. Dundar Yenilmez, A. Tuli and D. Du¨ zgu¨ nce Biochemistry, Health Science, Adana, TURKEY

P7–25 An efficient phosphorylation-free and ligase- free PCR-based method for multiple site- directed mutagenesis of up to six distal-sites simultaneously T. Y. Fang, J. W. Lin and X. G. Hung Food Science, National Taiwan Ocean University, Keelung, TAIWAN

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Melting curve analysis that combined with real-time PCR was introduced in 1997. Its a new method for DNA analysis espe- cially for genotyping and mutation scanning. Flourescence analy- sis of DNA melting is more sensitive and only nanogram amounts are needed. Cell free fetal DNA in maternal plasma is increasingly investigated in pregnancy in recent years. Genotyp- ing the mutation of cell free fetal DNA non-invasively is an important goal of prenatal diagnosis. In the present study we aimed to detect sickle cell anemia mutation (HbS) by high resolu- tion melting analysis in fetal DNA from maternal plasma. Mater- nal blood samples (5 ml) were collected into tubes containing EDTA; within 1 hour, the plasma was separated by centrifuga- tion at 1600 g and after 16 000 g. The supernatant was collected into fresh tubes and stored at -20(cid:2)C until use for DNA extrac- tion. LightCycler was used for real-time quantitative polymerase chain reaction and high resolution melting assay of 50 impreg- nate plasmas. HbS specific probes designed to detect the muta- tions. In this study, we established that 28 of the fetuses were Site-directed mutagenesis is a pivotal method in the study of the structure-function relationships of genes and proteins. Although several multiple site-directed mutagenesis methods have been reported, these methods require phosporylated mutagenic primers plus ligase to seal the nicks during the synthesis of the mutant strand and/or require the ligation of the mutated fragments into the restriction-digested vectors. Here we present an efficient phos- phorylation-free and ligase-free method for multiple mutations of up to six distal-sites simultaneously. This method consists of two stages of PCR. The multiple sets of megaprimers are synthesized from mutagenic primers, which are designed in either forward or reverse directions, in the first stage PCR, and then are used to amplify the whole plasmid template in the second stage PCR, resulting in the multiple-nicks-contained double-stranded mutant PCR product. Then the Dpn I digestion is applied to remove the template plasmid before transformation in an effort to increase

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the mutagenesis efficiency. This method could be extended to any plasmid DNA which is isolated from dam+ E. coli strains, and the results showed that the mutagenic efficiencies of quadruple and sextuple mutations were 80% and 40%, respectively.

P7–26 Novel two-step construction of base-modified nucleic acids. Application in electrochemical DNA sensing M. Fojta1, P. Horakova1, H. Pivonkova1, L. Havran1, P. Sebest1, M. Vrabel2, H. Cahova2, L. Kalachova2, J. Riedl2, V. Raindlova2 and M. Hocek2 1Department of Biophysical Chemistry and Molecular Oncology, Institute of Biophysics ASCR v.v.i., Brno, CZECH REPUBLIC, 2Institute of Organic Chemistry and Biochemistry, ASCR v.v.i., Gilead Sciences & IOCB Research Center, Prague, CZECH REPUBLIC

I.N.G.

sp. (39% identity). The sequence analysis showed that the protein contains a catalytic triad formed by Ser 163, Asp 263 and His 293, and the Ser of the active site is located in the conserved motif G-X-Ser-X-G, as in most esterases and lipases. The enzyme showed remarkable stability at low pH, against denaturing agents, like organic solvents and detergents. The protein cata- lyzed the hydrolysis of p-nitrophenyl (pNP) esters with different acyl chain length, confirming the esterase activity. The best sub- trate was p-NPacetate (C2) with KM and Vmax values of 17 lM and 360 lM/min, respectively. The enzyme is able to hydrolyse phenylacetate, and therefore the enzyme is classified as an arylest- erase. The esterase activity was shongly inhibited by the organo- phosphate Paraoxon, diethyl pyrocarbamate, and Pestatine A. These data showed that the serine, histidine and aspart are the aminoacids of catalytic triad. This was also confirmed by 3D structure model. The data obtained from this enzyme show a great potential for biotransformations of commercially interesting esters. Acknowledgements: This study was partially supported by MEC (BIO2007-62510), and ‘Grupo de Excelencia de la Regio´ n de Murcia’ and DGI Consejerı´ a de Educacio´ n y Cultura C.A. Murcia (09BIO2005/01-6470). is holder predoctoral research (FPU) grant from MEC, Spain. M.I.G.G. is a holder predoctoral research grant.(09B102005/01-6470)

P7–28 Abstract withdrawn

P7–29 BolA inhibits cell elongation and represses MreB, a bacterial actin homologue P. Freire, R. Moreira and C. Arraiano Biology – Control of Gene Expression Lab, ITQB, Lisboa, PORTUGAL

Electrochemical DNA sensing utilize various label-free (employ- ing intrinsic DNA electroactivity) as well as label-based detection strategies. The latter appear better suited for sequence-specific DNA analysis when reliable recognition of a specific nucleotide sequence among others is desirable. Labeled nucleic acids are usually prepared via multistep chemical synthesis of oligonucleo- tides (ON). Recently we have introduced a novel two-step con- struction of modified ON, relying in direct aqueous cross- coupling of halogenated deoxynucleoside triphosphates (dNTP) with various functional groups and enzymatic incorporation of the nucleotide conjugates into ON by DNA polymerases. Com- pared to the fully chemical ON synthesis, this approach is more versatile, allowing – once a set of modified dNTPs is available – an easy and instant construction of labeled ON bearing various tags (or their combinations) at specific positions using solely standard molecular biological tools. We synthesized modified dNTPs bearing electrochemically active labels at 7-position of 7- deaza purines or 5-position of pyrimidines. The base-coupled tags include ferrocene or [M(bpy)3]2+ (M = Ru or Os) complexes linked to the nucleobase via ethynyl bridge, as well as 3-nitro- or 3-aminophenyl groups. We thus obtained a complete set of four nucleobases bearing electrochemically distinguishable tags. More- over, electronic communication between the tags and nucleobases makes their redox potentials responding to the conjugate nucleo- base and to incorporation into DNA, thus offering another type of information to gain. All of these conjugates were successfully incorporated into ON and utilized in analytical applications such as SNP typing. Further applications of the proposed strategy in nanotechnology, ON self-assembly, chemical biology and biosen- sing is anticipated. Acknowledgements: This work was supported by GA ASCR (IAA400040901), GACR (203/09/0317) and MEYS CR (LC06035).

P7–27 Characterization and modelling of a new arylesterase from Gluconobacter oxydans 621H F. Garcı´ a-Carmona, A. Sa´ nchez-Ferrer, M. I. Garcı´ a-Garcı´ a and I. Navarro-Gonza´ lez Biochemistry and Molecular Biology-A, Universidad de Murcia, Murcia, SPAIN

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

In natural environments, most bacteria live attached to surfaces in structures known as biofilms. Overexpression of the morpho- gene bolA in Escherichia coli was shown to induce biofilm devel- opment while the deletion of this gene presents a consistent reduction in biofilm accumulation. The bolA gene is also highly induced in stress conditions and during stationary phase of growth causing a cellular round morphology when overexpressed (1, 2). bolA was shown to regulate the levels of PBPs 5 and 6 and of AmpC lactamase. In parallel to outer membrane proteins accessibility changes, the expression ratio of porins OmpC and OmpF was shown to vary according to bolA levels (3). Evidence shows that induction of bolA results in increased resistance of OM integrity to SDS and a decrease of cell sensitivity to vanco- mycin (3). Furthermore, our results show that the morphogene bolA is a repressor of cellular elongation and of mreB expression. bolA can affect the correct placement of the internal cytoskeleton and thus alter the maintenance of the normal rod shape of grow- ing cells, with the potential of affecting cell division. bolA overex- pression influences the mechanism by which bacteria elongate and retard cell growth rate. These effects occur mainly through a repression of the levels of mreB transcripts and protein by direct binding of BolA protein to mreB promoter sequence (4). Evi- dence provided establishes BolA as an important player in the control of cell elongation and formation of the cytoskeleton. This data opens a whole new role for BolA on the control of cell mor- phology as a regulator of essential structural elements in E. coli. References: 1. Freire P, et al. Biochimie 2006; 88: 341–346. 2. Santos JM, et al. Mol. Microbiol. 2006; 60: 177–188. from Gluconobacter oxydans The gene GOX0881 of 936Pb, 621H, was cloned, sequenced and overexpressed into Escherihia coli Rosetta strain. The deduced protein was a 35 kDa dimeric esterase with low homology to heroinesterases from Rhodococcus

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3. Freire P, et al. FEMS Microbiol. Lett. 2006; 260: 106–111. 4. Freire P, et al. J. Mol. Biol. 2009; 385: 1345–1351.

P7–30 Polysaccharides from wood-decay fungi of Phellinus and Inonotus genera G. K. Gomba1, A. Synytsya1, M. Novak1, M. Tomsovky2, A. Vesela1 and J. Copikova1 1Carbohydrate Chemistry and Technology, Institute of Chemical Technology, Prague, CZECH REPUBLIC, 2Faculty of Forestry and Wood Technology, Mendel University of Forestry and Agriculture in Brno, Brno, CZECH REPUBLIC

host-plant. The expression of TTSS effector protein gene (dspE) was specific host-dependently repressed, while non-host plant tis- sues induced the expression of dspE gene. We propose that E. ca- rotovora has a specific mechanism, controlling the serial secretion of TTSS-dependent proteins, essential for particular stage of pathogenesis, that is typical for specific plant-microbe interac- tions. The E. carotovora pectate lyase activity was up-regulated by plant components and was much more pronounced (to 3-fold) in presence of specific plant-host tissues. Thus, we disclosed that the QS system and production of virulence determinant are regu- lated by both plant non-specific tissue components and unconstit- utive metabolite of specific host-plant. We suppose that our model system will promote the identification of this signal fac- tors.

P7–32 Expression of a sir/nir gene from the cio operon is essential for growth on cyanide of Pseudomonas pseudoalcaligenes CECT5344 M. I. Guijo, R. Blasco and A. Quesada Bioquimica y Biologia Molecular, Facultad de Veterinaria, Caceres, SPAIN

Beta-1,3- D-glucans (also available with beta-1, 6- side chains) are naturally occurring polysaccharides available among other sources, in fungi. For years, they have been valued for their medicinal qualities with the ability to modulate the immune sys- tem. Beta-D-glucans from fruiting bodies of wood-decay fungi of the genera Phellinus sp. and Inonotus sp. (Europe and South Korea) were isolated by hot-water-extraction. Three fractions, water extract F1, alkali fraction F2 and water insoluble fraction F3 were obtained. Fractions F1 and F2 were dialyzed and then lyophilised. Structures of isolated fractions were characterised by FT-IR and NIR spectroscopy, NMR, DSC and TG. The infra red spectra of these products were compared using cluster analy- sis to determine the relationship between different species. Fur- ther, the molar ratio of neutral sugars was determined by HPAEC-PAD. Our results indicated the presence of alpha- and beta-glucans in either genera of wood-decay fungi. However, the genera Inonotus sp. has a considerably higher content of alpha- glucans as compared to Phellinus sp. and vice-versa, Phellinus sp. has a much higher content of beta-glucans as compared to Inono- tus sp. In all analysed samples, D-glucose has the most prevalent presence of neutral sugars. The presence of heteroglucans is sup- ported by D-mannose and D-galactose found in relatively lower levels. FT-IR spectra showed the presence of carboxylic func- tional groups indicating the probable presence of uronic acids. Acknowledgement: Czech Science Foundation project No. 521/07/J039 and Ministry of Education, Youth and Sport, pro- ject No 6046137305.

Cyanide is one of the most rapidly acting lethal poisons known to humankind. Its main molecular target is the cytochrome c oxi- dase, blocking irreversibly the aerobic respiration of mitochon- dria. The bacterium Pseudomonas pseudoalcaligenes CECT5344 tolerates and assimilates high cyanide concentrations using an assimilatory pathway, a cyanide-insensitive respiration and a high affinity iron uptake system. We have demonstrated the presence in the bacterium genome of a gene cluster coding for the cyto- chrome bd, a cyanide-insensitive terminal oxidase. The cio operon contains five genes, including cioAB (cytochrome bd) with a sin- gular organization. It starts upstream of cioAB with the coding sequences of a putative ferredoxin-dependent sulfite or nitrite reductase and spanning downstream two additional open reading frames that encode uncharacterized gene products. The sulfite/ nitrite reductase encoded in the cio operon (Sir/Nir), which has a chimeric structure, has never been described in this gene context, although it can be found in a related strain available through genome data bases. The mutagenesis of sir/nir has evidenced that function of this gene is not necessary for cyanide resistance on LB-medium, but surprisingly, it is essential for growth on mini- mal medium containing cyanide. One of our objectives is to eluci- date the role of Sir/Nir in this process. Acknowledgements: Authors wish to thank the funding by the Ministry of Science and Innovation (BIO2008-04542-C02-02) and the Regional Govern of Extremadura (PRI07A09).

P7–31 Plant-dependent regulation of Quorum Sensing system and virulence factors production in Erwinia carotovora subsp. atroseptica V. Gorshkov, O. Petrova, N. Mukhametshina and Y. Gogolev Kazan Institute of Biochemistry and Biophysics, Group of molecular biology, Kazan, RUSSIA

P7–33 Mutagenicity of essential oils of Coriandrium sativum L. and Cuminum cyminum L. in Salmonella/microsome test N. Hasimi,1 S. Ozdemir1, V. Tolan1 and S. Kizil2 1Biology, Dicle University, Diyarbakir, TURKEY, 2Field Crops, Dicle University, Diyarbakir, TURKEY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Phytopathogenic bacteria Erwinia carotovora is the causative agent of soft-rot disease in a range of host plants and responsible for ‘blackleg’ infection. We obtained data that quorum sensing system (QS) in plant pathogenic bacteria E. carotovora can be regulated by plant tissue component(s). Furthermore, the specific host-plant promotes the overexpression of bacterial gene of AHL-synthase, which is necessary for synthesis of N-acylhomo- serine lactones essential for intercellular communication. Because the induction of QS system should promote the production of virulence factors we have estimated the expression level or pro- duction of the main pathogenicity determinants [type III secre- tion system (TTSS) and pectate lyase] in presence of host- (potato) and non-host (tobacco) plant tissues. We observed the plant dependent induction of structural component (hrpA) and regulatory component (hrpL) of TTSS despite the specificity of Essential oils are concentrated, hydrophobic liquid containing volatile aroma compounds from plants, which are called aromatic herbs or aromatic plants. Various essential oils and their monot- erpenoid constituents have been widely used as fragrances in cos- metics, as flavouring food additives, as scenting agents in a variety of household products. The present study was undertaken to investigate the genotoxic potential of essential oils of Coriand- rium sativum L. and Cuminum cyminum L. by Salmonella/micro-

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some assay with TA98 and TA100 tester strains. The essential oils of C. sativum L. and C. cyminum L. were not to be found mutagenic on both strains of Salmonella.

P7–34 Antioxidant effects of some herb extract on heard and eye of chick embryo treated with adriamycin N. Hasimi1 and D. Musa2 1Biology, Dicle University, Diyarbakir, TURKEY, 2Biology, Harran University, Sanliurfa, TURKEY

of Agriculture 1B48068 (PUV) examine as many as hundreds of sequences in one unknown sam- ple of DNA during one analysis. The aim of our study was to find the way how to use the whole genomic DNA as an analyte in GMO analysis using DNA microarray. The whole genome labelling could make the analysis quite fast and easy. Our in house developed DNA chip is based on control DNA sequences for detection several plant species, regulation regions and con- struct-specific regions, which are characteristic for number of GM plants. We have tested two kinds of DNA probes – PCR products of specific genes and the oligonucleotides probes. We have also tested several different hybridization conditions and washing procedures and methods of labelling to elaborate opti- mised protocol. The results will be discussed along with possible application of the technology. Acknowledgement: The work was supported by projects of the Czech Ministry and CZ002700604.

P7–36 Abstract withdrawn

P7–37 The soluble complex formation between polyelectrolytes and bovin serum albumin in the presence of copper (II) in neutral aqueous media M. Karahan1 and Z. Mustafaeva2 1Chemistry, Art and Science Faculty, Yildiz Technical University, Istanbul, TURKEY, 2Bioengineering, Faculty of Chemistry and Metallurgy, Yildiz Technical University, Istanbul, TURKEY

Aim: Nigella sativa and Trigonella foenum graecum are medicinal herbs, used widely in Turkey to prevent many diseases as dia- betic, hypertensive and others. In this study we investigated the possible effects of methanol extract of the N. sativa and T. foe- num graecum on chick embryo rapid dividing cells treated with adriamycin. Materials and Methods: To investigate the effect of extract against free radicals caused by adriamycin in chick embryo eye and heart tissue, 480 fertile eggs of broiler chickens were divided into eight equal groups. Adriamycin (10 lg) was applied to each embryo before and after treatments with 4 and 8 mg N. sativa and T. foenum graecum. Eight and 19 days old embryos were lib- erated from egg shells washed with normal saline to remove all derbies and weighed, hearts were removed and weighed in sensi- tive balance. Slides were prepared from heart and eye of each embryo. Results: There is no toxic effect of adriamycin on eyes. Treat- ment with T. foenum graecum can increase the total body weight of embryo. But no protective against adriamycin toxicity. Treat- ment with 4 mg N. sativa can increase total body and heart weight also can protect embryos against cardiotoxicity of adria- mycin. Conclusions: Nigella sativa can reduce adriamycin induced car- diotoxicity and may be protective for adriamycin induced free radical damage in the heart tissue while there is no significant results in T. foenum graecum case. Histopathological observations did not shows any changes in heart tissue. So we suggest that protective effect of N. sativa treatment on adriamycin heart toxic- ity should be evaluated extensively.

P7–35 The new method for GMO detection: DNA microarrays with whole labelled genome J. Hodek1, J. Ovesna1 and K. Demnerova2 1Department of Molecular Biology, Crop Research Institute, Prague, CZECH REPUBLIC, 2Department of Biochemistry and Microbiology, Institute of Chemical Technology, Prague, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Reactions of polyelectrolytes (PE) with proteins in aqueous solu- tions have attracted great attention in the last decades. Polymer- protein complexes from as a result of the polyion chains with the oppositely charged groups of the protein molecule during these reactions. The extent of the interaction is found to be pH sensi- tive and dependent on the isoelectric points of the proteins. Such complexes represent a spesific class of polymer-protein com- pounds that have important applications in various areas (1). Polyelectrolytes of synthetic origin have been found to increase immunoresponse to the immunizing antigen and to produce an adjuvant effect. The use of PE as a carrier, which is firmly linked to microbial and viral antigens to form a stable complex (or con- jugate), not only increased by several orders of magnitude the immune responsiveness of the organism but also afforded effec- tive immune protection (2). In our study, the interaction between anionic PE (methyl vinyl ether maleik anhyride copolymer and polyacrilic acid) and bovin serum albumin in the presence of Cu2+ ions in aqueus solution (pH:7) were investigated with UV- visible, fluorescence and dynamic light scattering. Also the ter- nary polycomplex were investigated toxicity. The pattern of dis- tribution of Cu2+ ions between PE coil and of protein globules between polymer-metal complex particules appeared to follow the self-assembly principle. It shown that more stable water solu- ble ternary complex and non-toxicity. References: 1. Karahan M, Mustafaeva Z & Ozer H. Asian J. Chem. 2007; 19: 1837–1845. 2. Mustafaev M, et al. J. Appl. Polym. Sci. 1996; 62: 99–109. Handling with genetically modified organisms (GMOs) is within European Union under the strictly legislative control. In case of conventional food and feed products the labelling is mandatory if there is content traces of authorised GMOs below a limit of 0.9%. Consequently there must be some suitable analytical meth- ods for GMO analyses in food and feed products. Control labo- ratories keep at disposal a number of internationally validated methods recognised by ISO and validated through CRL JRC- ENGL. Use of microarrays technique for transgenes detection represents a new option in GMO analysis. This method allows to

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conclude

P7–38 Evaluation of chaperone-like activity of alginate: microcapsule and water-soluble forms N. Rezaii Neuroscience Research Center, Tehran, IRAN

Quantification of MMP-9 by zymography, western blotting and qRT-PCR was significantly increased in the tissue hyperplasia area compared with normal tissue. Treatment of Chol-R9 deliv- ered siRNA targeting MMP-9 showed significant decrease in- stent tissue hyperplasia. Conclusion: We that MMP-9 overexpression is responsible for tissue hyperplasia in this rat model and that gene therapy by Chol-R9 delivered siRNA targeting MMP-9 offers a novel approach to prevent in-stent restenosis.

P7–40 Esophageal stent placement and Evaluation of secondary tissue hyperplasia in a rat model E. Y. Kim1, Y. Y. Jung2, D. H. Shin3 and J. H. Shin4 1Medical Science, University of Ulsan College of Medicine, Seoul, SOUTH-KOREA, 2Radiology, Eulji General Hospital, Seoul, SOUTH-KOREA, 3Radiology, Asan Institute for Life Sciences, Seoul, SOUTH-KOREA, 4Radiology, Asan Medical Center, Seoul, SOUTH-KOREA

The use of enzymes at the industrial level is often limited by the low resistance of these proteins to technological conditions, especially the physico-chemical strains. Among all, protein aggre- gation is arguably the most common and troubling manifestation of protein instability. Alginate is regarded as a biocompatible, nontoxic, nonimmunogenic and biodegradable polymer, making it an attractive candidate for biomedical applications. To evalu- ate the chaperone-like activity of alginate stabilization and refolding of alkaline phosphatase (ALP) was investigated in the presence of alginate through two different approaches, the solu- ble form and microcapsule assisted methods. It was found that in the presence of microcapsules, ALP can be stabilized to a higher degree compared with the water-soluble form, whereas the dena- tured ALP is refolded with a higher yield through latter method. lower refolding yields of alginate beads compared with its soluble form may be the result of lower refolding rate of ALP upon elu- tion of the bound enzyme by dispersing the precipitate in NaCl which left the unfolded protein in an unsuitable environment, providing enough time for protein aggregation and leads finally to lower recovered activity compared with another method which applies soluble form of alginate. In addition in the case of algi- nate capsules, the choice of suitable divalent ion is essential for stability and refolding assistance.

P7–39 Suppression of tissue hyperplasia secondary to stent placement using cholesteryl oligo-D-arginine delivered siRNA targeting MMP-9: animal study in rat esophagus E. Y. Kim1, D. H. Shin2 and J. H. Shin3 1Medical Science, University of Ulsan College of Medicine, Seoul, SOUTH-KOREA, 2Radiology, Asan Institute for Life Sciences, Seoul, SOUTH-KOREA, 3Radiology, Asan Medical Center, Seoul, SOUTH-KOREA

Purpose: To evaluate the feasibility of bare stent placement and to evaluate the formation of granulation tissue caused by stent placement in a rat esophageal model. Materials and Methods: Twenty SD male rats were divided into four groups (group I – 4 mm diameter and larger cell space, group II – 5-mm diameter and larger cell space, group III – 5 mm diameter and smaller cell space, group IV – barbs added to group III stents). Three weeks after stent placement, incidence of stent migration was evaluated. Gross and histological findings were evaluated after sacrifice in groups of < 50% of stent migra- tion incidence. Results: Stent placement was technically successful in all rats with no early death until 3 week follow-up. No esophageal perfo- ration in all rats. Stent migration rate was 100%, 60%, 40%, and 0% in group I, II, III, and IV, respectively. The degree of inflammatory cell infiltration, papillary projection thickness, granulation tissue area, and percent of granulation tissue area were higher in group IV than in group III. Conclusion: Rat esophageal model was feasible and seemed to be efficient animal model to evaluate granulation tissue forma- tion caused by self-expanding bare metallic stent. In barbed stents, incidence of stent migration was the least without esopha- geal perforation, and formation of granulation tissue was excellent.

P7–41 Significance of protein order in thylakoid membranes for photosynthetic energy conversion H. Kirchhoff1, S. Haferkamp1, C. Mullineaux2 and P. Albertsson3 1Institute of Biological Chemistry, Washington State University, Pullman, WA, USA, 2School of Biological and Chemical Sciences, Queen Mary University London, London, UK, 3Department of Biochemistry, Lund University, Lund, SWEDEN

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Sessile land plants live in a highly fluctuating environment, which forced the evolution of sophisticated adaptation and repair mech- anisms of the photosynthetic apparatus localized in thylakoid membrane inside chloroplasts. These mechanisms are associated with structural reorganizations of photosynthetic protein com- Background and Purpose: Although esophageal stenting is the best palliative treatment for unresectable malignant strictures, there are many adverse events of stent placement, including new strictures caused by stent-induced tissue hyperplasia. The aim of this study was to establish the character, level, and expression of MMP-9 in tissue hyperplasia and to evaluate the efficacy of RNAi therapy using Chol-R9 delivered siRNA targeting MMP-9 for preventing tissue hyperplasia secondary to bare stent place- ment in a rat esophageal model. Materials and Methods: Esophageal stent placement (5 mm in diameter and 15 mm long) was performed in 10 rats. After 3 weeks, esophageal samples were subjected to histological exami- nation and quantification of MMP-9 by zymography, western blotting and qRT-PCR. Chol-R9 was synthesized and used to deliver MMP-9 targeted siRNA into rat esophagus. Ten rats underwent Chol-R9 delivered siRNA targeting MMP-9 at 1 week and 2 weeks after stent placement and were sacrificed at 3 weeks. Esophagus samples were analyzed. Results: In this rat model, immunohistochemical examination demonstrated increased MMP-9 expression in highly symptom- atic tissue hyperplasia with no difference in the level of MMP-2.

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Acknowledgements: This work was supported by GA AV KAN208130801, GA CR 522/07/0692 and 204/09/H002 References: 1. Palecek E et al. Talanta 2002; 56(5): 919–930. 2. Huska D et al. Electrophoresis 2008; 29(24): 4964–4971.

P7–43 Manipulation of glucosinolate synthesis S. Kopriva and S. G. Mugford Metabolic Biology, John Innes Centre, Norwich, UK

secondary metabolites

plexes within the thylakoid membrane, which require their mobil- ity. Furthermore certain electron transfer steps depend on lateral diffusion of small redox carriers (plastoquinone, plastocyanin). Thus the functionality, adaptation, repair as well as the biogene- sis and remodeling of thylakoid membranes require a high lateral mobility of its constituents. A conceptual problem in understand- ing lateral transport in thylakoids is that diffusion processes are expected to be severely restricted in this crowded membrane (pro- tein area 70–80%). We demonstrated by Computer simulations that plastoquinone as well as protein diffusion is expected to be too slow to support electron transport, adaptation and repair. In contrast protein diffusion measurements with the fluorescence after photobleaching technique on grana thylakoids reveal a very mobile protein complex fraction. We suggest that the reason for the difference between simulation and experiment is that simula- tions assume a pure random protein arrangement. We present evidence that proteins in native thylakoid membranes are non- random organized. Importantly the degree of order increase in stressed plants suggesting a crucial role of the protein arrange- ment. We will discuss advantages of protein ordering for the functionality and maintenance of photosynthetic membranes. Understanding the interplay between membrane architecture and for generating photosynthesis could be an important aspect robust crop plants or enhancing biomass/biofuel productivity.

Glucosinolates are plant involved in responses to biotic stress. The final step in glucosinolate synthesis is the transfer of a sulfo- group onto a desulfated precursor. The sulfate donor for this reaction, 3’-phosphoadenosine 5’-phospho- sulfate, is synthesized in two steps catalyzed by ATP sulfurylase and adenosine 5’-phosphosulfate kinase (APK). To assess to what extent APK controls the production of glucosinolates we analysed the corresponding gene family in Arabidopsis thaliana. Analysis of T-DNA insertion knockout lines for each of the four isoforms did not reveal any phenotypical alterations. However, when all six combinations of double mutants were compared, the apk1 apk2 plants were significantly smaller than wild-type plants. The levels of glucosinolates, a major class of sulfated secondary metabolites, were reduced approximately 5-fold in apk1 apk2 plants. The reduction in glucosinolates resulted in increased tran- script levels for genes involved in glucosinolate biosynthesis and accumulation of desulfated precursors. The data indicate that the APK1 and APK2 play a major role in control of synthesis of sec- ondary sulfated metabolites and that modulation of expression of this enzyme can strongly affect glucosinolate levels.

P7–42 Paramagnetic particles with specific sequences of nucleic acids as tools for detection heavy- metal-stress-induced genes’ expression in plants D. Huska1, O. Krystofova1, V. Adam1, J. Zehnalek1, P. Babula2, L. Havel3, L. Trnkova4 and R. Kizek1 1Department of Chemistry and Biochemistry, Mendel University of Agriculture and Forestry in Brno, Brno, CZECH REPUBLIC, 2Department of Natural Drugs, University of Veterinary and Phar- maceutical Sciences, Brno, CZECH REPUBLIC, 3Department of Plant Biology, Mendel University of Agriculture and Forestry in Brno, Brno, CZECH REPUBLIC, 4Department of Chemistry, Masaryk University, Brno, CZECH REPUBLIC

P7–44 On metalloresistance in silver hyperaccumulating Amanita strobiliformis P. Kotrba1, V. Urban1, P. L. Jedelsky2, J. Borovicka3, O. Daniel1, T. Macek4 and T. Ruml1 1Department of Biochemistry and Microbiology, Institute of Chemical Technology Prague, Prague, CZECH REPUBLIC, 2Department of Cell Biology, Faculty of Science, Charles University in Prague, Prague, CZECH REPUBLIC, 3Department of Nuclear Spectroscopy, Nuclear Physics Institute of AS CR, Rez near Prague, CZECH REPUBLIC, 4Department of Natural Products, Institute of Organic Chemistry and Biochemistry of AS CR, Prague, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Fungi are intimately involved in the cycling of elements and substrates. transformations of both organic and inorganic Despite numerous reports published over last five decades on macrofungal species capable to hyperaccumulate toxic metals, metalloids or noble metals, the mechanism employed in metal accumulation and detoxification remains largely unknown. Recently, hyperaccumulation of Ag by Amanita strobiliformis originating from non-metalliferous areas was observed [Borovi- cka et al. Mycol. Res. 2007; 111: 1339–1344], with the highest Ag content in fruit bodies of 1253 mg/kg. Chromatographic analyses of cell-free extracts of A. strobiliformis fruit bodies revealed absence of phytochelatins, known to detoxify heavy metals in plants, and the majority of Ag present in the complexes of molec- ular weight of 7 and 3 kDa with cysteine-rich peptide ligands. Partial sequence of a peptide liberated from the 7 kDa complex obtained from mass spectroscopy analysis allowed us to isolate The reliability of nucleic acids studies strongly depends on the accuracy of their isolation from biological material. New promis- ing technologies are based on (para) magnetic particles. These particles with 5 nm – 100 lm diameter having metal core (mostly gamma-Fe2O3 or Fe3O4) covered by chemical shells, which can be modified according to target molecules as specific protein or nucleic acids sequence. We used the advantageous properties of the particles and prepared particles modified by nucleic acids sequences complementary to heavy-metal-stress-induced genes such as phytochelatin synthase and glutathione reductase. Pri- marily we tested the particles on standard sequences and opti- mized experimental conditions. Square wave voltammetry in connection with adsorptive transfer stripping technique was uti- lized for detection of isolated nucleic acid. Under the optimized conditions we were able to determine down to nanogram of stan- dard sequence of nucleic acid. Further, we treated early somatic embryos of spruce and maize plants with cadmium(II) ions (0, 50, 100, 250 and 500 lM) for 14 days as biological samples, in which we determined expression of heavy-metal-stress-induced genes. It can be concluded that activity of phytochelatin synthase producing phytochelatins as low molecular mass peptides protect- ing a plant cell against heavy metal ions was enhancing in both plant model with increasing time of the treatment and concentra- tion of cadmium(II) ions.

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from cDNA library individual sequences encoding three isoforms of 34 amino acid residue metallothioneins (MTs) sharing 85% identity, dissimilar in sequence to recently described functional or predicted MTs of fungi, plants or animals, still containing char- acteristic Cys-X-Cys motifs. Knowledge about heavy metal (hyper) accumulation and tolerance mechanisms in ectomycorrhi- zal fungi would contribute to understanding of mobilization of metal species by these plant symbiots and lead to applications in bioprospection and in bioremediation of metal polluted soils, including construction of GM plants for phytoextraction. Acknowledgement: Funded by grants of Grant Agency of AS CR no. IAA600480801, Grant Agency of Czech Ministry of Edu- cation no. MSM 6046137305, MSM0021620858 and 1M06030.

P7–45 Characterization of microbial community after isolation from contaminated sediment J. Koubek, K. Jecna, O. Uhlik, P. Junkova, J. Lipov and M. Mackova Department of Biochemistry and Microbiology, Institute of Chemical Technology Prague, Prague, CZECH REPUBLIC

theless, little is known about the relevance of interleukin-27 (IL-27) for bladder tumor surveillance in rBCG immunotherapy. Here we investigated the ability of multicomponent vaccines in enhancing anti-tumor effects in vivo. Methods: Treatment with combined IL-27 and rBCG was exam- ined in syngeneic C3H mice. The delivery efficiency was detected by flow cytometry. Inhibition of tumor growth was monitored by bioluminescence-imaging system, with measurement of cytokines and phenotyping of infiltrating lymphocytes in tumors. Results: Increased immune cell infiltration and induction of apoptosis were noted after treatment with IL-27 plus rBCG DNA vaccines. Combination therapy with IL-27 and rBCG resulted in the highest long-term (> 90 days) tumor-free survival frequency of 78%. Animals that were tumor-free demonstrated tumor-specific protection after rechallenge with MBT-2 cells. IL- 27 alone and rBCG alone both delayed tumor progression, but only IL-27 plus rBCG significantly augmented long-term survival (65%) compared to the control group. At peak expansion, the combination treatment resulted in a 3- to 4-fold higher absolute number of circulating tumor antigen-specific CD8+ T cells than was stimulated by IL-27 or rBCG alone. Conclusions: Immunotherapy using IL-27 plus rBCG could be an attractive regimen for the treatment of bladder cancer. This approach presents new possibilities for the treatment of bladder carcinoma.

P7–47 Recombinant high mobility group box-1 peptide for delivery of nucleic acids S. Lee, J. S. Han, H. A. Kim and M. Lee Bioengineering, Hanyang University, Seoul, SOUTH-KOREA

sponsored by projects Monitoring of microbial populations, evaluation of microbial diversity in contaminated environments and taxonomical identifi- cation has aimed attention together with the increase of interest in the environment and environmental protection. Usually char- acterization of indigenous microorganisms is based on consortia cultivation, difficult isolation of pure cultures and then their bio- chemical and genetical description. The procedure of differentia- tion of isolates is usually long procedure. This work brings comparison of three different methods applicable for microbial identification in contaminated environments. Nine different strains originally isolated from the sediment contaminated with polychlorinated biphenyls were characterized. For the detection of bacterial biphenyl operon genes, polymerase chain reaction with specific primers was carried out. Biphenyl operon enables aerobic utilization of biphenyl as a source of carbon and energy. The characterisation itself was carried out by the set NEFERM- test 24, mass spectrometry MALDI-TOF and the sequencing of 16S rDNA. Use of NEFERMtest 24, which belongs to classical biochemical tests, showed its limited application. The method of mass spectrometry averred existence of four different bacterial species. All the isolates were identified after sequencing of 16S rDNA which proved existence of four different bacterial species amongst the isolates. This experiment showed the potential of MALDI-TOF for screening and distinguishing of different bacte- rial isolates from environmental samples. Acknowledgement: This work was NPVII 2B08031, MSM 6046137305, GACR 525/09/1058.

High mobility group box-1 (HMGB-1) is an abundant nuclear protein. HMGB1 is composed of high mobility group (HMG) box A, box B and acidic C-terminal region. Boxes A and B are DNA binding domains and have high contents of basic amino acids. In this study, a recombinant HMGB-1 box A (HMGB-1A) peptide was produced as a carrier of nucleic acids. The HMGB- 1A cDNA was amplified by RT-PCR using total RNA from 293 cells as a template. The HMGB-1A cDNA was cloned into the pET21a expression vector, resulting in construction of pET21a- HMGB-1A. pET21a-HMGB-1A was transformed into the BL21 strain and HMGB-1A was over-expressed by IPTG induction. HMGB-1A was purified using nickel affinity and ion exchange chromatography. The purified HMGB-1A was analyzed in SDS- PAGE. Gel retardation assay showed that HMGB-1A formed complexes with plasmid DNA or siRNA effectively. In transfec- tion assay, HMGB-1A delivered luciferase plasmid DNA into 293 cells. The transfection efficiency of HMGB-1A was compara- ble to that of poly-L-lysine (PLL). In addition, HMGB-1A deliv- ered anti-luciferase siRNA in CT-26 cells and reduced the internal luciferase expression. In MTT assay, HMGB-1A protein did not have any toxicity to cells, while PLL had much higher toxicity. Therefore, HMGB-1A may be useful for development of a non-toxic gene delivery carrier.

P7–46 Immunotherapy for human transitional cell carcinoma using Th1 cytokine interleukin-27 and recombinant BCG vaccine C. F. Lee1, D. S. Yu2, M. H. Tao3 and S. Y. Chang4 1Institute of Preventive Medicine, National Defense Medical Cen- ter, Taipei, TAIWAN, 2Department of Surgery, National Defense Medical Center, Taipei, TAIWAN, 3Academia Sinica, Institute of Biomedical Sciences, Taipei, TAIWAN, 4Department of Surgery, Tri-Service General Hospital, Taipei, TAIWAN

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Introduction: A crucial role for recombinant BCG (rBCG) immunotherapy of bladder carcinoma has been suggested. Never-

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P7–48 Flavonoid transformation by inducible Rhizopus nigricans enzymes M. Slana1, T. Makovec1, D. Zigon2 and H. Lenasi1 1Institute of Biochemistry, Medical Faculty, University of Ljublj- ana, Ljubljana, SLOVENIA, 2Institute Jozef Stefan, Ljubljana, SLOVENIA

P7–50 Generation of Escherichia coli strains with chromosomal integration of CP4 aroA genefor horizontal gene transfer studies M. Madyagol, S. Stuchlı´ k and J. Turna Department of Molecular Biology, Comenius University, Bratislava, SLOVAK REPUBLIC

the knowledge of detoxification of

A filamentous fungus Rhizopus nigricans has a large host range, it is found in soil/compost but it also thrives on storaged fruits and vegetables, thus it represents a serious trouble with its destructive effect to harvested crops. In the fungal inhabitations an array of flavonoids may be found. Flavonoids are involved in plant defence against pathogens, i.e. fungal growth inhibition or spore germination. On the other hand, flavonoid perception by microor- ganisms provokes the induction of enzymes converting these com- pounds into less toxic metabolites. The metabolism of flavonoids by R. nigricans has not been described so far, therefore we investi- gated the interaction of flavonoids with this fungus. We examined the impact of biochanin A, pinocembrin, 6-OH flavone, and chry- sin on R. nigricans. All tested compounds (0.3 mM) were able to inhibit fungal growth up to 100%, 64%, 38% and 22%, respec- tively. The fungus responded to the presence of flavonoids by their transformation into glucosylated products: 7-glucosyl-bioch- anin A, 5-glucosyl-pinocembrin, 6-glucosyl-flavone and 7-gluco- syl-chrysin, determined by HPLC/MS analyses. The conjugation products were less fungitoxic than aglycones, so the glucosylation of flavonoids by fungal enzymes represents a detoxification reac- tion. Bearing in mind the destructive effect of R. nigricans to har- vested crops, fungitoxic flavonoids by fungal enzymes may be of great importance when searching for a tool to prevent the transformation of fungitoxic flavonoids into less toxic compounds.

The ability to make precise genetic modifications to the bacterial chromosome and then to study the resulting phenotypic behavior is very important for functional studies of a large number of potentially new genes. Functional analysis of these genes requires simple and efficient gene manipulation methods, which allow tar- geted modifications of particular sequences in their chromosomal location. To test the effect of a mutation, the mutant allele is introduced into the cell where it can replace the wild-type (wt) gene by homologous recombination. The effect of the mutation can then be studied by expressing the gene at its native location, under normal conditions. Gene replacement on the E. coli chro- mosome can be done by a variety of techniques. In this study we have described construction of E. coli strain by homologous recombination between the chromosome and the plasmid carry- ing a mutant gene (aroA50) inactivated by the deletion of 350 bp from aroA CP4 and the plasmid segregation allowing the simple and efficient isolation of mutants via a selection for the aroA50 in the (now chromosomal) mutant gene. We have used a plasmid-based test system to demonstrate integration of aroA CP4 deletant mutant into the wild type E. coli strains. This method consisted of a two-step gene-replacement technique: (i) chromosomal integration of a plasmid carrying an internal dele- tion in the gene of interest (aroA50) and (ii) excision of the vec- tor resulting in gene replacement. We have successfully integrated aroA CP4 deletant mutant into chromosome using a thermosensi- tive integration vector pMAK705 to facilitate the gene replace- ment. This plasmid is stably maintained in wild type E. coli strains at 28(cid:2)C but is unstable at 42–44(cid:2)C. E. coli mutants were constructed carrying a aroA50 CP4 geneusing a phenotypic selec- tion. This strain with chromosomal integrated aroA deletant will be used for the study of horizontal gene transfer (HGT) in the future work. The gene replacement method used in our studies is optimized and is available for the construction of mutants for any gene in E. coli.

P7–49 Metabolic engineering of Chlorella zofingiensis (Chlorophyta) for enhanced biosynthesis of astaxanthin J. Liu, J. Huang and F. Chen School of Biological Sciences, The University of Hong Kong, Hong Kong, CHINA

P7–51 Interaction of a bacterial dirhamnolipid with phosphatidylcholine membranes: a biophysical study M. Sa´ nchez, F. J. Aranda, J. A. Teruel and A. Ortiz Bioquı´mica y Biologı´a Molecular-A, Universidad de Murcia, Murcia, SPAIN

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Astaxanthin is a commercially important ketocarotenoid synthe- sized only in some organisms. The unicellular green alga Chlorella zofingiensis can heterotrophically grow very fast and produce a moderate amount of astaxanthin. To further enhance the produc- tion of astaxanthin in C. zofingiensis, we genetically manipulated the biosynthetic pathway of carotenoids in the alga. First, we iso- lated and characterized the phytoene desaturase (PDS) gene from a number of C. zofingiensis mutants that are resistant to the herbi- cide norflurazon. In vitro assay revealed that one PDS mutant with a single amino acid alteration (L516F) exhibited about 25-fold greater resistance against norflurazon and 30% higher activity of converting phytoene to zeta-carotene than its wild counterpart. Second, the mutated PDS gene was introduced into wild C. zofingi- ensis via biolistic approach. Transformants harboring the mutated PDS gene accumulated up to 28% and 38% higher astaxanthin than the wild-type cells upon induction of glucose and high light irradiation respectively. The enhanced accumulation of astaxan- thin in the engineered cells was revealed to be related to the increase of PDS transcript. Our study clearly shows that the mutated PDS gene is a useful selectable marker that can be used for genetic engineering of carotenoid biosynthesis in C. zofingiensis and possibly in other important green algae. In addition, the engi- neered C. zofingiensis might serve as an alternative source of natu- ral astaxanthin on a large scale. Biosurfactants constitute a group of surface active compounds which are synthesized by a number of microorganisms. These amphiphilic compounds present a wide structural diversity. Because of their special properties, mainly low toxicity and biode- gradable character, it is of great interest to characterize new bio- surfactants, in order to evaluate their use as potential alternatives to chemically synthesized compounds. Pseudomonas aeruginosa is an environmental microorganism and opportunistic pathogen which produces rhamnolipids when grown under the appropriate physicochemical conditions. Rhamnolipids are a group of glyco- lipid biosurfactants composed of a hydrophilic head, which is formed by one or two rhamnose molecules, called respectively monorhamnolipid and dirhamnolipid, and a hydrophobic tail con-

283

Abstracts

Poster Presentations

P7–53 Production and partial characterization of alpha amylase by thermophilic Bacillus sp. under solid-state fermentation using some agro-residue as substrate F. Matpan and S. Ozdemir Biology, Dicle University, Diyarbakir, TURKEY

The effect of incubation time and different concentration of pep- tone, yeast extract and starch on a-amylase production of ther- mophilic Bacillus sp. were investigated in solid-state fermentation (SSF) by using rice husk (RH) as a substrate. The incubation time was 72 hour. Optimum temperature of a-amylase activity of thermophilic Bacillus sp. was found to be 70(cid:2)C. Alpha-amylase showed good stability at 60, 70 and 80(cid:2)C, maintaining 85%, 70% and 35% of the original activity after 2 hour of incubation for these temperatures. The enzyme was optimally active at pH 6.0 and stable in the pH range of 6.0–8.0. The maximum a-amy- lase production in the presence of RH as substrate was observed as 3468 U/mg with peptone followed by yeast extract (3206 U/ mg) and starch (3022 U/mg) at 0.5% concentration.

taining one or two fatty acids. Rhamnolipids represent one of the most important classes of biosurfactants because of various advan- tageous characteristics. Concerning its production, high yields are obtained as compared to other biosurfactants; furthermore, several raw materials, like used oils or wastes from the food industry, can be used as a source of carbon. Due to the above mentioned reasons it is of great interest to study the interactions of these compounds with biological membranes. In this work, FTIR spectroscopy and fluorescence polarization were used to show that a bacterial dir- hamnolipid interacts with phospholipid membranes composed of DPPC, altering both the acyl chain and the interfacial region of the bilayer. Incorporation of increasing amounts of dirhamnolipid into 2H-DPPC membranes broadened the transition and shifted the transition temperature toward lower values, according to the effect on the CD2 stretching vibration. Examination of the 13C=O stretching band of 13C-DPPC indicated that, both below and above the phase transition, dirhamnolipid produced a shift of the band frequency toward higher values, indicating a strong dehydration of the phospholipid C=O groups, and therefore of the interfacial region of the membrane. The effects on DPH and TMA-DPH fluo- rescence polarization provided additional support to hypothesize on the location of dirhamnolipid within the bilayer. The results shown here could help to explain some of the interesting mem- brane-related biological actions of rhamnolipids reported before.

P7–54 Alpha-amylase production and characterization by a newly isolated Bacillus subtilis under submerged fermentation (SmF) S. Ozdemir1, K. Guven1, F. Matpan1 and Z. Baysal2 1Biology, Dicle University, Diyarbakir, TURKEY, 2Chemistry, Dicle University, Diyarbakir, TURKEY

P7–52 Potential of fungal nitrilases as biocatalysts L. Martinkova1, O. Kaplan1, K. Bezouska2, O. Benada3, V. Vejvoda1, D. Kubac1 and V. Kren1 1Laboratory of Biotransformation, Institute of Microbiology, Prague, CZECH REPUBLIC, 2Department of Biochemistry, Faculty of Science, Charles University Prague, Prague, CZECH REPUBLIC, 3Laboratory of Molecular Structure Characterization, Institute of Microbiology, Prague, CZECH REPUBLIC

Among the different culture medium the basal medium A, con- taining 0.2% beef extract, 0.2% peptone and 0.1% sodium chlo- ride showed maximum production of extra-cellular a-amylase from Bacillus subtilis. The results on the time-course studies on alpha-amylase production was obtained at 24th hour of incuba- tion. Partial purified enzyme showed maximum activity at 60(cid:2)C with optimum pH 6.0. The enzyme showed good stability at 50 and 60(cid:2)C, and maintained 57% and 40% of the original activity after 2 hour of incubation respectively. Amylase was stable in the pH range of 5.0–7.0. At pH 5.0 and 7.0 alpha amylase retained 89% and 90% of its maximum activity, respectively. Bacillus sub- tilis a-amylase was activated by Ca2+ and Mg2+ (relative activity 113% and 105%, respectively).

P7–55 Isolation and partial characterization of alpha- amylase from Klebsiella pneumoniae and Enterobacter sp. F. Matpan and S. Aguloglu Fincan Biology, Dicle University, Diyarbakir, TURKEY

IAA500200708, FT- concept LC06010, OC09046, research

Nitrilases, members of the nitrilase superfamily, have attracted increasing attention due to their potential in biocatalysis and bio- remediation. Our aim is to explore filamentous fungi as a rich source of these enzymes. According to sequence databases, more than 20 nitrilases occur in filamentous fungi but the biochemical and catalytic properties of most of them are unknown. Our work consisted in the purification and characterization of nitrilases from native producers, mainly strains of genera Aspergillus and Fusarium, and from the heterologous producer E. coli, and in their biotechnological applications for the manufacture of hetero- cyclic carboxylic acids (nicotinic acid, isonicotinic acid) (1). To this end the enzymes were immobilized by ion exchange, hydro- phobic interactions or cross-linking enzyme aggregates. The unique structural properties of these enzymes – subunit aggrega- tion and formation of extended helices of > 1 MDa – were stud- ied by analytical centrifugation and electron microscopy. We compare the benefits and drawbacks of fungal nitrilases with those of well-described nitrilases from bacteria and plants. Acknowledgement: National projects Inst. TA5/043, AV0Z50200510 (Institute of Microbiology). Reference: 1. Martinkova L, Vejvoda V, Kaplan O, Kren V, Bezouska K & Cantarella M (2009) Nitrilases from filamentous fungi. In Modern Biocatalysis (Fessner W-D, & Anthonsen T, eds), pp. 229–245, Wiley-VCH, Weinheim.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Today, using microorganisms as biotechnological sources of industrial enzymes is quite important. Amylases are one of the most important industrial enzymes. Alpha-amylase [(E.C 3.2.1.1), (alpha-1,4 D-glucanohydrolase)] is used in the food industry, starch liquefaction, baking, chemistry, detergents, textile, medi- cine and paper industry. In present study, Klebsiella pneumoniae and Enterobacter sp. were used for extracellular alpha-amylase source. Activity of alpha-amylase optimum pH, temperature and time course according to Bernfeld method for these bacteria were determined. Extracellular alpha-amylase activity for K. pneumo- niae for alpha-amylase optimum pH, temperature and time course were determined as 8.0, 40(cid:2)C and 24th hour (1690 U/mg) and for Enterobacter sp. determined as 7.0, 40(cid:2)C and 18th hour, respectively (1536 U/mg).

284

Poster Presentations

Abstracts

P7–56 Involvement of fumarases and malate-quinone oxidoreductases in metabolic adaptation of Pseudomonas pseudoalcaligenes CECT5344 to cyanide F. Merchan, M. I. Igeno, G. Becerra and R. Blasco Bioquimica y Biologia Molecular, Facultad de Veterinaria, Caceres, SPAIN

high (Spain), tolerates assimilates

to increase the shelf life for food products, in substitution to non-edible, non-degradable plastic material. These edible films are able to produce a controlled atmosphere, controlling gas and water permeability. In order to give more protection, nisin addi- tion to a corn starch-galactomannan film was evaluated in con- trolling Listeria monocitogenes. The performance of nisin against Listeria monocitogenes, in different edible films prepared with a galactomannan-corn starch blend, containing glycerol as a plasti- cizer was tested. The edible films with nisin at 0.1 and 0.25 mg/ ml in citrate buffer (50 mM, pH 5.0) showed dose dependent effect decreasing the ufcs after 24 and 48 hours of time exposure. When the distinct treatments was compared (control and nisin- containing films), statistical differences (p < 0.05) in the number of ufcs were detected. Nisin showed effectiveness against L. monocitogenes when blended with galactomannan and corn starch to different samples of edible films. Acknowledgement: Supported by Capes, CNPq, FUNCAP, a- VALNATURA, RENOR.

P7–58 Crosslinking aggregates of acetyl xylan esterase from Bacillus pumilus: higher stability in biotechnological applications S. Montoro-Garcı´ a, J. Navarro-Fernandez, F. Garcı´ a-Carmona and A. Sa´ nchez-Ferrer Biochemistry and Molecular Biology-A, Universidad de Murcia, Murcia, SPAIN

Residues with high concentrations of cyanide and cyano-metal complexes are generated in mining, metallurgic and jewellery industries. In spite of its high toxicity, cyanide may be used as a nitrogen source by some microorganisms. The bacterium Pseudo- monas pseudoalcaligenes CECT5344, which was isolated in Co´ r- doba cyanide and concentrations under alkaline conditions (pH 9–10), thus avoid- ing its volatilization as HCN, and makes it a good candidate for the biological treatment of industrial wastes contaminated with cyanide. Due to cyanide toxicity those organisms able to degrade it, need first to develop specific resistance mechanisms. Among them we have found a cyanide-inducible aerobic respiration insensitive to cyanide. Concerning to the cyanide assimilation pathway, it is remarkable the fact that in this bacterium L-malate is an excellent electron donor of bacterial respiration (without NAD+), producing oxaloacetate. Oxaloacetate reacts with cya- nide producing the corresponding cyanohydrin. In this work we study the role and regulation of the two malate-quinone oxidore- ductase (MQO) genes found in the draft of the bacterium gen- ome. Fumarate is also substrate of the aerobic respiration of this bacterium catalyzed by cell-free extracts, giving that the fumarase activity converts fumarate into malate. We also have found two fumarase genes in the draft of the genome, one probably contain- ing Fe, and the other without iron and probably regulated by fur. That is the reason why we propose as objectives to know the role of the fumarases in cyanide metabolism by P. pseudoalcalig- enes CECT5344. Acknowledgements: The authors thank the financial support from the Ministerio de Ciencia e Innovacio´ n.-BIO2008-04542- C02-02, and Junta de Extremadura PRI07A09.

the precipitant

P7–57 Corn starch-galactomannan-nisin based edible film antimicrobial properties C. A. Soares1, L. M. Pastrana Castro2, M. L. Ru´ a2, A. C. Monteiro-Moreira3, W. P. Felix1 and R. A. Moreira3 1Bioquı´mica, Universidade Estadual do Ceara´ – UECE, Fortaleza, BRAZIL, 2Quimica Alimentaria, Universidad de Vigo – Facultad de Ciencias, Ourense, SPAIN, 3Pharmacy, Universidade de Fortaleza – UNIFOR, Fortaleza, BRAZIL

exhibit higher KM,

growthin in importance

eubacterium Bacillus pumilus CECT 5072 The mesophilic expresses a hypothetical acetyl xylan esterase (AXE). The resulted protein was over-expressed in a soluble form into Escher- ichia coli Rosetta (DE3) (pLys), and purified. Recombinant acetyl xylan esterase was found to have deacetylase activity towards a number of acetylated substrates, including cephalosporin C and 7-aminocephalosporanic acid; therefore it was classified as a Cephalosporin-C deacetylase (EC 3.1.1.41). The result enzyme presents alkaliphilic properties, which is often required in indus- trial applications where harsh conditions exist. Immobilization of this enzyme via cross-linking is attractive because the final pro- tein is highly active and stable biocatalyst. Consecutive optimiza- tion of type and cross-linker concentration resulted in CLEAs showing higher operational stability compared to the soluble enzyme over a broad range of pH and tempera- ture. The aggregation method was improved up to 87% activity yield, even avoiding diffusion limitations. A remarkable change of affinity was found with the inmobilization for both substrates. 70.81 mM and lower Vmax CLEAs 14.04 lmoles/min/mg towards 7ACA, compared to soluble form affinity. Physical structure of CLEAs was studied under opti- mized conditions with SEM. Such immobilized enzymes are expected to reduce costs in the deacetylation of cephalosporins, resulting in a highly valuable biocatalyst for the industrial syn- thesis of semisynthetic b-lactam antibiotics. Acknowledgements: This study was partially supported by MEC (BIO2007-62510), ‘Grupos Excelencia Regio´ n de Murcia’ and DGI Consejerı´ a Educacio´ n Cultura CARM. (09BIO2005/01- 6470). S.M.G. is a holder of a predoctoral grant (FPU) from Fundacio´ n Se´ neca, Murcia, Spain. J.N.F is a holder predoctoral (FPI) grant from MEC, Spain.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Packaging is an important sector of the world industry, repre- senting 2% of the GNProduct of developed countries, and is con- tinuously and size. Preservation processes of foods still depend on effective packaging. So the modified-atmosphere packaging makes possible that several food products can be sold fresh or only chilled following the increased demand on self-life extension, product safety, cost-efficiency and consumers convenience. The ability of galactomannan edible films to regulate the migration of moisture, lipids, and gases, can be used to improve the quality and extend the shelf life of food- stuffs. Galactomannans, heteropolysaccharides constituted by a b-(1-4)-linked D-mannose backbone with D-galactosyl residues a- (1–6)-linked ramification showing a unique high viscosity charac- teristic. The food safe Adenanthera pavonina seed endosperm galactomannan-corn starch blend coating films have been tested

285

Abstracts

Poster Presentations

infiltration. Expressed

P7–59 The in vivo StreptoTag method: a new technique for isolation of RNA binding proteins M. Mu¨ llner1, S. Glanz1, W. H. Gu¨ nzburg1, S. Indik1, C. Hohenadl1 and J. Dangerfield2 1Pathobiology, Institute for Virology, Vienna, AUSTRIA, 2Austrianova Singapore, Singapore, SINGAPORE

inducible RbcS promoter. Several genetic constructs were designed and prepared and the possible expression of desired pro- teins in tobacco plants was studied by transient expression via agrobacterial oxygenases His/BphC, BphC/GUS, BphC/LUC and His/ISPTOL were then detected by Western blot or histochemically. The next step involved prepara- tion of transgenic plants. BphC gene was transferred into plant genome of N. tabacum by agrobacterial infection. The presence of transgenic DNA and expressed proteins was studied using sev- eral techniques. There has been also proven the higher resistance of transgenic plants to 2,3-dihydroxybiphenyl. TodC1C2 genes are transferred nowadays into plant genome of N. tabacum and L. ussitatissimum. Acknowledgement: MSMT 1M06030 and ME-09024-BIOA- ROM MSM 6046137305.

(RRE). Standardisation of element

P7–61 Analysis of polysaccharide from Bifidobacterium G. Novik1, E. Szwajcer Dey2 and A. Gamian3 1Belarus Collection of Microorganisms, Institute of Microbiology, National Academy of Sciences of Belarus, Minsk, BELARUS, 2Pure and Applied Biochemistry, Lund University, Lund, SWEDEN, 3Immunology of Infectious Diseases, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, POLAND

RNA-protein interactions play a key role in fundamental pro- cesses of all living organisms. A number of methods are available to identify RNA binding proteins in vitro; however, many often lead to experimental artefacts. The StreptoTag method employs in vitro transcribed RNA target sequences which are tagged with an RNA aptamer (STag) specifically binding to streptomycin. High- and low-affinity RNA binding proteins from crude cell extracts may bind to the chromatographically immobilised target RNA and are then co-eluted by addition of streptomycin. We have now adapted the system for in vivo applications. Therefore, STag-RNA is expressed in transfected eukaryotic cells allowing RNA-protein interactions in physiological conditions thereby sig- nificantly reducing unspecific bindings. For a proof of principle, we tried to isolate the HIV-1 RNA binding protein Rev from HEK293 cells by expressing STag-RNA harbouring the Rev responsive the method included demonstration of high-level target RNA synthesis in tranfected cells and successful high affinity interaction of recom- binant Rev with immobilised STag-RRE-RNA. In a next step, target RNA and Rev protein will be co-expressed in HEK293 cells and chromatographically purified. In conclusion, an opti- mised in vivo StreptoTag method will facilitate isolation and iden- tification of currently unknown RNA binding proteins as well as further protein interacting factors.

P7–60 Introduction of bacterial genes for dioxygenases into plant genome to increase phytoremediation abilities of aromatic pollutants M. Novakova1, M. Novakova2, M. Mackova1, M. Mackova2, Z. Chrastilova1, J. Viktorova1, M. Sylvestre3, T. Macek2 and T. Macek1 1Department of Biochemistry and Microbiology, Institute of Chemical Technology Prague, Prague, CZECH REPUBLIC, 2Department of Organic Chemistry and Biochemistry CAS, Department of Natural Products, Prague, CZECH REPUBLIC, 3INRS-Quebec, Montreal, QC, CANADA

Bifidobacteria are thought to be important for human health and their participation in the functions of the intestine, taxonomy and ecology had been reviewed. Several reports concerned the composi- tion of the bifidobacterial cell wall and the structure of cell wall polysaccharides. It was shown that the polysaccharide components present an anti-tumour and immunopotentiating activities. In this study we purified and analysed the extracellular polysaccharides of Bifidobacterium adolescentis 94 BIM, B. bifidum 791 and B. longum 379M. Morphological studies these strains showed that cells are either long (0.5 · 8 nm), sometimes branched and have wide ends, or short and coccoid. We observed, using the electron microscopy, the existence of the intercellular links with the aid of different extracellular structures – cell wall evaginations, capsule-like mate- rial or microfibrillae, similar to the other strains of this species. Aiming at understanding of the molecular basis for the intercellu- lar links, we have been investigating surface polysaccharides of these strains. Cells grown in various media produced similar poly- saccharides with the same sugars, but some differences occurred in their molar ratios. These polysaccharides differ from previously reported polysaccharides of B. adolescentis YIT 4011 and B. adole- scentis M101-4. The first one contained glucose and 6-deoxytalose with small amount of glycopeptide and the second one had glucose and galactose as major constituents and a galactofuranose as minor compound. Cell wall polysaccharides composed of glucose and galactose were also found in B. infantis and B. longum. Varia- tion of dietary environment of bifidobacteria might change the structure of surface polysaccharides, thus modulate their antigenic- ity and possibly other, including probiotic activities.

P7–62 Pestivirus diagnosis by RT-PCR and ELISA H. Albayrak1, S. Okur Gumusova2, E. Ozan1 and Z. Yazici2 1Virology, Veterinary Control and Research Institute, Samsun, TURKEY, 2Virology, Veterinary Faculty, Ondokuz Mayis University, Samsun, TURKEY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

In this study, 42 aborted fetuses (21 lambs and 21 calves) were investigated for pestivirus by RT-PCR and ELISA at 10 different provinces in Black Sea Region in Turkey. We used to suspen- Phytoremediation using transgenic plants can provide a useful, cheap and effective method for decontamination of the environ- ment. The aim of this work is to construct genetically modified plants with increased capabilities to degrade organic pollutants such as polychlorinated biphenyls, toluene, TCE and other organic pollutants. Therefore bacterial genes were chosen to clone into plant of Nicotiana tabacum and Linum ussitatissimum. Chosen genes of environmental importance are genes of bacterial dioxygenases – bphC and todC1C2 genes. BphC gene encodes 2,3- dihydroxybiphenyl-1,2-dioxygenase which cleaves the aromatic ring of dihydroxybiphenyl and was cloned in fusion with gene for beta-glucuronidase (GUS), luciferase (LUC) and with histi- dine tail, under the CaMV 35S promoter. The todC1C2 genes were chosen to clone into plants to produce oxygenase ISPTOL (with histidine tail), a component of bacterial toluene dioxygen- ase that can oxidize toluene and other organic pollutants, both genes either under the constitutive CaMV 35S promoter or

286

Poster Presentations

Abstracts

sions of brain, lung and liver tissues of aborted fetuses in the tests. At the end of the ELISA test was determinated 28.57% pestivirus in both lambs and calves. Besides, RT-PCR test was detected 66.66% Border diseases virus (BDV) in lambs and 28.57% Bovine viral diarrhea virus (BVDV) in calves fetuses. As a result of, BVDV was diagnosed the equal sensitivity with pesti- virus antigen ELISA test and RT-PCR test in aborted calves.

P7–63 First investigation of West Nile virus by rRT-PCR in Turkey H. Albayrak1, S. Okur Gumusova2, Z. Yazici2 and E. Ozan1 1Virology, Veterinary Control and Research Institute, Samsun, TURKEY, 2Virology, Veterinary Faculty, Ondokuz Mayis University, Samsun, TURKEY

lines and to inhibit protein kinase activity,

Kinetics of the hemolysis process was followed by the release of K+ and hemoglobin from erythrocytes. K+ release induced by 0.030 mM trehalose lipid was completed after 20–30 minutes. At this point hemoglobin release began, taking ca. 60 minutes to be completed. Thus, hemolysis was a slow process, and K+ release clearly preceded haemoglobin release. Our results suggest that trehalose lipid induced erythrocyte permeabilization to small sol- utes, without the destruction of the membrane. An osmotic-pro- tection experiment was conducted to get further insight into the mechanism of hemolysis. Osmotic protectants (PEG of various molecular weigths), with a diameter equal or larger than 25 a¨ ng- strom, impeded trehalose lipid-induced hemolysis. These results trehalose lipid incorporates into the erythrocyte suggest that membrane, forming defects or ‘pores’, which allows leakage of small solutes, like K+, but not of larger polymers, like hemoglo- bin. Altogether indicate that trehalose lipid-induced hemolysis occurs through an osmotic-lytic mechanism. Since succinoyl tre- halose lipids have been shown to induce differentiation of leu- kaemia cell it is feasible that most of these biological actions relate to their per- turbing actions on the phospholipid membrane, like we have shown here.

In this study, a total of 120 horse sera samples equally collected, four province from both coastal and inland Blacksea Region in Turkey. The sera were investigated for West Nile virus (WNV) presence by Taqman-based real-time reverse transcriptase poly- merase chain reaction assay (rRT-PCR). End of the study, WNV wasn’t detected in horse sera samples. The results are indicated that there is no evidence of WNV in horses in the Blacksea Region of Turkey.

P7–64 Diagnosis to Bovine Herpes Virus-1 (BHV-1) in Turkey by ELISA S. O. Gumusova1, H. Albayrak2, E. O¨ zan2 and Z. Yazici1 1Virology, Ondokuz Mayis University Veterinary Faculty, Samsun, TURKEY, 2Virology, Veterinary Control and Research Institute, Samsun, TURKEY

P7–66 Phytase from Bacillus strain isolated from rhizosphere of Acacia cyanophylla Lindley H. U. Ozturk1, A. A. Denizci1, S. Dincer2, A. Ogan3, A. Erarslan4 and D. Kazan1,5 1Enzyme and Microbial Biotechnology, Tubitak Marmara Research Center Genetic Engineering and Biotechnology Institute, Kocaeli, TURKEY, 2Department of Biology, Cukurova University Faculty of Science and Letters, Adana, TURKEY, 3Department of Chemistry, Marmara University Faculty of Art and Science, Istan- bul, TURKEY, 4Department of Chemistry, Kocaeli University Fac- ulty of Arts and Sciences, Kocaeli, TURKEY, 5Bioengineering Department, Marmara University Faculty of Engineering, Istanbul, TURKEY

In this study, seroprevalace of Bovine Herpes Virus-1 (BHV-1) infection was investigated in cattle in private farms in 4 provinces (Sinop, Samsun, Amasya, Tokat) and their towns at Middle Blacksea Region in Turkey. For this aim, serum samples ran- domly collected from 501 cattle was tested by Enzyme Linked Immunosorbent Assay (ELISA). The antibodies were determinat- ed 44.71% (224/501) in cattle against to BHV-1.

P7–65 Mechanism of the hemolytic activity of a Rhodococcus sp. trehalose lipid biosurfactant A. Ortiz, A. Zaragoza, J. A. Teruel and F. J. Aranda Bioquı´mica y Biologı´a Molecular-A, Universidad de Murcia, Murcia, SPAIN

Phytases (myo-inositol hexakisphosphate phosphohydrolase) are a special class of phosphatase that catalyze the sequential hydro- lysis of phytate, the major storage form of phosphate in grains and oil seed, to less phosphorylated myo-inositol derivatives and inorganic phosphate. In recent years, phytases have been studied intensively because of the great interest in its application as feed additive, processing of human food, synthesis of lower inositol importance. Bacillus phytases have phosphates, and ecological considerable potential in commercial and environmental applica- tions because of their desirable activity profile under neutral pH, higher thermal stability during animal feed-pelleting process in which temperature can reach to 80–100(cid:2)C, and strict substrate specificity. In our study, various phytase producer candidates were isolated from different local ecosystems from C¸ ukurova region in Turkey. The supernatant of culture broth was tested for the best phytase activity. Bacillus strain, isolated from the rhizosphere of Acacia cyanophylla Lindley showed the highest phytase activity production and therefore chosen for phytase enzyme producer. Taxonomic characterization of strain such as morphological and biochemical characters, cellular fatty acid analyses, 16S rRNA genes and %GC mol content was deter- mined. For an efficient phytase production, different carbon and nitrogen sources as well as environmental conditions (pH, tem- perature, etc.) were optimized.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Biosurfactants are surface active amphiphilic compounds of bio- logical origin, mainly produced by microorganisms. These com- pounds are encountering new interesting applications because they are more biodegradable and environmentally friendly than chemically synthesized surfactants, and display various interest- ing biological activities. In addition, many of these compounds can be produced through biotechnological processes, using wast- ing raw materials as carbon source, which is an added value. In the search for new compounds an important step is the charac- terization of their physicochemical and biological activities. We show here that a trehalose lipid biosurfactant produced by Rho- dococcus sp. caused human erythrocytes hemolysis. Hemolysis took place at concentrations both above and below the cmc (ca. 0.080 mM), i.e., at sublytic concentrations of the biosurfactant.

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P7–67 Teaching Biotechnology through a laboratory exercise that integrates concepts of Enzymology and Molecular Biology A. S. Martins1, N. B. Pinhal1, R. Soler2, L. M. C. Paiva1, A. D. Panek1 and C. L. A. Paiva2 1Department of Genetics and Molecular Biology, Instituto Biomedi- co/UNIRIO, Rio de Janeiro, BRAZIL, 2Departamento de Bioqui- mica, Instituto de Quı´mica, Rio de Janeiro, BRAZIL

ing that the affinity of substrate was weaker. The activity of free and immobilised Rapidase C80 was maximum at pH 4.2, while the optimum temperature was shifted from 55 to 50(cid:2)C. The acti- vation energy of reaction was also calculated. Thermal stability was not significantly altered by immobilisation. Free and immo- bilised preparation reduced the viscosity of banana juice from 2.27 to 0.81 and 1.05 mm2/s, respectively, after 30 minutes at 50(cid:2)C. Furthermore, the immobilised enzyme could be re-used through 3 cycles and the efficiency loss in viscosity reduction was found to be only 7.1%.

P7–69 Identification of trichothecene- and moniliformin-producing Fusarium species based on DNA microarray L. Pavlatova1, J. Ovesna1, J. Hodek1 and D. Novotny2 1Department of Molecular biology, Crop Research Institute, Pra- gue 6, CZECH REPUBLIC, 2Department of Mycology, Crop Research Institute, Prague 6, CZECH REPUBLIC

The aim of this poster is to present a laboratory exercise for teaching Biotechnology through an approach that integrates con- cepts of Enzymology and Molecular Biology. This novel labora- tory exercise is based on the extraction of trehalase (EC 3.21.28) from a transformed Escherichia coli strain Mph2 (carrying the plasmid pTRE11 that harbors the trehalase gene TreA+) and immobilization of the enzyme on chitin particles. The students participate in steps such as bacterium cultivation and transforma- tion, protein extraction from E. coli periplasmic space, prepara- tion of chitin particles and enzyme immobilization and they can discuss the vast biotechnological applications of trehalose. Treha- lase can be easily released by osmotic shock and it is not neces- sary to purify it from the osmotic shock fluid (OSF) because it is free of any other glycosidase. Therefore, OSF was immobilized on chitin without any step of trehalase purification. This lab exercise also teaches how to determine the kinetic parameters (Michaelis constant- Km and maximum velocity- Vm) for free and immobilized enzymes. The determination of kinetic parame- ters using the Michaelis-Menten model is not a novelty. Never- theless, an experimental class that includes the comparison of these parameters for free and immobilized enzymes, using the students´ own experimental data, would be extremely useful for teaching the subject. They can identify and compare these param- eters and discuss the differences and similarities among them. This discussion points towards issues such as enzyme conforma- tion and structural rigidity related to changes in kinetic parame- ters mainly Km. This Laboratory Exercise was designed for graduate students that have a good background in Biochemistry and have joined courses involving Biotechnology.

Fusarium is one of the most potent plant pathogenic fungi worldwide and produce wide range of secondary metabolites such as trichothecenes, moniliformin, zearalenone and enniatins. The aim of our work was to test DNA microarray for identification trichothecene-and moniliformin-producing Fusarium species in our condition on the species which mostly occure in Czech Republic. We prepared in-house DNA microarray by using alde- hyde glass slides and oligonucleotiedes with modification on 5’ end by amino group. The sequences of oligonucleotide probes were assumed from literature and were developed on well known sequence of translation elongation factor (TEF-1a). Afterwards we amplified part of (TEF-1a) by using one pair of specific prim- ers for fusarium conservative genome seat. Obtained PCR prod- ucts were fluorescent labelled using BioPrime Total Genomic Labelling System (Invitrogen). We also tested whole genome DNA labelling and hybridized products against the DNA Micro- array but we didn’t receive expected specific signals. Optimisation of the protocol should be the next step of the procedure. We show that our in-house DNA microarray is suitable for identifica- tion and detection of fusarium species occuring in the Czech Republic. Acknowledgement: The work was supported by the project of the Ministry of Agriculture of the Czech Republic (PUV) NAZV 1G 46068 and CZ002700604

P7–68 Properties of polygalacturonase immobilised on alginate beads for the clarification of banana juice M. D. Busto, K. E. Garcia-Tramontin, N. Ortega, D. Palacios and M. Perez-Mateos Biotechnology and Food Science, University of Burgos, Burgos, SPAIN

P7–70 Micro-extraction of hydrolyzed free and conjugated sterols for their determination using HPLC M. Pavlik1, V. Siglerova1, Z. Wimmer1, H. Sovova2, M. Sajfrtova2 and D. Pavlikova3 1Isotope Laboratory, Institute of Experimental Botany CAS, Prague 4, CZECH REPUBLIC, 2Separation Processes, Institute of Chemical Process Fundamentals CAS, Prague 6, CZECH REPUBLIC, 3Agro-Environmental Chemistry and Plant Nutrition, Czech University of Life Sciences Prague, Prague 6, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Pectinases play an important role in the food industry to improve the extraction yield or to decrase the viscosity of fruit juice. For industrial application, the ability to make enzymes stable and re- usable has meant that immobilised enzyme have attracted a great deal of attention. Other benefits obtained as well, include better operational control, flexibility of reactor design, and ease of product recovery without catalyst contamination. In this study, the commercial pectinases Rapidase C80 (Gist-Brocades), Pectin- ex 3XL (Novozyme), Biopectinase CCM (Quest International) and Grindamyl 3PA Pectinase (Danisco Ingredients) containing polyg- alacturonase were immobilised on alginate beads to clarify banana juice. Soluble and immobilised polygalacturonase fol- lowed Michaelis-Menten kinetics. Rapidase C80 and Pectinex 3XL showed values of Km similar to those of the free enzyme, while the apparent Km of the Biopectinase and Grindamyl im- mobilised was higher than that of the enzyme in solution, indicat- The increasing concentration of phytosterols in plants is possible to evaluate as positive aspect for reducing plasma total and low density lipoprotein cholesterol concentration. Several studies sug- gest that these sterols offer a protection from the most common cancers. Phytosterols are used by numbers of producers with the growing interest. In the tested procedures for determination of free and conjugated sterols a quantity of an only 1 mg of isolated

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Abstracts

sample was needed for the HPLC analysis. The sample can be prepared using either the alkaline hydrolysis of steryl esters or the acid hydrolysis of steryl glycosides, which both result in obtaining free sterols. The procedure designed with no need of neutralization of the hydrolysate is an important advantage. The designed procedure has been based on employing the only alka- line hydrolysis for transformation of esters to free sterols (and si- toindosides to steryl glycosides). Steryl glycosides and free sterols can be directly analyzed using HPLC at a wavelength 210 nm. Reduced number of analyses in contrast to the original procedure is needed. Acknowledgement: A financial support: A project (Suprafyt) 2B06024 (MSMT CR) and GA CR 104/07/0977.

salts, e.g. sulphate, phosphate and nitrate, as a source of nitro- gen. From complex substrates, pepton, extracts, cornsteep, malt wort, etc. are suitable. For the synthesis of phosphoproteids, nu- cleoproteids, phospholipids, nucleic acids and phosphoryl‘s poly- saccharides, microorganisms utilize phosphor in the inorganic phosphate forms. Because of the synthesis of sulphur amino acids and glutathione, the medium for yeast is also supplemented with ammonium sulphate. The most commonly growth factors for this type of microorganisms are biotin and pantothen acid. In order to achieve as high biomass yield as possible, the most important is to ensure aerobic cultivation, which is limited by an amount of carbon. Too high content of carbon source namely leads to a change of aerobic cultivation to fermentation. Besides a good choice of the productive strain and suitable composition of the medium, the optimization of physical conditions of a cultivation process, e.g. pH, temperature and aeration, is very important.

P7–71 Cloning of structural and nonstructural genes of Potato leafroll virus (PLRV) for antibodies production and its safety detection H. Plchova1, N. Cerovska1, T. Moravec1 and P. Dedic2 1Virology, Institute of Experimental Botany v.v.i. CAS, Prague 6, CZECH REPUBLIC, 2Virology, Potato Research Institute, X Havlickuv Brod, CZECH REPUBLIC

P7–73 Biodiversity of catalases and peroxidases of the microbial isolates from toxic environment J. Godocikova, M. Buckova, M. Zamocky and B. Polek Department of microbiology, Institute of Molecular Biology, Bratislava, SLOVAK REPUBLIC

Potato leafroll virus (PLRV) is the type member of the genus Po- lerovirus which belongs to the family Luteoviridae. The RNA genome of PLRV consists of a 3 5.9 kb positive-sense single- stranded RNA and encodes eight main open reading frames (ORFs) numbered from 0 to 7. PLRV is considered one of the most damaging potato viruses and is worldwide distributed. One way how to manage PLRV is to use virus-free certified seeds. Enzyme-linked immunosorbent assay (ELISA) is a sensitive method for PLRV detection. To obtain antibodies for safety detection of PLRV, we sequenced complete genome of three Czech PLRV isolates and chose one nonstructural gene (ORF2– encodes motifs typical for RNA-dependent RNA polymerases) and one structural gene (ORF3 – encodes coat protein) for clon- ing into bacterial expression vectors pET-45b(+) and pMPM4W. Only the middle part of ORF2 cloned into pET-45b(+) as a fusion with His-tag was possible to expressed in Rosetta-gami 2(DE3) cells, because of the high content of codons rarely used in E. coli. The full-length ORF3 was cloned into pMPM4W. The recombinant viral proteins expressed in bacterial cells have great potential as an alternative source of antigens for raising specific antibodies to plant viruses, which can be produced in large quan- tities and can be adapted for specific aplications. Acknowledgement: This research was supported by the grant No. 1M06030 of the Ministry of Education, Youth and Sports of the Czech Republic and the grant No. QH71123 of the Grant Agency of Ministry of Agriculture of the Czech Republic. Regulation of catalase-peroxidase activities including alternation in expression of isozymes and biodiversity in sequences of katG isolates surviving in toxic environ- was essential for microbial ment. We have evaluated the role of heme catalase encoded by cat-1 gene from the soil bacterium Comamonas terrigena N3H in the response to various forms of oxidative stress. Results indicate that this constitutively expressed catalase represents the major source for the defense of Comamonas terrigena against toxic per- oxides. The Comamonas cells express also a second–bigger cata- lase isozyme but its regulation is probably more complicated. The sequence analysis confirmed the presence of highly conserved catalase sequence motifs in two environmental strains of Coma- monas terrigena. We have furthermore found remarkable molecu- lar biodiversity in sequences of katG genes from strains of Comamonas terrigena and Comamonas testosteroni isolated from sludge of a wastewater treatment plant. We have concomitantly observed a several-fold increase in both catalase and peroxidase activities upon various forms of oxidative stress in these bacteria. Our results underline the importance of katG expression in the defense mechanism against oxidative stress in proteobacteria. Biodiversity of Comamonadaceae katG genes clearly demon- strates an adaptive form of eubacterial evolution to various forms of oxidative stress in the natural and contaminated envi- ronment. Acknowledgement: This work was supported by the grant VEGA 2/0084/08 and APVV-0444–07.

P7–72 An influence of medium components on yeast biomass formation A. Pola´ kova´ , E. Szabova´ and D. Urminska´ Department of Biochemistry and Biotechnology, Slovak Agricultural University, Nitra, SLOVAK REPUBLIC

P7–74 Biofilm in Listeria monocytogenes and its relation to luxS S. Purkrtova, T. Pilchova, K. Demnerova and J. Pazlarova Department of Biochemistry and Microbiology, Institute of Chemical Technology Prague, Prague 6, CZECH REPUBLIC

cultivation aerobic ensure which of

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

The yeast for biomass formation requires medium with suitable water activity, carbon and nitrogen sources, the biogenous ele- ments and microelements as well as growth factors in the conce- these trations, microorganisms. Chemoheterotrophic organisms, such as yeast, most frequently utilize carbon and nitrogen in the organic molec- ular forms, which are also sources of hydrogen. The most usable is carbon in hexose sugars and sucrose. Yeast uses ammonium Listeria monocytogenes is one of the most dangerous pathogens in the food processing industry and eating food contaminated with this bacterium could lead to serious disease – listeriosis. Food could be contaminated on food processing surfaces, if cleaning and disinfection are not perfect and regular, because lis- terias can create in such conditions structured community – bio- films, in which they overcome unfavourable conditions and take

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among tested bacteria. PFGE is suitable for pathogenic bacteria characterization analysis.

P7–76 Catheter Associated Bacteriuria in different Human Patients groups subjected to Cystoscopy S. K. Arif1, A. R. Al-Najar2, R. a. M. Al-Obaidi3, C. Hagemann4 and H. M. Said5 1Biology Department, University of Sulaimani College of Science, Sulaimani-Kurdistan Region, IRAQ, 2Department of Medical Microbiology, Al-Mustansryiah University College of Medicine, Baghdad, IRAQ, 3Department of Microbiology, University of Sulaimani College of Veterinary Medicine, Sulaimani-Kurdistan Region, IRAQ, 4Tumor biology Laboratory Department of Neurosurgery, University of Wu¨rzburg Medical Faculty, Wuerzburg, GERMANY, 5Department of Radiation Oncology, University of Wu¨rzburg Medical Faculty, Wuerzburg, GERMANY

needed nutrients. A type strain of Listeria monocytogenes CCM 7202 (ATCC 13932) and an industry isolate were compared in their ability to grow and to form biofilm in different media and at different temperatures on a model system using microtiter plates COSTAR 3797. Buffered peptone water (BPW), saline solution, brain heart infusion (undiluted, 10X diluted), BPW with 0.5% glucose and/or 5% NaCl, BPW with 0.05% glucose and/or 0.5% NaCl were tested. BPW with 0.05% glucose in 30(cid:2)C was found to be the optimal for forming biofilm contrary the other temperatures 8, 25 and 37(cid:2)C. More than 10 other isolates of Lis- teria monocytogenes from raw milk and food were tested for the ability forming biofilm in these optimal conditions. It is known that strains of Listeria monocytogenes whose luxS was partly deleted are able to form much more biofilm. To study this gene and its relation to biofilm forming polymerase chain reaction amplifying sequence containing luxS was designed and carried out with the DNA from tested isolates. Products were studied by restriction cutting and by sequencing. Acknowledgement: This project was supported by MSˇ MT 2B08050.

P7–75 Bacterial biofilms in patients with indwelling urinary catheters S. K. Arif1, A. R. Al-Najar2, R. a. M. Al-Obaidi3, C. Hagemann4 and H. M. Said5 1Biology Department, University of Sulaimani College of Science, Sulaimani-Kurdistan Region, IRAQ, 2Department of Medical Microbiology, Al-Mustansryiah University College of Medicine, Baghdad, IRAQ, 3Department of Medical Microbiology, University of Sulaimani College of Veterinary Medicine, Sulaimani-Kurdistan- Region, IRAQ, 4Tumor biology Laboratory Department of Neuro- surgery, University of Wu¨rzburg Medical Faculty, Wuerzburg, GERMANY, 5Department of Radiation Oncology, University of Wu¨rzburg Medical Faculty, Wuerzburg, GERMANY

Introduction: Urinary catheters are inserted in millions patients in acute-care hospitals. Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in hospitals, comprising > 40% of all institutionally acquired infections & the 2nd nosocomial bloodstream infection cause. Here, we investigate the catheter role in pre and post operative bacteriuria in patient’s undergone cystoscopy. Materials and Methods: 154 patients admitted to Surgical Spe- cialties Hospital in Baghdad for Diagnostic cystoscopy, transure- thral resection of bladder tumor (TURT) and Transurethral Resection of Prostate (TURP) were included. Patient’s age, sex and surgery duration were studied as bacteriuria risk factors. 462 urine samples, 158 cystoscope swabs, 65 irrigation samples and 112 antiseptic samples were investigated to determine bacteriuria incidence, causative agents and possible contamination source during operation. Results: In non catheterized patients 26.3% of the patients had bacteriuria, 90% of catheterized patients showed bacteriuia pre operatively (p < 0.05). Patients with pre operative catheterization developed bacteriuria post operatively, while 49% of patients with no previous catheterization history developed post operative bacteriuria (p < 0.05). Post operative bacteriuria in patients undergone surgery less than 45 minutes was 49% which was sig- nificantly less than those undergone surgery more than 45 min- utes 80% (p < 0.05). Patient’s age and sex were found to be not significantly related to post operative bacteriuria. Escherichia coli was the most common organism isolated in pre, per and post operative samples in TURT and TURP group. Conclusions: Catheterization and surgery duration > 45 min- utes were found to be the most important risk factors for bacteri- uria post operatively when performed in the hospitals in the region. Local enviromental factors should be taken in concern when novel patients treatment concepts recognizing this facts are established and applied.

P7–77 Characterization and immobilization of a new sialic acid aldolase from Lactobacillus plantarum WCFS1 A. Sa´ nchez-Ferrer, A. B. Lo´ pez-Rodrı´ guez, G. Sa´ nchez-Carro´ n, A. Sola-Carvajal and F. Garcı´ a-Carmona Biochemistry and Molecular Biology-A, Universidad de Murcia, Murcia, SPAIN

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Introduction: Bacterial biofilm help clinicians explain some chal- lenging areas in pathogenesis, diagnosis, and urinary tract infec- tions (UTI) treatment. Microscopic observation confirmed that many types of UTIs (e.g., catheter-associated infections, struvite urolithiasis, continuous ambulatory peritoneal dialysis, and chronic prostatitis) are biofilms associated. Genetic and molecu- lar biofilm formation basis in human pathogenic bacterial strains is multifaceted. Biofilm formation needs 2 properties: cells adher- ence to surface & accumulation to form multilayered cell clusters. Matrials and Methods: We aimed causative agents isolation and identification of pre, per and post operative bacteriuria, bio- film formation on microtiter plates and urinary catheter, biofilm formation inhibition via chemical surfactants and biofilm antibi- otic susceptibility to antimicrobial agents. Ionic surfactant forma- tion analysis (SDS) and anionic surfactant (Tween 80) at 0.2% in TSB in addition to biofilm antibiotic susceptibility assay & bacte- rial isolates molecular analysis via Pulse Field Gel Electrophore- sis (PFGE) & parallel biochemical & serologic samples analysis was done. Results: Bacteriuria incidence in controls was 28%, E. coli were 10%, S. aureus 8%, P. aeruginosa 2% and 1% S. faecalis. SDS significantly inhibited biofilm formation on microtiter plate wells. Catheterization is most important risk factor for bacteriuria pre & post operatively. Antibiotics application did not eradicate bac- terial biofilm on catheter surface. Most isolated uropathogens were, E. coli, S. aureus and K. pneumoniae. Conclusions: Most isolated uropathogens were, E. coli, S. aur- eus and K. pneumoniae. Pseudomonas aeruginosa showed highest biofilm size, biofilm growth ratio and biofilm formation rate Sialic acid aldolases (EC 4.1.3.3, NAL) are type I aldolases that catalyze the reversible aldol cleavage of N-acetylneuraminic acid

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Abstracts

P7–79 Complex of multi-haem cytochrome with flavin photoinjector of electron as a prototype of electron conductng nanodevice A. Slutsky, M. Kritsky, T. Lyudnikova, T. Tikhonova and V. Popov Biochemistry, A. N. Bach Institute of Biochemistry, Moscow, RUSSIA

(Neu5Ac) to form pyruvate and N-acetyl-D-mannosamine with the equilibrium favoring the Neu5Ac cleavage. NAL has been biotechnologically used for the synthesis of Neu5Ac and its ana- logs and its X-ray structure solved. In this work, the gene nanA, of 879 pb, from Lactobacillus plantarum WCFS1was cloned and overexpressed into Rosetta Escherichia coli strain. The cloned putative protein was a 37 kDa monomeric aldolase with low homology with that from E. coli (38% identity). Its correspond- ing model showed the presence of the reactive lysine (Lys163), and the other relevant active site amino acids (Asp189, Glu190 and Ser206). The enzyme showed pH optimum about pH 7.3– 7.5 and remarkable temperature stability above 60ordm;C, with a thermal activation. The protein catalyzed the hydrolysis of Neu5Ac with KM and Vmax values of 1.7 mM and 60.2 lM/min- utes, respectively. Immobilization of this enzyme was carried out via cross-linking, giving rise to an active and stable biocatalyst. Consecutive optimization of the precipitant type and cross-linker concentration resulted in CLEAs showing good operational stability. Such immobilized enzymes are promising biocatalysts for the chemoenzymatic synthesis of sialic acids and their deriva- tives. Acknowledgements: This study was partially supported by MEC (BIO2007-62510), and ‘Grupos Excelencia Regio´ n de Mur- cia’ and DGI Consejerı´ a Educacio´ n y Cultura C.A. Murcia (09BIO2005/01-6470). A.L.R., G.S.C.and A.S.C. are holders of predoctoral grants from Universidad de Murcia, from MEC (FPU), and from 09B102005/01-6470, respectively.

The redox proteins such as cytochromes are considered today as a biomaterial to design electron conducting-nanodevices. Here we communicate the application of photochemical method to inject electrons to the electron-transfer chain of multi-haem nitrite reductase purified from the cell free extracts of haloalkaliphilic bacteria Thioalkalivibrio nitratireducense (TvNiR), exists in solu- tion as a stable, highly symmetrical hexamer, which binds 48 ha- ems. The redox potential values of eight cytochrome c-type haems composing an electron transport chain in each subunit vary from -10 to -310 mV. This chain furnishes electrons to per- form of catalytic functions in active center such as nitrite reduc- tion to ammonia. To study the electron donor activity of reduced flavins (riboflavin, FMN, FAD) and pteridines, the oxidized mol- ecules were subjected to photochemical reduction in the deaerat- ed samples, just prior to addition of TvNiR protein. In process of dark incubation only FMNH2 reduced electron transfer chain and, in presence of nitrite, induced its catalytic reduction. The oxidized forms of flavins and of 6,7-dimethylpterine (but not of lumazine) sensitized photooxidation of a high potential electron donor (Na2-EDTA) and acted as a photoswitch injecting elec- trons to TvNiR haemes and then to catalytic center and the reduction substrate. The electron injection into the protein abso- lutely depended on excitation of photosensitizer. When exposed to alternating irradiation and dark and in the presence of DMP sensitizer in microaerated medium (ca. 2% of atmospheric O2 pp), TvNiR demonstrated a periodic photoreduction and dark reoxidation of its haemes. Acknowledgement: This work is supported by RFBR grants 07-04-00460a, 07-04-01559a.

P7–78 Development of a ‘photoswitchable’ restriction endonuclease B. Schierling1, A. J. Noel1, L. H. Thi2, W. Wende1 and A. Pingoud1 1Institute of Biochemistry, Justus-Liebig-University, Giessen, GERMANY, 2Chemistry Department, Moscow State University, Moscow, RUSSIA

P7–80 Colloidal nanoparticles as a label for antibody in rapid immunodetection – comparison with the ELISA Z. Smidova, M. Blazkova, P. Rauch and L. Fukal Department of Biochemistry and Microbiology, Institute of Chemical Technology, Prague 6, CZECH REPUBLIC

The engineering of proteins in order to obtain light-responsive characteristics is in focus of photochemical research, because the possibility to regulate protein function quickly and of reversibly, which is difficult to achieve by other means. As a proof of principle for promising applications in future, we tried to generate a restriction endonuclease that can be turned on and off by irradiation with light, using the single chain (sc) variant of the restriction enzyme PvuII. ‘Photoswitchable’ pro- teins have been pro duced using azobenzene crosslinkers that can be isomerized between the trans- and cis-state and vice versa by illumination at specific wavelengths or by thermal relaxation from the cis- to the more stable trans-state. Up to now, a 7-fold higher activity could be obtained with a variant of scPvuII modified by two intramolecular crosslinks with 4,4¢- azobenzene-dimaleimide in the cis configuration than in the trans configuration. Switching was shown to be fully reversible. The higher activity of the scPvuII variant with the azobenzene group in the cis state was shown to be due to an increase mainly in Vmax. Acknowledgement: The work was partially supported by DFG program ‘International Research Training Groups’ (grant RFBR-DFG 08-04-91974).

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Nanoparticles can be a very useful tool to accelerate and simplify the immunoanalysis. The aim of the work was to develop such an immunoassay for the pesticide thiabendazole and to compare its parameters with the ELISA technique. Antibodies were labeled with colloidal carbon and gold and subsequently used to set up the immunochromatographic detection procedure. This was developed in a way that immunoreactants migrate horizon- tally through the porous membrane strip and are specifically cap- tured by their reaction partners in the final strip area. The detection limit of the method for the pesticide thiabendazole is 0.1ng/mL. The method was tested as a rapid detection tool for thiabendazole determination in fruit, baby food and fruit juices. Artificially contaminated model samples were used. Into the anal- ysis a diluted sample or a supernatant of the homogenized sam- ple, respectively, was applied. It was found out that the negative sample matrix effect was larger in fresh fruit and baby food than in fruit juices, therefore the former ones had to be more diluted. The achieved detection limit of the method in the majority of the

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tested matrix types enables to recognize exceeding the maximum residue limit given by the legislative, therefore this method is suit- able for food monitoring and control. In the work the achieved detection limits for particular matrix types using the immuno- chromatographic method and the ELISA will also be presented. Acknowledgement: This work was supported by the Czech Grant Agency No.525/07/0618 and by Ministry of Education of the Czech Republic, MSM6046137305.

and antiphlogistic antioxidant antitumor,

providing satiety, fullness and depth of aroma and flavour. Due to low contents of bitter components Pleurotus ostreatus bitter- ness is masked by high contents of sweet amino acids and soluble carbohydrates. For this reason the components of umami taste as well as sweet components provide the natural, typical taste of edible mushrooms. From the imunoactive matters isolated from microorganisms, plants or animals, beta-glucans have the prior position. These natural carbohydrates extracted from fruiting bodies of Pleurotus ostreatus have the significant immunostimu- lating effect on human organism. Valuable are their antimicro- bial, influences. Therefore, they are of great importance in prevention, prophy- laxis and supportive therapy of some civilized diseases like ath- erosclerosis, diabetes and some types of cancer.

P7–81 The influence of different cis-acting elements on expression of target protein in plant using viral vector A. Sokolova, P. Ivanov and J. Atabekov Virology, Moscow State University, Moscow, RUSSIA

P7–83 The importance of several Arabidopsis chromatin genes in transformation and/or transgene expression G. N. Tenea1, J. Spantzel2, L. Y. Lee2, Y. Zhou2, H. Oltmanns2 and S. B. Gelvin2 1Institute of Genetics, University of Bucharest, Bucharest, ROMANIA, 2Department of Biological Sciences, Purdue University, West Lafayette, IN, USA

Stable nuclear transformation is the most popular for protein expression in plants but transient systems are becoming more widespread. One of them utilizes viral vectors based on genomes of (+)-RNA-containing viruses. We tested the influence of three different cis-acting elements in the context of Turnip vein-clearing tobamovirus on the expression of human Granulocyte Colony- Stimulating Factor (G-CSF). Several viral vectors coding for those elements in all possible combinations were constructed, cloned into binary vector and used for infections of Nicotiana benthamiana leaves via agroinfiltration. Replication cycle of (+)- RNA viruses take place in cytoplasm, but in case of agroinjection transcription of T-DNA in nucleus creates the problem of effi- cient processing and export of vector RNA. Application of syn- thetic intron in the sequence of target gene significantly increased the expression of G-CSF. Another element consisted of nucleo- tide sequence coding for the first 26 aminoacids from viral coat protein (CP) that formed fusion with N-terminus of G-CSF. This modification improved synthesis of target protein as well. One might suppose that conservation of 5’-nontranslated sequence of corresponding subgenomic RNA and nucleotide context of initial codon helps to imitate the natural conditions of CP translation. We proved that intron and N-terminal CP sequence are function- ing independently from each other. When both elements are pre- sented in the vector positive effects are summarized so the level of expression is at least 4–5 times higher than during control infection. The last element – sequence of 6xHis tag did not affect expression.

P7–82 Pleurotus ostreatus, a source of nutritive and sensory effective matters E. Szabova´ , D. Urminska´ and A. Pola´ kova´ Department of Biochemistry and Biotechnology, Slovak Agricultural University, Nitra, SLOVAK REPUBLIC

Previous work from our laboratory indicated an essential role for Arabidopsis histone genes in T-DNA integration (2). RNAi tar- geted against 109 Arabidopsis chromatin genes further demon- strated a role for other chromatin proteins, such as SGA1, in Agrobacterium-mediated transformation (1). We investigated the effects of over-expressing numerous Arabidopsis histone cDNAs and an anti-silencing factor A (SGA1) cDNA on transformation and transgene expression. Transgenic Arabidopsis plants contain- ing additional copies of cDNAs encoding histone H2A (HTA), histone H4 (HFO), or SGA1 displayed increased susceptibility to transformation. Over-expression of all tested histone H2B (HTB) and most histone H3 (HTR) cDNAs did not increase transforma- tion. A parallel increase in transient gene expression was observed when the histone HTA or HFO cDNAs were co-trans- fected, together with a plant active gusA gene, into tobacco pro- toplasts. Using RT-PCR, we also detected an increase in gusA transcripts when the histone HTA1 cDNA was over-expressed in protoplasts. No such increase in gusA activity was seen when a SGA1 cDNA was co-transfected with a gusA gene into BY-2 protoplasts. These results suggest that over-expression of HTA and HFO increases transformation by stimulating transgene expression, whereas over-expression of SGA1 increases transfor- mation by a different mechanism. Over-expression of histone or SGA1 cDNAs does not increase expression of a previously inte- grated transgene, nor could HTA1 reverse silencing. These data suggest that histones may increase transgene expression by work- ing directly on the promoter of incoming DNA, or that histones may play a role in stabilizing transgene DNA (and thereby trans- gene expression) during the initial stages of transformation. References: 1. Crane Y.M. and Gelvin S.B.

(2007) RNAi-mediated gene silencing reveals involvement of Arabidopsis chromatin-related genes in Agrobacterium-mediated root transformation, PNAS, 104(38):15156–15161.

2. Mysore K.S., Nam J., Gelvin S.B. (2000a) An Arabidopsis histone H2A mutant is deficient in Agrobacterium T-DNA integration. Proc Natl Acad Sci USA 97: 948/953.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Mushrooms are nutritionally valuable food. Oyster mushroom (Pleurotus ostreatus) is the third largest fungi in the world, whose commercial production is achieved by solid-state fermentation using different lignocellulosic by-products. Fruiting bodies of mushrooms are appreciated mainly for their specific taste attri- butes, aroma, content of proteins, mineral matters, fibres, vita- for mins and other biologically active substances important human organism. Besides soluble carbohydrates and 5‘-nucleo- tides free amino acids are also among stable taste components of edible mushrooms. Besides other aminoacids, all for human essential and semiessential ones also occur free in Pleurotus ostre- atus. In addition to four known tastes, i.e. sweet, sour, salty and bitter, umami taste is given as the fifth one. Taste is characterized like this caused by the presence of amino acid, glutamic acid. The umami taste is depicted as spicy, hot, savoury and earthy,

292

Poster Presentations

Abstracts

P7–84 Multiple generation of novel monoclonal antibodies based on B-cell targeting M. Tomita, K. Furuta, Y. Sano and K. Tsumoto Division of Chemistry for Materials Graduate School of Engineering, Mie University, Tsu, JAPAN

biphenyl. The most effective inducers were naringin, 4-hydroxy- coumarin, caffeic and ferulic acids. R-(+)-limonen, coumarin and morin proved very poor induction activity. These results show, that biodegradation and use plant for induction of bacte- rial degradation is promising attitude for decontamination of PCB contaminated enviroment, especially soil on big areas or places where excavation is undesirable.

P7–86 Stable isotope probing in bioremediation: identification of (polychlorinated) biphenyl- metabolizing bacteria in contaminated soil O. Uhlik1, K. Jecna1, P. Stursa1, M. Mackova1, C. Vlcek2, T. Macek3, O. Uhlik3 and T. Macek1 1Department of Biochemistry and Microbiology, Institute of Chem- ical Technology, Prague, CZECH REPUBLIC, 2Department of Genomics and Bioinformatics, Institute of Molecular Genetics AS CR, Prague, CZECH REPUBLIC, 3IOCB & ICT Joint Laboratory, Institute of Organic Chemistry and Biochemistry, Prague, CZECH REPUBLIC

Introduction: Hybridoma technology was originally established in 1975 by somatically fusing antibody-producing B lymphocytes with cancerous myeloma cells. It has since become clear that monoclonal antibodies (mAbs) are highly specific for the antigens of interest. Several practical methods have been shown to gener- ate hybridoma cells. However, the conventional approach lacks selective fusion of aimed B lymphocytes with myeloma cells. Recently we have reported antigen-based B-cell targeting that features obvious advantages in terms of selective fusion of desired cell pairs. In the present study, we focused on effectively yielding novel mAbs against multiple antigens based on this new technology. Methods: After a mouse was immunized by multiple antigens, sensitized B lymphocytes were pre-selected by each antigen on the basis of immunoglobulin receptors on B lymphocytes. Anti- gen-selected B lymphocytes were brought together with myeloma cells by harnessing biotin/avidin interactions. Finally, B lympho- cyte-myeloma cell complexes were selectively fused by electrical pulses. Hybridoma cells secreting mAbs against aimed antigens were screened by ELISA method. Results: B-cell targeting was verified for its application to multi- ple generation of mAbs against desired antigens. At least three to five antigens are available for this new technology. Intriguingly, even homologous antigens were employed for immunization, pre- selection of B lymphocytes by an aimed antigen resulted in selec- tive production of mAbs against the target antigen. Conclusions: These results demonstrate that B-cell targeting is applicable for simultaneous generation of mAbs against multiple antigens with high selectivity. This advanced technology could contribute to effectively generating novel mAbs against various antigens in short term.

P7–85 Secondary metabolites of plants and their contribution to bacterial degradation of xenobiotics L. Trbolova´ 1, V. Dudkova´ 1, M. Mackova´ 1, M. Mackova´ 2, T. Macek2 and T. Macek1 1Department of Biochemistry and Microbiology, Institute of Chemical Technology, Prague, CZECH REPUBLIC, 2Institute of Organic Chemistry and Biochemistry AS CR, Prague, CZECH REPUBLIC

Bacteria are the major contributors to biological remediation of contaminated sites. The knowledge of the bacterial diversity had been greatly extended by enabling researchers to study to date uncultured bacteria besides the cultured ones. However, linking community structure with function was allowed only after DNA- based stable isotope probing (DNA-SIP) had been introduced. The exploitation of 13C-labeled substrates in bioremediation stud- ies allows for a deeper insight into the biodegradative processes occurring in the environment. This knowledge is necessary in order to ensure efficient bioremediation of pollutants. In our study, DNA-SIP was used to identify active (polychlorinated) biphenyl-metabolizing bacteria in non-vegetated and horseradish- vegetated soil contaminated by polychlorinated biphenyls (PCBs). 16S rRNA gene clone libraries constructed from 13C-DNA fol- lowing 3, 7, and 14 days incubation of the matrices with 13C- biphenyl suggest that biphenyl is catabolized mainly by Hydrog- enophaga spp. in the horseradish rhizosphere and by Paenibacillus spp. in the non-vegetated soil. Members of Achromobacter, Vari- ovorax, Methylovorus, Methylophilus, or some unclassified bacte- ria at the genus level were also detected to have derived carbon from biphenyl. Only one organism, Achromobacter spp., was detected by cultivation methods as well. In silico digestion of the sequences in the clone libraries was used to predict terminal restriction fragments (T-RFs) that were matched to the peaks in the T-RFLP profiles in order to indicate the relative abundance of taxa. Functional gene analysis performed on 13C-DNA follow- ing 3 days incubation revealed that deduced amino acid sequences of the biphenyl dioxygenase alpha subunit (BphA) are similar to the one of Pseudomonas alcaligenes B-357. Thus the spectrum of the PCB congeners that can be degraded by these enzymes is likely to be similar to that of the strain B-357. Our results indicate that plants may affect the spectrum of bacteria within the site. They also confirm the importance of SIP as a method that allows for revealing truly active members of the community, including those yet uncultured. Acknowledgement: This work was supported by projects MSMT NPVII 2B0 8031 and 2B0 6156, MSM 6046137305, and Z 40550506.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Polychlorinated biphenyls (PCB) are one of the most toxic and persistent contaminants in the enviroment. The physical and chemical methods for decontamination are expensive and compli- cated. Biodegradation of PCBs, is a promising attitude, because it appears like a frugal and cheap method. The induction of bac- terial degradation is provided by the substances of similar struc- ture to biphenyl which is commonly used in laboratory. This work is focused on determination of the effect of substances, pro- duced by plants and exuded by roots, on bacterial metabolism of PCB. The bacterial strains were isolated from rhisosphere of plants black nightshade and horseradish and from nonvegetated soil, both contaminated by PCB. The PCB degradation ability of bacterial strains was measured with different types of plant com- pounds previously identified in plant tissue which can work as in- ducers of bacterial degradation. Chosen compounds were generally better inducers of bacterial PCB degradation than

293

Abstracts

Poster Presentations

P7–87 ELISA and PAGE analysis of protein determinants from cereal and pseudocereal grain causing human coeliac disease D. Urminska´ 1, P. Socha1 and A. Vollmannova´ 2 1Department of Biochemistry and Biotechnology, Slovak Agricul- tural University, Nitra, SLOVAK REPUBLIC, 2Department of Chemistry, Slovak Agricultural University, Nitra, SLOVAK REPUBLIC

the

where DH 17.87% and 13.08%. Subtilisin Carlsberg was found to be least suitable for hydrolysis of wheat albumins and globu- lins, reaching DH 1.73%. Gliadins and glutenins were best hydrolyzed by subtilisin Carlsberg, DH obtained was 7.13% and 8.49%. Trypsin (DH 0.51%), papain (DH 0.33%) and Deuterol- ysin (0.54%) showed the least hydrolysis efficiency in gliadin hy- drolysates while trypsin (DH 3.24%), papain (DH 3.12%) and pepsin (DH 2.69%) indicated the same efficieny in glutenin hy- drolysates. 20 kDa partially split and an increased content of low-molecular peptides with Mr 1046–555 Da and Mr 555– 369 Da was observed. Hydrolysates of glutenins as well as those of gliadins contained a majority of peptides with Mr > 1046 Da were detected. In the hydrolysates of gliadins peptides with Mr < 20 kDa, peptides with Mr 14.2–6.5 kDa and low-molecu- lar peptides with Mr > From electrophoretic analysis of the hy- drolysates of albumins and globulins it follows that a mixture of peptides with a broad interval of molecular weights was pro- duced. Most products of the hydrolysates of albumins and globu- lins had a molecular weight up to 29 kDa. A smaller amount of peptides showed the molecular weight above 45 kDa. By using the method SE–HPLC G2000 of albumin and globulin hydroly- sates fraction of high-molecular peptides with Mr < 1046 Da. From RP–HPLC analysis it follows that the hydroly- sates of albumins and globulins, and of glutenins contained a greater amount of hydrophilic peptides but the hydrolysates of gliadins had more hydrophobic peptides.

P7–89 The potential use of Bacillus subtilis for bioremediation of the environment contaminated with heavy metals J. Urminska´ 1, D. Urminska´ 2, A. Vollmannova´ 3 and T. To´ th3 1Department of Environmentalism and Zoology, Slovak Agricul- tural University, Nitra, SLOVAK REPUBLIC, 2Department of Biochemistry and Biotechnology, Slovak Agricultural University, Nitra, SLOVAK REPUBLIC, 3Department of Chemistry, Slovak Agricultural University, Nitra, SLOVAK REPUBLIC

Coeliac disease is a disease caused by an increased sensitivity of some human beings to the presence of the allergen gluten pro- teins of cereals, namely wheat, rye, barley, and oats. An intoler- ance to gluten is mostly the lifelong matter. Gliadins, proteins of wheat grain with a relatively low molecular weight of about 30 kDa are the most toxic fraction. The toxic hypothesis sup- poses that it is a genetic disorder of metabolism connected with the activity disorder of enzymes hydrolyzing gluten in the small intestine. These peptidases split low molecular peptides which are produced in the beginning of gluten and/or gliadin digestion. From amino-acid analysis it folows, that peptides are rich in pro- line and glutamine. In literature it is showed the presence of coe- liac active tetrapeptide fragments, namely Pro-Ser-Glu-Glu and Pro-Glu-Glu-Gln are common to all allergen fragments. If these toxic peptides cannot continue to metabolise, they accumulate in the mucose of the small intestine and after reaching certain con- centration, they attack enterocytes of the mucose and thus gluten sensitive enteropathy develops. On the other hand, storage pro- teins of other cereal crops, such as zeins of maize, panicins of millet, cephalins of sorghum, and oryzeins of rice, show no coe- liac activity. The reactivity of peptide fragments of gliadins is supposed to be dependent on the conformation of polypeptide, i.e. its secondary structure. For determination of presence of aler- gic proteins are used protein fractionation by specific solutions, SDS – PAGE and A – PAGE methods. These methods are not sufficient because not all amount of prolamine proteins has an allergic activity. Only the specific immunochemical reaction between antibody and antigen is taken the right results. The results of immunochemical tests using ELISA-assays prepared on the basis of monoclonal antibodies are taken into account when a decision is made on suitability of raw materials and foods for gluten-free diet. The problem of the preparation of gluten-free food is that gluten proteins are found not only in standard cereal products (bread, pasta) but also in a wide assortment of food- stuffs amd additives, such as isotonic drinks, malt flavourings, dressings, ketchup, wheat starch, emulgators, ice creams, sugar substitutes, freeze-dried soups, stabilisers, powder foods, spice mixture, chocolate, sweets, beer, meat produce and others, as well as in cosmetics and drugs.

P7–88 Proteolysis as a way of food processing E. Sendrejova´ 1, D. Urminska´ 1, M. Kamac2 and A. Kosinska2 1Department of Biochemistry and Biotechnology, Slovak Agricul- tural University, Nitra, SLOVAK REPUBLIC, 2Division of Food Science, Instytute of Animal Reproduction and Food Research, Olsztyn, POLAND

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

To prepare enzymatic hydrolysates of wheat proteins is the way how to improve nutritional value and eliminate antinutritive alle- genic substances from wheat grain. Individual protein fractions of wheat grain were hydrolyzed by commercially available micro- bial, plant and animal peptidases and the course of enzymatic hydrolysis was studied by determining a hydrolysis degree, using the pH-stat and OPA methods. Wheat albumins and globulins were best hydrolyzed by Deuterolysin and Aspergillopepsin II, The territory of Banska´ Sˇ tiavnica region belongs to the contami- nated areas of the Slovakia. Among the present polluters is an aluminium manufacturing plant in Ziar nad Hronom, while in the past it was an extensive mining activity, which had a signifi- cant adverse impact on the environment of Sˇ tiavnica and its environs. The aim of the work was to analyse the chemical com- position of bottom sediments of selected water reservoirs in Sˇ tiavnica region and to assess the suitability of the use of bac- teria of the genus Bacillus as a positive concentration bioindica- tor or a bioremediation organism, using model experiments. Enzyme activities of five strains of Bacillus subtilis differing in amylasis and endopeptidases in cultivation in an optimum the production of medium were studied. Heavy metal salts in the concentrations of 0.01, 0.05, 0.1 and 0.5% (w) were added to the optimum medium. After 48 h of submersion cultivation, activities of produced extracellular enzymes in the cultivating medium were determined. In the environs of Sˇ tiavnica being investigated the cadmium contents of bottom sediments in the Windsachta were found to exceed the maximum allowable concentration by 30– 68% (concentrations of 15.6–20.12 mgÆkg-1 dry mater, were deter- mined) and Velˇ ka´ Richnˇ ava by 9.75% (13.17 mgÆkg-1 dry mater, were determined). At the same time these sediments showed higher amounts of lead, but their limit concentration level has not been exceeded. According to the results from measuring, cad- mium and lead were added to the cultivating medium for Bacillus subtilis. The presence of cadmium in the -amylase production, especially in the a cultivating medium caused a decrease in strain

294

Poster Presentations

Abstracts

P7–91 Transformation of chlorobenzoic acids by plant-bacteria associations B. Vrchotova1, M. Mackova1 and T. Macek2 1Department of Biochemistry and Microbiology, Institute of Chem- ical Technology, Prague, CZECH REPUBLIC, 2Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, CZECH REPUBLIC

of Bacillus subtilis BS-2 (16.2 times). The presence of lead has resulted in a 5.65 fold decrease in the activity of the strain of BS- 1. The activity of endopeptidases was markedly inhibited by cad- mium. We have observed a 7.55 fold activity reduction in the strain of BS-3. The reaction of the BS-2 strain to the presence of lead was found to be the most sensitive; it decresed 5.06 fold. Comparison of a gradual decrease in enzyme activity due to the increased cadmium and lead concentrations in the cultivating medium indicate that bacteria of the genus Bacillus subtilis are capable of tolerating the concentrations of these heavy metals up to 0.5%. Therefore, we think that Bacillus subtilis may be used for bioremediation processes, i.e. for eliminating toxic elements from the environment.

P7–90 Biotransformation of trans-2-hexenal to trans- 2-hexenol by recombinant ADH and cofactor regeneration by recombinant FDH. P. Utekal1, S. Stuchlik1, P. Kois2, L. Panciova1 and J. Turna1 1Molecular biology, Comenius University Faculty Natural Sciences, Bratislava, SLOVAK REPUBLIC, 2Organic Chemistry, Comenius University Faculty Natural Sciences, Bratislava, SLOVAK REPUBLIC

Plants thank to their wide-range enzymatic systems are able to detoxify various organic compounds and at natural conditions they participate on degradation of different xenobiotics. In mutual cooperation with other organisms present in particular ecosystem they are able to remove also substituted compounds including halogenated componds, sulphonated, nitrated ones. The aim of this work was to find out whether the higher plants associated with bacterial strains isolated from contaminated area are able to metabolize chlorobenzoic acids and compare effi- ciency of transformation with system consisted of plants or bac- teria alone. Solanum nigrum (black nightshade) was used as a model plant and two bacterial strains previously identified as Pseudomonas species were used in co-cultivation experiments. Defined amount of microbial cells and tested chlorobenzoate were added to the medium with one month old plants. During 14 days of incubation the samples of media were collected and concentration of chlorobenzoic acid was determined using HPLC. Control samples consisting of plant or bacteria alone in media with chlorobenzoic acid were cultivated at the same conditions. At the end of the experiment the amount of bacterial cells, mass of plant biomass and concentration of chlorobenzoic acid in roots and upper ground parts of plant biomass were measured. The metabolisation of chlorobenzoic acids depended on number of chlorine atoms and their position on benzoic ring. Microogan- isms alone and plant-bacteria association were able to transform 2-, 3- and 2,5-dichlorobenzoic acids by 100% during 14 days. Plant-bacterial consortia transformed above mentioned chloro- benzoic acids faster then plants or microorganisms alone. On the other hand metabolisation of 3,5-di and 2,3,5-trichlorobenzoate were not proved in any of all tested options. Acknowledgement: The work was sponsored by the grants Centrum 1M06011, VZ MSM 6046137305 and NPVII 2B06156.

from an economic

P7–92 Production of monoclonal antibodies against different Turkish BVDV antigens (TR-15 and TR-21 ) by a single fusion F. Yucel1, E. A. Akcael1, R. Demirgan1 and K. Yesilbag2 1Genetic Engineering and Biotechnology Institute, TUBITAK Marmara Research Center, Gebze, TURKEY, 2Virology Section, Uludag University Faculty of Veterinary Medicine, Go¨ru¨kle/Bursa, TURKEY

C-6 aldehydes and alcohols contribute to the fresh green odor in plants and are widely used in perfumes and in food technology. Important member of this family is trans-2-hexenol. It can be produced by transformation of trans-2-hexenal which is extracted from plant tissue. Carbonyl compounds such as aldehydes and alcohols are reduced by chemical methods in industry but it is not appropriate for production of compounds used in food industry. Therefore, in recent decades biocatalysis is used for these purposes. The enzymes suitable for reduction of aldehydes are oxidoreductases, which catalyze the reduction of carbonyl groups of aldehydes and alcohols. The most suitable enzyme from this class is alcoholdehydrogenase (ADH), which is the last enzyme involved in lipoxygenase pathway in several species of higher plants, which converts natural trans-2-hexenal to trans-2- hexenol. In the case of redox reactions catalyzed by oxidoreduc- tases, which often require stoichiometric oxidation or reduction of costly coenzymes such as NAD(P) and FAD, efficient coen- zyme recycling must be accomplished if a given application is to be practical standpoint. A variety of approaches for efficient coenzyme recycling have been put forth over the years. These have generally entailed either enzymatic, chemical or electrochemical regeneration of the reduced or oxi- dized coenzyme being consumed in the enzymatic reaction of interest. Use of additional reaction which regenerates NAD(P) back to NAD(P)H can result in significant decrease of coenzyme cost. Formate dehydrogenase (EC 1.2.1.2, FDH) is one of the frequently used biocatalysts for NADH regeneration. In this work we focused on the biotransformation of trans-2-hexenal to trans-2-hexenol using isolated recombinant ADH from Saccharo- myces cerevisiae for redox rection and recombinant FDH from Candida boidinii for regeneration of cofactors, both expressed in Escherichia coli.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Bovine viral diarrhoea virus (BVDV) is one of the most signifi- cant viral diseases in cattle. BVDV infections occur at a high rate in cattle population in Turkey and cause economical losses. Monoclonal antibodies are usefull tools for direct detection of the presence of BVDV antigens in infective samples. The aim of this study is to generate monoclonal antibodies against Turkish strains of BVDV and to use for diagnostic purposes. Two BVDV isolates (TR-15 and TR-21) from Turkey belonging to different genetic groups were selected from a virus collection including more than 60 isolates obtained between 1997 and 2005. These viruses were separately used for immunization of Balb/c mice as 100lg dose. Lymphocytes from spleen of two mice immunized with the different BVDV isolates were simultaneously fused with F0 myeloma cells by using PEG4000 in a single fusion. Fused

295

Abstracts

Poster Presentations

many cases and prenatal diagnosis. In this study, we determined abnormal hemoglobin mutation such as often come across Hb S b6(A3)Glu fi Val (GAG fi GTG) and Hb D Los Angeles b121(GH4)Glu fi Gln (GAA fi CAA) alongside rare muta- tions that Hb D Iran b22(B4)Glu fi Gln (GAA fi CAA), Hb E Sascatoon b22(B4)Glu fi Lys (GAA fi AAA), Hb G Coushatta b22(B4)Glu fi Ala (GAA fi GCA), Hb Hamadan and Hb O Arab (GGC fi CGC) b56(D7)Gly fi Arg b121(GH4)Glu fi Lys (GAA fi AAA) by microarray.

cells were incubated in selective HAT medium. Positive hybrid clones were determined with indirect ELISA, then cloned and produced by cultivation. ELISA tests showed that among 1632 wells 5 hybrids (5D6, 13G1, 11H6, 2F6, 11H5) reacted with both TR-15 and TR-21 isolate. These monoclonal antibodies did not cross react with the other bovine proteins and MBDK cell cul- ture lysate. By isotyping studies, it is determined that 5D6 anti- body is as IgG type and the others are IgM types. The produced antibodies, demonstrated to have a higher affinity towards TR-15 compared to TR-21. This approach is thought to be a time- and labor-saving technique for producing monoclonal antibodies. Acknowledgement: This work is financially supported by ‘Production and TUBITAK-TOVAG (Project no: 107-O-019), Diagnostic Use of Monoclonal Antibodies against Bovine Viral Diarrhoea Virus’.

P7–95 Plant uptake of hexabromocyclododecane (HBCD) J. Zlamalikova1, K. Demnerova1, M. Mackova1, J. Hajslova2, J. Pulkrabova2, P. Hradkova2, M. Napravnikova2, T. Macek3 and H. Stiborova1 1Department of Biochemistry and Microbiology, The Institute of Chemical Technology, Prague, CZECH REPUBLIC, 2Department of Food Chemistry and Analysis, The Institute of Chemical Tech- nology, Prague, CZECH REPUBLIC, 3T. Macek Research Team, The Institute of Organic Chemistry and Biochemistry AS CR v.v.i., Prague, CZECH REPUBLIC

P7–93 Cases with Hb Hamadan (ß 56, Gly–Arg, GGC > CGC) defined by a novel designed microarray reporter in cukurova region S. Yuzbasioglu Ariyurek and A. Kiymet Department of Biochemistry, Cukurova University Faculty of Med- icine, Adana, TURKEY

Hb Hamadan was formed due to changing the Gly residue to Arg, induced by a GGC fi CGC mutation at codon 56 of the b gene. This hemoglobin elutes like Hb A2 on HPLC analysis with hemoglobin variant column and its abnormal band moves like Hb S on alkaline elektrophoresis. So that it’s easily cause mistakes and could lead to wrong diagnoses. We first observation Hb Hamadan mutation by sequencing in Cukurova region. In this study, we designed novel reporter to Hb Hamadan mutation for microarray and than determined cases with homozygous and heterozygous Hb Hamadan and combine with IVS I-110 muta- tion.

P7–94 Abnormal hemoglobin established cukurova region determine by microarray S. Yuzbasioglu Ariyurek, S. Seydel and K. Aksoy Biochemistry, Health Science, Adana, TURKEY

Hexabromocyclododecane (HBCD) is used as additive flame retardant. HBCD due to its chemical and physical properties belongs to persistent organic pollutants (POPs) – it is a persis- tent, bioaccumulative and toxic compound. It is reported to be absorbed from the gastrointestinal tract but relevant toxicity studies are lacking. According to available data it is dangerous especially to the liver and also thyroid hyperplasia and inhibition of oogenesis was described. Sewage sludge from fifteen Czech sewage treatment plants have been analysed for HBCD. The lev- els ranged between 0 n.d.–126.4 ng/g dw. HBCD has been also detected in river sediments near sewage treatment plants. In the Czech Republic is produced 175x103 tonnes of sewage sludge in a year. 86% of this amount is land-applied. But there are no information about bioaccumulation and biotransformation of HBCD by plants. The objective of this study was to find out if plants are able to accumulate HBCD from contaminated sewage sludge and from soil substrate enriched by its addition. We have chosen Nicotiana tabacum and Solanum nigrum as model plants. HBCD uptake, ecotoxicity and changes in microbial diversity were also evaluated. Acknowledgements: This work was sponsored by the grants MSMT NPVII 2B06151, GA CR 104/08/P188 and MSM 6046137305.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

The thalassemias and abnormal hemoglobines are common in our country as is worldwide. Their discrimination and true defini- tion important especially Cukurova region that carry on premari- tal scenning programe. Conventional method must be improve and benefit from new technology for screnning study that do too

296

Poster Presentations

Abstracts

P8 From Biochemistry to Medicine

P8–1 Tamarindus indica plant as a source of potent antioxidants with potential hypolipidaemic properties A. Abdul Aziz, S. Mat Junit and N. Razali Molecular Medicine, University of Malaya, Kuala Lumpur, MALAYSIA

to control (8.1±1.01 U/mg versus 6.06±0.4 U/mg; p < 0.05). In the presence of troglitazone, PON1 activity was slightly increased (6.29±0.91 U/mg), but this increase was not significant. Both atorvastatin and simvastatin increased PON1 activity although it was not significant (5.13±0.71 U/mg and 6.4±1.5 U/mg respec- tively, versus. 4.91±0.49 U/mg control). In this study, it was observed that PPARc ligands especially pioglitazone increased the activity of PON1. Definition of interactions between hypolipi- demic drugs and anti-atherogenic molecules is important in the prevention and cure of atherosclerosis.

P8–3 The effect of LPS incubation periods on nitric oxide levels in RAW 264.7 macrophage cell line A. Akinci1, A. Karabay1, T. O¨ zkan2, A. Sunguroglu3 and F. Aktan1 1Biochemistry, Ankara University Faculty of Pharmacy, Ankara, TURKEY, 2Biotechnology, Ankara University Institute of Biotech- nology, Ankara, TURKEY, 3Medical Biology, Ankara University Faculty of Medicine, Ankara, TURKEY

The tamarind plant (Tamarindus indica) possesses various medici- nal properties and animal studies had demonstrated its hypolipi- daemic effects. A microarray analyses conducted by our group also revealed its potential to regulate lipid metabolism. The anti- oxidant properties of this plant are relatively unknown and are useful in view of its hypolipidaemic properties. Here, we reported extensive analyses of T. indicawhere we measured the total phe- nolic content and antioxidant activities of the methanol, ethyl acetate and hexane extracts of the leaves, stems, flesh and seeds of this plant. Overall, results showed the methanol extract to con- tain the highest antioxidant activities and polyphenolic content in the order of methanol > ethyl acetate > hexane. The methanol extract of T. indica leaves showed strong ferric reducing proper- ties and had the highest scavenging abilities against the synthetic DPPH and ABTS radicals as well as superoxide anion and nitric oxide radicals when compared to the stems, flesh and seeds extracted with methanol, ethyl acetate and hexane. The same extract also contained the highest polyphenolic content, suggest- ing that the observed antioxidant activities were possibly contrib- uted by the polyphenolics. In most instances, the antioxidant activities were comparable to, if not higher, than rutin, a pure polyphenolic compound used as positive control. Generally, the order of the antioxidant/polyphenolic potency of various parts of T. indica was leaves > seed > stem > flesh. These results indi- cate that the high antioxidant properties of this plant can com- plement its hypolipidaemic action and this is particularly useful in preventing oxidative damage in general and LDL oxidation, more specifically.

Lipopolysacharide (LPS), is a conserved outer membrane compo- nent of gram negative bacteria and induce iNOS (inducible nitric oxide synthase) gene expression in RAW 264.7 macrophage cell line. Excessive nitric oxide produced by iNOS plays a significant role in many pathological processes. In this study, two main experiments have been conducted. Firstly, RAW 264.7 macro- phage cells were incubated for 2, 4, 6, 18, 20, 22 and 24 hours with LPS at 1 lg/ml and the nitrite concentrations in the medium were determined. At the 2nd, 4th and 6th hours, no statistically significant differences between the nitrite levels of the control and LPS groups could be observed. Only after the 18th hour, control group and the LPS group have been observed to have statistically significant nitrite level differences (p < 0.0001). Secondly, cells are induced by 1lg/ml LPS for 20 hours. Then the medium con- taining LPS has been replaced by a fresh medium, and the nitric oxide production has been observed at 1st, 5th and 20th hours. 1st hour observation did not show any statistically meaningful results. 5th and 20th hours observations showed that nitric oxide production increased with respect to the control group although no LPS was available in the medium (p < 0.05, p < 0.01 respec- tively). This situation showed that although no LPS were avail- able in the medium, the activated iNOS continues nitric oxide synthesis in the LPS induced cells.

P8–2 Paraoxonase1 activity is increased by PPARgamma agonists in HepG2 cell line F. Akbiyik, T. Bayrak, S. Cevik, A. L. Dogan and E. Demirpence Department of Biochemistry, Hacettepe University Faculty of Medicine, Ankara, TURKEY

P8–4 Biochemical and in vitro studies on bee (apis mellifera) venom R. Albulescu1, L. Albulescu2, D. Popescu2 and E. Raducan2 1Pharmaceutical Biotechnology, National Institute for Chemical Pharmaceutical R&D, Bucharest, ROMANIA, 2Biochemsitry and Proteomics Laboratory, Victor Babes National Institute of Pathol- ogy, Bucharest, ROMANIA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Low-density lipoprotein (LDL), when modified by free radicals (ox-LDL), induce atherosclerosis. In contrast to ox-LDL, high- is able to prevent atherosclerosis density lipoprotein (HDL) through a protein with antioxidant properties, paraoxonase 1 (PON1). PON1 is incorporated to HDL after being synthesized in liver.The aim of this study is to investigate the effect of hypo- lipidemic drugs such as PPARa, PPARc ligands and hyroxym- ethylglutaryl CoA reductase inhibitors (statins) on the activity of PON1 in human hepatocellular carcinoma (HepG2) cell line. Cul- tured cells were incubated in the presence of PPARa ligands (fe- nofibrate and WY-14,643), PPARc ligands (pioglitazone and troglitazone) andstatin drugs (atorvastatin and simvastatin)atap- propriate doses and periods. Ethanol and DMSO were used as controls. At the end of the incubation, cells were harvested. The activity of PON1 was measured spectrophotometrically using phenylacetate as substrate. In HepG2 cells, PON1 activity was significantly increased in the presence of pioglitazone compared Bee venom is considered an alternative therapeutic agent in sev- eral inflammatory diseases. Protein and polypeptide composition of several samples was analyzed using electrophoretic and mass spectrometric techniques. Ability to modulate expression of cyto- kines in vitro was investigated on Jurkat cell and peripheral lym- phocyte cultures. 9 samples, differentiated by time and place of collection were analyzed. Sample time and storage conditions

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P8–6 New mechanism of HIV resistance: investigating cleavage site mutations in Gag D. Andersson1, N. M. Van Maarseveen2, M. Nijhuis2 and J. Konvalinka1 1Department of Biochemistry, Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences, Prague 6, CZECH REPUBLIC, 2Department of medical microbiology, University medical center, Utrecht, The Netherlands

were also introduced as variables during the studies. Electropho- retic analysis was performed on Protein 80 Chips using Agilent 2100 bioanalyser, and resulted in a relevant quantitative profile for proteins ranging 4.5–80 kDa. Variation in protein expression levels between samples were found, although several key protein components showed a very small variation between different specimens. Mass spectrometric analysis was performed by SEL- DI-TOF (Bio-RAD) using several types of standard chips. A good resolution of venom potential bioactive peptides could be obtained, as well as a protein profile that confirms the electro- phoretic results. Mass spectrometric analysis proves to be sensi- tive and highly reproducible, yet, due to different chemistries of the chip surface further investigations are required to provide complete protein profiling. The combination of the two analytical techniques represents a valuable tool in quality and authenticity control of natural products. Cytokine modulating activity was evaluated by multiplex – xMAP technology (Luminex 200) on culture supernatants from Jurkat and peripheral lymphocytes cul- tures. Statistically significant modulation of cytokine release was found for IL-1b, IL6, IL8, TNF alpha in bee venom treated cul- tures. Further investigations may be needed to provide a proof of therapeutical efficacy.

Inhibitors of HIV protease are important antiviral drugs. How- ever, their effect is compromised by rapid resistance development. Normally, selection of mutations in the protease coding region is followed by mutations in the HIV polyprotein cleavage sites that compensate for the impaired proteolytic activity of the mutated protease. In a recent paper (Nijhuis et al. 2007) mutations in the NC-p1 cleavage site of HIV Gag that can cause resistance to pro- tease inhibitor drugs were described. Interestingly, these muta- tions were selected without prior mutations in the HIV-1 protease. In order to gain insight into the underlying mechanism that causes this resistance we designed 13-mer substrates (ER- QAN-FLGKIWPS, wild type) mimicking the NC-p1 cleavage site of the wild-type and mutated virus strains from patients as well from in vitro evolution experiments. The peptides were as digested with wild type HIV-1 protease, with subsequent separa- tion of substrates and products using HPLC. Integration of peak area was performed in order to quantify the concentrations. The peptide ERQAN-FLGETWPS showed a 2.4 fold higher activity than the wild type peptide, which correlates with the increased replicative capacity in vitro of the corresponding mutated virus. For this peptide a unique cleavage pattern was observed, yielding four products. The identities of these products were determined using LC-MS and preparative HPLC. The consequences of these findings for resistance development in HIV will be discussed.

P8–5 Plant polyphenols: in vitro studies on cytokine release and cell signaling R. Albulescu1, G. Caraene1, C. Nichita1, V. Vulturescu1, L. Albulescu2, E. Codorean2 and C. Tanase2 1Pharmaceutical Biotechnology, National Institute for Chemical Pharmaceutical R&D, Bucharest, ROMANIA, 2Biochemistry and Proteomics Laboratory, Victor Babes National Institute, Bucharest, ROMANIA

P8–7 The adrenal chromaffin cell dysfunction in MSG-obese rats A. E. Andreazzi1, P. B. Marangon2, A. Chaves2, E. Goulart2, L. Pinheiro2, S. Silva2, R. M. Garcia2 and P. C. Mathias1 1Celular Biology and Genetics, Biology, Maringa´, BRAZIL, 2Biology, Biology, Juiz de Fora, BRAZIL

it

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Neonatal treatment with monosodium L-glutamate (MSG) causes obesity in rodents. MSG animals present low sympathetic activ- ity. It was also showed that catecholamine secretion is increased is currently in response to muscarinic stimulation; while, accepted as nicotinic ones play the major role. Therefore, the present work investigated the adrenomedullary dysfunction in MSG-obese rats. During the first 5 days after birth, MSG (4 mg/ g body weight) was injected subcutaneously on male Wistar rats. Controls received saline. On the 90th day the animals were sacri- ficed and the adrenal glands removed. The obesity was estimated weighting the retroperitoneal and periepididymal fat pads. Right adrenal glands were used to measure total catecholamine content (tCC). Left glands were used in Western Blotting to evaluate tyrosine hydroxylase (TH) enzyme expression and, also, to incu- bate the isolated medullae with different concentrations of carba- chol (Cch) in the absence or in the presence of atropine. The catecholamines were quantified by fluorometry. MSG treatment enhanced 57.2% fat pad weight. MSG-rats had an increase of 40.3% in tCC. TH expression decreased 37.6% in MSG rats. Catecholamine basal secretion was similar in both groups. Cch stimulated a dose-dependent manner catecholamine release with higher magnitude in obese than in lean ones. Atropine blocked completely the cholinergic response from MSG-obese rats. In the Aim: The purpose of the study was the assessment of the efficacy of several selective polyphenol complexes to modulate the secre- tion levels of signaling molecules (cytokines and chemokines) and signal transduction in vitro. Experimental methods: Cell cultures Jurkat cells, 3T3 cells and peripheral lymphocytes were used for the evaluation of modula- tory effects of poly-phenol complexes on signaling molecule release and signal transduction. Exposure to poly-phenol com- plexes over a range of 10-5–10-10M was performed in triplicate; higher concentrations were used for the estimation of potential cytotoxic effects, while lower levels for efficacy. End points were represented by cytokine/chemokine levels and kinetics in culture supernatants (1), signal transduction molecules in cell lysates (2), cell viability. Cell viability and proliferation were measured by MTS and LDH assays, while end-point sets 1 and 2 were deter- mined by multiplex assays using xMAP/Luminex arrays. Results: Based on MTS and LDH assays, the products did not produce statistically significant cytotoxic effects in the applied concentration range. On 3T3 cells, the compounds demonstrated a significant effect on TNFalpha induced apoptosis. Dose depen- dent modulation in cytokine/chemokine and signal transduction profiles were demonstrated for the tested compounds; association with some antitumor agents demonstrated for some of the com- pounds a significant potentiating effect. Conclusions: Multiplexed xMAP arrays in combination with in vitro assays provide a reliable and sensitive tool for efficacy assessment of therapeutical molecules. Some of the selective poly- phenol complexes demonstrated significant capacity to enhance therapeutical efficacy, proved by specific modulation of signaling molecules and cell responses.

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control group, the atropine inhibited weakly the catecholamine secretion. The higher tCC in MSG rats is due, at least in part, to low sympathoadrenal activity, which also explain the high sensi- tivity to Cch, probably due the muscarinic receptors up regula- tion mechanism.

P8–10 Protective effect of diallyl sulfide against benzene induced chemical carcinogenesis in kidney S. Arslan1 and E. Arinc2 1Biology, Pamukkale University, Denizli, TURKEY, 2Biological Sciences, Middle East Technical University, Ankara, TURKEY

P8–8 Conditions for targeted transfection of bone marrow mesenchymal adherent stem cells with genetically engineerined recombinat collagen type I genes J. Aniol1, M. Tarnowski2, A. Szydlo1 and A. L. Sieron1 1General and Molecular Biology and Genetics, Faculty of Medicine in Katowice Medical University of Silesia, Katowice, POLAND, 2Stem Cell Biology Program at James Graham Brown Cancer Center, University of Louisville, Louisville, KY, USA

Diallyl Sulfide (DAS) is one of the organosulfur compounds derived from garlic. It has cancer protective effects in various animal models, primarily through modification of carcinogen detoxification enzymes, such as cytochrome P450 enzymes. DAS reduces the activity and expression of CYP2E1 which is involved in metabolism of many low molecular pro-carcinogens such as benzene and nitrosamines. Benzene is an occupational and envi- ronmental toxicant that causes acute myelogenous leukemia. N- nitrosamines are an important class of multitarget environmental carcinogens known to cause cancer upon metabolic activation in humans and in animals at multiple sites such as liver, kidney, and lung. Metabolism of benzene and nitrosamines by CYP2E1 is required for their carcinogenic action. In this respect, the aim of this study is to investigate whether DAS prevents bioactivation thus, possible carcinogenic effect of benzene by decreasing CYP2E1 protein amount and its associated enzyme activity. DAS was administered intragastrically to benzene-treated rabbits in two different doses and conditions (before or after benzene treat- ment). Renal CYP2E1 associated p-nitrophenol hydroxylase activity and CYP2E1 protein level was determined. 3-fold increase in renal p-nitrophenol hydroxylase activity by benzene pretreatment was decreased to its basal level by DAS treatment. Similarly, increased CYP2E1 protein level by benzene treatment was abolished completely by DAS. In conclusion, DAS may reverse not only hazardous action of benzene but also possible carcinogenicity of NDMA by decreasing CYP2E1 protein amount and its associated enzyme activity in rabbit kidney. These results suggest that DAS may have potent chemopreven- tive properties in CYP2E1 induced chemical carcinogenesis.

A hope for curing systemic genetic diseases is a gene therapy, a stem cell therapy or combination of both, the gene and the stem cell therapy. One example in human is brittle bone disease (Osteogenesis Imperfecta), which is caused by mutations in COL1A1 or COL1A2 genes and affects the production of type I procollagen and quality of its fibrils. Up to date no cure is for OI, except for orthopedic treatment or prevention against bone breaks with orthopedic equipment. Here, we investigated the approach based on combination of gene and the stem cell ther- apy by examining conditions for targeting rat genes col1a1 and col1a2 in mesenchymal stem cells with homologous sequences from corresponding human genes. A five different hybrid DNA constructs comprising of isogenous sequences of rat and human collagen genes were prepared. Each sequence contained homo- logues fragments of different length in order to determine their efficiency for homologous recombination. Constructs of hybrid DNA were introduced by lipofection, with different carriers, into the rat mesenchymal stem cells isolated from bone marrow. The cells were cotransfected with the neomycin resistant gene (G418) in a separate DNA construct. The G418 resistant cell clones were screened by for targeted incorporation of human sequences in the rat cells. The incorporation of the hybrid DNAs was verified by endonuclease restriction cleavage followed by Southern blot anal- ysis. A production of collagen type I and its secretion from the clones was analyzed by collagen type I quantitation and its im- munodetection in the culture medium.

P8–11 The effects of some neuroleptic drugs on Paraoxonase enzyme activity purified from human serum and in Hep3B cells A. Avcikurt1, M. Aydin1, D. Demir2, H. Yildirim1, S. Eryilmaz1, S. Aydogan Turkoglu1, F. Kockar1 and S. Sinan1 1Biology, Faculty of Arts and Sciences, Balikesir, TURKEY, 2Chemistry, Faculty of Arts and Sciences, Balikesir, TURKEY

P8–9 Antioxidant activity of whey and whey hydrolysates N. Arda1, E. Onay Ucar1, M. Pekmez1, A. M. Yilmaz2 and A. S. Yalcin2 1Molecular Biology and Genetics, Istanbul University Faculty of Science, Istanbul, TURKEY, 2Biochemistry, Marmara University School of Medicine, Istanbul, TURKEY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Antioxidant capacity of whey and hydrolysed whey solutions sep- arated by Sephadex G-50 column chromatography were investi- gated by three testing methods. Superoxide scavenging activity, lipid peroxidation inhibitory activity and cuprac ion reducing activity of the fractions were determined. Antioxidant activity was enhanced by the treatment of whey solution with proteolytic enzymes. Superoxide scavenging activity and lipid peroxidation inhibitory activity of all fractions were higher than that of the whey solution. The second fraction of trypsin-treated whey is the most effective superoxide scavenger and cuprac ion reductant whereas the first fraction of pepsin-treated whey exhibited the highest lipid peroxidation inhibitory activity. Members of the serum paraoxonase (PON) family have been identified in mammals and other vertebrates, and in inverta- brates. PONs exhibit a wide range of physiological important hy- drolytical activities, including drug metabolism and detoxification of nerve agents. PON1 and PON3 reside on high-density lipopro- tein (HDL, good cholesterol) and are involved in the prevention of atherosclerosis. However, several investigations reported that the enzyme is inhibited by some substances including especially transient metals and aliphatic alcohols. In this study, effects of zuclopenthixol, haloperidol and fluoksatin HCl on purified human serum paraoxonase 1 enzyme activity and human hepa- toma cell line (Hep3B) paraoxonase activity were investigated, in vitro. Serum and hepatoma cell paraoxonase activity was determined spectrofotometrically using paraoxan as a substrate according to a method of Gan et al. For these studies, human serum paraoxonase was purified using ammonium sulfate precipi- tation and hydrophobic interaction chromatography (Sepharose- 4B, L-tyrosine, 1-Napthylamine). In addition, effects of these

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of identification beta-lactamases neuroleptic drugs on paraoxonase, produced by human hepatoma cell line (Hep3B), which is a model of human liver paraoxonase, in vitro. Inhibition effects of three different drugs on paraoxonase were determined using % of esterase activity by plotting concen- tration of drugs. IC50 values of the drugs exhibiting inhibition effects were found by means of these graphs

P8–12 Heme oxygenase activity in lymphocytes of renal transplantation patients treated with Cyclosporin/Tacrolimus F. Bakar1, K. Keven2, A. Tuzuner3, B. Erbay2 and S. Nebioglu1 1Biochemistry, Ankara University Faculty of Pharmacy, Ankara, TURKEY, 2Nephrology, Ankara University Faculty of Medicine, Ankara, TURKEY, 3Surgery, Ankara University Faculty of Medicine, Ankara, TURKEY

susceptibility of these bacteria to 5 usual disinfectants (Oxigenon, Combi instruments, Virkon, Biguacid, Big spray) and 13 newly synthesized chemical compounds with potential disinfectant activ- ity was investigated by an adapted disk diffusion techniques. The genotypic (blaTEM-like, blaSHV-like, blaOXA-like, blaCTX-M-like), sulfonamides (sul1, sul2 and sul3), tetracyclines (tet alleles) and disinfectants resis- tance (qacE) encoding genes was performed by simple and multi- plex-PCR. Results: The E. coli strains exhibited 98% ampicillin resistance, 2.7% strains being also resistant to 3rd generation cephalosporins (cephtazidime). The PCR analysis evidenced the blaTEM1 in 11 strains, all strains being negative for blaSHV, blaOXA and blaCTX were. The tetA, tetB, tetC si tetD genes have been evi- denced in different combinations, the most frequent being tetA gene (80%). The tetH, tetE, tetI, tetG, tetY, tetZ si tet30 were not detected in the tested strains. The sulphametoxazole resistant strains exhibited dfrA genes, and the quinolones resistance was encoded by sull1 and sull2 genes. Concerning their susceptibility to usual disinfectants, the tested strains exhibited high resistance to Oxigenon and to Virkon and were susceptible to Biguacid, Big spray and Combi instruments, the last one being the most effec- tive, active at low concentrations against all tested enterobacterial strains. Concerning the newly synthesized compounds, they exhibited a very low antimicrobial activity. The phenotypic resis- tance to disinfectants was accompanied by the presence of qacE gene.

P8–14 Molecular mechanisms of homeostatic effect of hemorphins in pathophysiology of different diseases N. Barkhudaryan1, H. Zakaryan1, H. Stepanyan2, O. Hunanyan1 and F. Sarukhanyan1 1Neuropeptides Biochemistry, H.Buniatian Institute of Biochemis- try NAS RA, Yerevan, ARMENIA, 2Cancer Chemotherapy, Insti- tute of Fine Organic Chemistry NAS RA, Yerevan, ARMENIA

Renal transplantation is the preferred therapy for patients with end-stage renal disease. It’s known that the antioxidant defense system deteriorated in patients with renal failure. Heme oxygen- ase (HO) is the rate-controlling enzyme of the predominant path- way of hepatic heme degradation. It has an important role in degradation of heme and protects cells from the harmful effects of free heme molecule. HO, oxidatively degrades protoheme IX to biliverdin and carbon monoxide. In mammals, biliverdin is further converted into bilirubin, that’s an endogenous radical scavenger through the action of BVR. The aim of our study was to investigate the alterations of HO activity to evaluate oxidative stress in renal transplant recipients receiving Cyclosporin (CsA) and Tacrolimus (FK506). Hence, renal transplantation patients treated with CsA (n = 6) and FK506 (n = 12) were studied and the samples were collected before transplantation (BT), at the 60th (AT60) and 120th day (AT120). 12 healthy donors formed our control group. HO activity was performed spectrophotomet- rically. In CsA group, HO activity has significantly increased at the 60th day compared to control and BT. On the other hand we didn’t observe any significant changes in FK506 group. The inhi- bition of bilirubin formation leads to oxidative damage in the organism. In conclusion, the increase of HO activity may be the sign that the antioxidant system is more favourable in CsA trea- ted group at the late terms of transplantation. Acknowledgement: The study was supported by Ankara Uni- versity Research Foundation (2001-08-03-028), Ankara Univer- sity Biotechnology Institute (2001-K-120-240(35) and TUBITAK SBAG-AYD-438.

P8–13 Phenotypic and geneticresistance markers in Escherichia coli isolated from the hospital environment M. M. Mitache1, M. C. Chifiriuc2, E. Panus3 and V. Lazar2 1Microbiology Immunology, Public Health Authority, Bucharest, ROMANIA, 2Microbiology Immunology, University of Bucharest, Bucharest, ROMANIA, 3Public health Authority, Constanta, Bucharest, ROMANIA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Purpose: The phenotypic and genetic analysis of the antibiotic and disinfectants resistance features in Escherichia coli isolated from the hospital environment. Material and Methods: 100 Enterobacteriaceae strains belong- ing to E. coli isolated from different surfaces in the hospital envi- ronment were identified by biochemical and serological tests. Disc diffusion susceptibility test, microplate broth dilution tech- nique and phenotypic screening tests for the presence of beta-lac- tamses were used to investigate the antibiotic susceptibility. The The effect of intraperitoneal (ip) administration (1mg/kg) of syn- thetic LVV-hemorphin-7 and hemorphin-7 on rat endotoxic stress model and rat model of sarcoma-45 was studied. Earlier we dem- onstrated that hemorphins were able to modulate calcineurin activity in vitro by binding with calmodulin (CaM). In present study we examine if hemorphin may affect calcineurin activity in vivo. The enzyme activity was measured in plasma and brain by a spectrofluorimetric assay using 4-methylumbelliferyl phosphate as a substrate (Anthony et al., 1986). The influence of hemorphins on superoxide dismutase (SOD) activity in plasma and brain dur- ing mentioned pathologies was also measured using SOD assay kit. Stress was induced by ip injection of rats (n = 10) with lipo- polysaccharide (LPS) (0.5 mg/kg). In experimental group rats (n = 10) received LPS + hemorphin simultaneously. Control rats (n = 10) received saline. 3 hours later animals were sacri- ficed. Rats inoculated with sarcoma-45 received ip administration of hemorphin during 7 days. Our results indicate that calcineurin activity in response to LPS-induced stress was significantly increased (2.2 fold) and after treatment with hemorphin the activ- ity of enzyme was significantly decreased (1.8 fold) becoming close to the activity of calcineurin in control group. The activity of calcineurin in rats with sarcoma-45 was down regulated and after treatment with hemorphin was restored becomig close to the level of enzyme in control group. The SOD activity in both models was down regulated and restored after treatment with hemorphin. It is proposed that hemorphins by restoration of both calcineurin and SOD activity in vivo act as homeostatic agents.

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P8–15 Myelin basic protein epitope library: path to diagnostics and treatment of Multiple Sclerosis A. Belogurov, I. Kurkova, N. Ponomarenko and A. Gabibov Biocatalysis, Institute of Bioorganic Chemistry, Moscow, RUSSIA

ing to large signaling complexes of about 20 MDa containing G proteins, beta-arrestin, phosphorylated dynamin, Src tyroxine kinases, Vav, Rac1, Grb2 and Ras. Acknowledgement: Supported by grants from Ministry of Education of Czech Republic (MSM 21620808 and 1M0505), Czech Grant Agency (303/09/0477 and 305/09/H008), and by EU Spine 2 project (contract LSHG-CT-2006-03/220).

P8–17 Antitumor effect of plant nuclease TBN1 in vitro A. Bilahorkova1, P. Lipovova1, T. Podzimek1, L. Zmatlikova1, B. Kralova1, J. Matousek2 and L. Orctova2 1Department of Biochemistry and Microbiology, Institute of Chem- ical Technology, Prague 6, CZECH REPUBLIC, 2Department of Plant Molecular Biology, Academy of Science, Ceske Budejovice, CZECH REPUBLIC

Background: The pathologic role of autoantibodies in autoim- mune disease is widely accepted. Direct penetration of autoanti- bodies through the blood–brain barrier and their co-localization with neural tissue-specific autoantigens may explain their possible contribution in the neurodegeneration. Objectives: The aim of this study was to determine myelin basic protein (MBP) epitopes specific for the autoantibodies in Multi- ple Sclerosis (MS) and compare these data with those from other neuronal disorders (OND) and rodent models of MS leading to the generation of new diagnostic and prognostic criteria. Methods: We constructed a MBP-derived recombinant ‘epitope library’ covering the entire molecule. We used ELISA and PAGE/SELDI-MS assays to define the epitope binding/cleaving activities of autoantibodies isolated from sera of 26 MS patients, 22 OND patients, 11 healthy individuals, mice and rats with induced experimental autoimmune encephalomyelitis (EAE). Results: The levels of autoantibodies to MBP fragments 48–70 and 85–170 as well as whole MBP and myelin oligodendrocyte glycoprotein molecules were significantly higher in sera of MS patients than healthy donors. In contrast, selective reactivity to the two MBP fragments 43–68 and 146–170 distinguished the OND and MS patients. Catalytic antibodies were found in 77% progressive-, 85% relapsing-remitting MS patients, 9% OND patients and no healthy donors. In terms of binding DA rats were the most similar rodent model to MS. Rat’s treatment by the respective peptides seems very promising and significantly reduces disease onset. Application to practice: Thus, using myelin basic protein epi- tope library approach we determined specific characteristics of MS autoantibodies compared to OND and healthy donors. These data may serve as additional biomarker of disease progression and may open new paths in MS treatment.

supported by MSMT: At the present time, the number of people dying of cancer is increasing every year. Although we have a lot of cancer drugs, new antitumor substances are still needed because of tumor diversity and frequent tumor drug resistance. In sixties of the last century it was found that a RNase from a frog Rana pipiens has antitumor effect and this had evoked the interest in nuclease research. In past decades, especially animal nucleases have been studied. Todays effort is focused on plant nucleases, which are predicted to have less immune responses in mammals than ani- mal nucleases. This difference is probably caused by different affinity to nuclease inhibitor protein, which is present in all mam- mals. The aim of this work is to investigate antitumor effect of tomato bifunctional nuclease TBN1 on tumor cell line ML-2 (myeloid leukemia) and HeLa (cervical cancer). Our results revealed that native TBN1 is N-glycosylated protein. However, we found that enzyme is active in both glycosylated and non-gly- cosylated forms. The PEGylated form of TBN1 nuclease was pre- pared to improve pharmacokinetic properties and the influence of this modification was tested on the cell line too. We com- pared antitumor effect of TBN1 with bovine-seminal RNase (BS-RNase), whose effect is known to be significant. The micro- culture tetrazolium assay was used for antitumor activity evalua- tion. Acknowledgement: This work is MSM6046137305 and GACR 521/09/1214.

P8–16 Ligand mimetics for activating receptors of natural killer cells are molecular triggers causing lymphocyte activation and permanent protection against tumors K. Bezouska1, K. Krenek1, H. Mrazek1, A. Fiserova2, K. Vladimir3 and K. Daniel3 1Biochemistry, Charles University Prague, Praha 4, CZECH REPUBLIC, 2Immunology, Institute of Microbiology, Praha 2, CZECH REPUBLIC, 3Biotransformation, Institute of Micro- biology, Praha 4, CZECH REPUBLIC

P8–18 Increased serum sTRAIL level following BevacizumAb treatment is correlated with patient survival in metastatic colon cancer A. Bisgin1, C. Aydin1, E. Terzioglu2, A. D. Yalcin2, A. Kargi3, B. Savas3 and S. Sanlioglu4 1Human Gene Therapy Unit and Department of Medical Biology and Genetics, Akdeniz University Faculty of Medicine, Antalya, TURKEY, 2Department of Rheumatology Allergy and Immunol- ogy, Akdeniz University Faculty of Medicine, Antalya, TURKEY, 3Department of Medical Oncology, Akdeniz University Faculty of Medicine, Antalya, TURKEY, 4Human Gene Therapy Unit and Department of Medical Biology and Genetics, Akdeniz University Faculty of Medicine, Antalya, TURKEY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Activating lectin-type receptors on natural killer (NK) cells such as CD161 (NKR-P1) have been shown to react with N-acetyl-D- glucosamine (GlcNAc) conjugates resulting in partial protection against tumors in animal models. We describe here novel com- pounds linking two GlcNAc residues to alkyl via thiourea bonds. GlcNAc decyl dimers can efficiently precipitate the A isoform of NKR-P1 in both rat and mouse NK cells, and activate NK cells at concentrations as low as 10-10 M. When administered into melanoma bearing mice, GlcNAc dimers can provide a perma- nent protection in 70% of animals. This is due to activation of NKT cells, and subsequent tumor infiltration by CD8+ T cell. Unexpected signaling efficiency of GlcNAc dimers is explained by sequential cooperative engagement of the target receptor lead- Colon/rectum cancer is the third most common cancer and the second leading cause of cancer-related death. In recent years, BevacizumAb, a novel humanized monoclonal antibody directed against vascular endothelial growth factor (VEGF) has found widespread clinical use as an angiogenesis inhibitor for certain types of metastatic cancers. Treatment with bevacizumAb with/

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Poster Presentations

Acknowledgement: We gratefully acknowledge funding by PMU0206K to HH and LBK, O¨ AD Czech Republic/Austria Project Nr. 072006 and Project Nr. CZ02/2008.

P8–20 Branched Neurotensin peptides for tumor personalized therapy J. Brunetti1, C. Falciani1, B. Lelli1, S. Pileri1, A. Cappelli1, A. Pini1, N. Ravenni1, L. Bencini2, R. Moretti2, M. Scatizzi3, S. Menichetti4 and L. Bracci1 1Molecular Biology, University of Siena, Siena, ITALY, 23rd Divi- sion of General and Oncologic Surgery, Careggi Regional and Uni- versity Hospital, Florence, ITALY, 3UO Surgery 2, Valdarno Hospital, Arezzo, ITALY, 4Organic Chemistry, University of Florence, Florence, ITALY

retain binding efficiency of

without the combination of other chemotherapeutics inhibited VEGF receptor activation and vascular permeability that eventu- ally led to tumor cell apoptosis. However, recently used RECIST parameters are not adequate to document the differences in treat- ment response. Therefore, there is a need for a sensitive, specific and reliable serum marker to monitor the therapeutic response. The purpose of our study was to evaluate the possible use of sol- factor-related apoptosis-inducing ligand uble tumor necrosis (sTRAIL) and VEGF concentrations as markers for treatment efficacy and prognosis in patients with metastatic colon cancer. sTRAIL and VEGF levels were measured by ELISA in the serum of 16 subjects with metastatic colon cancer who also received BevacizumAb therapy and 10 healthy control individu- als. Patients’ sera were analyzed before and 3 months after Bev- acizumAb therapy. The serum sTRAIL concentrations of patients before the therapy were similar to those of controls and seven out of sixteen patients’ sTRAIL levels were increased after the treatment. The other nine patients who showed no increase in sTRAIL levels died after 8 months. Therefore, we wanted to know whether there is any correlation between sTRAIL levels and survival. Our study demonstrated that elevated sTRAIL lev- els after the BevacizumAb treatment are significantly associated with increased survival. Not surprisingly, the serum VEGF levels were decreased in all patients who received BevacizumAb therapy. As a conclusion, serum sTRAIL levels can be used to assess prognosis of metastatic colon cancer patients following the treatment.

P8–19 Ribosomal protein rpL10 and translation associated pathomechanisms in autism H. Breitenbach-Koller1, A. Chiochetti2, J. Wang1, J. Zhou1, S. Pfeifenberger1, O. Haubenreisser1, J. Kellermann3, F. Lottspeich3, K. Richter1, A. Pichowa4, M. Breitenbach1 and S. M. Klauck2 1Cell biology/Genetics, University Salzburg, Salzburg, AUSTRIA, 2Molekular Genome Analysis, DKFZ Heidelberg, Heidelberg, GERMANY, 3Biochemistry, MPI Munich, Munich, GERMANY, 4Cell biology, CAS Prague, Prague, CZECH REPUBLIC

Specific targeting of tumor-associated antigens selectively over- expressed by tumor cells is the goal of modern cancer therapy aimed at overcoming non-specific toxicity of most anticancer drugs. The finding that membrane receptors for endogenous pep- tides like Neurotensin, Somatostatin and many others are over- expressed in different human tumors, suggested the use of pep- tides as tumor-targeting agents. We demonstrated that branched peptides corresponding linear sequences and have a longer half-life. Our goal is to produce branched peptides that can be used for tumor cell tracing and for personalized therapy, by selective binding to specific receptors on cancer cells and delivering of functional units to the same tumor cells. Neurotensin (NT) and its active fragment (NT8-13), synthe- sized as tetra-branched dendrimers, can be conjugated with dif- ferent functional units, such as chromophores or chemotherapic moieties. Fluorophore-conjugated branched NT peptides allow discrimination between tumor and healthy tissues in human biop- sies from colon and pancreas adenocarcinomas and can be used to measure tumor versus healthy peptide binding in each patient, giving an indication of possible efficiency of NT-mediated ther- apy. NT branched molecules, when conjugated to drugs, allow selective tumor cell killing and also by-passing of innate drug resistance in colon, pancreas and prostate tumor cell lines. The NT-drug tetra-branched peptide was tested on animal models. Results indicate promising features of branched NT for selective tumor targeting. The approach proved promising for personal- ized therapy of tumors that over-express NT receptors, such as colon, pancreas and prostate carcinoma, and might be applied to different tumor targets.

P8–21 Fluorimetric investigation of polyelectrolyte- influenza virus antigen conjugates Y. Budama Battal, Z. O. Ozdemir, M. Topuzogullari, E. Karabulut and Z. Mustafaeva Bioengineering Department, Yildiz Technical University, Istanbul, TURKEY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Autism is comprised of a clinically heterogeneous group of neu- rological disorders which present with impaired social relation- ship, impaired language and restricted mental capacities. Single gene disorders identified in autistic patients offer a promising route to investigate on the cellular level in model organisms the associated pathomechanisms. Here we present functional analysis of human mutant alleles of ribosomal protein (RP) RPL10, L206M and H213Q in the model system Saccharomyces cerevisi- ae. On the assembled ribosome, large subunit RP rpL10 and small subunit RP rpS6 line the mRNA entrance channel. 2D- DIGE analysis identified differentially expressed proteins in the heterozygous RPL10/rpl10D strain and in yeast strains expressing the L206M and H213Q alleles. Loss of RPL10 is accompanied by increased expression of a 35 kD phospho-protein and ectopic expression of the L206M and H213Q alleles is associated with differential expression of regulators of ribosome biosynthesis. Two-hybrid analysis showed that the L206M and H213Q alleles bind more strongly to a TGFß modulating enzyme than the wild type human rpL10. Interestingly, we had shown before that rpL10 specifically is involved in translation of secretory proteins. Together, these results suggest that in the neural cells rpl10 may be involved in differential mRNA translation targeting the secre- tory pathway. If so, this would be a crucial contribution to the process of synaptic plasticity, which is severely affected in autistic patients. Introduction: Influenza viral infections are a significant global public health concern because of the morbidity and mortality associated with the acute respiratory disease, associated second- ary complications and pandemic threat. The viral peptides of influenza disease are Hemagglutinin 98–106 (YPYDVPDYA) and Hemagglutinin 91–108 (WSKAFSNCYPYDVPDYASL). These peptides were synthesized and N terminus of HA 91–108 were modified with tryptophan amino acid. Fluorimetric techniques have recently been used to study peptide-PE conjugates. From the fluorescent emission spectra shift of aromatic amino acid resi- dues in proteins or peptides, it is possible to localize the interac- tion between protein and PE at certain protein domains.

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Poster Presentations

Abstracts

P8–23 Antibiotic- photosensitizer conjugates- a novel class of antibacterial molecules R. Cahan1, N. Swissa1, G. Gelerman2 and Y. Nitzan3 1Chemical Engineering and Biotechnology, Ariel University Center of Samaria, Ariel, ISRAEL, 2Biological Chemistry, Ariel University Center of Samaria, Ariel, ISRAEL, 3Life Sciences, Bar-Ilan University, Ramat-Gan, ISRAEL

Methods: We investigate covalent binding mechanism of syn- thetic peptides which include tryptophan and tyrosine residues in the peptide sequence with different polyelectrolytes (polyacrylic acid, derivative of poly(1-azabicyclo[4.2.0] octane) and poly(N- isopropyl acrylamide)) depending upon the concentration ratio of components by fluorescence method. Hemagglutinin 98–106 and Hemagglutinin 91–108 antigenic peptides are synthesized by microwave-assisted solid phase peptide synthesis method. Pep- tide-polymer covalent conjugations are performed in PBS media. Covalent conjugations are carried out by carbodiimide method. Water soluble carbodiimide, carbodiimide hydrochloride (1-ethyl- 3-(3-dimethylaminopropyl)) is used for this method, activation of carbonyl groups of synthetic polymer which is the binding area of peptides that includes free amino groups. Analysis of conju- gates were performed with fluorimetric methods depending on tryptophan and tyrosine residues of peptides, FT-IR and the other spectroscopic methods. Results and Conclusions: From the analysis of peptide-PE conjugates, it is possible to discuss the mechanism of the conju- gate formation and structure of forming particles.

P8–22 Cytotoxicity and genotoxicity of commercial and novel binary titanium alloys M. Burlibasa1, G. Tanase1, A. Dumitriu1, D. Bodnar1, T. Bodnar1, D. Mocuta1 and L. Burlibasa2 1Oral Implantology, UMP Carol Davila, Bucharest, ROMANIA, 2Genetics Department, University of Bucharest, Bucharest, ROMANIA

The bacterial resistance to variety of antibiotics has become a severe problem in the treatment of pathogenic diseases. This problem opened intensive research on the effect of photosensitiz- ers against bacterial cells. On this research we examined the effect of the combination of Penicillanic Acid (PA) and the photosensi- tizer Rose Bengal (RB) and the combination of the antibiotic Kanamycin (KAN) and Rose Bengal on bacterial cells. In addi- tion we examined the effect of conjugates composed of RB and each of the antibacterial agents PA or KAN. We have synthe- sized the following conjugates: Rose Bengal-Penicillanic Acid (RBPA); Rose Bengal-Linker-Penicillanic Acid (RBLPA); Rose Bengal-Linker-Kanamycin (RBLKAN). These conjugates were synthesized by an amide bond between the precursors (RB and PA or KAN) with or without a linker. Their activity was exam- ined on Staphylococcus aureus the Gram positive and Escherichia coli the Gram negative bacteria. The MIC of RBPA, RBLPA and RBLKAN on Staph. aureus was 0.195 lM, 0.156 lM and 0.004 lM respectively. The MIC of RBPA, RBLPA and RBL- KAN on E. coli was 1.56 lM, 2.5 lM and 0.156 lM respec- tively. It was found that RBLKAN and RBLPA are bactericidal for E. coli as well as for Staph. aureus. Scanning electron micros- copy analysis showed damage to E. coli as well as Staph. that were treated with these conjugates. In this research we demon- strate for the first time that the combined treatments of antibiot- ics and photosensitizers as well as the conjugates that composed of these molecules have antibacterial effect against pathogenic cells.

P8–24 Identifying pharmacogenetic candidates for statin-induced muscle toxicity using yeast S. Callegari1, J. Bellon2, R. McKinnon1, S. Andrews1 and M. de Barros Lopes1 1Sansom Institute, University of South Australia, Adelaide, AUSTRALIA, 2The Australian Wine Research Institute, Adelaide, AUSTRALIA

lacking. This

We assessed the biological response to several novel titanium alloys that have promising physical properties for biomedical applications. Two commercial titanium alloys Super-TIX800 and Ti-6Al-4V, and two experimental titanium alloys Ti-13Cr-3Cu and Ti-1.5Si-5Cu were tested. Commercially pure titanium CP Ti: ASTM grade 2 and Teflon (polytetrafluoroethylene) were used as positive controls. Genotoxic and cytotoxic potential of titanium alloys were evaluated in a metabolically competent, mouse osteoblastic cells line MC3T3-E1. The specimens were cleaned and disinfected, and then each cleaned specimen was placed in direct contact with osteoblastic cells line for 72 hours. Genotoxic potential was determined by evaluating DNA strand breaks using single cell gel electrophoresis (SCGE) or the comet assay and induction of cytogenetic damage using cytokinesis- blocked micronucleus (MN) assay, cytotoxicity was determined by measuring the retention of supravital stain, neutral red, by the lysosomes using the neutral red retention (NRR) assay. Cytotox- icity and genotoxicity of the metals tested was not statistically different compared to the CP Ti and Teflon controls (p > 0.05). These novel titanium alloys pose cytotoxic risks no greater than many other commonly used alloys, including commercially pure titanium. The promising short-term biocompatibility of these Ti alloys is probably due to their excellent corrosion resistance under static conditions, even in biological environments. These favourable biocompatibility results show that these novel alloys have promise for use in dental restorations.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Statins are among the most highly prescribed drugs in the world and their use has affected a revolution in the treatment of hyper- cholesterolemia. Despite their effectiveness, as many as 10% of patients experience muscle pain or fatigue and in more serious cases, develop myalgia, rhabdomyolysis and liver toxicity. Cur- rently, data on pharmacogenetic candidates relating to statin tox- icity is the simple unicellular study exploits eukaryote Saccharomyces cerevisiae to identify and characterise genes that influence statin sensitivity. S. cerevisiae demonstrates drastic morphological modification and growth inhibition in response to statins. Moreover, cells undergo a loss of mitochon- drial function, which may be related to muscle toxicity. The addi- tion of mevalonate, lipids or isoprenyl precursors suppresses this response. Genome-wide screens, of S. cerevisiae deletion mutants exposed to statins have identified a number of genes whose inac- tivation increases cell sensitivity to statins. Based on the presence of a human ortholog containing Single Nucleotide Polymor- phisms (SNP’s), which are conserved in the yeast gene, several of the most statin sensitive strains were chosen. The sensitivity of these deletion mutants to statin was confirmed, leading to the

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Poster Presentations

Our data also showed no effects of type of diabetes and sex on serum calcium levels. In conclusion, observed increase in potas- sium levels in serum of older Type 2 diabetics is probably related to impaired glucose tolerance and other conditions associated with this type diabetes, such as insulin-resistance, hypertension, and cardiovascular complications. selection of the MEF2 gene as a potential pharmacogenetic can- didate. The yeast MEF2 gene encodes a mitochondrial elongation factor, with significant homology to the human EFG2 gene. SNPs have been created in the yeast MEF2 gene that correspond with it’s human ortholog and results to date demonstrate that at least one SNP has been shown to confer statin sensitivity.

P8–27 Evaluation of antioxidant activity of ethanol extract of Hypericum retusum and its protective effect on protein oxidation B. Ceken, G. Kizil, M. Yavuz and M. Kizil Chemistry, Dicle University, Diyarbakir, TURKEY

P8–25 S-homocysteinylated LDL dose-dependently induce oxidative stress and cell damage in human endothelial cells C. Carru, A. Zinellu, S. Sotgia, B. Scanu, M. Sanna, E. Pisanu, G. Pintus, A. M. Posadino, A. Cossu, B. Sanna and L. Deiana Biomedical Sciences, University of Sassari, Sassari, ITALY

different homocysteine treated with

investigation, our data suggest that

Alterations of functions and oxidative damages, induced by inter- actions with modified LDL on vascular endothelial cell, represent the early stages of the development of atherogenesis. Despite the abundance of evidence on the other covalent modifications of LDL, S-homocysteinylation remains largely unstudied. Since Hcy has been reported to induce cell injury through ROS generation, we investigated whether exposure of human endothelial cells (HECs) to S-homocysteinylated LDL (Hcy-S-LDL) results in the production of intracellular ROS. For this purpose HECs were incubated either with native-LDL (N-LDL) or with LDL previ- ously concentrations (between 1 and 100 lmol/L) and the generation of intracellular ROS was monitored. We found that the intracellular ROS con- tent increases with the levels of LDL homocysteinylation suggest- ing a pro-oxidant effect of Hcy-S-LDL. To test whether Hcy-S- LDL could affect cell proliferation, we examined the rate of DNA synthesis (measured by [3H] thymidine incorporation) in HECs exposed either to N-LDL or Hcy-S-LDL. Hcy-S-LDL induced a significant decrease in [3H] thymidine incorporation suggesting an effect on cell proliferation. This effect was evident when LDL were incubated with Hcy at concentration as low as 10 lmol/L. Hcy-S-LDL also significantly decreased cells viability by more than 20% compared to N-LDL. Although the mecha- nism by which Hcy-S-LDL affects cellular physiology needs fur- ther intracellular ROS production might be responsible for the observed changes in cell proliferation and viability. Oxidative damage of protein, DNA and lipids by oxidants has been implicated in a number of pathological conditions such as cancer, aging and cardiovascular diseases1. Flavonoids are natu- ral substances with variable phenolic structures. Much of the attention given to flavonoid compounds comes from results of epidemiological studies, which suggest that high fruit and vegeta- ble consumption is associated with decreased risk of several types including breast, colon, pancreas and prostate can- of cancer, cer2.Various extracts of different Hypericum species have been examined for their antiinflammatory, antimicrobial, antioxidant and hypolipidemic activity3,4. In the present study, the total anti- oxidant activity and the protective effect of Hypericum retusum (HR) on protein oxidation induced by Fenton reaction system were investigated. Total antioxidant activity of ethanol extract of HR was tested by using ferric thiocyanate (FTC) and thiobarbi- turic acid (TBA) methods. Antioxidative activity of HR extract was found to be comparable with vitamin E. We also studied the ability of ethanol extract of HR to prevent oxidative damage to bovine serum albumin (BSA) induced by Fenton reaction system (Fe+3/H2O2/Ascorbic acid), as assayed by carbonyl content and polyacrylamide gel electrophoresis. The oxidative damage of BSA, induced by hydroxyl radical in an acellular system, was prevented in the range of 50–1000 mg/ml of HR extract. Thus, from the results obtained we observed that Hypericum retusum extract showed significant antioxidant activity and protects pro- teins from free radical induced oxidative damage. References: 1. Kalpana KB, Srinivason M & Menon VP. Mol. Cell Biochem. 2009; 323: 21.

2. Ross J-A & Kasum CM. Annu. Rev. Nutr. 2002; 22: 19. 3. Sokmen A, Jones MB & Ertu¨ rk M. Phytother Res. 1999; 13: 355. 4. Kizil G, Toker Z, O¨ zen HC¸ & Aytekin C¸ . Phytother Res. 2004; 18: 339.

P8–26 Effects of type of diabetes on potassium and calcium levels in serum of elderly patients S. Semiz1, M. Malenica1, B. Cico2 and T. Dujic2 1Department of biochemistry and clinical analysis, Faculty of phar- macy, Sarajevo, BOSNIA AND HERZEGOVINA, 2Biochemistry lab, General hospital, Sarajevo, BOSNIA AND HERZEGOVINA

P8–28 Non-viral gene therapy of diabetic nephropathy P. Celec1, R. Pa´ lffy2, M. Behuliak2 and R. Gardlı´ k2 1Department of Molecular Biology, Comenius University, Bratislava, SLOVAK REPUBLIC, 2Institute of Pathophysiology, Comenius University, Bratislava, SLOVAK REPUBLIC

trend of

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Diabetic patients are known to be prone to development of hy- perkalemia and to alterations in calcium homeostasis. However, the current status of changes in kalemia and calcemia in diabetics under clinical settings remains obscure. In this work, serum potassium and calcium levels were analysed in 40 patients with Type 1 and 40 patients with Type 2 diabetes. Average age of tested population was 64 and 65 years, respectively. Interestingly, results of this study have demonstrated a significant correlation between potassium and glucose levels in Type 2 diabetics, with a increased kalemia in these patients versus general healthy controls. Furthermore, there was a significant increase in serum potassium levels in female patients with Type 2 diabetes as compared to their male counterparts (p < 0.03). Similar changes in serum potassium levels were not observed in Type 1 diabetics. The pathogenesis of diabetic nephropathy is complex and involves alterations of angiogenesis and inflammation. The aim of our study was to analyze the effects of anti-inflammatory and anti-angiogenic gene therapy using non-viral vectors in an experi- mental model of diabetes mellitus. Plasmid DNA (pcDNA3, 100 ug) was electroporated into hindlimb muscles of adult male Wistar rats with streptozotocin-induced diabetes. Angiomotin and 7ND – a deletion mutant of monocyte chemoattractant peptide 1 were cloned in the constructs. After 3 months, renal

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Abstracts

factor common in all E. faecalis strains causing amyloid arthrop- athy. None of the gelatinase negative strains caused the amyloid arthropathy in any of the chickens. In conclusion; it was sug- gested that enterococcal gelatinase plays a role in the develop- ment of experimental amyloid arthropathy and the gelatinase positive E. faecalis strains may cause amyloid arthropathy which is characterized by arthritis, lameness and growth retardation.

function and morphology, oxidative and carbonyl stress markers and expression of target genes were assessed. Despite severe dia- betes with high glycemia and profound bodyweight decrease, in this model no significant changes of renal functions were found. Both gene therapy approaches improved renal histology and decreased the expression of angiogenic and inflammatory genes. Anti-angiogenic and anti-inflammatory gene therapy improved some aspects of experimental diabetic nephropathy. Potential synergistic effects should be tested in further studies.

P8–31 Comparison of effect of vanadocene dichloride and classic cytostatic drug cisplatin in MOLT-4 leukemia cell line J. Cmielova1, P. Krejcirikova1, D. Muthna1, P. Siman1, R. Havelek1, M. Rezacova1 and J. Vinklarek2 1Faculty of Medicine in Hradec Kra´love´, Department of Medical Biochemistry, Hradec Kra´love´, CZECH REPUBLIC, 2Department of General and Inorganic Chemistry, University of Pardubice, Pardubice, CZECH REPUBLIC

P8–29 Determination of serum sialic acid and ceruloplasmin concentrations in Toxoplasma gondii seropositive dogs C. Nisbet1, S. Cenesiz1, T. Oncel2, G. F. Yarim1 and E. Handemir3 1Ondokuz Mayis University Veterinary Faculty, Biochemistry, Samsun, TURKEY, 2Pendik Veterinary Control and Research Institute, Parasitology, Istanbul, TURKEY, 3Konya Veterinary Control and Research Institute, Parasitology, Konya, TURKEY

(10.28 ± 1.63 mg/dl)

Summary: This study aimed to investigate serum total sialic acid (TSA) and ceruloplasmin (CP) levels and their possible correla- tion in Toxoplasma gondii (T. gondii) seropositive dogs. The study was conducted on the dogs that had been kept in dog- houses located in the province of Istanbul. Based upon IFAT test results, 15 T. gondii seronegative dogs and 25 seropositive infected dogs were included in the study. Blood samples were col- lected from each dog and serums were obtained. Subsequently, TSA and CP concentrations were measured. For serum TSA con- centration, no significant difference was found between control (15.97 ± 0.55 mg/dl) (17.20 ± 0.55 mg/dl) and infected dogs (p > 0.05). On the other hand, serum CP concentration was sig- nificantly higher in seropositive dogs (16.49 ± 1.11 mg/dl) com- pared to control dogs (p < 0.01). No correlation was found between serum TSA and serum CP. Absence of a significant difference between groups for serum TSA might be related to that the disease was in chronic stage, and the severity of cellular damage might have been decreased. Elevation of serum CP in T. gondii infected dogs is most likely related to cellular damage as a consequence of infection. We sug- gest that serum CP can be used as an additional prognostic parameter in toxoplasmosis in support to other serologic parame- ters.

Aim: The aim of the present study was to determine differences between routinely used cytostatic drug cisplatin and a new com- pound vanadocene dichloride in MOLT-4 leukemia cell line. Methods: We used WST test to evaluate toxicity of cytostatics (IC50 value) and trypan blue exclusion technique to determine cell viability. Changes in protein expression have been analyzed by Western blotting. Results: IC50 value for cisplatin was determined as 8.5 lmol/l and 125 lmol/l for vanadocene dichloride after 24 hours long treatment. Forty-eight hours long incubation with cytostatics in mentioned concentration leads to eradication of cell culture. Changes in protein expression of p53 and its phosphorylations on serine 15 and 392 were evaluated. Twenty-four hours long cis- platin treatment (10 lmol/l and 5 lmol/l) leads to increased level of p53. In the same time period, phosphorylation of p53 on ser- ine 15 occurs, mainly after 5 lM cisplatin treatment. Phosphory- lation of p53 on serine 392 was observed only after 5 lM cisplatin. No induction of p21 was detected during the whole per- iod of treatment with cisplatin. Comparing to cisplatin, vanado- cene dichloride causes no changes in expression of p53 and in its phosphorylation status, whereas induces increased expression of p21 in the concentration of 125 lmol/l after 24 hours long treat- ment. Conclusion: Cisplatin increases p53 expression and its phospho- rylations on serines 15 and 392, whereas vanadocene dichloride induces p21 expression even if no previous p53 activation was observed. Acknowledgement: This work was supported by The Ministry of Education of Czech Republic, project MSM 0021620820.

P8–30 The role of enterococcal virulence factors on experimental amyloid arthropathy in chickens A. Ciftci1 and K. S. Diker2 1Ondokuz Mayis University Veterinary Faculty, Microbiology, Samsun, TURKEY, 2Ankara University Veterinary Faculty, Microbiology, Ankara, TURKEY

P8–32 Bacteria mediated gene therapy in experimental colitis in mice R. Palffy1, E. Comajova1, M. Behuliak1, R. Gardlik1, P. Ja´ ni2 and P. Celec3 1Comenius University, Pathophysiology, Bratislava, SLOVAK REPUBLIC, 2Associated Health Care Center, Pathology, Krnov, CZECH REPUBLIC, 3Comenius University, Molecular Biology, Bratislava, SLOVAK REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

In this study, it was aimed to determine the role of enterococcal virulence factors on the development of amyloid arthropathy in chickens. Enterococcus faecalis strains phenotypically variable in gelatinase, aggregation substance and cytolisine traits were inocu- lated intraarticularly to chickens of 2 weeks and intravenously to chickens of 5 weeks. In intraarticular inoculation groups, amy- loid arthropathy occurred in 65.2% of gelatinase positive strains, 30.4% of aggregation substance positive strains and 22.2% of cy- tolisine positive strains. In intravenous inoculation groups, bilat- eral amyloid arthropathy occurred in 75% of gelatinase positive strains, 50% of aggregation substance positive strains and 26.6% of cytolisine positive strains. Gelatinase was the only virulence Background and Aim: Bacterial gene therapy bactofection is a simple and cheap method to deliver plasmid DNA into targeted tissue. We hypothesized that oral in vivo bactofection can be a simple and easy way to influence the course of inflammatory bowel diseases. The aim of this study was to prove the effects of antioxidative and anti-inflammatory gene therapy in dextran sul- fate sodium treated mice.

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P8–34 Short peptides with antimicrobial effect isolated from plants I. Dolezilkova1,2, M. Mackova1, T. Macek1,2, T. Neubauerova1,2, M. Sanda3 and M. Kralova1 1Department of Biochemistry and Microbiology, Institute of Chem- ical Technology, Prague, CZECH REPUBLIC, 2Department of Natural Compounds, Institute of Organic Chemistry and Biochemistry, Prague, CZECH REPUBLIC, 3Department of Mass Spectrometry, Institute of Organic Chemistry and Biochemistry, Prague, CZECH REPUBLIC

Methods: Attenuated bacteria Salmonella typhimurium SL7207 carrying plasmids with genes encoding Cu–Zn superoxide dismu- tase and an N-terminal deletion mutant of monocyte chemoattr- actant protein-1 were prepared. Male Balb/c mice had ad libitum access to 1% dextran sulfate sodium solution instead of drinking water during 10 days (mild model of colitis) and were daily fed with 20 Mio bacteria via gastric gavage. Fecal consistency, clini- cal status, food and water intake were monitored. After 10 days samples were taken and markers of oxidative stress and inflam- matory cytokine levels were measured. Colonic tissue was scored histologically by a blinded investigator. Results: Dextran sulfate sodium treatment significantly increased the levels of inflammatory cytokines and malondialdehyde in colon homogenates. In comparison with untreated group, bacte- rial gene therapy lowered the histological colitis score. Conclusions: Treatment with 1% dextran sulfate sodium solu- tion induced mild inflammation in Balb/c mice. There was no effect of gene therapy on inflammatory cytokines levels. How- ever, the histological parameters of colon were improved by the antioxidative and anti-inflammatory gene therapy using attenu- ated salmonella as a vector.

P8–33 Biochemical and histoenzymic studies on side- effects occurring in rats after long-lasting administration of GnRH analogs: dalarelin and cetrorelix P. Czekaj, A. Suszka-Switek, K. Wrona-Bogus, A. Bryzek, D. Plewka, R. Skowronek, W. Maruszczyk, K. Smorzyk and R. Wiaderkiewicz Department of Histology, Faculty of Medicine in Katowice Medical University of Silesia, Katowice, POLAND

Antimicrobial peptides have many of the desirable features of a novel antibiotic class. To date, more than 800 peptides have been described. Antimicrobial peptides are a class of small, positively charged peptides known for their broad-spectrum of antimicrobial activity. These peptides have also been shown to possess anti-viral and anti-cancer activity and, most recently, the ability to modulate the innate immune response. The aim of this study is isolation and characterization of small peptides with antimicrobial activity from plants. Antimicrobial peptides were isolated by precipitation and separated by FPLC, SPE or RP-HPLC and then characterized by tricine electrophoresis and mass spectrometry. Antimicrobial and antifungal activity was evaluated using bacterial strains G– – Esc- herichia coli, Pseudomonas aeruginosa, Agrobacterium rhizogenes, Citrobacter sp.; G+ – Micrococcus luteus, Staphylococcus aureus, Bacillus subtilis, B. megatherium, B. cereus, B. pumillus, Streptococ- cus equii zooepidermicus, Enterococcus faecalis, Arthrobacter urea- faciens and fungi Fusarium culmorum, Cladosporium herbarum, Aspergillus terreus, Trichoderma virens, Aspergillus oryzae, Aure- obasidium pollulans, Geotrichum candidum and Alternaria sp. The activity of isolated peptides is tested by screening diffusion test or by the bacterial growth evaluation on bioscreen apparatus. At pres- ent we have isolated fractions with activity from the species: tobacco, horse-radish, pasqueflower seeds, spinachand from horse- radish and tomato tissue cultures with activity against fungi Clados- porium herbarum, Alternaria sp. and bacteria Escherichia coli, Pseu- domonas aeruginosa, Staphylococcus aureus, Bacillus megatherium, Micrococcus luteus, Streptococcus equii zooepidermicus, Enterococ- cus faecalis and Citrobacter sp. The mass spectrometry shows the presence of antimicrobial peptide precursors in tissue cultures. Acknowledgement: The project was supported by grant VZ MSM 6046137305.

P8–35 Screening of the antimicrobial activity of some new dibenzothiepine derivatives O. Dracea1, C. E. Stecoza2, M. C. Chifiriuc3, L. C. Delcaru3, C. Iordache3, A. M. Israil3, I. Chirita2, D. C. Nuta2 and C. Badiceanu2 1I.N.C.D.M.I. Cantacuzino, Microbial Culture Collection, Bucha- rest, ROMANIA, 2Department of Pharmaceutical Chemistry, University of Medicine and Pharmacy Carol Davila, Bucharest, ROMANIA, 3I.N.D.C.M.I. Cantacuzino, Microbiology, Bucharest, ROMANIA

dehydrogenase, succinic

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Objectives: The aim of this study was to evaluate the in vitro antimicrobial activity of some newly synthesized compounds, O-acyloximino-dibenzo[b,e]thiepins and O-acyloximino-dibenzo [b,e]thiepin-5,5-dioxides. Methods: The new dibenzothiepine derivatives were obtained in a five-steps synthesis and their structures and purity were con- firmed by elemental and spectral analysis. The qualitative screen- ing of the susceptibility spectra of different microbial strains to these compounds was performed by three adapted diffusion methods: paper filter disk impregnation with the tested sub- Synthetic analogs of GnRH are presently used for treatment of many diseases. They lead to ‘down regulation’ or desensitization of hypophysis, resulting in repression of hypothalamic-pituitary- gonadal axis. However the mechanisms of GnRH analogs’ action and side-effects caused by long-term pharmacotherapy and their reversibility are poorly recognized. The aim of the study was to investigate whether a long-term administration of a new GnRH receptor agonist – dalarelin, and antagonist – cetrorelix causes changes in blood, pituitary and hepatic enzyme activities, and modifies the expression of GnRH receptor in hypophysis, and in what degree these changes are reversible. The study was per- formed on Sprague–Dawley adult rat females administered intra- peritoneally with dalarelin or cetrorelix at a dose of 6 lg/kg of b.w. Tissues were taken after 1, 2, 3 months of treatment and 1, 2, and 4 weeks after finishing administration. In blood, activity of alanine and aspartate aminotransferases, lactate dehydroge- nase, creatine kinase, glutamate dehydrogenase, and triglyceride, cholesterol and HDL concentration (colorimetric and kinetic methods) and levels of gonadotropins and sex hormones (radio- immunoassay) were measured. Pituitary and hepatic slices were analyzed morphologically (H-E staining) and histochemically tetrazolium (glucose-6-phosphatase, reductase, lactate dehydrogenase, adenosine triphosphatase and acid phosphatase activities quantified by densitometry). Expres- sion of GnRH receptor was detected immunohistochemically and by RT-PCR. Temporary increase in the activity of blood amin- otransferases and LDH, and hepatic activity of enzymes responsi- ble for aerobic respiration and gluconeogenesis were observed. A decrease in lysosomal activity and membrane transport was shown. Most of these functional changes were reversible after analogs cessation, but in time- and analog-dependent manner.

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in order to establish the minimal

stances solutions, the disposal of tested solutions in agar wells and the spotting of tested solutions on solid medium seeded with microbial inoculums. The quantitative assay of the antimicrobial activity was performed by binary microdilution method, in 96 inhibitory multi-well plates, (Staphylococcus concentration (MIC) against Gram-positive aureus, Bacillus subtilis) Gram-negative (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa) bacteria and as well as Candida albicans and Aspergillus niger, using both reference and clinical, multidrug resistant strains. Results: The original tested dibenzothiepine derivatives exhibited various levels of antimicrobial activity (MIC from 125 to 15.6 lg/ml) on Gram-pozitive, Gram-negative and fungal strains. Conclusion: Our studies demonstrated that all the tested dib- enzothiepine derivatives exhibit selective and effective antimicro- bial properties that could lead to the selection and use of these compounds as efficient antimicrobial agents.

cessing of glycoproteins, reflecting in wide clinical spectrum. The second most frequent type of CDGs, CDG-Ic is caused by muta- tions in hALG6 gene that encode Man9GlcNAc2-PP-Dol a1,3- glucosyltransferase. More than fifteen mutations in hALG6 caus- ing CDG-Ic are known so far. With the purpose to determine the frequencies of various mutations/polymorphisms in hALG6 gene in Croatian population we screened 600 blood samples collected from healthy Croatian habitants. One of the studied mutation was 391C>T resulting in amino acid substitution Y131H, which is considered to be a direct cause of CDG-Ic when present in homozygous form. Using TaqMan SNP genotyping assay for detection of heterozygotes for Y131H mutation we surprisingly detected eight homozygotes. Since the samples were obtained from healthy persons, we employed direct sequencing for check- ing these weird results. It has revealed three novel mutation in exon 5 of ALG6 gene (383T>C, 390G>A and 429G>C) and two novel polymorphisms (IVS5 + 17C>T and IVS5 + 34G>A) in downstream intervening sequence. It seems that pres- ence of these until now unknown mutation caused incorrect/ inappropriate binding of the designed primers and probes thus yielding false positive results. The base substitution T>C on the position 383 does not cause amino acid substitution, but 390G>A and 429G>C cause substitution of V128A and K143N, respectively. Additional functional studies on novel mutations 383T/C and 429G/C will be undertaken to establish if they can cause CDG-Ic or they are just polymorphisms that contribute to genetic diversity.

P8–36 Simultaneous fluorogenic derivatization and boronate affinity enrichment of 3-nitrotyrosine containing peptides: application for sequence- specific quantitation of protein nitration E. Dremina, V. Sharov, X. Li, J. Stobaugh and C. Scho¨ neich University of Kansas, Pharmaceutical Chemistry, Lawrence, KS, USA

tagging reagent,

P8–38 Yellow mealworm Tenebrio molitor as a source of specific peptidases for treatment of celiac disease E. Elpidina1, I. Goptar2 and I. Filippova2 1A.N. Belozersky Intitute for Physico-Chemical Biology, Plant Proteins, Moscow, RUSSIA, 2Moscow State University, Chemical Faculty, Moscow, RUSSIA

in of peptides derivatization 3-NT residues

Protein tyrosine (Tyr) nitration to 3-nitrotyrosine (3-NT) is a nitric oxide (NO)-dependent post-translational modification asso- ciated with a number of diseases and biological aging. In order to correlate protein Tyr nitration mechanistically with specific physiological and pathological conditions, it is important to iden- tify protein target sequences and to quantify protein-3-NT resi- dues on these proteins. We synthesized and characterized a novel bi-functional (3R,4S)-1-(4-(aminomethyl)phen- ylsulfonyl)pyrrolidine-3,4-diol (APPD), for the selective fluoro- and genic simultaneous affinity enrichment. A synthetic 3-NT-containing peptide was employed as a model for method optimization. The derivatized peptide shows strong retention on a synthetic boro- nate affinity column, containing sulfonamide-phenylboronic acid, under mild chromatographic conditions, affording a route to enrich the derivatized peptide for fluorescence detection and MS identification and quantitation. Further, this method was success- fully tested for sequence-specific analysis of 3-NT containing pro- teins from cultured C2C12 cells exposed to peroxynitrite. Tandem MS analysis identified chemical structures of the fluores- cent derivatives at low levels of protein modification (below 0.01 % on individual protein) due to enrichment of low abundant 3- NT containing peptides from milligrams of total cell/tissue pro- tein digests. Our novel APPD tagging approach is suitable for proteomic characterization of protein nitration in tissues, and for relative quantitation of nitrated sequences based on application of stable isotope-coded tagging reagents.

P8–37 Five novel mutations in hALG6 gene encoding Man9GlcNAc2-PP-Dol a1,3-glucosyltransferase S. Supraha Goreta, S. Dabelic and J. Dumic Faculty of Pharmacy and Biochemistry University of Zagreb, Department of Biochemistry and Molecular Biology, Zagreb, CROATIA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Congenital disorders of glycosylation (CDGs) are a growing group of genetic disorders caused by deficient assembly or pro- Yellow mealworm, Tenebrio molitor L. (Coleoptera: Tenebrioni- dae), is a pest of stored grains and grain products. The main food proteins of T. molitor are proline- and glutamine-rich wheat gliadins, so general nutritional requirements of T. molitor are in many respects similar to those of warm-blooded animals. Grains constitute also one of the main foodstuffs of the people in many countries, so the pathway of grain products digestion in T. moli- tor is of special interest due to a possibility to use this knowledge in medicine for treatment of celiac disease. Celiac disease (celiac sprue) is a widespread (but underdiagnosed) autoimmune type enteropathy of the small intestine. It is induced by ingested wheat gliadins or similar proteins (gluten) from rye and barley seeds. Genetically susceptible individuals develop an inflammatory response to proteolytically resistant Pro- and Gln-rich gluten peptides (epitopes), resulting in villous atrophy, crypt hyperpla- sia, and malabsorption of nutrients. The only effective treatment currently available to a patient is life-long strict dietary exclusion of gluten-containing products. According to our and literary data mechanisms of prolamines digestion differ in T. molitor and human. Our work is devoted to a search of specific digestive peptidases in T. molitor that can hydrolyze Pro- and Gln-rich gluten peptides resistant to human peptidases. A search for pro- line specific digestive peptidases in T. molitor larvae revealed three different enzymes: prolyl carboxypeptidase (PCP, EC 3.4.16.2), dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5), and prolidase (EC 3.4.13.19). All enzymes were isolated and charac- terized. All peptidases are soluble enzymes. PCP and DPP VI are secreted digestive peptidases, and prolidase is an intracellular enzyme presumably involved in the last stage of digestion. PCP is

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supported by Russian

P8–40 Structural and functional studies of cytosolic 5’-Nucleotidase II D. N. Filoni1, R. Pesi1, M. Camici1, S. Allegrini2 and M. G. Tozzi3 1Unita’ di Biochimica, Biologia Universita’ degli Studi di Pisa, Pisa, ITALY, 2Unita’ di Biochimica, Scienze del Farmaco Univer- sita’ di Sassari, Sassari, ITALY, 3Unita’ di Biochimica, Biologia Universita’ degli Studi di Pisa, Pisa, ITALY

discovered for the first time among animal digestive peptidases and is the second (after human) cloned and sequenced PCP. Glu- tamine specific activity belongs to the main digestive peptidase of this insect – cysteine cathepsin L. A scheme is suggested describ- ing complete hydrolysis of gliadins by endopeptidase cathepsin L and three proline specific exopeptidases. This complex of T. moli- tor digestive peptidases has a potential for oral administration to treat celiac disease. Acknowledgement: This work was Foundation for Basic Research (Grant No 09-04-01449-a` ). (cN-II), 5’-nucleotidase/phosphotransferase

P8–39 Transcriptional regulation of human INSIG2, an endoplasmic reticulum membrane protein involved in lipid metabolism regulation A. Fernandez-Alvarez, C. Cucarella and M. Casado Instituto de Biomedicina de Valencia IBV-CISC, Unidad de patologia metabolica y experimental, Valencia, SPAIN

Cytosolic acting through the formation of a phospho-enzyme intermediate, is spe- cific for oxypurine monophosphates. cN-II is an enzyme of great importance: cN-II knocked-down ADF die by apoptosis and 10- fold overexpression of cN-II has negative effects on human cell survival. cN-II is involved in the regulation of intracellular con- centration of IMP, AMP, GMP and also PRPP, therefore in the regulation of purine de novo synthesis and salvage. cN-II is clini- cally interesting, since it might be involved in prodrug cellular metabolism. Besides, mRNA level of cN-II has a prognostic value in adult AML. In the light of this, any findings concerning how cN-II activity is regulated will offer new possible therapeuti- cal approaches. To verify how cN-II is regulated by its effectors (Ap4A, ATP, ADP, BPG, Pi), which seem to influence subunit aggregation, we choose a site-directed mutagenesis approach, cre- ating point mutants in the hypothetic effector sites and at the subunit interface as resolved by cN-II crystal structure studies by Wallde´ n et al. The activity, kinetic properties and molecular mass of the recombinant, mutated cN-II were measured by radiochem- ical/colorimetric assays and gel filtration. Our preliminary results indicate that the enzyme possesses at least two different activa- tion sites as suggested by crystal structure, with different specific- ity for activators and that mutations on the interface result in a shift to a dimeric form still bearing catalytic activity. This experi- mental branch could be of big help in the tailoring of chemother- apy on patients enzyme expression level.

P8–41 Radiation exposure: cellular damage and defense mechanisms F. Folgosa1,2, R. Silva2, P. Tavares1, F. Raposo2 and A. Pereira2 1REQUIMTE – Departamento de Quimica, CQFB – Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, PORTU- GAL, 2CEFITEC – Departamento de Fisica, Faculdade de Cien- cias e Tecnologia, Universidade Nova de Lisboa, PORTUGAL

Sterol regulatory- element-binding proteins (SREBPs) are tran- scription factors with a central role in cholesterol and fatty acid metabolism. Alterations in its functionality have been implicated in the development of several human metabolic diseases such as obesity or type 2 diabetes, which have emerged as global health crises in recent years. SREBPs are synthesized as inactive precur- sors embedded in ER membranes until they are transported to the Golgi apparatus where they are activated by proteolysis. The ER- to-Golgi migration of SREBPs is crucially dependent on its inter- action with two other membrane proteins SREBP cleavage-acti- vating protein (SCAP) and insulin-induced gene (INSIG), both regulated by cell sterol levels. Two INSIG isoforms are known, designated INSIG1 and INSIG2. In humans, both proteins are 59% identical but they differ in their mode of regulation. Whereas transcription of INSIG1 gene in cultured cells requires nuclear SREBPs, INSIG2 activity seems to be constitutive and does not require active SREBP. Transcription of INSIG2 in mice is con- trolled by two promoters that give rise to alternative mRNA tran- scripts named INSIG2a and INSIG2b. INSIG2b is ubiquitous but INSIG2a is exclusively expressed in liver and is down-regu- lated by insulin. The objective was to characterize the human INSIG2 promoter that has not been previously studied. Our results show that INSIG2a transcript is not present in human liver, rat liver or in human derived cell lines. However, this tran- script is present in hamster origin CHO cell line and, as previously described, in mouse liver. Luciferase reporter assays allowed us to enclose the minimal human INSIG2 promoter to 300 bp from the transcription start site. Pointed mutations demonstrated the pres- ence of two GGA sites required for optimal expression. Eletroph- oretic mobility shift, siRNAs and ChIP assays have shown that Elk1 and Sap1a proteins are able to bind to these sites, although Sap1a is the only one active in vivo. This activation is dependent of its phosphorylation status via Ras/MAPK pathway but is inde- pendent of one of its common partners SRF. In conclusion, our data demonstrate for the first time, that members of Ets family of transcription factors are involved in the regulation of human INSIG2 expression and therefore of SREBP processing.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Ionizing radiation, by definition, is a type of radiation that contains enough energy to displace electrons and break chemical bonds, meaning that it is able to remove, at least, one electron from an atom or molecule, creating radical species such as reactive oxygen species (ROS). ROS can be divided in radical (e.g. superoxide, hydroxyl or peroxyl) and non-radical (e.g. hydrogen peroxide, ozone or singlet oxygen). At higher concentrations these species will promote dam- age to cellular structures by oxidizing lipids, reducing sugars and amino acids or causing a large number of purine and pyrimidine modifications (1–3). To overcome this problem, nature developed protection/repair mechanisms that enable organisms to survive to this type of threats. In bacteria, these mechanisms are mainly com- posed by enzymes that either eliminate the ROS (such as superoxide reductases or dismutases) or act as scavenger for metals (such as ferritins) preventing Fenton chemistry. However, there are other proteins that are able to perform both of these functions. The so called Dps proteins (DNA protection from starved cells) are able not only to act as iron chelator but also use hydrogen peroxide as a co-substrate. As a result, these enzymes act as a defense mechanism to DNA in cells that are under stress conditions (4,5). In humans,

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the major concern focuses on the occupational exposure to ionizing radiation. This has particular relevance if we consider that in certain jobs such as airline pilots or radiologists, the exposure to this type of radiation is very high. Extreme exposure conditions can lead to severe damage in cellular structures (DNA, proteins and others) and, therefore, to diseases such as cancer. However, defense mecha- nisms similar to the ones found in bacterial systems are also present in humans. In this work we propose to study not only the effect of occupational exposure to ionizing radiation in an airline pilots’ pop- ulation but also the biochemical response to that exposure. Acknowledgement: FF would like to acknowledge to Fundacao para a Ciencia e Tecnologia for the grant SFRH/BPD/48430/2008. References: 1. Imlay JA Annu Rev Biochem 2008; 77: 755–776. 2. Valko M, et al., Chem Biol Interact 2006; 160: 1–40. 3. Collins AR Bioessays 1999; 21: 238–246. 4. Pereira AS, et al., Eur J Inorg Chem 2007; 18: 2569–2581. 5. Zhao G, et al., J Biol Chem 2002; 277: 27689–27696.

it

is lethal to insect larvae and is broadly cytolytic to vertebrate as well as invertebrate cells. In this research Cyt1Aa crystals were purified from Bacillus thuringiensis IPS78/11 harboring the pHT- cyAp20 plasmid. The Cyt1Aa crystals were released from the lysed cells separated from the spores and cell debris, then solubi- lized, and proteolytically activated by proteinase K. The activity of the proteolytical form of Cyt1Aa (23–24 kDa) was examined on pathogenic Gram negative Escherichia coli (E. coli) as well as on Gram positive Staphylococcus aureus (S. aureus) bacteria. The Cyt1Aa minimal inhibitory concentration (MIC) for E. coli and S. aureus was 1.25 and 5 lg/ml, respectively. It was found that Cyt1Aa is bacteriocidic for E. coli, whereas it is bacteriostatic for S. aureus. The penetration of Cyt1Aa into these two bacterial cells was demonstrated by Western blot analysis using antibodies against the whole d-endotoxin crystal and by fluorescent labeled Cyt1Aa. The results of these experiments show that Cyt1Aa mol- ecules penetrate the cytoplasmic membrane. In a purpose to targeting the Cyt1Aa specifically examine the possibility of toward prokaryotic cells, we prepared Cyt1Aa-siderophore conju- gate. The siderophore is an iron carrier that binds iron and trans- ports specifically into the bacterial cells. The Cyt1Aa- siderophore-Fe conjugate showed less hemolytic activity for red blood cells than the free Cyt1Aa. All the experiments have been preformed on Pseudomonas mutant bacterium that did not pro- duce endogenous siderophore at low Fe media (0.5 M SSM with the chelator EDDA 50 lg/ml). From the results obtained the influence of the conjugate did not revealed cytolytic effect. More- over, when Pseudomonas putida has been treated with the conju- gate there was an increase in the cells growth. We assume that the cells had used the Fe from the conjugate without its penetra- tion, another possibility is that the conjugate penetrated to the cells but the Cyt1Aa lost its cytolytic nature while preparing the conjugate.

P8–42 Functional expression and characterization of cathepsin B and L from the gut of the tick Ixodes ricinus Z. Franta1, H. Penickova1, J. Dvorak2, E. I. Schneider3, M. Horn4, M. Mares4, D. Sojka1, J. H. McKerrow2, C. R. Caffrey2 and P. Kopacek1 1Biology Centre ASCR, Institute of Parasitology, Ceske Budejo- vice, CZECH REPUBLIC, 2UCSF, Sandler Center for Basic Research in Parasitic Diseases, San Francisco, CA, USA, 3UCSF, Department of Pharmaceutical Chemistry, San Francisco, CA, USA, 4ASCR, Institute of Organic Chemistry and Biochemistry, Praha, CZECH REPUBLIC

P8–44 The effect of PAMAM dendrimers on human SK-OV-3 ovarian carcinoma cells T. Gabryelak1, A. Rucinska1, K. Maczynska1, B. Klajnert1 and S. Rozalska2 1Department of General Biophysics, University of Lodz, Lodz, POLAND, 2Department of Industrial Microbiology and Biotechnology, University of Lodz, Lodz, POLAND

is supported by the grant Ticks differ from hematophagous insects in intracellular localiza- tion of hemoglobin proteolysis. We have recently described in the hard tick Ixodes ricinus that this process occurs at acidic pH and as is maintained by a machinery of gut-associated cysteine and aspartic proteases similar to blood feeding platyhelmints and nematodes. In this work, we functionally expressed in Pichia pas- toris and characterized two components of tick hemoglobinolytic cascade, namely IrCB and IrCL (cysteine proteases of CA fam- ily). Using a positional scanning of synthetic combinatorial library we identified the P1–P4 specifities of both enzymes. IrCL displays a strict acidic optimum at pH 4, while IrCB shows broader pH range. Both fusion enzymes were demonstrated to digest bovine hemoglobin and albumin in vitro. Expression pro- files of both enzymes were strongly upregulated during blood meal uptake. Biochemical properties of recombinant enzymes accord with endogenous activities in tick gut homogenate. Anti- bodies raised against recombinant enzymes were used to co-local- ize IrCB and IrCL inside the digestive cells of tick gut. Acknowledgement: This project IAA600960910 from GA ASCR and Research Center LC06009.

P8–43 Effect of Cyt1Aa toxin from Bacillus thuringiensis israelensis and the conjugate Cyt1Aa-siderophore on bacterial cells H. Friman1, R. Cahan1 and Y. Nitzan2 1Ariel University Center of Samaria, Chemical Engineering and Biotechnology, Ariel, ISRAEL, 2Bar-Ilan University, The Mina and Everard Goodman Faculty of Life Sciences, Ramt-Gan, ISRAEL

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Cyt1Aa is a d-endotoxin protein which is produced by Bacillus thuringiensis israelensis. It is a membrane pore-forming toxin that Dendrimers are relatively new, hyperbranched macromolecules prepared by a stepwise interactive synthesis. Polyamidoamine (PAMAM) dendrimers are the first dendrimer family that was commercialized. Pharmaceutical applications of dendrimers including the encapsulation and solubilization of drugs, using dendrimers as carriers of drugs, DNA or oligonucleotides, have been intensively studied. The aim of this study was to compare the effect of amino- and carboxy-terminated PAMAM dendrimers on cultured human ovarian cancer SK-OV-3 cells. We used two generations of dendrimers: 3.5 and 4. The cells were exposed to various concentrations of dendrimers (ranging from 0.5 to 300 lM) for 24 hours. The cytotoxicity of dendrimers was studied by MTT assay immediately after the incubation with dendrimers or 24 hours after removing the dendrimer from the medium. The morphological changes of SK-OV-3 cells were examined by a con- focal laser scanning microscope. Mitochondrial membrane poten- tial changes were monitored using a fluorescent probe JC-1. The level of reactive oxygen species (ROS) was estimated by DCFH- DA assay. Obtained results showed that anionic PAMAM G3.5 did not demonstrate a cytotoxic activity in vitro. In contrast, the cationic PAMAM G4 dendrimer exerted multiple suppressive effects on SK-OV-3 cells, including proliferation inhibition, induction of apoptosis and collapse of mitochondrial membrane

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potential. In conclusion, our preliminary in vitro studies demon- strated the antitumor effect of PAMAM G4 dendrimer on human ovarian cancer cells. These results provide the basis for further investigations of PAMAM G4 dendrimer as a potential medical candidate for a cancer therapy. Acknowledgement: Project ‘Biological properties and biomedi- cal applications of dendrimers’ operated within the Foundation for Polish Science Team Programme co-financed by the EU European Regional Development Fund.

N-terminal domain as the antigen. Coat protein (CP) of Alter- nanthera mosaic virus (AltMV) served as a carrier in plant expression systems. It is capable of forming pseudovirions under native conditions without genomic RNA. C-terminal part of CP was fused to short and full-length versions of M2E peptide. Additional constructs contained 6 · His tag for purification from plant extracts. Modified genes were cloned into binary vector and expressed in Nicotiana benthamiana leaves via agroinfiltration. Chimeric CPs were localized in soluble fraction, we confirmed their stability and high level of accumulation. Functions of these proteins and exposed epitopes, for example the ability of self- assembly either in vitro or in vivo, were checked by immunologi- cal methods and electronic microscopy. In order to increase the level of synthesis in comparison with initial binary plasmids, mutant CP genes were transferred to viral vectors based on cDNA of related potexvirus Potato virus X. Wild-type coat pro- tein gene was changed to modified AltMV CP genes. Most of the constructs lacked triple block of movement protein genes, but in the control vectors they were retained. We inoculated N. benth- amiana plants with our chimeric viruses that allowed us to test the assembly of virions in plant cell and efficiency of cell-to-cell and systemic movement, which influences the productivity of tar- get protein.

P8–45 TNFalpha, IL1beta, IL13 and IFNgamma effects on the cell death of the A549 lung carcinoma cells V. Galani1, G. Chondrogiannis1, M. Kastamoulas1, A. Sfikas2, G. Vartholomatos3, T. Markopoulou4, D. Arvanitis5, E. Kolettas4 and P. Kanavaros1 1Faculty of Medicine, Department of Anatomy-Histology, Univer- sity of Ioannina, Ioannina, GREECE, 2Faculty of Medicine, Department of Anatomy-Histology and Department of Physiology, University of Ioannina, Ioannina, GREECE, 3University Hospital of Ioannina, Laboratory of Hematology, Ioannina, GREECE, 4Faculty of Medicine, Department of Physiology, University of Ioannina, Ioannina, GREECE, 5Faculty of Medicine, Department of Anatomy, University of Thessalia Larissa, GREECE

P8–47 Antiproliferative activity of purine nucleoside phosphorylase multisubstrate analogue inhibitors containing difluoromethylene phosphonic acid against leukaemia and lymphoma cells L. Glavas-Obrovac1, M. Suver2, S. Hikishima3, M. Hashimoto3, L. Magnowska4 and A. Bzowska4 1Department of Clinical Chemistry and Biochemistry, School of Medicine University J.J. Strossmayer of Osijek, Osijek, CROA- TIA, 2University Hospital Osijek, Scientific Unit for Clinical- Medical Research, Osijek, CROATIA, 3Department of Chemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo, JAPAN, 4Department of Biophysics, Institute of Experimental Physics, Warsaw University, Warsaw, POLAND

The effects of TNFa, IL1b, IL13, IFNc and Fas on cell death of A549 cells were investigated. Flow cytometry: IFNc treatment increased cell death, IL1b treatment decreased cell death whereas TNFa and IL13 treatment did not alter cell death in comparison to untreated cells. The combinations Fas/CH11-TNFa, CH11- IL13, CH11-IL1b, CH11-IL13-TNFa, CH11-IFNc-TNFa and CH11-IFNc-IL13 decreased cell death whereas the combination CH11-IFNc increased cell death in comparison to CH11. TNFa or IL1b or IL13 pretreatment decreased whereas IFNc pretreat- ment increased CH11-induced cell death. TNFa and IL1b anti- cell death effects were attenuated by BAY-117082 (NF-kB inhibi- tor) and IL-13 and IL1b anti-cell death effects were attenuated by LY-294002 (PI3-K inhibitor). In A549 cells with NF-jB sup- pression, TNFa or IL1b pretreatment did not induce significant alterations in CH11-induced cell death. Western blot: The cleaved form of PARP1 protein was detected in CH11-treated cells. The TRAF1 protein and decreased Ik-Ba protein expression were observed in TNFa-treated cells without NF-jB suppression. Decreased p27 protein expression was observed in CH11-IFNc treated cells. No alterations of p53, Bcl2, Bcl-xl, Bax, Bak and Bad expression were detected suggesting that these proteins do not play a major role in cell survival events mediated by TNFa, IL1b, IL13 and IFNc in A549 cells. The present results show that TNFa, IL1b and IL-13 attenuate whereas IFNc enhance the pro-cell death effects of Fas/CD95 on A549 cells. The anti-cell death effects of TNFa, IL1b and IL-13 were mediated, at least partially, by the NF-kB and PI3-K pathways.

P8–46 Plant expression system for presenting M2E antigen from Influenza virus as the potexvirus coat protein fusion T. Gasanova, L. Tuylkina, P. Ivanov and J. Atabekov Moscow State University, Virology, Moscow, RUSSIA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Matrix protein M2 of Influenza virus A is a promising candidate for immunotherapy because its sequence is conservative between different strains as opposed to variable neuraminidase and hemagglutinin genes. We chose 24 aminoacids-epitope M2E from Purine nucleoside phosphorylase (PNP, E.C.2.4.2.1) catalyzes the reversible phosphorolysis of nucleosides and deoxynucleosides which is an important step of purine catabolism pathway. PNP is crucial for the integrity of the immune system, since PNP defi- ciency in humans leads to defective T-cell response. Hence, potent PNP inhibitors that would be able to cause effects similar to PNP deficiency are expected to be useful in treatment of type IV autoimmune diseases, T-cell proliferative disorders, and T-cell haematological malignancies. In our search for compounds effi- cient as PNP inhibitors, five compounds with guanine moiety, three with hypoxanthine moiety, and five compounds with 9-de- azaguanine moiety, all connected by a linker with difluoromethyl- ene phosphonic acid were synthesized and tested for PNP inhibitory potential in vitro on calf spleen and human erythrocyte PNPs, respectively. The antiproliferative potential of analogues against normal lymphocytes and seven human cell lines derived from haematological malignancies (K562, JURKAT, MOLT, HuT78, Raji, CCRF-CEM, and HL-60) using MTT test was also investigated. Obtained results show that all analogues inhibit mammalian PNPs with inhibition constants ranging from 5 to 70 nM. Screening of their in vitro anti-leukaemia and anti-lym- phoma activity brings evidence that compounds with 9-deazagua- nine moiety has better inhibitory potential in comparison with guanine and hypoxanthine analogues. No differences were observed between the effects on the growth of tumour cells

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sensitive to inhibition of PNP activity, such as human adult T-cell leukaemia and lymphoma cells, and other leukaemia and lymphoma cells of B-cell, or non-T-, non-B-cell lineages.

P8–48 In vitro antiproliferative activity of a series of novel genistein glycoconjugates against human cancer cell lines A. Gogler1, A. Rusin1, M. Glowala-Kosinska1, K. Kedzior1, A. Gruca1, J. Zawisza2, W. Szeja2, G. Grynkiewicz3 and Z. Krawczyk1 1Department of Tumor Biology, Maria Sklodowska-Curie Memo- rial Cancer Center and Institute of Oncology Gliwice, Gliwice, POLAND, 2Department of Organic Chemistry Bioorganic Chemis- try and Biotechnology, Silesian University of Technology, Gliwice, POLAND, 3Department of Chemistry, Pharmaceutical Research Institute, Warsaw, POLAND

(Virco) assay. One of the six clones contained no DRV-associ- ated mutations and also carried the D30N and I50L mutations, which may confer increased DRV susceptibility. In order to ana- lyze the mechanism of resistance development to DRV on a molecular level, we prepared pure recombinant PRs derived from these patient samples. We characterized the PRs in terms of enzy- matic activity, thermodynamics of protein/ligand binding, and X- ray structure analysis. Enzymatic analysis shows an increase in the relative Ki values by 3–4 orders of magnitude for some of the mutants accompanied by a corresponding increase in vitality values. However, some mutant PRs remain sensitive to the com- pound, even though they accumulated as many as 20 mutations, including those responsible for cross-resistance to most other PIs. Thermodynamic analysis of one of the most resistant PRs revealed an increase in binding enthalpy of 10.7 kcal/mol com- pared to wild-type protease. This significant change can be explained by rearrangement of hydrogen bonds, as observed in the structure of the PR/DRV complex solved by X-ray crystal- lography.

P8–50 In vitro anti-diabetic properties and cytotoxicity of four plants used to treat diabetes in South Africa T. Koekemoer1, D. Grierson2, M. van de Venter1 and S. Roux1 1Department of Biochemistry and Microbiology, Nelson Mandela Metropolitan University, Port Elizabeth, SOUTH AFRICA, 2Department of Botany, University of Fort Hare, Alice, SOUTH AFRICA

This work was undertaken to assess an antiproliferative activity in vitro of a series of novel synthetic genistein glycoconjugates. 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay was used to determine the cytotoxicity of compounds against DU145 and HCT116 cells. Genistein gly- coconjugates showed diverse antiproliferative potency against tested human tumor cell lines. Among the series, Ram-3 exhib- ited the most potent growth-inhibitory activity against cancer cells. Cell cycle analysis with flow cytometry showed that antipro- liferative activity of Ram-3 was mediated by arrestment of cells in the G2/M phase of the cell cycle. Cellular response to Ram-3 treatment was assessed also by immunofluorescence microtubule array. We have found Ram-3 caused mitotic spindle disorganiza- leading to cell tion and aberrant chromosomes distribution, death. TUNEL staining and DNA agarose gel electrophoresis revealed that Ram-3 induced apoptosis in DU145 and HCT116 cells. In summary, we found that compound Ram-3 exerted con- siderable antiproliferative activity against human cancer cells and appeared a promising candidate for possible chemotherapeutic use, further modification as a lead compound, and an object for structure – activity relationship studies.

P8–49 Molecular characterization of clinical isolates of HIV PRs resistant to Darunavir K. Grantz Saskova1, M. Kozisek1, P. Rezacova1, J. Brynda1, T. Yashina2 and R. M. Kagan2 1Institute of Organic Chemistry and Biochemistry AS CR v.v.i., Biochemistry, Prague, CZECH REPUBLIC, 2Department of Infectious Diseases, Specialty Laboratories, Valencia, CA, USA

Medicinal plants are often used as adjunctive therapy with con- ventional medications, raising the possibility for synergistic or antagonistic interaction. Since numerous therapeutic targets are exploited to treat diabetes, knowledge on the mechanism by which herbal extracts exert their effect would be beneficial when traditional medicine is combined or used as an alternative to con- ventional drugs. Ethnobotanical surveys conducted in South Africa indicate that Artemisia afra, Ruta graveolens, Sutherlandia frutescens and Leonotis ocymifolia are the most popular plants used to treat diabetes, however their precise mechanism of action is unknown. Various in vitro models designed to simulate specific therapeutic targets were used to screen plant extracts for poten- tial anti-diabetic activity. The assays included a-glucosidase inhi- bition, DPPIV inhibition, antioxidant activity, glucose uptake and PPAR-c agonist activity. In addition, potential hepatotoxic- ity was assessed using HepG2/C3A cells. None of the plants showed any significant a-glucosidase inhibitory activity nor could they significantly stimulate adipocyte differentiation. The antioxi- dant capacity of the different plant extracts varied depending on the assay used. A. afra and S. frutescens had the best metal che- lating and oxygen radical scavenging activity while only R. graveolens showed H2O2 scavenging activity. Both aqueous and ethanolic extracts prepared from S. frutescens demonstrated DPPIV inhibition, however the concentration required to achieve 50% inhibition suggests that this activity has little physiological relevance. Except for A. afra, none of the plants were toxic to cultured hepatocytes even at a concentration as high as 1 mg/ml. Further studies are required to identify the metabolic targets of these plants.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

HIV protease (HIV PR) is one of the key targets for anti-HIV treatment, and protease inhibitors (PIs) are potent antiviral drugs. However, resistance toward clinically available protease inhibitors (PIs) plays an important role in treatment failure. Development of novel PIs active against a wide range of resistant PR species is therefore still needed. Darunavir (DRV) is a sec- ond-generation HIV PR inhibitor designed to overcome antiviral drug resistance. In vitro studies have indicated a strong genetic barrier to the development of DRV resistance. To date, there is no report of an HIV PR species derived from a DRV-treated patient associated with high resistance toward the drug. We obtained six DNA clones from clinical samples submitted to a US reference laboratory for HIV-1 resistance testing. These clones carried as many as 21 amino acid exchanges in compari- son to the wild-type enzyme and had high levels of predicted phe- notypic resistance to DRV according to the VirtualPhenotype(cid:4)

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in processes that occur at the site of inflammation. Thus, a phar- macological manipulation on this enzyme might open new ways for treatment of a wide range of diseases associated with the development of inflammatory state. Acknowledgement: This work was supported by grant No. N301 067 31/2018 from the Ministry of Science and Higher Edu- cation (Poland).

P8–51 The monitorisation of the oxidative status of the patients with uveal melanoma in the dynamic of the oncological treatment M. I. Gruia1, D. Glavan1, G. Murgoi1 and I. Gruia2 1Institute of Oncology, Biochemistry and Radiobiology, Bucharest, ROMANIA, 2University of Bucharest, Optics, Bucharest, ROMANIA

P8–53 The influence of kinins on leukocyte adhesion I. Guevara-Lora1, J. Skrzeczynska-Moncznik2 and A. Kozik1 1Jagiellonian University Faculty of Biochemistry Biophysics and Biotechnology, Analytical Biochemistry, Krako´w, POLAND, 2Jagiellonian University Faculty of Biochemistry Biophysics and Biotechnology, Immunology, Krako´w, POLAND

Background: Uveal melanoma is the most common primary intraocular malignant tumor in adults. Overall mortality rate remains high because of the development of metastatic disease, which is highly resistant to systemic therapy. Improved under- standing of the molecular pathogenesis of cancer has led to a new generation of therapeutic agents that interfere with a specific path- way critical in tumor development or progression. Although no specific genes have been linked to the pathogenesis of uveal mela- noma, which differs from that of cutaneous melanoma, progress has been made in identifying potential targets involved in uveal melanoma apoptosis, proliferation, invasion, metastasis and angio- genesis. The aim of our clinical study is to establish the mecha- nisms between the free radicals of oxygen that are involved in the cellular cytotoxicity as a measurement of the treatment efficiency. Materials and methods: We investigated 44 patients in the dynamic of 2 years oncological treatment. In the serum we moni- tored the oxidative stress index obtained by the fraction of lipid peroxidation reaction, the copper oxidase activity and the total thiol groups. Results: The obtained data indicate an increase of the oxidative stress index in the first part of the treatment, followed by a sta- tionary of the level even when the patients have a good response to the treatment. Conclusions: Our results suggest the installation of a systemic cytotoxicity due to the adaptative mechanisms of the tumoral presence.

During inflammatory processes, leukocytes adhere to the vascular endothelium through cell-cell and extracellular matrix protein-cell interactions. Kinins are well known strong mediators of inflam- mation but the effect of these peptides on leukocyte adhesion has not been well recognized. In this work, we characterize the influ- ence of bradykinin (BK) or des-Arg10-kallidin (DAKD) on adhe- sion of human neutrophils and U937 cells, differentiated with phorbol ester or retinoic acid, to extracellular proteins or to the endothelium. The cells were treated with peptides at 1 nM or 1 lM concentration and analyzed for adhesion to fibrinogen- or fibronectin-coated microplate or to HMEC-1 cells. Both the dif- ferentiated U937 cells and neutrophils showed an increased adhe- sion to extracellular matrix proteins after BK and DAKD treatment. The cell adhesion to fibronectin was stronger after pretreatment with higher peptide concentration and this effect was enhanced upon co-incubation with cytokines. Similar results were obtained for adhesion to HMEC-1 cells. A flow cytometry analysis of membrane proteins, CD11b and CD18, which are responsible for adhesion in the cells pre-treated with BK or DAKD was also performed. The kinins, at higher concentrations, caused a moderate increase of the number of CD11b- or CD18- positive cells that was augmented in the presence of cytokines. Taken together, the results obtained suggest regulatory effects of kinins on leukocyte adhesion, which may be important for the mechanisms of cell interactions during inflammation. Acknowledgement: This work was supported by grant No. N301 067 31/2018 from the Ministry of Science and Higher Edu- cation (Poland).

P8–52 The regulation of inflammatory processes by a carboxypeptidase M inhibitor I. Guevara-Lora1, B. Augustynek1, K. Stalinska2 and A. Kozik1 1Jagiellonian University Faculty of Biochemistry Biophysics and Biotechnology, Analytical Biochemistry, Krako´w, POLAND, 2Jagiellonian University Faculty of Biochemistry Biophysics and Biotechnology, Cellular Biochemistry, Krako´w, POLAND

P8–54 Potential antiviral role of p53 in HCV-infected patients N. Hamdi1, M. El-Serafy2, G. Esmat3, W. Alaakel3 and A. I. Abdelaziz4 1German university in Cairo, Molecular Pathology, Cairo, EGYPT, 2Cairo University, Tropical Medicine, Cairo, EGYPT, 3Cairo University, Tropical Medicine and Hepatology, Cairo, EGYPT, 4German University in Cairo, Molecular Pathology, Cairo, EGYPT

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Kinins (bradykinin-related peptides) stimulate several inflamma- tory processes by a modulation of the release of other pro-infla- matory mediators (cytokines, leukotriens and prostaglandins). Among numerous kinin-inactivating peptidases, the carboxypep- tidases N and M (CPN and CPM) can produce metabolites known as strong stimulators of inflammation. We characterized the influence of a CPM specific inhibitor, DL-2-mercaptomethyl- 3-guanidinoethylthiopropanoic acid (MGTA), on the regulation of kinin receptor expression in human microvascular endothelial cells (HMEC) and in U937 cells. These two model cell types pos- sessed the CPM activity that was regulated by cytokines. The expression of kinin receptor genes was analyzed after stimulation with bradykinin (BK), alone or upon a co-incubation with inter- leukin 1b (IL1b) and MGTA. BK was able to induce the expres- sion of B1 receptor in HMEC and U937 cells. However, a decrease of this expression was observed after MGTA co-incuba- tion with BK and with BK/IL1b. The gene and protein expres- sion of IL1b was augmented by BK stimulation and this effect was reversed by MGTA. A mediation of selected transcription factors (NFkB, AP-1 or STATs) confirmed the effect described above. Our results suggest a regulatory role of carboxypeptidases Background: p53 is recently implicated in antiviral defense by activation of interferon a/b (IFN a/b) pathway. In vitro studies working with viral infected cells showed that p53 gene promoter possesses interferon stimulated response elements (ISREs) that are transcriptionally induced by IFNa/b. Furthermore, p53 regu- lates intracellular HCV replication through transcriptional activa- tion of the interferon regulatory factor 9 (IRF9) that further stimulates the transcription of interferon stimulated genes (ISGs). These findings were further confirmed in HCV-infected mice but have never been studied in HCV-infected patients.

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P8–56 Mutagenic analysis of human serine racemase H. Hoffman, J. Jiraskova and J. Konvalinka Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences, Biochemistry, Prague 6, CZECH REPUBLIC

compared to healthy controls

Aim: Peripheral blood mononuclear cells (PBMCs) are reservoirs for viral replication and strong IFN producers. We aimed to investigate the antiviral function of p53 in PBMCs of HCV- infected patients by correlating p53 mRNA expression with that of MxA which is an ISG, stimulated by interferon, mediating an antiviral effect. Methods: Blood samples were collected from healthy volunteers and HCV-patient’s before IFN therapy. PBMCs were isolated, total cellular RNA was extracted and cDNA was synthesized. TaqMan real-time PCR was used for relative quantitation (RQ) of mRNA expression levels. Results: Marked p53 upregulation (more than 1.5-fold increase) (2.718 ± 0.156; n = 32; p < 0.0001) was accompanied by signif- icant elevation of MxA gene expression (7.821 ± 1.318; n = 32; p = 0.0215) (1.039 ± 0.127; n = 6) (1.413 ± 0.501; n = 8). While MxA expression was not significantly elevated (3.442 ± 1.125; n = 10; p = 0.149) upon mild p53 upregulation (1.4367 ± 0.064; n = 10; p = 0.0075). Conclusion: For the first time, we demonstrate the upregulation of p53 in PBMCs of HCV-untreated patients with the transcrip- tional activation of the downstream interferon stimulated gene- MxA that further supports the antiviral role of p53.

recombinant protein for

D-serine is present at high concentrations in the mammalian ner- vous system, where it serves as a co-agonist of N-methyl-D- aspartate (NMDA) type glutamate receptors. Aberrant regulation of D-serine biosynthesis has been implicated in a variety of neu- ropathologies, including schizophrenia, Alzheimer’s disease, and amyotrophic lateral sclerosis. D-serine is synthesized from its enantiomer by the pyridoxal-5’-phosphate dependent enzyme ser- ine racemase, and glutamatergic neurotransmission in serine race- mase knock-out mice is significantly altered. Serine racemase therefore holds great promise as a pharmaceutical target for treatment of a variety of pathologies, yet relatively little is known about its overall structure and catalytic mechanism. We have developed a recombinant Escherichia coli based expression system that enables us to obtain modest quantities of purified human serine racemase. The recombinant enzyme catalyzes the intercon- version of L- and D-serine as well as the elimination of serine to pyruvate. In order to identify the amino acid residues critical for serine racemase function and in hopes of affording a high yield of structural studies, we subjected human serine racemase to both site-directed and random muta- genesis. The resulting library of mutants was analyzed for soluble expression in our E. coli based system. The soluble mutants were then analyzed for their ability to racemize serine. This approach allowed us to identify several amino acid residues necessary for serine racemase folding and/or activity, and the results of this screening are summarized here.

P8–55 Differences in vanadocene dichloride and cisplatin effect on apoptosis induction in MOLT-4 leukemia cells and human lymphocytes of peripheral blood R. Havelek1, P. Siman1, P. Krejcirikova1, L. Zarybnicka2, Z. Sinkorova2, J. Vinklarek3 and M. Rezacova1 1Faculty of Medicine in Hradec Kralove, Medical Biochemistry, Hradec Kralove, CZECH REPUBLIC, 2Faculty of Military Health Science, Radiobiology, Hradec Kralove, CZECH REPUB- LIC, 3Faculty of Chemical Technology, General and Inorganic Chemistry, Pardubice, CZECH REPUBLIC

P8–57 A potential antiangiogenic properties of brassinosteroids L. Hoffmannova1, S. Zahler2, L. Kohout3 and M. Strnad1 1Palacky University, Laboratory of Growth Regulators, Olomouc, CZECH REPUBLIC, 2Department of Pharmacy Centre of Drug Research – Pharmaceutical Biology, Ludwig-Maximilians Univer- sity, Munich, GERMANY, 3Institute of Organic Chemistry and Biochemistry ASCR, Prague, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Aim: To detect difference in cell death induction caused by potential cytostatic vanadocene dichloride and established cyto- static cisplatin in MOLT-4 leukemia cells and human peripheral lymphocytes. Methods: Cells were treated for 1 hour with drugs, after 24/ 72 hours cytotoxic effect was evaluated using WST-1. Cell viabil- ity was detected by trypan blue. For apoptosis detection we used flow cytometry (Annexin V/propidium iodide). Activity of casp- ases 3 was determined by Caspase-Glo(cid:3)3/7. Results: For IC50 value of vanadocene was lymphocytes 106 lmol/l, for cisplatin 231 lmol/l after 72 hours of incubation after treatment. Seventy-two hours after exposure to vanadocene (1 mM, resp. 0.5 mM) almost all lymphocytes were apoptotic (98, resp. 95%). Almost twice lower apoptosis induction was caused by cisplatin in the same concentrations (42, resp. 29%). For MOLT-4 IC50 value for vanadocene was 843 lmol/l, for cis- platin 41 lmol/l. Both cytostatics in concentrations 1 mM, resp. 0.5 mM caused eradication of the culture, however significantly lower apoptosis was detected after vanadocene in concentrations below 0.2 mM (compared to cisplatin). IC50 value for 24-hours incubation of MOLT-4 with cisplatin was long continuous 8.5 lmol/l and 125 lmol/l for vanadocene. In concentrations cor- responding to IC50 cisplatin caused pronounced activation of caspase 3, while vanadocene did not. Conclusion: Vanadocene induces significantly higher apoptosis of lymphocytes compared to cisplatin. In MOLT-4 cells, both the drugs in 1 mM resp. 0.5 mM caused eradication of the culture, but more pronounced apoptosis induction by cisplatin was observed after concentrations below 0.2 mM. Acknowledgement: Supported by project MSM 0021620820. Phytohormones brassinosteroids play an important role in hor- mone signaling and physiological response in plant organism. They regulate various types of processes, such as growth and development, e.g. increase of number of cells and their elonga- tion. Brassinosteroids enhance the disease resistance against abi- otic and biotic stresses in plants. The cellular and molecular mechanisms of action of these phytohormones in animal and human cells are still almost unknown. Only the cytotoxic effect on animal and human cancer cells derived from tumors were esti- mated recently in vitro. The effects of brassinosteroids and their synthetic derivatives were studied on endothelial primary cells human umbilical vein endothelial cells (HUVEC) and human mammary epithelial cells (HMEC). Methods used for detection of changes in viability, proliferation, migration and apoptosis were: proliferation assay (staining with crystal violet), migration assay, tube formation and flow cytometry. Both natural brassi- nosteroids and their synthetic analogues inhibit proliferation and migration of human endothelial cells and cause apoptosis. Espe- cially the inhibition of migration plays a crucial role in develop- ment of new blood vessels, formation of new centres of tumor and spreading. These results should help us to understand the potential antiangiogenic effect of brassinosteroids on endothelial cells.

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in two ways. On the one hand, it either causes death of certain transformed cells or may increase their sensitivity to doxorubicin. On the other hand, lovastatin attenuates some side effects of this chemotherapeutic agent.

P8–60 Effects of antitumor drug, vinorelbine, on chromatin components in solution E. Chamani and A. Rabbani-Chadegani I.B.B, Biochemistry, Tehran, IRAN

P8–58 Hemoglobin proteolysis in blood-feeding ticks: mapping of multienzyme cascade by functional proteomics M. Horn1, M. Nussbaumerova1, M. Sanda2, Z. Kovarova1, J. Srba1, Z. Franta3, D. Sojka3, P. Kopacek3 and M. Mares1 1Department of Biochemistry and Molecular Biology, Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague 6, CZECH REPUBLIC, 2Department of Mass Spectrome- try, Institute of Organic Chemistry and Biochemistry, Czech Acad- emy of Sciences, Prague 6, CZECH REPUBLIC, 3Department of Molecular Ecology of Parasites, Institute of Parasitology Biology Centre of the Academy of Sciences of the Czech Republic, Ceske Budejovice, CZECH REPUBLIC

Hemoglobin digestion is essential for the survival of blood-feed- ing parasites. Using chemical tools, we deconvoluted the intracel- lular hemoglobinolytic cascade in the tick Ixodes ricinus, a vector of Lyme disease and encephalitis. A set of peptidases in the tick gut tissue was demonstrated through imaging with specific activ- ity-based probes and activity profiling with peptidic substrates/ inhibitors. This peptidase network is induced upon blood feeding and degrades hemoglobin at acidic pH. Selective inhibitors were applied to dissect the roles of the individual peptidases and deter- mine the peptidase-specific cleavage map of the hemoglobin mol- ecule. The degradation pathway is initiated by endopeptidases of aspartic and cysteine class (cathepsin D and L supported by legu- main) and continued by cysteine amino- and carboxy-dipeptidas- es (cathepsin C and B). The identified enzymes are potential targets to developing novel anti-tick vaccines. Acknowledgements: The work was supported by grants no. 203/09/1585 (Grant Agency of the Czech Republic) and IAA600960910 (Grant Agency of the Academy of Sciences of the Czech Republic), by research projects no. Z60220518 and Z40550506, and Research Center No. LC06009. Vinorelbine belongs to vinca alkaloid family widely used as an anticancer drug in cancer therapy. Previous studies have shown that vinorelbine exerts its anticancer activity by inhibition of microtubule polymerization and mitotic spindle formation. Chro- matin is a nucleoprotein complex containing DNA, histones and non-histone proteins. Therefore, chromatin but not DNA is the main target for anticancer drugs in the cell nucleus. In the present study chromatin was isolated from hepatocytes nuclei and sub- jected to micrococcal nuclease digestion procedure. Chromatin and histone proteins were incubated with different concentrations of vinorelbine, and after desired periods of time the samples were ana- lyzed employing thermal denaturation, UV/VIS, and fluorescence spectroscopy techniques. The results showed that interaction of vinorelbine with DNA decreased the absorbance at 260 and 215 nm. The fluorescence emission intensity was decreased as drug concentration was increased indicating quenching of the drug chro- mospheres with the DNA molecule. Although Tm of DNA was slightly affected in the presence of vinorelbine, its binding to chro- matin shifted DNA thermal denaturation to higher temperatures. The affinity of vinorelbine to isolated histone proteins was higher than others confirming that vinorelbine also binds to histone pro- teins. From the results presented above it is concluded that apart from microtubules, chromatin can also be considered as a new tar- get for vinorelbine and inhibition of DNA and RNA synthesis may be a result of vinorelbine’s effect on chromatin.

P8–59 Lovastatin attenuates doxorubicin cytotoxic side effects in vivo J. Huelsenbeck, C. Henninger and G. Fritz Universitaetsmedizin Mainz, Toxicology, Mainz, GERMANY

P8–61 A novel platinum blue complex containing sulfur-donor as a potential antitumor active drug S. B. Isgor and S. Ozalp Yaman Atilim University, Chemistry Group, Ankara, TURKEY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) are widely used in the treatment of hypercho- lesterolemia. Statin treatment causes apoptosis in certain tumour types such as acute myelogenous leukemia. Furthermore, statin cotreatment potentiates the effect of chemotherapeutic drugs (e.g. cisplatin, doxorubicin) on transformed cells and can reverse chemoresistance. Recently, we demonstrated that statin treatment protects human umbilical vein endothelial cells (HUVEC) from the cytotoxic effects of doxorubicin. Here, we extend these find- ings to the in vivo situation. Balb/C mice were treated with doxo- rubicin, either alone or in cotreatment with lovastatin. After 3 weeks, the mice were sacrificed. Analysis of serum parameters revealed that doxorubicin caused an increase of the level of tro- ponin I, as well as GLDH and GPT, indicative of heart and liver damage, respectively. Furthermore, the mRNA level of the fibro- sis marker connective tissue growth factor (CTGF) was elevated in heart and liver of doxorubicin treated mice. Cotreatment with lovastatin reduced these effects of doxorubicin significantly. Nei- ther troponin I. nor GLDH nor GPT serum levels were elevated. The CTGF mRNA level was nearly reduced to the level in con- trol mice. In conclusion, lovastatin attenuated the cytotoxic effects of doxorubicin on non transformed cells in vivo. Regard- ing cancer treatment using doxorubicin, lovastatin is thus useful Cisplatin, the well known platinum drug, has been used for can- cer therapy since 1978 and so far found quite effective against testicular and ovarian cancer. However, the success of cisplatin chemotherapy is limited by some serious clinical issues such as nephrotoxicity, ototoxicity, neuropathy, myelosuppression and tumor resistance developed against the therapy, as well as some common side effects such as nausea and vomitting. A family of deeply colored (intense blue or purple) platinum compounds, usually called platinum blues, has received great attention due to their potential to overcome these clinical issues that reduces the effectiveness of cisplatin based chemotherapy, disease prognosis as well as the patient’s quality of life. A member of this family, the platinum pyrimidine-blue derivatives, when compared with cisplatin, has been found to have a high index of antitumor activ- ity with low nephrotoxicity to design and synthesis of more effec- tive, less toxic and orally available platinum drugs. In the present study, as part of our efforts to more effective platinum drug design and synthesis, the use of a sulphur donor ligand 2-amino- thiophenol (2-atp), to synthesize a novel platinum blue complex is shown for the first time. This new platin blue complex has been successfully tested by electrochemical and electrophoretic

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methods for DNA binding studies to verify its ability to bind and cleave the DNA in a dose-dependent manner. The inhibitory effect of platinum blue complex on glutathione-S-transferase (GST), one of the most important enzymes involved in drug con- jugation and biotransformation reactions have also been verified. The overall analysis of this novel platinum complex, therefore, suggests that the complex can be used as a potential platinum based antitumor agent since its highly possible effectiveness in a combined therapy and its strength against drug resistance mecha- nism based on thiol conjugation.

7-pentoxyresorufin-O-dealkylase

eczema and expelling digestive tract worms in Turkey. Despite its wide usage, there is no available information about their actions on CYP450 dependent drug metabolism. In this respect, the aim of this study is to determine the effect of water extracts of Cycla- men trochopterantum on hepatic CYP3A and CYP2B enzymes that are mainly involved in drug metabolism. Male Wistar rats were treated with four different dose of cyclamen tuberose water extract for 10 consecutive days. The catalytic activities of hepatic CYP3A and CYP2B were measured using specific probe sub- strates. CYP3A dependent Erythromycin N-demethylase activity was increased in all groups between 1.4–2.6-fold. Similarly, CYP2B dependent (PROD) activity was significantly induced in four groups (p < 0.005). Data of the presented study clearly suggest that cyclamen con- tains constituents changing the activities of CYP450 enzymes. Therefore, it is expected that people using this plant as a herbal remedy may have alteration of drug clearance and clinical drug toxicity due to induction of CYP3A and CYP2B enzymes.

P8–62 Pro-oxidant and anti-oxidant properties of mitochondrial matrix-targeted ubiquinones MitoQ10 and SkQ1 P. Jezek, J. Jezek, A. Dlaskova and L. Plecita-Hlavata Institute of Physiology AS CR v.v.i., Membrane Transport Biophysics, Prague 4, CZECH REPUBLIC

P8–64 The effect of lipopolysaccharide on blood brain barrier permeability of acute hyperglycemic rats during epileptic attacks H. Yorulmaz1, B. Seker2, E. Kaptan3 and B. Oztas4 1Halic University, School of Nursing, Istanbul, TURKEY, 2Department of Physiology, Faculty of Dentistry, Istanbul University, Istanbul, TURKEY, 3Department of Biology, Faculty of Science, Istanbul University, Istanbul, TURKEY, 4Department of Physiology, Istanbul Faculty of Medicine, Istanbul University, Istanbul, TURKEY

(antimycin, inhibitors

Oxidative stress of mitochondrial origin (elevated mitochondrial superoxide production) belongs to major factors determining aging and oxidative-stress related-diseases. Mitochondria-targeted antioxidants, coenzyme Q analogs, MitoQ10 and SkQ1, may pre- vent or cure these pathological conditions. To elucidate and com- pare their pro- and anti-oxidant action, we studied their effects on HepG2 cell respiration, mitochondrial network morphology, and rates of superoxide release to the mitochondrial matrix (Jm). Addition of MitoQ10 to rotenone-inhibited HEP-G2 cells sharply decreases Jm and increases cell respiration. Thenoyltrifluoroace- tone, a Complex II inhibitor, together with rotenone completely inhibited HEPG2 cell respiration and kept high Jm, while subse- quent MitoQ10 addition restored again respiration in an antimy- cin- (stigmatelin- and myxothiazol-)-dependent manner, but did not prevent high Jm. Thus MitoQ10 is able to accept electrons prior to the rotenone-bound Q-site and a reverse mode of Com- plex II is required to regenerate the reduced MitoQ10H2 to Mi- stigmatelin and toQ10. Complex III myxothiazol) prevented high Jm in HEP-G2 cells induced with either rotenone plus MitoQ10, or with rotenone, MitoQ10, plus thenoyltrifluoroacetone. Sole MitoQ10 also increased basal Jm in HEPG2 cells. Effects of all reagents combinations on Jm were more pronounced in HEPG2 cells than in the effects on H2O2 formation in isolated liver mitochondria. SkQ1 acted similarly. In conclusion, MitoQ10 possesses a pro-oxidant role when added to intact mitochondrial respiratory chain, whereas its antioxidant role is striking when Complex I- derived superoxide generation increases due to retardation of electron flow within the Complex I. Acknowledgement: Supported by grants No. NR/9183 - 3, IAA500110701, and 303/07/0105. (p < 0.01), IL-10

The purpose of this study is to examine the effect of lipopolysac- charide (LPS) on blood brain barrier (BBB) permeability during epileptic attacks occurring in acute hyperglycemia. In addition, we have aimed in examining the changes observed in coloring of Zonula Occludens-1 and Glial Fibrillary Acidic Protein as well as the changes in the release of serum TNF-a, and IL-10, IL- 12 + p40 cytokines under artificial conditions. The attacks occurring in acute hyperglycemia caused a remarkable increase of BBB permeability (p < 0.01). While LPS administration pro- vided protective effects during epileptic attacks on BBB perme- ability (p < 0.01), it did not cause a significant change in BBB permeability during epileptic attacks occurring in acute hypergly- cemia (p > 0.05). Coloring power of Zonula Occludens-1 in hyperglycemic rats was observed to reduce. Coloring power of Glial Fibrillary Acidic Protein in hyperglycemic rats was not observed to change. It was determined that TNF-a levels in LPS, in LPS, LPS + PTZ groups levels LPS + PTZ, LPS + acute hyperglycemia + PTZ, (p < 0.01), IL-12 + p40 in groups treatment with PTZ (p < 0.05), increased significantly. As a result, although LPS caused an increase in immune reactivity of Zonula Occludens-1 and Glial Fibrillary Acidic Protein and in TNF-a and IL-10 levels, it was determined that LPS did not display a protective effect on BBB permeability during epileptic attacks occurring in acute hyperglycemia.

P8–63 Possible implications of Cyclamen trochopterantum on human therapeutics M. Kapdag, S. Arslan, O. Ozgun, M. Oztas, M. Ural, O. Dusen and A. Sen Pamukkale, Biology, Denizli, TURKEY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Cytochrome P450 (CYP450) enzymes are major players in the phase I oxidative metabolism of a wide range of xenobiotics and endobiotics. Among them, CYP3A is the most abundant hepatic and intestinal CYP450 isozyme that metabolizes more than 50% of marketed drugs. CYP2B is another isozyme that metabolizes several important pharmaceutics. CYP enzyme activities and expression can be profoundly altered by many herbs. Cyclamen is one of the widely used herbs for treatment of hemorrhoids and

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Poster Presentations

P8–65 Effect of methylsulfonylmethane on proliferation and nitric oxide production in LPS activated RAW 264.7 cells A. Z. Karabay1, A. Akinci1, T. Ozkan2, A. Sunguroglu3, F. Aktan1 and Z. Buyukbingol1 1Faculty of Pharmacy, Biochemistry, Ankara University, Ankara, TURKEY, 2Biotechnology Institute, Biotechnology, Ankara University, Ankara, TURKEY, 3Faculty of Medicine, Medical Biology, Ankara University, Ankara, TURKEY

cultures, conversions in liver homogenate and pharmacokinetics in rabbits. After incubation of TZV with cell cultures or peroral administration into rabbits, the emergence of its metabolite (M) was observed. We suggested that M might be a product of reduc- tion of TZV nitro group into amino group with the formation of 2-methylthio-6-amino-1,2,4-triazolo[5,1-s]-1,2,4-triazine-7(4)-on (AMTZV). The reference sample AMTZV was synthesized and its identity with M was confirmed by UV and mass spectra and HPLC. AMTZV was shown to be non-toxic, inactive against influenza virus and, probably, it is a product of TZV utilization ensuring its excretion from the body. Pharmacokinetic parame- ters of TZV such as Tmax, T1/2, AUC, and bioavailability of TZV were calculated. Acknowledgement: aRussian patent No. 2005120250/2006: O. N. Chupakhin, V. L. Rusinov et al., ‘Sodium salt of 2-methyl- thio-6-nitro-1,2,4-triazolo-[5,1-s]-1,2,4-triazin-7(4)-on, dihydrate with antiviral activity’. The work was supported by the RFBR, project No. 08-04-00552 and Federal program ‘Living Systems’ (State contract 02.512.11.2237).

P8–67 Changes of cardiac Ca2+-ATPase (SERCA 2a) heat production, phospholamban and mitochondrial respiration promoted by cold exposure and thyroid hormone alterations L. A. Ketzer1, A. P. Arruda1, D. P. Carvalho2 and L. de Meis1 1Medical Biochemistry Institute, Federal University of Rio de Janeiro, Rio de Janeiro, BRAZIL, 2Institute of Biophysics Carlos Chagas Filho, Federal University of Rio de Janeiro, Rio de Janeiro, BRAZIL

Methylsulfonylmethane (MSM) is a natural organosulfur com- pound that is generally used in combination with glucosamine and chondroitine sulphate for helping to cure osteoarthritis. When activated, macrophages transcriptionally express iNOS that executes long-time and extensive NO production. Abnormal release of NO may lead to inflammation and tissue injury. There- interventions that aim to control poten- fore, pharmacological tially harmful pro-inflammatory activity of macrophages present promising treatment options. In this study, MSM was tested in vitro on viability and nitrite production in LPS (lipopolysac- charide) activated RAW 264.7 cells. RAW 264.7 cells were cul- tured in RPMI 1640 medium supplemented with fetal bovine serum, L-Glutamine and antibiotics at 37(cid:2)C in a humidified atmosphere of 5% CO2 in air. Cells (4 · 104) were seeded to 96- well plates and co-incubated with different concentrations of MSM for 1 hour. Following co-incubation, cells were incubated in the absence or presence of LPS (1 lg/ml) for 24 hours. Prolif- eration of cells was estimated by MTT test. NO levels were deter- mined spectrophotometrically with Griess Assay. LPS induced NO production was reduced by MSM in a dose dependent man- ner. We found significant reductions of nitrite levels in LPS induced cells that were treated with MSM at concentrations of 12 mM, 16 mM, 20 mM, 24 mM. After 24 hours of incubation with LPS, proliferation decreased significantly and MSM induced cell proliferation in LPS-activated cells dose dependently (p < 0.001). This study shows that MSM decreases NO levels and increases proliferation in LPS induced RAW 264.7 macro- phages. This study shows that MSM may have the potential to be used as an alternative for disorders related with NO production.

P8–66 A novel inhibitor of influenza A and B viruses based on azolo-1,2,4-triazines: antiviral properties, metabolism and pharmacokinetics I. Karpenko1, S. Deev2, O. Chupakhin2, O. Kiselev3, A. Ivanov1, O. Smirnova1, S. Kochetkov1 and M. Kukhanova1 1Engelhardt Institute of Molecular Biology RAS, Russian Academy of Sciences, Moscow, RUSSIA, 2Postovsky Institute of Organic Synthesis, Ural Division of Russian Academy of Sciences, Ekater- inburg, RUSSIA, 3Research Institute of Influenza, Russian Acad- emy of Medical Sciences, St. Petersburg, RUSSIA

Short-term cold exposure promotes a small but significant rise of serum T3 in both euthyroid rats and rabbits and the heart is an important target of T3 action. In this report we measured changes in SERCA 2a and phospholamban (PLB) in rabbit hearts from hypo- and hyperthyroid animals, and compared them with modifications induced by short- and long- term cold expo- increases sure. Short-term cold exposure, like hyperthyroidism, SERCA 2a expression in the heart. The total PLB content does not change in hyperthyroidism, but short cold exposure promotes a significant decrease in total PLB and an increase in the ratio of phosphorylated to total PLB. The temperature of a given tissue depends on the balance between the heat provided by blood cir- culation and the rate of heat production by the tissue. In an attempt to evaluate the heat contribution of cardiac tissue, we measured mitochondrial respiration in permeabilized cardiac muscle and heat produced by cardiac sarcoplasmic reticulum (SR) during Ca2+ transport. We observed that in both hyperthy- roidism and short cold exposure of euthyroid rabbits, there was an increase in oxygen consumption and heat production during Ca2+ transport by cardiac SR. In contrast to hyperthyroidism, in hypothyroid rabbits both the respiration rate and the heat derived from Ca2+ transport were decreased. The heart changes detected during short cold exposure were abolished after cold adaptation, indicating that the transient rise in serum T3 during short cold exposure might play a role in these cardiac alterations.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

rimantadine-resistant Influenza A and B viruses are continuing causes of morbidity and mortality on an annual basis. Despite the influenza virus importance as human pathogen, the antiviral drugs approved to combat influenza virus infections are currently limited. Recently a novel drug triazavirine (2-methylthio-6-nitro-1,2,4-triazolo[5,1- s]-1,2,4-triazine-7(4)-on, TZV) was shown to be effective against human influenza A and B viruses as well as highly pathogenic avian influenza A virus strain H5N1a. TZV activity was similar to that of a currently used drug rimantadine against influenza A virus and exceeded it against strains. Herein, we studied TZV metabolism in HEK 293T and Huh7 cell

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Abstracts

P8–68 Genotypic method for checking and predicting acyclovir-resistance on the level of human alphaherpesviruses thymidine kinases V. Khrustalev, E. Barkovsky and D. Zadorozniy Belarussian State Medical University, General Chemistry, Minsk, BELARUS

and GLUT4 were reduced in type-2 diabetic patients. In leuko- cytes of diabetic patients, GLUT1 and GLUT4, protein and mRNA were unchanged, but GLUT3 protein and mRNA levels were down-regulated compared to those of healthy controls. Conclusions: Elevated glucose concentration affects leukocyte GLUT expression. Decreased expression of GLUT isoforms in leukocytes may be responsible for diminished activation of dia- betic leukocytes. These situations possibly contribute to a predis- position to infection and to a decreased immune response in diabetes.

P8–70 Easy-to-use and rapid detection of potential tumour disease marker metallothionein by using of PVDF membranes and chicken antibodies S. Krizkova1, V. Adam1, T. Eckschlager2 and R. Kizek1 1Department of Chemistry and Biochemistry, Mendel University of Agriculture and Forestry in Brno, Brno, CZECH REPUBLIC, 2Department of Paediatric Haematology and Oncology, Charles University, Prague, CZECH REPUBLIC

There are 108 nucleotide sequences of human herpesvirus type 1 (HSV1) thymidine kinase (TK) in GenBank for today. We ana- lyzed all amino acid substitutions in them and collected the data- base of critical (causing acyclovir resistance) and polymorphic substitutions. The same kinds of databases have been collected by us for human herpesvirus type 2 (HSV2) and human herpesvi- rus type 3 (VZV) thymidine kinases, but numbers of critical and polymorphic substitutions in them are much lower yet. Original computer algorithms (‘HSV1TK’, ‘HSV2TK’ and ‘VZVTK’) have been created for checking acyclovir-resistance of these three al- phaherpesviruses by the way of comparison between amino acid substitutions in each concrete sequence coding for TK with sub- stitutions from database. To use our algorithm one should paste the nucleotide sequence coding for TK in the subsequent cell on ‘sequence2’ list and see the result of genotypic method appliance on ‘results’ and ‘information’ lists. For the prediction of acyclo- vir-resistance in case if substitutions in the sequence have not been included in database we proposed the following method. TK is likely to be resistant to acyclovir in case if its active centers or conserved regions are affected and substitutions in active cen- ters or conserved regions have not been found in phylogeny. We created one more database with all phylogenetically neutral sub- stitutions in conserved regions of TK from all the members of simplexvirus genus and varicellovirus genus that can be useful for the prediction of acyclovir-resistance. Our algorithms written in form of MS Excel spreadsheets are available on web site: www.barkovsky.hotmail.ru.

Metallothioneins (MT) are low-molecular-mass proteins capable to bind heavy metals. They are involved in transporting and detoxifying of metal ions. Based on recently published papers their role in carcinogenesis can be considered. A lot of methods different in the analytical approach and character of information obtained can be used for metallothionein detection, such as chro- matographic, electrophoretic and immunological ones. The aim of this work is to determine metallothionein in blood serum sam- ples from patients with tumour disease and in cell lines of human fibroblasts (healthy and tumour) exposed to Cd(II) by using dot immunobinding assay and Western blotting. The results obtained are compared with those measured by differential pulse voltam- metry Brdicka reaction. We proposed a new methodological approach for visualization of MT on PVDF membranes by using both mouse E9 and chicken antibodies. We optimized whole approach and found the most suitable experimental conditions as follows: chicken yolk antibodies, PVDF membrane and 3-amino- ethyl-9-carbazole (AEC) as chromogenic substrate. Under these conditions we estimated detection limit as 3 pg of MT/1 ll. The optimal approach was further utilized for detection of MT level in two human fibroblast cell lines and in samples obtained from children with medulloblastoma. Level of MT in patients’ samples was 6 ± 3 lM. The results were in good agreement with electro- chemical method. Acknowledgement: This work was supported by GA AV IAA401990701 and Liga proti rakovine. References: 1. Fabrik I, et al., Electroanalysis 20 2008; 14: 1521–1532. 2. Krizkova S, et al., Electroanalysis 21 2009; 3–5: 640–644.

P8–69 Type 2 diabetes down-regulates glucose transporter proteins and genes of the human blood leukocytes D. Kipmen-Korgun1, S. Bilmen-Sarikcioglu2, H. Altunbas3, R. Demir4 and E. T. Korgun4 1Department of Biochemistry, Akdeniz University, Antalya, TURKEY, 2Central Laboratory, Akdeniz University, Antalya, TURKEY, 3Division of Endocrinology and Metabolism, Department of Internal Medicine, Akdeniz University, Antalya, TURKEY, 4Department of Histology and Embryology, Akdeniz University, Antalya, TURKEY

P8–71 Poly-2-hydroxyethylmethacrylate polymeric gels synthesis with different structured hydroxyapatite S. Canim, K. Kizilbey and Z. Mustafaeva Akdeste Bioengineering Department, Yildiz Technical University, Istanbul, TURKEY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

The structures of natural hard tissues (bones and teeth) are gen- erally formed with hydroxyapatite and collagen. Many interesting studies have been made on development of synthetic bone materi- als to improve the biomechanical and economical qualities. Synthetic hydroxyapatite, which is a mineral part of bone, has Objective: White blood cells are essential in mediating immune and inflammatory responses. A prominent feature of these cells during activation of the immune function is increased glucose uti- lization, and this is dependent on the functioning of specific glu- cose transporter (GLUT) isoforms. The few data available on leukocyte glucose transporter expression are limited to type-2 diabetes mellitus, and nothing is known about its regulation. Material and methods: Peripheral blood was drawn from 35 healthy controls and 35 diabetic subjects. Expression of GLUT1, GLUT3 and GLUT4 was determined in the leukocytes of healthy individuals and diabetic patients by flow cytometry, Western blot and semi-quantitative RT PCR. Results: GLUT3 was decreased in granulocytes, lymphocytes and monocytes from diabetic patients. In monocytes, GLUT3

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the presence of low concentrations of metal ions. The mechanism of Cu(II)-mediated polycomplexes were investigated by UV spec- trophotometry, FT-IR and HPLC. References: 1. Mustafaev M, Functionally Biopolymer Systems, Sigma, J of Engineering and Natural Sciences 2004.

2. Mustafaev M, Yu¨ cel F, Cirakolu B, Bermek E, Immunol Lett. Immune response to progesterone involved in Cu(2+)-medi- ated polyanion-protein complex–antigen specificity and affinity of hybridoma clones, 1996; Sep;52(2–3): 63–8.

P8–73 A comparative study of enzymatic activity and tissue expression of mouse and human glutamate carboxypeptidase II T. Knedlik, P. Sacha, K. Hlouchova, J. Starkova, M. Rovenska and J. Konvalinka Institute of Organic Chemistry and Biochemistry AS CR v.v.i., Biochemistry, Prague, CZECH REPUBLIC

excellent biocompatibility and important bioactivity (1). As well as organic chemicals can absorb hydroxyapatite; it is also used as a catalyzer, ion exchanger, biosensor and bioceramic in biomedi- cal field (2). In recent studies, HA-silica composite type materials were produced by the diffusion of calcium ions to the gel matrix (3). Biocomposite synthesis was made with similar method by using gelatin and agarose gels (4). Biocomposite development in the presence of synthetic polymers is the most hopeful system (5). In this study, different structured hydroxyapatite combinations, which include HEMA monomer, were synthesized by using N,N- dimethyl-p-toluidin as a starter and EGDMA as a cross-linker. Hydroxyl groups were converted to carboxyl groups by polymer modification in synthesized, linear and polymeric gel structured pHEMA and different component pHEMA-HA combinations. As a result of this conversion, composites were developed with different-OH/-COOH ratios. The structures of synthesized poly- mers and composites were investigated by physicochemical meth- ods. The connection of carboxyl groups with peptides was performed with thionyl chloride in the polymeric gel based HA composites; and biocomposites with different compositions that have adhesion property to osteoblast cells were synthesized. References: 1. Ratner BD, Hoffman AS, Schoen FJ, Lemons JE. Biomateri- als Science 1996; ISBN:0-12-582460-2, Academic press.

2. Bohner M, Int J Injury Care 2000; 31: 37–40. 3. Villacampa AI, Garcia-Ruiz JM, J Crystal Growth 2000; 211: 111–115.

4. Itoh S, et al., Biomaterials 2002; 23: 3919–3926. 5. Ramakrishna S, Mayer J, Wintermantel E, W Leong K, Com- pos Sci Technol 2001; 61: 1189–1224.

P8–72 Progesterone-containing polyelectrolyte complexes K. Kizilbey, S. Derman and Z. Mustafaeva Akdeste Yildiz Technical University, Bioengineering Department, Istanbul, TURKEY

Glutamate carboxypeptidase II (GCPII) is a membrane metallo- peptidase expressed in various human tissues, e.g. brain, prostate and kidney. In brain it cleaves the neurotransmitter N-acetyl-L- aspartyl-L-glutamate (NAAG) thus releasing free L-glutamate into the synaptic cleft. The function of GCPII in prostate is unknown but it is overexpressed in prostate cancer and vascula- ture of most of the solid tumors. Therefore, it could become a perspective target for treatment of prostate cancer as well as neu- ronal disorders associated with increased glutamate neurotoxicity. For the development and testing of new drugs and therapeutics, it is necessary to have an appropriate animal model. While mouse is been used as a model by most experimentators, no detailed comparison of mouse and human GCPII enzymes regarding their enzymatic activity, inhibition profile and expres- sion has been performed yet. Possible variation in expression and activity profiles among the human and mouse orthologs are very relevant for the development of novel GCPII-based anticancer drugs. We prepared, expressed, purified and tested recombinant mouse GCPII and compared it to its human ortholog. Substrate specificity was determined using analogs of the endogenous pep- tide substrate, N-acetyl-aspartyl-glutamate (NAAG). The activity and inhibition of mouse GCPII were characterized by radioenzy- matic assay. We also analyzed GCPII expression in mouse tissues by Western blotting using specific monoclonal antibody.

In order

P8–74 Structure-activity relationships of gliadin peptides in coeliac disease P. Kocna1, Z. Vanickova1, J. Hlavata2 and K. Topinkova2 1Institute of Clinical Chemistry and Laboratory Diagnostics, Laboratory of Gastroenterology, Prague, CZECH REPUBLIC, 2Institute of Chemical Technology, Biochemistry, Prague, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Steroid hormones (estradiol, progesterone and testosterone) are widely used as contraceptive, anti-inflammatory, and anticancer drugs, and in general have extremely low immunogenicity. Estro- gen acts as a regulator of various physiological processes in the body, progesterone is important in preparing the uterus for the implantation of the blastocytes and in maintaining pregnancy. Therapeutic application of progesterone is the treatment of cer- tain types of endocrine dysfunction such as amenorhea and dys- to elicit an immune functional uterine bleedings. response to such antigens, small hapten molecules must be cou- pled to carrier structures. Recently, the possibility of construction of such complete synthetic immunogens was demonstrated by the direct conjugation of steroid hormones (estradiol and progester- one), anticancer betuline and functional polypeptides (Hepatitis B surface antigen and Foot-and-Mouth Disease Virus VP1 pro- tein epitopes) with different polyelectrolytes (1). The immuno- genic properties of water soluble (PAA-Cu2+-BSA) and colloidal (PAA-Cu2+-BSA.P) polycomplexes were investigated, and the specificity of antibodies produced was analyzed. Polycomplexes containing progesterone appeared to possess a high steroid-spe- cific immunogenic activity. A comparative study of immunogenic properties of polycomplexes versus BSA.P+ incomplete Freund’s adjuvant (IFA) mixtures revealed differences in regards to the specificity of antibody production (2). In the present study, cova- lent conjugates of bovine serum albumin (BSA) with Progester- one (P) hormone were synthesized by carbodiimide coupling. Ternary complexes of BSA-P conjugate with nontoxic copoly- mers of acrylic acid with N-vinyl-2-pyrrolidone were prepared in Coeliac disease (CD) is a chronic autoimmune inflammatory intestinal disease with known environmental trigger – gluten and a strong genetic component. Coeliac-toxic gliadin peptides result by proteolytic digestion of gluten proteins and peptides contain- ing the sequences Pro-Ser-Gln-Gln and Gln-Gln-Gln-Pro and may be involved in the pathogenesis of coeliac disease. We pub- lished in 1988 alpha-gliadin fragments acquired by peptic-tryptic- protease digestion (PTP-aGli). These PTP peptides were analysed by RP-HPLC, MALDI-TOF and BLAST and three important sequences QPFPQPQLP, QLQPFPQPQ and RPQQPYPQPQPQ were detected. In previous papers we demonstrated the opioid

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Abstracts

P8–76 Loss of LMAN1 glycoprotein carrier function in microsatellite unstable colorectal tumors J. Kopitz, N. Roeckel, R. Gromes, G. Patsos and J. Gebert University of Heidelberg, Applied Tumor Biology, Heidelberg, GERMANY

i.e.

activity of PTP-aGli investigated in vitro by the contraction of guinea pig ileum. The tendency to form a beta-turn in alpha-glia- din was estimated using the B-cell determinant prediction pro- gram based on the Chou and Fasman probability of beta-turn formation. By means of solid-phase synthesis several alpha-glia- din peptides were obtained in 1991, and their toxicity was tested using the foetal chick duodenum. Our last studies are focused on deamidated gliadin peptides in CD diagnosis. Tissue transgluta- minase epitopes in ELISA tests are blocked by PTP-aGli frag- ments, and antibodies of coeliac patients cannot bind as we published in 2006. We compared ELISA methods of Inova (iD- GPA, iDGPG) and Euroimmun kits with deamidated gliadin peptides (PLQPEQPFP and PEQLPQFEE) to ELISA with puri- fied alpha-gliadin as antigen. Deamidated gliadin peptides could increase sensitivities, specificities and accuracies of coeliac disease serology screening. Results of this study suggest as optimal screening modality a combination of anti-tissue transglutaminase IgA and Inova iDGPA and iDGPG ELISA antibodies. Gliadin peptide sequences have important role in the etiopathogenesis, diagnosis, and probably in the therapy of coeliac disease as well.

P8–75 mRNA quantitative analysis and study of the L-DOPA Decarboxylase (DDC) gene in colon cancer C. Kontos1, I. Papadopoulos2, E. Fragoulis1 and A. Scorilas1 1Department of Biochemistry and Molecular Biology, University of Athens, Athens, GREECE, 24th Surgery Department, University of Athens, Athens, GREECE

inactivation seems

length variations of short Microsatellite instability (MSI), repetitive DNA sequences, occurs somatically and is tumor cell specific in more than 90% of hereditary non-polyposis colorectal cancers (HNPCC) and in about 15% of sporadic cancers of dif- ferent organs due to loss of cellular DNA mismatch repair func- tion. Thus MSI positive cancers represent a substantial part of the total world wide cancer burden. Several target genes affected by MSI have been shown to contribute to MSI tumorigenesis by interfering with different processes, including cell senescence, cell differentiation, apoptosis, and the escape of immune surveillance. Despite the obvious significance of altered glycoprotein synthesis, transport and secretion in colorectal cancer (CRC), aberrant gly- cosylation and glycosylation pathways have not been investigated in MSI CRC. Using a combined bioinformatics, molecular and biochemical approach we identified MSI-target genes encoding proteins of the glycosylation machinery. Among 28 candidate genes, the LMAN1/ERGIC53 gene that encodes a carrier of spe- cific proteins from the endoplasmic reticulum to the ERGIC showed particularily high frameshift mutation frequencies of a A9 coding repeat in MSI CRC cells (52%), adenomas (45%) and carinomas (40%). Biallelic LMAN1 mutations were identified in MSI CRC cells as well as in primary tumor tissues. Loss of LMAN1 expression was observed in LMAN1-deficient tumors and correlated with biallelic mutation status. Functional analysis of LMAN1-deficient MSI CRC cells revealed severely reduced transport and secretion of the known LMAN1 client protein alpha1-antitrypsin (A1AT). Lectin-FACS analyses indicated changes in cell surface glycosylation upon reconstitution of LMAN1 expression in LMAN1-deficient cell lines. Our results suggest that genetic alterations in LMAN1 might be involved in tumorigenesis. Several lines of evidence support this hypothesis. The high mutation frequency predicts LMAN1 as a MSI target gene whose mutational to be positively selected during carcinogenesis. Biallelic mutational inactivation of LMAN1 in adenomas and carcinomas points to its potential tumor suppressor function. LMAN-deficient cell lines show loss of secretion of A1AT, an inhibitor of angiogenesis and tumor growth. Our current work focuses on the identification of yet unknown client glycoproteins of LMAN1, either secreted or plasma membrane-associated, that might contribute to tumor development.

P8–77 Structural studying of conformational interplay within two-module thrombin binding DNA aptamers A. Kopylov1, A. Golovin2, R. Reshetnikov2, T. Turchaninov3, A. Yuminova3, V. Spiridonova4 and A. Arutyunyan4 1Belozersky Institute, Chemistry, Moscow, RUSSIA, 2Moscow State University, Bioengineering and Bioinformatics, Moscow, RUSSIA, 3Moscow State University, Chemistry, Moscow, RUSSIA, 4Moscow State University, Belozersky Institute, Moscow, RUSSIA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

thrombin-binding DNA aptamer Introduction: L-DOPA decarboxylase (DDC), which catalyses the decarboxylation of L-DOPA to dopamine, was found to be involved in cancer pathobiology. The aim of this study was to determine the mRNA expression of DDC in colon cancer cells and to analyse its prognostic significance in colon cancer patients. Methods: Total RNA was extracted from tissues of 95 colon cancer patients as well as from DLD-1, Caco-2, HT-29, and HCT-116 cell lines. cDNA was prepared by reverse transcription and highly sensitive quantitative real-time PCR (qRT-PCR) was performed for DDC using the SYBR(cid:3) Green chemistry. Calcula- tions were made using the comparative CT (2–CT) method. Results: The detection limit of relative DDC expression in colon cancer cells was found to be 0.04 mRNA copies/103 GAPDH mRNA copies (c/Kc). DDC overexpression was found to be more frequent in patients with well-differentiated tumours (p = 0.011). Cox regression analysis revealed that DDC expression is posi- tively related to progression-free and overall survival (p = 0.021 and p = 0.047, respectively). Kaplan–Meier survival curves also demonstrated that patients with DDC-positive colon tumours have significantly longer PFS (p = 0.009) and OS (p = 0.027). Conclusions: Our results suggest that the DDC gene is involved in cancer progression and may be considered as a new biomarker for prognosis and monitoring of colon cancer. Acknowledgements: The project was supported by a PENED grant #03ED178, co-funded by the European Union – European Social Funds (75%), National Resources (25%) – Ministry of Development – General Secretariat for Research & Technology of Greece, and ELPEN A.E. thought EPAN.M.8.3 – 3rd Com- munity Support Framework. Molecular dynamics simulation (MD) using super-computer clus- ters has been applied to explore structural details of several deriv- atives of (TBA) having G-quadruplex structural module. MD was used to evaluate two different models of conventional 15-mer TBA (15-TBA) that were

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Poster Presentations

P8–79 Effect of diabetes on viability and mobility of murine endothelial progenitor cells J. Kotlinowski1, A. Grochot-Przeczek1, M. Kozakowska1, A. Sierpniowska2, R. Derlacz3, J. Dulak1 and A. Jozkowicz1 1Jagiellonian University Faculty of Biochemistry Biophysics and Biotechnology, Medical Biotechnology, Krako´w, POLAND, 2Jag- iellonian University Faculty of Biochemistry Biophysics and Bio- technology, Biophysics, Krako´w, POLAND, 3Adamed Ltd, R&D Department, Pienko´w, POLAND

temperature

based on the controversial NMR and X-ray data; they are distin- guished by a number of key features, mainly by the chain polar- ity. MD comparison of these models revealed that NMR model has more definite structure. Taking G-quadruplex as one struc- tural module, another double-strained module has been computer added to 15-TBA to create a model of two-modular 31-mer TBA (31-TBA). Variants of 31-TBA models have been simulated, each consists of two modules connected by flexible hinge region (each DNA strand has two nucleotides within the internal loop). It turned out that G-quadruplex module is able to stabilize neigh- boring module structures which has very low melting tempera- ture. Comparative stability of G-quadruplex structures have been studied by circular dichroism spectra. G-quadruplex structure is a target, as well as a part of different kind of potential drugs, therefore this study could be applied for rational drug design especially for the thrombin inhibitors. Microfluidic chip capillary electrophoresis data (Experion) on affinity of aptamers to human thrombin are also presented. Acknowledgement: The work has been supported by grants Rosnauka 02.512.11.2242.1-08; RFBR 08-04-01244a.

P8–78 Fibrinogen Rokycany - molecular defect in fibrinogen Bbeta chain 351 Asn/Lys R. Kotlı´ n1, Z. Reicheltova´ 1, J. Suttnar1, T. Riedel1, Z. Zena´ hlı´ kova´ 2, J. Kvasnicka2, M. Mal3 and J. E. Dyr1 1Department of Biochemistry, Institute of Hematology and Blood Transfusion, Prague 2, CZECH REPUBLIC, 2General University Hospital and Charles University Faculty of Medicine 1, Throm- botic Center, Prague 2, CZECH REPUBLIC, 3Department of Cardiology, Motol University Hospital, Prague 5, CZECH REPUBLIC

Type 2 diabetes mellitus may lead to severe complications caused by micro- and macro-angiopathies. They result from dysfunction of endothelial cells due to chronic hyperglycaemia. It has been shown that endothelial progenitor cells (EPC) may play a vital role in vascular biology, through induction of neovascularization in ischemic tissues or incorporation into injured vessels. Our aim was to check how thiazolidinediones, the insulin-sensitizers, affect functions of EPC isolated from healthy and diabetic mice. Exper- iments were performed in the 10-week old wild type and db/db mice. Some diabetic animals were fed with rosiglitazone (10 mg/ kg for 14 days), which ameliorated hyperglycaemia. EPC were purified from the bone marrow and cultured in vitro for 7– 10 days. Staining of apoptotic cells with Annexin-V demonstrated that EPC isolated from the wild type mice were significantly more resistant to oxidative stress induced by H2O2 than cells iso- lated from db/db counterparts. Noteworthy, EPC obtained from rosiglitazone-treated diabetic individuals showed a tendency for improved viability after exposure to H2O2. On the other hand, the same treatment did not influence the increased sensitivity of EPC from db/db mice to apoptosis induced by supplementation of culture media with high concentration of glucose (25 mM). Additionally, EPCs from the untreated or rosiglitazone-treated db/db mice displayed significantly reduced migratory capabilities, which could be fully restored by rosiglitazone (10 lM) added in vitro. This effect was PPARc-dependent, as it was reversed by supplementation of cells with PPARc antagonist, GW9662. Thus, rosiglitazone may improve viability and migration of EPC iso- lated from diabetic mice.

P8–80 Interaction of flavonoids with butyrylcholinesterase Z. Kovarik1, G. Sinko1, M. Katalinic1 and G. Rusak2 1Institute for Medical Research and Occupational Health, Biochemistry and Organic Analytical Chemistry Unit, Zagreb, CROATIA, 2Faculty of Science, Department of Biology, University of Zagreb, Zagreb, CROATIA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Butyrylcholinesterase (BChE; 3.1.1.8) is a serine hydrolase that belongs to the lipase/protease family with a/b-hydrolase fold. Although its exact physiological function remains unclear, it is known that BChE is responsible for the metabolism of various drugs. It can serve as a co-regulator of cholinergic neurotrans- mission and at high neurotransmitter acetylcholine concentration BChE can efficently hydrolyse it. This is why the inhibition of BChE appears to be of an interest in treating diseases having symptoms of reduced acetylcholine levels, such as the Alzheimer disease. We evaluated the BChE inhibition by eight selected flavonoids: galangin, kaempferol, quercetin, myricetin, fisetin, ap- igenin, luteolin and rutin; belonging to a large family of biologi- cally active polyphenolic compounds found in many plants and plant-derived products that are components of everyday human diet (fruits, vegetables, chocolates, herbs, red wine, tea, beer, etc.). All tested flavonoids reversibly inhibited BChE and the evaluated enzyme-inhibitor dissociation constants (Ki) ranged Introduction: Hereditary hypofibrinogenemia is a disease that can be associated with either hemorrhagic complications or thrombosis. A 28-year-old female presented with a low fibrinogen level and hypofibrinogenemia was suspected. Methods: Routine coagulation tests were performed with STA- R coagulation analyzer. Total fibrinogen level was measured using immunoturbidimetric assay. Fibrin polymerization was measured by addition of either thrombin or reptilase to the patient plasma at 365 nm. The purified genomic DNA was amplified by a PCR using specific primers and dideoxysequencing was performed with CEQ 8000 analyzer. Results: The patient presented with low fibrinogen level as deter- mined by Clauss and immunoturbidimetric method. She did not indicate any haemorrhagic or thrombotic complications. Fibrin polymerization and fibrinolysis were found to be normal. There was no evidence of the presence of the mutant chain in plasma by SDS PAGE and Western blot. DNA analysis revealed hetero- zygous substitution Asn351Lys in the fibrinogen Bb chain. The mutation is situated in the inverse type c turn (Bb 350–352) in the bC domain and the replacement of neutral side chain of asparagine by positive charged side chain of lysine may change the conformation of the bC domain, which may lead to either impaired fibrinogen assembly or secretion. Conclusion: The patient was found to bear a novel heterozygous point mutation Bb Asn351Lys, which may be the direct cause of hypofibrinogenemia. Acknowledgement: This work was supported by a grant of The Grant Agency of the Czech Academy of Sciences nr. KAN200670701 and by grant of IGA MZd nr. NS 9636-3/2008.

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P8–82 Study of the effect of a promoter polymorphism in the heme oxygenase-1 gene on multiple sclerosis L. Kralik1, E. Krasulova2, M. Hrdinka1, M. Tyblova2, E. Havrdova2 and P. Martasek1 1Department of Pediatrics, First Faculty of Medicine, Charles University, Prague 2, CZECH REPUBLIC, 2Department of Neurology, First Faculty of Medicine, Charles University, Prague 2, CZECH REPUBLIC

from 10 lM to 500 lM. The inhibition potency increased in the following order: rutin < luteolin < fisetin £ myricetin £ querce- docking tin < kaempferol £ apigenin < galangin. Molecular revealed a multiple modes of interactions between flavonoids and BChE active site amino acids. It pointed out involvement of flavonoids multiple hydroxide groups in creating hydrogen-bonds with gorge amino acids, and aromatic rings of flavonoids involved in p–p interactions with i.e. Trp82, Phe329 and Tyr332. Also, OH group from b-hydroxy ketone motif in two condensed rings can create an intramolecular hydrogen bond with carbonyl oxygen, meaning that it is less probable this OH group will form H-bonds with the gorge residues. With the lowest observed Ki value of 10 lM, galangin was pointed out as a promising lead in the search for new BChE inhibitors.

P8–81 Kinetic, structural and thermodynamic characterization of HIV protease with insertion in the flap region isolated from an HIV-positive patient M. Kozisek1, K. Grantz Saskova1, P. Rezacova1, J. Brynda1, N. M. van Maarseveen2, M. Nijhuis2 and J. Konvalinka1 1Institute of Organic Chemistry and Biochemistry AS CR v.v.i., Biochemistry, Prague, CZECH REPUBLIC, 2Department of Virology, Eijkman-Winkler Institute, Utrecht, THE NETHER- LANDS

Heme oxygenase-1 (HO-1) is a stress protein that degrades heme to biliverdin, iron, and carbon monoxide (CO). HO-1 has anti- oxidant and anti-inflammatory effects. A (GT)n dinucleotide repeat polymorphism in the HO-1 promoter modulates HO-1 gene expression. Short (<25) GT repeats are associated with HO-1 up-regulation. Multiple sclerosis (MS) is an autoimmune disorder resulting in CNS demyelination and neurodegeneration. HO-1 or exposure to its end product CO counters autoimmune neuroinflammation in experimental mice model of MS, and might have therapeutic potential for the treatment of MS. In this study, we investigated the association of the HO-1 promoter (GT)n repeat polymorphism with MS. We determined the number of GT repeats in the HO-1 promoter in 291 patients with MS. We used data from previous studies as a control group. We com- pared the frequencies of short (<25) repeat (class S) and long (‡25) repeat (class L) alleles. Genotype distributions of S/S, S/L and L/L in patients were 8.9%, 49.5% and 41.6%, which was similar to the distribution in controls with 11.5%, 44.5% and 44.0%. No significant difference between these two groups was found. Thus, the (GT)n dinucleotide repeat polymorphism in the HO-1 promoter is not directly associated with MS. Comparison of the course of MS disease in patient groups homoallelic for either S/S or L/L may add yield differences in the expression of the diseases symptoms between these two groups, suggesting that therapeutic intervention with HO-1 inducers may be possible. Acknowledgement: Supported by Grants: IGA MZ 1A8713-5 and MSMT 0021620806.

P8–83 The extracellular matrix proteins markedly affect viability and proliferation of glomerular podocytes in vitro J. Krtil1, J. Platenik1, W. Brima1 and T. Zima2 1First Faculty of Medicine, Institute of Medical Biochemistry, Charles University in Prague, Prague 2, CZECH REPUBLIC, 2First Faculty of Medicine, Department of Clinical Biochemistry and Laboratory Medicine, Charles University in Prague, Prague 2, CZECH REPUBLIC

HIV protease represents a prime target for rational drug design, and protease inhibitors are powerful antiviral drugs that signifi- cantly decrease patient mortality. However, they exert a powerful selection pressure on the virus, which results in appearance of viral strains less sensitive to the inhibitors. Resistance to virostat- ics is regarded a major obstacle in the treatment of HIV positive patients. In addition to resistant mutations, insertions in the genes encoding reverse transcriptase and protease were described. It is well known that the inserts in RT play an important role in development of drug resistance; their function in PR has been described recently (Kozisek et al., J Virol 2008). We identified an amino acid insertion at position 35 of HIV PR isolated from a patient treated by protease inhibitors for a prolonged period of time, and we set out to characterize the contribution of this inser- tion to viral resistance on the molecular level. Resistant PR vari- ants with or without the insertion at position 35 were cloned in E. coli, purified and enzymologically characterized using a chro- mogenic peptide substrate and a panel of inhibitors. We found that the E35EE insertion does not significantly decrease the cata- lytic activity of the enzyme but brings about an approximately 10-fold increase in the relative inhibition constant for some prote- ase inhibitors. X-ray structures of the resistant PR-inhibitor com- plexes were determined to 1.8 A˚ resolution with very good structural factors. Thermal analysis by differential scanning calo- rimetry (DSC) was applied to determine the thermal stability of proteases with and without insertion. We conclude that amino acid insertions into the PR sequence in the vicinity of the binding cleft have a stabilization effect on the structure comparable to active site mutations (Todd et al., Biochemistry 2000).

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Podocytes form the outer layer of glomerular filtration barrier. Their damage leads to proteinuria and glomerulosclerosis. Podo- cyte cell cultures are typically grown on collagen I, which how- ever is absent from healthy glomerular basement membrane (GBM). We study how different types of extracellular matrix (ECM) proteins affect properties of cultured podocytes. Primary rat podocytes after the first passage were seeded at 8000 cell/cm2 on different surface: collagen I, collagen IV, ECM protein extract, laminin, and bare plastic. The culture status was assessed 8 hours after plating (adhesion), during 5 days of growth with 10% serum (proliferation), and finally after 3 days with 0.5% serum (starvation). We counted number of adherent cells, assessed the cellular metabolic activity (MTT test), proliferation (BrdU incorporation), and cellular differentiation (expression of WT1, synaptopodin, and CD2AP). The initial adhesion of cells

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was similar on all kinds of cultivation surface. Cellular prolifera- tion on culture day 3 decreased in the order: ECM extract, colla- gen IV/laminin, collagen I and bare plastic. The differentiation markers were already expressed during the proliferation phase on all surfaces except the bare plastic. Serum starvation decreased number of cells on all surfaces, but ECM extract or pure laminin apparently supported the cells better than the collagens. The ECM extract (collagen IV – laminin – heparan sulfate) should best mimick the physiological GBM and in this study provided the optimal conditions for growth of cultured podocytes. This issue can be also important in vivo as in many glomerulopathies the podocyte injury is associated with GBM alteration.

and ex vivo to characterize mildronate as a pharmacological inhibitor of CrAT. The biochemical measurements and molecular docking were applied to give insights into the CrAT binding with mildronate as well as to assess the effects after treatment in vivo by a cardioprotective dose of mildronate. Purified CrAT was used for activity assays in vitro and mice heart homogenates were used for activity assays after 20-day mildronate (200 mg/kg i/p) treatment. After long term mildronate administration CrAT activity was statistically significantly increased in mildronate trea- ted mice hearts for 40%. In contrast, in vitro mildronate inhibits CrAT in a competitive manner (Ki 1.6 mM) through binding to the carnitine binding site and the bound conformation of mildro- nate closely resembles that of carnitine, except the orientation of the trimethylammonium group, which in the mildronate molecule is exposed to the solvent. Thus, our results show that even though mildronate acts as a weak competitive inhibitor of CrAT in vitro, the long term mildronate treatment in vivo induces an increase in activity of the enzyme in heart tissues, which might affect cardiac energy metabolism pathways.

P8–84 Determination of telomeres length due to the process of human aging M. Kudela1, J. Sajdok1, F. Pudil1, J. Zidkova1, A. Pilin2, M. Smetakova1, H. Marikova1, M. Jancikova1 and F. Auinger1 1Institute of chemical technology Prague, Biochemistry and Microbiology, Prague, CZECH REPUBLIC, 2Charles University Prague, Forensic medicine and toxicology, Prague, CZECH REPUBLIC

P8–86 A novel field of action for suberoylanilide hydroxamic acid (SAHA) in leukemic cells: regulation of cellular adhesivity to extracellular matrix K. Kuzelova´ , M. Pluskalova´ , D. Grebenova´ and Z. Hrkal Department of Cellular Biochemistry, Institute of Hematology and Blood Transfusion, Prague, CZECH REPUBLIC

and We attempted to describe a relationship between the telomere length and human age in different types of human tissues. We suggested a new method to determine telomere length in micro- well plates – the Twin Probe. This method is based on hybridiza- tion of two different fluorescence probes to DNA immobilized on NucleoLink NH plates. The telomeres length is expressed as a ratio of both fluorescence signals. We also measured telomeres length using the electrophoresis of TRF in agarose gel (reference method). Analogous with the Twin Probe we worked on the elec- trophoresis of TRF in agarose gel. We proved that restriction enzymes HinfI and RsaI are suitable for fragmentation of DNA. We also verified the accuracy of this method by using multiple analysis of one sample. Comparing various DNA samples obtained from individuals at different age, we found out that mean value of telomere length in muscular tissue is age indepen- dent. It confirms hypothesis that telomere length differs in the beginning of individual evolution. The data dispersion, represent- ing replication history of the cell, showed a little increase with age. The age was correlated to difference of telomere length between two tissues, not to absolute telomere length as was done in previous studies. Acknowledgement: Funding by GACR 523/06/0439 MSM 6046137305.

Suberoylanilide hydroxamic acid (SAHA) is the first member of the family of histone deacetylase (HDAC) inhibitors that has been approved in the USA for clinical use. However, its mechanism of action remains largely unclear as its effects are pleiotropic and usu- ally strongly cell type-dependent. We revealed that in addition to the known consequences of SAHA treatment (cell cycle arrest and cell death, usually with apoptotic features), the cell adhesion proper- ties are also affected. An increase in the cell adhesivity to fibronectin occurs already at subtoxic SAHA concentrations together with an increase in the expression level of integrin b1 and paxillin, an impor- tant component of focal adhesion complexes. These changes were observed in six different human hematopoietic cell lines but not in normal lymphocytes. Surprisingly, the increase in the cell adhesivity does not depend on the activity of the protein ROCK that regulates the cytoskeleton structure and adhesion to extracellular matrix in adherent cells. We also found no correlation between the adhesivity to fibronectin on one hand and the extent of actin polymerization or the activity of ROCK target proteins (LIM kinase, cofilin) on the other hand. At higher SAHA doses, the adhesivity of cells to fibro- nectin returns to lower values, probably in relation to the induction of cell death, which involves cell detachment from the extracellular matrix in its early phase. Acknowledgements: This work was supported by grant No 301/09/1026 from the Grant Agency of the Czech Republic and by grant NR/9243-3 from the Grant Agency of the Ministry of Health, Czech Republic.

P8–85 Characterization of mildronate as an inhibitor of carnitine acetyltransferase J. Kuka1, K. Zinovjevs2, E. Skapare1 and E. Liepinsh3 1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Riga Stradins University, Riga, LATVIA, 2Department of Physical Organic Chemistry, Latvian Institute of Organic Synthesis, Riga, LATVIA, 3Latvian Institute of Organic Synthesis, Laboratory of Pharmaceutical Pharmacology, Riga, LATVIA

P8–87 Cyclin: a novel target for anticancer drug discovery? S. Lapenna1, F. Caporuscio2, M. Botta2 and A. Giordano1 1CROM – Oncology Research Centre ‘‘Fiorentino Lo Vuolo’’, Medicinal Chemistry, Mercogliano (AV), ITALY, 2University of Siena, Dipt. Farmaco Chimico Tecnologico, Siena, ITALY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Carnitine acetyltransferase (CrAT; EC 2.3.1.7) catalyzes the reversible transfer of acetyl groups between acetyl-coenzyme A (acetyl-CoA) and L-carnitine, and regulates the cellular pool of CoA and availability of activated acetyl groups for cellular energy metabolism. The inhibitor of carnitine biosynthesis, car- dioprotective compound mildronate (3-(2,2,2-trimethylhydrazini- um)-propionate) is structural analogue of carnitine. In this study, the effects of mildronate on CrAT activity were evaluated in vitro The eukaryote cell cycle is regulated by the activities of different cyclins and cyclin-dependent kinases (Cdks). In cancer cells, these

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P8–89 Antitumor Effects of Bacillus Calmette–Guerin Recombinant Protein PStS-3 on Xenografted Murine Bladder Carcinoma C. F. Lee1, D. S. Yu2, S. Y. Chang3 and J. Y. Jiang1 1National Defense Medical Center, Institute of Preventive Medi- cine, Taipei, TAIWAN, 2Department of Surgery, National Defense Medical Center, Taipei, TAIWAN, 3Department of Surgery, Tri-Service General Hospital, Taipei, TAIWAN

proteins are frequently misregulated. While Cdk targets have been explored in drug discovery, selective inhibition of cyclins could represent an alternative strategy for anticancer therapy. The aim of this work was to develop a multistep computational protocol for the generation of a structure-based pharmacophore model and in silico screening of a large number of chemicals for binding to cyclin A. We took advantage of the solved crystal structures of cyclin A/Cdk2 in complex with several peptides to analyse the underlying peptide-cyclin A interactions and generate a pharmacophore model to be used to perform a virtual screen- ing of a large library of chemicals. Molecules that matched the pharmacophore model were filtered on the bases of predicted sol- ubility and permeability in order to remove ‘non-druglike’ chemi- cals. Subsequently, compounds were scored by their capability of undertaking profitable interactions with cyclin A, and a small subset of molecules was selected for in vitro testing. This work provides a rational approach for the identification of small-mole- cule lead inhibitors of cyclin A, and thereby sets the basis for the investigation of cyclins as prospective targets in anticancer drug discovery.

P8–88 Lyophilisation of human phenylalanine hydroxylase: use of additives to preserve the biological function of a human enzyme P. R. Lino1, A. J. Almeida2, I. T. de Almeida1 and P. Leandro1 1iMed.UL, Metabolism and Genetics Group, Lisbon, PORTUGAL, 2iMed.UL, Nanomedicine and Drug Delivery Systems Group, Lisbon, PORTUGAL

Introduction: Intravesical immunotherapy with live Mycobacte- rium bovis bacillus Calmette–Guerin (BCG) is the treatment of choice for superficial bladder cancers after transurethral bladder tumor resection. Nevertheless, adverse effects are common. Here we investigated the efficacy of recombinant BCG, PStS-3, in inducing cytokines production and suppressing orthotopic blad- der tumor growth in mice. Methods: The efficiency of protein expression was detected. Dif- ferent dosages of PStS-3 were instilled intravesically after intrave- sical implantation of MBT-2 cells. Systemic cytokine responses, tumor growth and cumulative survival rates were monitored. Results: In vitro expression of recombinant PStS-3 was efficient in our system. Treatment with 150 lg and 250 lg of PStS-3 sig- nificantly inhibited orthotopic tumor growth in mice when com- pared with control and 100 lg treatment groups in terms of tumor taking rate, bladder tumor burden, and mortality rates. Meanwhile, marked increased serum INF-c level with limited but significant increase of serum IL-18 was observed in mice treated with 150 lg and 250 lg PStS-3 when compared with the control and 100 lg-treated groups. Conclusion: Highly immunopotent recombinant PStS-3 was pro- duced that elicited immune responses with a high serum INF-c inhibited tumor growth and prolonged the survival of level, tumor bearing mice. Thus, immunotherapy using intravesical recombinant PStS-3 may be an alternative BCG regimen for the treatment of bladder carcinoma.

is still

P8–90 CYP1A1, 1A2, and 1B1 expression in breast cancer MCF7 cells treated with active components of cabbage juices B. Licznerska and W. Baer-Dubowska University of Medical Sciences, Pharmaceutical Biochemistry, Poznan, POLAND

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

supported by project Recent epidemiological migrant studies showed that consumption of raw or short cooked cabbage and sauerkraut is connected with significant reduction of breast cancer incidences. Conventional treatment of breast cancer is expensive and for advanced cancer ultimately unsuccessful. Chemoprevention, the early intervention by usage of agents inhibiting the development of invasive cancer, is a good alternative. Estrogens are important in the proliferation of breast carcinoma of which most is estrogen dependent. Estro- gens are metabolised mainly by 1A1, 1A2 and 1B1 isoforms of CYP450. The aim of the study was to investigate the effects of juices and its active components – cabbage and sauerkraut indole-3-carbinol (I3C), 3,3’-diindolylmethane (DIM) and sulfo- raphane (SUL) on the expression profile of CYP1A1, CYP1A2, and CYP1B1 in human breast cancer estrogen dependent MCF7 cell line. Cells were treated with these compounds at the concen- trations relevant to those observed in human plasma. The screen- ing of cDNA from total RNA extracted from cell cultures after 72 hours of incubation was performed using real-time PCR assay with specific primers for each gene. The increased expression of all analyzed CYPs was found as a result of cabbage juices or The development of recombinant protein pharmaceuticals became a reality with the advances in the area of molecular biol- ogy and biotechnology. However, their use to treat human dis- eases limited because therapeutic proteins are large physicochemically unstable macromolecules that in solution are very susceptible to lose their biological function. Alternative approaches involve their use in the dry state. However, during drying proteins are subjected to harsh conditions through which is activity can be severely reduced. To overcome these problems protective agents are usually required to prevent protein instabil- ity during the drying process. The aim of our work was to inves- tigate the role of several additives (glycerol, mannitol, trehalose, sucrose and glucose) on the preservation of recombinant human phenylalanine hydroxylase (hPAH; EC 1.14.16.1) biological func- tion during the lyophilisation process. This homotetrameric enzyme is of particular interest in human health since its impair- ment results in phenylketonuria (MIM 261600), the most fre- inherited disorder of amino acid metabolism. The quent recombinant hPAH was produced in a prokaryotic expression system and purified by IMAC. The yield, catalytic activity, oligo- merization state and deamidation pattern were followed during time storage. From the tested lyoprotectors trehalose maintained about 85% of enzyme activity while sucrose and glucose, allowed a catalytic activity preservation of about 58% and 68%, respec- tively, whereas, glycerol and mannitol were unable to preserve PAH activity during lyophilisation. The tested sugars maintained the oligomeric profile of recombinant hPAH, without changes in the deamidation pattern. Our data strongly suggest that trehalose is a promising hPAH lyoprotector thus opening new strategies to phenylketonuria treatment using enzyme reposition therapy. Acknowledgement: This work was PTDC/QUI/64023/2006 (FCT).

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indoles (I3C and DIM) treatment. Sauerkraut juice has stronger effect than raw cabbage juice, and DIM stronger than I3C. SUL slightly increase CYP1B1 expression but decrease CYP1A2. Since up-regulation of CYP1A andCYP1A2 causes the reduction of active estrogens, the application of cabbage juices (especially sau- erkraut) and its active components (especially indoles) could be well-founded as preventive agents against breast cancer develop- ment.

study, we observed firstly the protective effect of AF-1 against pulmonary fibrosis. Mice were subjected to intratracheal adminis- tration of bleomycin (5 mg/kg) to establish the fibrotic model and received AF-1 (15 mg/kg) by intratracheal administration 3 days later. The mRNA expression level of precollagen III and precollagen I in lung tissue were quantified by RT-PCR at 14th day and 28th day, respectively. The bleomycin-induced increase of precollagen III and precollagen I mRNA expression were sig- nificantly down-regulated by AF-1. The collagen deposition detected by Masson staining and hydroxyproline content was decreased by AF-1. All together, our results showed that AF-1 could reduce the degree of lung damage and fibrosis. In conclu- sion, AF-1 has a protective effect on bleomycin-induced lung fibrosis and it might prove useful as an add-on therapy for the treatment of fibrotic disorders of lung such as idiopathic pulmo- nary fibrosis, a disease that still represents a major challenge to medical treatment.

P8–91 Antimicrobial evaluation of newly synthesized dibenzoxepins C. Limban1, A. V. Missir1, G. M. Nitulescu1, I. C. Chirita1, C. Delcaru Larion2, A. M. Israil2 and M. C. Chifiriuc2 1Faculty of Pharmacy "Carol Davila" University of Medicine and Pharmacy, Pharmaceutical Chemistry, Bucharest, ROMANIA, 2Institute of Research and Development for Microbiology and Immunology Cantacuzino, Microbiology, Bucharest, ROMANIA

P8–93 Caspase-independent NMDA receptor triggered neuronal cell death could be prevented by mitochondria preservation by glycine N. Lobysheva1, A. Selin2, A. Tonshin1, L. Yaguzhinsky1 and Y. Nartsissov2 1Belozersky Intitute of Physico-Chemical Biology Moscow State University, Bioenergetics Department, Moscow, RUSSIA, 2Insti- tute of Cytochemistry and Molecular Pharmacology, Bioenergetics Department, Moscow, RUSSIA

Objective: The authors designed and synthesized new dibenzoxe- pins compounds in order to be used as an alternative antimicro- bial treatment as a solution to the high level of bacterial antibioresistance. Methods: The original dibenzoxepin compounds were obtained by state of the art methods, solubilized in dimethyl sulfoxide and tested for their antimicrobial activity by qualitative and quantita- tive methods. The qualitative screening was performed by three adapted diffusion methods: impregnation of paper filter disk with the tested substance solutions, disposal of the tested solutions in agar wells and spotting of the tested solutions on solid medium seeded with microbial inoculum. The quantitative assay was per- formed using broth microdilution method in order to establish the minimal inhibitory concentration (MIC). The antimicrobial activity was tested against Gram-negative (Escherichia coli, Pro- teus vulgaris, Morganella morganii, Salmonella arizonae, Pseudo- monas aeruginosa, Klebsiella planticola) and Gram-positive (Staphylococcus aureus) strains. After selecting the compounds with the highest antimicrobial activity, we investigated the sub- stances influence upon the expression of bacteria virulence factors using different culture media. Results: We found that 12 of the 26 tested compounds expressed specific antimicrobial activity, the highest activity being noticed on: E. coli, K. planticola, S. arizonae and Ps. aeruginosa (MICs 62.5 lg/ml) strains. When tested for the expression of the viru- lence factors, by comparison with the control, the strains sub- missed to subinhibitory dosis of different tested antimicrobial substances exhibited only very little differences. Conclusion: The new synthesized compounds with dibenzoxepin nucleus proved to have specific antimicrobial activity and may be regarded as potential antimicrobial agents.

P8–92 Effective anti-fibrosis responses on mice model of lung fibrosis by intratracheally administered antiflammin-1 W. Liu, Z. Luo, C. Li, J. Han, W. Liu, L. Wang, D. Feng and J. Wan Xiangya School of Medicine, Physiology, Changsha, CHINA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Cerebral blood flow reduction during ischemic stroke causes depletion of oxygen and glucose. Either necrosis or apoptosis, occurred by caspase-dependent or -independent ways, were observed in zone of ischemia. We studied whether the level of glucose plays a causal role in determination of neuronal cell death signaling pathway under anoxia and the ability of glycine to prevent neurodegeneration. Using in vitro model of brain cor- tex tissue slices surviving under anoxia, we demonstrated that ele- vation of glucose concentration from 5 mM to 10 mM switches programmed cell death mechanism from caspase-independent to caspase-dependent manner. In absence of glucose in incubation medium of brain slices necrosis was observed. Caspase-indepen- dent apoptosis occurred in the presence of 5 mM glucose in the incubation medium; it was observed by internucleosomal DNA fragmentation after 24 hours. Cell death was evoked by the selec- tive NMDA receptor activation as it was prevented by 10 lM of MK-801, non-competitive agonist of NMDA receptor. On the contrary, apoptosis that occurred in the presence of 10 mM of glucose in the incubation medium of brain slices was accompa- nied by caspase 3 activation but not cytochrome c release. It was mediated apparently via activation of AMPA- and kainate recep- tors. Amino acid glycine (5 mM) and PTP-inhibitor cyclosporine A (10 lM) inhibited apoptosis realized through NMDA receptor activation (5 mM of glucose) while they were ineffective in the case of non-NMDA mediated cell death (10 mM of glucose). Moreover 5 mM of glycine reduced mitochondrial impairment after 1 hour incubation of brain cortex slices under anoxia. Apparently glycine and cyclosporine A could prevent release of pro-apoptotic mitochondrial factors such as AIF, which plays a crucial role in caspase-independent cell death pathway, and hence prevent apoptosis development. On the other hand, cell death development in case of 10 mM of glucose realized through non- mitochondria caspase 3 activation pathway. We conclude that the concentration of glucose is the crucial factor in determination of cell death pathway. Apoptosis induced via NMDA receptor activation develops through the caspase-independent pathway Antiflammin-1 (AF-1,MQMKKVLDS) is a nonapeptide that has a powerful anti-inflammatory effect. It acts as its original full length protein Clara Cell Secretory Protein (CCSP) and may dis- play potent clinical application value. Our previous studies indi- cated that Clara cells on the terminal bronchioles significantly inhibited pulmonary fibrosis induced by bleomycin. In present

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and involves release of pro-apoptotic mitochondrial factors. Gly- cine neuroprotective action in case of NMDA receptor mediated apoptosis is associated with its ability to prevent mitochondrial impairments.

lean breeds. The leptin gene is 4.4-fold overexpressed in Manga- litza in comparison with the Duroc breed (p < 0.001), respec- tively 6.7-fold in the case of the Large White breed (p < 0.001). The difference between the expression levels of the leptin gene in animals from the Duroc and Large White breeds was not signifi- cant (p > 0.05). The overexpresion of the leptin gene in the Mangalitza breed could be explained by a resistance to leptin in the case of this breed; this resistance contributes to the reduced reproduction function in the Mangalitza breed in comparison to the Duroc and Large White breeds.

P8–94 Expression and purification of microbial galactofuranosyltransferases T. Lunackova1, B. Kralova1, R. Daniellou2, V. Ferrieres2 and Z. Purkrtova1 1Institute of Chemical Technology Prague, Department of Bio- chemistry and Microbiology, Prague 6, CZECH REPUBLIC, 2Ecole Nationale Superieure de Chimie de Rennes, CNRS UMR 6226, Rennes Cedex 7, FRANCE

P8–96 Colorimetric method for alkylresorcinols determination in plasma M. Mania, A. Dabrowska, M. Korycinska and A. Kozubek Department of Lipids and Liposomes, University of Wroclaw Faculty of Biotechnology, Wroclaw, POLAND

(mean ± SEM) of

Nowadays, the increasing antibiotic resistance of many important human pathogens has led to expansion in searching for new anti- microbial therapies based on innovative therapeutic strategies and mechanisms. One of promising non-conventional antimicro- bial sites of action is a sugar galactofuranose. While pyranoic configuration of d-galactose is widely distributed in nature, including in mammalian glycoconjugates, furanoic configuration of d-galactose is much less abundant, having only been found in microorganisms, such as bacteria, protozoa and fungi. Moreover, galactofuranose has been shown to be present in numerous struc- tures considered to be essential for virulence in many pathogenic organisms and has been reported as highly antigenic and immu- nodominant. Microorganisms possess special group of enzymes, galactofuranosyltransferases, which enable incorporation of d-ga- lactofuranose on their cell surface or into their cell wall. Compre- hension of this enzyme group opens the door to use of the galactofuranose as antimicrobial target. We focus our attention on galactofuranosyltransferases involved in the biosynthesis of cell surface glycoconjugates of two important human pathogens: Mycobacterium spp. (namely M. tuberculosis and M. smegmatis) and Leishmania spp., especially L. major. The first phase of study was aimed at expression and purification of studied enzyme. The second phase of project revealed preliminary results of enzyme activity specification. Acknowledgement: This research was partly supported by the Ministry of Education, Youth and Sports of the Czech Republic (MSM 6046137305).

Epidemiologic studies support the theory that whole grains may prevent heart diseases, cancers, diabetes and obesity (1). To con- firm the theory there is a need for development of a biomarker for whole grains and grain components. Candidates for such a marker are 5-n-alky(en)lresorcinols – phenolic lipids characteristic for the outer layers of grains (2,3). A colorimetric method with diazonium salt Fast Blue B · Zn salt was established to identify alkylresorcinols in the extracts from human and animal plasma. In this study, two extraction solvents were compared, diethyl (2:1 v/v) with the 2.5 times ether and chloroform/methanol higher extraction rate for the second one. The yield of recovery was 6.4% higher for the samples without incubation, compared to 16 hours incubation at 37(cid:2)C. The mean yield of recovery of the alkylresorcinols added to human plasma varied from 375% to 3100%. After a single intake of wheat bran rich in al- kylresorcinols, the profile of plasma concentration of alkylresorci- nols were similar in the subjects (n = 4), with the first peak after 31 hour and the second peak after 34 or 36 hours and maxi- mum concentrations 0.41 ± 0.09 lg/ml (31080 ± 235 nmol/l ) and 1.24 ± 0.09 (33237 ± 249 nmol/l). Acknowledgement: This work was supported by Ministry of Science and Higher Education Grant No. N312 049 32/2699. References: 1. Liu RH. J Cereal Sci 2007; 46: 207–219. 2. Ross AB, Kamal-Eldin A, A˚ man P. Nutr Rev 2004; 62: 81–95. 3. van Dam RM, Hu FB. Am J Clin Nutr 2008; 87: 797–798.

P8–95 The overexpression of leptin gene in high fat Mangalitza breed M. A. Manea, S. E. Georgescu, S. E. M. Kevorkian, M. Zaulet, M. Costache and A. Dinischiotu University of Bucharest, Biochemistry and Molecular Biology, Bucharest, ROMANIA

P8–97 Increased electrochemical potential at plasma membrane drives fungicidal activity of amiodarone L. Maresova1, R. Rao2 and H. Sychrova1 1Department of Membrane Transport, Institute of Physiology AS CR v.v.i., Prague 4, CZECH REPUBLIC, 2Department of Physiology, Johns Hopkins University School of Medicine, Baltimore MD, USA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Obesity stems from an abnormal accumulation of fat in the white adipose tissue. Research, employing genetic models of obesity, has emphasized the major role of leptin as a controller of obesity. Leptin is thought to be an important regulator of appetite, energy metabolism and reproduction. The swine from the Man- galitza breed represent an ideal animal model for the study of obesity due to their high backfat and reduced reproduction func- tion. Our objective was to assess the porcine leptin mRNA levels in the adipose tissues of the fat pig breed (Mangalitza) versus lean pig breeds (Large White and Duroc) using the Real-Time PCR technique. As a reference gene we used the ribosomal pro- tein L32 gene (RPL32) and glyceraldehyde 3-phosphate dehydro- genase gene (GAPDH). In the case of the Mangalitza breed the results showed a high level of leptin mRNA in contrast with the The budding yeast Saccharomyces cerevisiae has been widely used as a model organism both for studying eukaryotic cell biology and for investigating the effects of chemical compounds and drugs. We tested amiodarone, an ion channel blocker in clinical use as an antiarrhythmic agent, which exhibits broad-range fungi- cidal effects, shown previously to be interconnected with induced influx of Ca2+. To better understand the mechanistic basis of its antifungal activity, we assessed the effect of the drug on mem- brane potential in wild type yeast, and in mutants with known perturbations of membrane potential (tok1D,pma1-105). We show

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Poster Presentations

that low concentrations of amiodarone elicit an immediate, dose- dependent hyperpolarization of the membrane. At higher doses, hyperpolarization is transient and is followed by depolarization, coincident with loss in cell viability. Proton and alkali-metal cat- ion transporters play reciprocal roles in membrane polarization, depending on the availability of glucose. Acknowledgement: This work was supported by the Czech grants MSMT LC531 and KJB500110701.

P8–98 Effects of human POR variation on heme oxygenase I activity C. C. Marohnic1, W. J. Huber III2, J. R. Reed2, P. Marta´ sek3, J. Alam4, W. L. Backes2 and B. S. Masters1 1University of Texas Health Science Center at San Antonio, Bio- chemistry, San Antonio, TX, USA, 2Louisiana State University Health Science Center, Pharmacology, New Orleans, LA, USA, 3Charles University School of Medicine, Pediatrics, Prague, CZECH REPUBLIC, 4Louisiana State University Health Science Center, Biochemistry, New Orleans, LA, USA

cose. For this, we studied the effect of some natural mineral waters and some of their constituents on the apical uptake of 14C-butyrate (14C-BT) and 3H-O-methyl-D-glucose (3H-OMG) by Caco-2 cells. The apical uptake of 14C-BT (20 mM) by Caco-2 cells was significantly increased after an acute (20 minutes) expo- sure to 1% (v/v) distilled water, and, comparatively to distilled water, it was decreased after an acute exposure to Pedras Salga- das(cid:3) 1% (v/v) and Melgac¸ o(cid:3) 5% (v/v), and increased by Vidag- o(cid:3) 5% (v/v). Moreover, it was increased by chronic (48 hours) exposure of the cells to Vidago(cid:3) or Melgac¸ o(cid:3) waters [5% (v/v)]. Moreover, uptake of 14C-BT was reduced after acute (20 min- utes) exposure to MgCl2 (100 and 1000 mg/l Mg2+), MgSO4 (1000 mg/l Mg2+) and CaCl2 (100 and 1000 mg/l Ca2+). The apical uptake of 3H-OMG (10 lM) by Caco-2 cells was signifi- cantly reduced by acute (20 minutes) exposure to Melgac¸ o(cid:3) water [1% (v/v)], when compared with distilled water. Also, chronic (48 hours) exposure to Pedras Salgadas(cid:3) [5% (v/v) increased uptake, and chronic exposure to Melgac¸ o(cid:3) (5% (v/v)] decreased it. Finally, uptake of 3H-OMG was decreased after acute (20 minutes) exposure to MgSO4 (1000 mg/l Ca2+) and NaF (0.1 and 1 mg/l F-). In conclusion, uptake of 14C-BT and3H-OMG by Caco-2 cells is differently modulated by distinct natural mineral waters. Acknowledgement: Supported by FCT (PTDC/SAU-FCF/ 67805/2006).

syndrome (OMIM#201750), Antley–Bixler

P8–100 Modulation of folic acid uptake in BeWo cells by cannabinoids. F. Martel, J. R. Araujo and P. Goncalves Faculty of Medicine of Porto, Dept. of Biochemistry, Porto, POR- TUGAL

Genetic mutations (>50) and polymorphisms (>300) in human POR, encoding NADPH-cytochrome P450 oxidoreductase (CY- POR), have been the subject of intense investigation in recent years. CYPOR inactivating mutations can result in POR defi- (ABS, ciency OMIM#207410), adrenal hyperplasia, and polycystic ovary syn- drome. The effects of POR variation on CYPOR-dependent heme oxygenase I (HO-1), may exacerbate known P450 deficien- cies by impeding degradation of heme, itself a toxic pro-oxidant. Several disease states, including atherosclerosis and cancer have been correlated with loss of the inducible cytoprotective function of HO-1. Recombinantly expressed and purified full-length human CYPOR and HO-1, reconstituted in DLPC vesicles, were assayed for the conversion of heme to bilirubin. The human CY- POR variants examined were: WT, A115V, Y181D, P228L, M263V, A287P, R457H, Y459H, and V492E. Each variant exhibited decreased bilirubin production and lower HO-1 affinity, relative to WT. The activities of Y181D, Y459H, and V492E (all flavin deficient) were partially restored by the addition of FAD and FMN to the reaction. In conclusion, human POR mutations, shown previously to impact P450 activities, also diminished HO- 1 activity, to varying degrees. Consequently, altered HO-1 func- tion may contribute to the phenotypic abnormalities observed with POR deficiency. Differentiating the cumulative effects of POR variation on a multitude of P450s and heme oxygenase will contribute to a better understanding of the pathology of CYPOR deficiencies. Acknowledgements: Supported by US National Institute of Environmental Health Sciences ES004344 (WLB) and National Institute of General Medical Sciences GM081568 (BSSM, The Robert A. Welch Distinguished Professor in Chemistry, AQ- 0012).

The aim of this study was to investigate the influence of cannabi- noids (CB) on the uptake of folic acid (FA) by the choriocarci- noma cell line (BeWo cells). Acutely, anandamide caused a 15% decrease in 3H-FA uptake (pH 7.5). Moreover, tetrahydrocan- nabinol (THC) caused a 30% increase, and AM630 a 15% decrease (pH 6.5). Neither the inhibitory effect of anandamide nor the stimulatory effect of THC were changed by the CB receptor type 1 or type 2 antagonists (AM251 and AM630, respectively). Chronically (48 hours), THC and AM251 decreased by 20% the uptake of 3H-FA, and anandamide and AM630 increased it by 30% (pH 7.5). Moreover, CP 55 940 increased uptake of 3H-FA by 30% (pH 6.5). RT-PCR analysis showed that the mRNA levels of the Reduced Folate Transporter 1 increased by 9% after chronic treatment with AM630, and the mRNA levels of the Proton-coupled Folate Transporter decreased by 17% and increased by 18% after chronic treatment with THC and AM251, respectively. In conclusion, uptake of 3H-FA by BeWo cells is significantly, although not very mark- edly, changed by several distinct CB receptor agonists and antag- onists, either after acute and after chronic exposure of the cells. The acute effects of CB receptor agonists do not seem to be CB receptor-mediated, and with a few exceptions the chronic effects do not seem to be related to changes in the expression levels of FA transporters. Acknowledgement: Supported by FCT (PTDC/SAU-FCF/ 67805/2006).

P8–99 Effect of some natural mineral waters upon butyrate and glucose uptake by Caco-2 cells F. Martel, P. Goncalves and J. R. Araujo Faculty of Medicine of Porto, Dept. of Biochemistry, Porto, PORTUGAL

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

The aim of this study was to investigate the influence of natural mineral waters on the intestinal absorption of butyrate and glu-

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P8–101 Expression and localization of CYP1A2, CYP2D1 and CYP3A1 in rat liver and CYP2D4 in rat brain after neuroleptic treatment K. Wrona-Bogus1, W. Maruszczyk1, A. Bryzek1, D. Plewka1, M. Krzystanek2, P. Czekaj1, A. Wiaderkiewicz1, R. Skowronek1 and R. Wiaderkiewicz1 1Faculty of Medicine in Katowice Medical University of Silesia, Department of Histology, Katowice, POLAND, 2Faculty of Medi- cine in Katowice Medical University of Silesia, Department and Clinic of Psychiatry and Psychotherapy, Katowice, POLAND

obstructive pulmonary disease (COPD), a disease associated with an immune as well as an oxidant/anti-oxidant imbalance. There- fore hsp70-2 A/G (+1267), hsp70-hom T/C (+2437) and TNFa G/A (+489) gene polymorphisms were analysed in patients with COPD by PCR–RFLP procedure. Polymorphisms of the HOX-1 gene regarding to the number of GT repeats in 5’-flanking region were analysed with sequencing of PCR products. The obtained data showed that there were no statistically significant differences in the genotype distributions of hsp70-2 (Chi square test, p = 0.186), hsp70-hom (Chi square test, p = 0.859) and TNFa (Chi square test, p = 0.299) between patients (31) with COPD and control subjects (48). Likewise there was no statistically sig- nificant difference in number of GT repeats in HOX-1 gene between patients with COPD and control subjects (Chi square test, p = 0.907).

P8–103 Antioxidant enzymes activities in rat kidney after cold stress exposure N. Cikcikoglu Yildirim1, M. Yurekli1, F. Matpan2 and N. Yildirim3 1Inonu University, Molecular Biology, Malatya, TURKEY, 2Dicle University, Biology, Diyarbakir, TURKEY, 3Dicle University, Bio- technology, Diyarbakir, TURKEY

grant MNiSW no by Psychotropic drugs are extensively metabolized by cytochrome P450 enzymes and are therefore susceptible to metabolically based drug interactions with other psychotropic agents or with compounds used for the treatment of concomitant somatic ill- nesses. If compared to formerly used antipsychotics, which are potent inhibitors of CYP2D and may therefore impair the elimi- nation of substrates for this isoform, novel antipsychotics are only weak inhibitors of CYP isoenzymes in vitro at therapeutic concentrations. The aim of the study was to compare to what degree haloperidol (classic neuroleptic), clozapine and olanzapine (representing the second generation of antipsychotics released for clinical use) influence the expression and localization of CYP1A2, CYP2D1 and CYP3A1 within the liver and CYP2D4 in the brain of rats. Groups of adult male Wistar rats (200–210 g) were injected intraperitoneally for 4 weeks at doses of: olanzapine 5 mg/kg, clozapine 10 mg/kg, and haloperidol 1 mg/kg b.w., respectively. The expression of studied CYP isoforms was detected by RT-PCR, Western blotting and immunohistochemis- try. It was shown that all analyzed antipsychotics changed expression of studied CYP isoforms in the liver in drug-depen- dent manner. In case of haloperidol expression of investigated CYP was limited to zone 3. In the brain, treatment with antipsy- chotics resulted with increased activity of CYP2D4 in granular cells (haloperidol) or both in granular as well as pyramidal cells of hippocampal formation (olanzapine and clozapine). It was concluded that classic and new antipsychotics in different way change expression pattern and localization of cytochromes P450 in the liver and hippocampal formation. Acknowledgement: Supported 2P05D09130.

Stressful conditions lead to the formation of excessive free radi- cals that are major internal threat to cellular homeostasis of aero- bic organism. The elevation of endogenous corticosterone due to the stress response has been reported to accelerate the generation of free radicals. Free radicals inhibited the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX) enzymes. Mammalian cells have developed antioxidant defense systems to prevent oxidative damage and to allow sur- vival in an aerobic environment. Cells can respond to oxidants with SOD, CAT and GPX. The main goal of the present study was to investigate the effects of cold stress on antioxidant enzyme activities in rat kidney. Twelve male Wistar albino rats (8 months old, 190–240 g) were used. The rats were divided into the follow- ing two experimental groups: control group (n = 6) and cold stress group (n = 6). In cold stress treatment group, animals were exposed +10(cid:2)C cold during a week. The activity of CAT was determined spectrophotometrically. The activities of SOD and GPX were determined by using The Cayman Chemical Assay kit. In cold stress groups CAT activities were significantly decreased in kidney (p < 0.05), however, SOD and GPX activi- ties were increased in kidney (p > 0.05). In this study, it was determined that cold stress can disrupt the balance in an oxidant/ antioxidant system and cause oxidative damage to kidney tissue by altering the enzymatic and non-enzymatic antioxidant status.

P8–102 Hsp70, TNFalpha and hsp32 gene polymorphisms and COPD M. Matokanovic1, A. Malic2, A. Turk1, I. Cepelak1, S. Popovic-Grle3 and K. Barisic1 1Faculty of Pharmacy and Biochemistry, Department of Medical Biochemistry and Haematology, Zagreb, CROATIA, 2Psychiatric Hospital ‘‘Sveti Ivan’’, Zagreb, CROATIA, 3Clinical Hospital for lung disease, Zagreb, CROATIA

P8–104 Chromatographic and electrophoretic study of gastric mucins of different origin E. Miarkova1, H. Muselova1, Z. Kucerova1 and M. Ticha1,2 11st Faculty of Medicine Charles University in Prague, Institute of Pathophysiology and CEH, Prague 2, CZECH REPUBLIC, 2Faculty of Science Charles University in Prague, Department of Biochemistry, Prague 2, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Genes encoding members of the heat shock protein (Hsp) 70 family, hsp70-1, hsp70-2 and hsp-hom, and gene encoding tumor necrosis factor alpha (TNFa) lie in the class III region of the human major histocompatibility complex (MHC). Hsp70-1 and hsp70-2 genes encode the major heat and stress inducible Hsp70. Hsp70-hom encodes a protein that is highly related to Hsp70-1, but is not heat inducible. TNFa encodes pro-inflammatory cyto- kine, which plays an important role in inflammatory processes. Hsp32 gene (HOX-1) encodes inducible heme oxygenase-1, enzyme that catalyzes the degradation of heme, providing cellular protection against oxidative mediated injury. Polymorphisms of stress (hsp70 family and hsp32) and tumor necrosis factor alpha (TNFa) genes have been reported to be related to chronic Gastric mucins are high molecular weight extracellular proteins that are present in the mucosal secretions covering epithelial cell surface. They are highly glycosylated consisting of 75–85% sac- charides (O-glycosidically linked) and remaining 20–25% peptide chains. Investigation of gastric mucins is difficult not only due to their high molecular weight and a high degree of glycosylation,

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P8–106 Mechanism of action of three novel dicationic bis(phenylamidine) derivatives K. Miskovic1, I. Stolic2, I. Piantanida3, M. Baus Loncar1, M. Bajic2 and L. Glavas-Obrovac4 1Faculty of Medicine University of J.J. Strossmayer, Department of Medicinal biology, Osijek, CROATIA, 2Faculty of Veterinary Medicine University of Zagreb, Department of Chemistry and Bio- chemistry, Zagreb, CROATIA, 3Department of Organic Chemistry and Biochemistry, Institute Ruder Boskovic, Zagreb, CROATIA, 4Faculty of Medicine University Hospital Osijek, Department of Clinical Chemistry and Biochemistry and Department of Nuclear Medicine and Radiation Protection, Osijek, CROATIA

(1), dihydrochloride

but also due to their polydispersity and conformational changes induced by different factors. The physical state of gastric mucin plays an important role in gastric diseases. Structural studies on gastric mucins were mostly performed using separated subunits of glycoproteins or glycoproteins isolated under degrading condi- tions. In the present study we have used different native gastric mucins (dog, rat), including human samples from patients with gastric diseases, without any degradation. Samples of mucins were obtained from supernatant after homogenization of dis- sected mucosa in phosphate buffer. The samples were compared using size exclusion chromatography and PAGE in the presence of SDS. A distribution of peptide and saccharide components of gastric mucins depending on their size was investigated. Size exclusion chromatographic profile as well as electrophoretic pat- tern of studied mucins depended on their origin. In the case of human gastric mucins, differences in these parameters were observed in patients with ulcer diseases and gastric cancer. Acknowledgements: This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (grant MSM 0021620806 and project CEH LC 06044) and by the Czech Science Foundation (grant 203/09/0857).

P8–105 Prostate cancer stem cells of LNCaP and DU-145 cell lines as targets for combined anticancer therapy J. Miloszewska1, M. Przybyszewska1, H. Trembacz1, M. Gos1 and A. Kutner2 1Cancer Center Institute of Oncology, Cell Biology Department, Warsaw, POLAND, 2Pharmaceutical Research Institute, Chemical Minisynthesis Department, Warsaw, POLAND

Three novel DNA minor groove binders: 2,5-bis(4-amidinophe- nyl)-3,4-ethylenedioxy-thiophene 2,5-bis [4-(N-isopropylamidino)phenyl)]-3,4-ethylene-dioxythiophene dihy- drochloride (2) and 2,5-bis[4-(2-imidazolino)phenyl]-3,4-ethylene- dioxythiophene dihydrochloride (3) have been synthesized and evaluated for antiproliferative activity. According to the UV/VIS titrations compounds 1–3 show high affinity (Ks > 105 M-1) toward ds-DNA, and accordingly cause strong thermal stabilisa- tion of DNA (nTm > 12(cid:2)C). For all studied compounds circular dichroism (CD) titrations strongly support DNA minor groove binding as a dominant interaction. Furthermore, compounds 1–3 and DNA- minor groove binder Hoechst 33258, at concentration of 10-4 to 10-7 M, were tested against panel of seven solid tumour cell lines (MCF-7, NCI-H358, CaCo-2, HEp-2, HeLa, AGS, and MiaPaCa). Compound 3 exhibits the best antiproliferative effi- ciency at all tested doses compared to control. By monitoring internalization dynamics we notice that compound 3 penetrates into the live HeLa cell and localises to the nucleus after 90 min- utes of incubation. Cell cycle analysis showed that compound 3 at equitoxic concentration (5 · 10-5 M) arrested HeLa cells in G1 and G2/M phases at all three tested times (24, 48, 72 hours) at statistically significant level (p < 0.05), at variance to the effect observed for Hoechst 33258, which stopped cell cycle in S phase under the same conditions.

P8–107 Selection of DNA aptamers specifically interacting with fibrillar form of the yeast sup35 protein E. Surina1, E. Morozkina1, A. Marchenko11, A. Antipin2, O. Mitkevich3, V. Kushnirov3, M. Ter-Avanesyan3 and S. Benevolensky1 1Bach Institute of Biochemistry, Biochemistry, Moscow, RUSSIA, 2GosNIIgenetika State Federal Unitary Enterprise, Biochemistry, Moscow, RUSSIA, 3Russian Cardiology Research and Production Center Moscow Russia, Biochemistry, Moscow, RUSSIA

supplementary treatment with vitamin D3 or

Numerous studies suggested that tumours comprise a population of cancer stem cells with a capacity to maintain tumour growth. These cells are resistant to standard chemotherapeutic drugs. As a result, conventional chemical treatments often fail because qui- escent tumour stem cells survive and contribute to the repopula- tion of the tumour. By treating a cancer cell line by doxorubicin or other standard chemotherapeutics at the lethal dose (LD) 75– 90, cancer stem cell-enriched population can be obtained. We examined the CD133+ population of the LNCaP and DU-145 human prostate cancer cell lines to study substances that can induce differentiation or apoptosis of cancer stem cells. The LNCaP and DU-145 cell lines were treated in vitro with doxoru- bicin at LD 90. After doxorubicin treatment, both cell lines pre- sented the elevated level of CD133 expression, as assessed by the real-time PCR. The fraction of doxorubicin-resistant cells was treated with an active form of vitamin D3, the differantiation and antiproliferative agent (a ligand for the VDR receptor expressed by the LNCaP and DU-145 cell lines) or with the specific tyro- sine kinase inhibitor. Vitamin D3 had a strong effect on plating efficiency of the LNCaP and DU-145 cells, and on their survival assessed in a cytotoxicity test (MTT). Also the tyrosine kinase inhibitor demonstrated a noticeable effect on LNCaP cell growth assessed by colony formation and cytotoxicity assays. The com- lines showed bined treatment of human prostate cancer cell that tyrosine kinase inhibitor reduced the growth potential of the prostate can- cer cells more efficiently than the chemotherapeutic treatment alone. Our results suggest that adjuvant treatment of patients with prostate cancer might diminish the number of chemo- and radiotherapy-resistant cells and potentially improve patients’ outcomes.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Prions are a unique class of infectious agents whose infectivity is related solely to protein. The PrPC refers to the endogenous prion protein, while PrPSc refers to the misfolded form of PrPC. The PrPSchas been implicated in a number of diseases, including bovine spongiform encephalopathy in cattle and Creutzfeldt–Ja- kob disease in humans. The infectious form PrPSc can catalyze conversion of the normal cellular form PrPC into PrPSc. A related phenomenon was described in Saccharomyces cerevisiae. Yeast prions and Sup35 prion protein, in particular, represent an effi- cient model system for development of approaches for diagnos- tics and therapy of prion diseases. SELEX (Systematic Evolution of Ligands by Exponential enrichment) is the procedure of selec- tion from an enormous mixture of short DNA or RNA oligonu- cleotides with randomized sequence those that can specifically

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P8–109 Antimicrobial evaluation of a novel class of synthetic derivatives in order to find potential new therapeutic agents L. Morusciag1, A. V. Missir1, G. M. Nitulescu1, I. C. Chirita1, D. C. Nuta1, C. E. Stecoza1, L. M. Ditu2 and G. Mihaescu2 1Faculty of Pharmacy ‘‘Carol Davila’’ University of Medicine and Pharmacy, Pharmaceutical Chemistry Department, Bucharest, ROMANIA, 2University of Bucharest Faculty of Biology, Micro- gen Centre, Bucharest, ROMANIA

and with high affinity bind to the ligand of interest. Single- stranded DNA aptamers interacting with fibrils formed by the Sup35 protein of Saccharomyces cerevisiae were obtained by the SELEX procedure. Specificity of interaction with Sup35 is inves- tigated for 10 from the total of 40 selected aptamers. It is shown that nine aptamers bind to the fibrillar Sup35 protein and not to its monomeric form. The rate of dissociation constant of apt- amer-fiber complex varies from 0.1 to 1 mkM for different apta- mers. Selected aptamers can be used to study prionization of Sup35, and become a base for the development of diagnostic kits and therapeutic means against prion diseases. Acknowledgement: This work was supported by the ISTC 2750, CRDF2859 and RFBR 08-04-0062; 07-04-91105.

P8–108 Antimicrobial activity screening of new 9- fluorenone derivatives towards some pathogen bacteria strains implicated in skin and mucosal lesions L. Morusciag1, L. M. Ditu2, G. M. Nitulescu1, S. Maracineanu2, C. Bleotu3 and G. Mihaescu2 1Faculty of Pharmacy "Carol Davila" University of Medicine and Pharmacy, Pharmaceutical Chemistry Department, Bucharest, ROMANIA, 2University of Bucharest Faculty of Biology, Micro- gen Centre, Bucharest, ROMANIA, 3Virusology Institute St. Nico- lau, Bucharest, ROMANIA

Objectives: In a time of emerging bacterial resistance there is need for novel original antibacterial agents. For the purpose of developing alternative antimicrobial substances we synthesized a new cluster of compounds, thioureides of the 2-phenethylbenzoic acid. Methods: The new compounds were synthesized by state of the art chemical methods and analyzed by 1H-NMR, 13C-NMR, IR spectra and elemental analysis to certify their structures and pur- ity. QSAR studies were performed to find the relationship between the chemical structure and the activity. The antimicro- bial activity assay was performed using the binary microdilution method in liquid medium (Mueller Hinton broth for bacteria and YPG broth for fungus) placed in 96-well plates, in order to establish the minimum inhibitory concentration. The expression of soluble virulence factors was determined and the cellular sub- strate adherence capacity was tested on HeLa cells. In order to the compounds cytotoxicity we used HeLa cells culture test (ECACC #93021013), adherent cells were maintained in DMEM (Dulbecco’s Modified Essential Medium -Sigma) culture medium supplemented with 10% fetal calf serum – Sigma and incubated at 37oC in humid atmosphere with 5% CO2. Results: The new synthesized compounds showed variable levels of antimicrobial activity depending on the taxonomic strains characteristics and the phenotypic resistance. The analyzed com- pounds present both antibacterial and antifungal activity. Some of these thioureides influenced the adhesines activity and the adherence capacity to the inert and cellular substrate. The cyto- toxicity assay results for the tested substances show reduced lev- els of cytotoxicity. Conclusion: New compounds were developed and may contri- bute to new alternative antimicrobial agents.

P8–110 Lipid peroxidation as a source of carbon monoxide in obstructive cholestasis L. Muchova1, M. Lenicek1, K. Vanova1, J. Zelenka1, M. Juklova1, M. Vejrazka2, R. J. Wong3, H. J. Vreman3 and L. Vitek1 1Charles University, Institute of Clinical Chemistry and Laboratory Diagnostics, Prague, CZECH REPUBLIC, 2Charles University, Institute of Medical Biochemistry, Prague, CZECH REPUBLIC, 3Stanford University School of Medicine, Department of Pediat- rics, Stanford, CA, USA

Aim: Screening of the antimicrobial activity of new original com- pounds towards some pathogen bacteria strains implicated in skin and mucosal lesions to develop new treatment strategies against multiresistant strains. Methods: New 9-fluorenone derivatives were prepared as poten- tial new antiseptics and were tested on eight bacterial strains iso- lated from skin and mucosal lesions: Bacillus sp., Acinetobacter baumanii, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae and three Staphylococcus aureus strains. The antimi- crobial activity assay was performed using two adapted disc dif- fusion method: spot method and paper filter disk impregnated method (using tested solutions at 10 mg/ml concentration, in DMSO – Dimethyl Sulfoxide). In order to establish the MIC (minimum inhibitory concentration) value for all tested com- pounds we used the binary microdilution method in liquid med- ium (Mueller Hinton broth) placed in 96-well plates, started with 1 mg/ml concentration. The expression of soluble virulence fac- tors by the bacterial pathogen strains, after cocultivation in the presence of the tested compounds at the MIC value, was deter- mined using different specific media. The cellular substrate adher- ence capacity was tested on HeLa cells in the tested compounds presence at the MIC value. Results: Two of the analyzed compounds presented antibacterial activity towards all the tested bacterial strains at 10 mg/ml con- centration and the MIC value were between 1 mg/ml and 0.5 mg/ml. The interference with the expression of the virulence factor was not evident. The tested compounds can be used as a good antiseptic substance with skin application or as disinfec- tants for inert surfaces.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Increased heme oxygenase (HO) has been shown to protect liver from oxidative, toxic and inflammatory insults through the eleva- tion of its bioactive products, carbon monoxide (CO) and biliru- bin. Serum bilirubin and bile acids are also elevated in obstructive cholestasis. Lipid peroxidation is an alternative source of CO. Our objective was to assess the roles of HO, biliru- bin, CO, and bile acids on oxidative stress in an animal model of obstructive cholestasis. Adult female Wistar rats underwent bile duct ligation (BDL, n = 7) or sham operation (n = 6). After 5 days, animals were sacrificed. Serum cholestatic markers and carboxyhemoglobin (COHb) were measured. Lipid peroxidation,

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Poster Presentations

P8–112 In vivo effect of enrofloxacin (EF) on paraoxonase, catalase, peroxidase and carbonic anhydrase activities on brown trout (Salmo trutta fario) H. Nadaroglu1, F. Koc2, N. Demir3 and Y. Demir4 1University of Ataturk, Oltu Vocational Trainig School, Erzurum, TURKEY, 2University of Ataturk, Department of Pharmacology and Toxicology Faculty of Veterinary Medicine, Erzurum, TUR- KEY, 3University of Ataturk, College of Arts and Sciences Chem- istry Department, Erzurum, TURKEY, 4University of Ataturk, College of Education Chemistry Department, Erzurum, TURKEY

hydroxyalkenals, CO, and HO activity levels were measured in liver sonicates. Peroxyl radical scavenging capacities in sera and liver homogenates were determined. Compared to sham-operated controls, liver HO activity in BDL rats significantly decreased 39% (p < 0.001). Furthermore, lipid peroxidation and hydrox- yalkenal production increased in livers of BDL animals (179 ± 37% and 123 ± 12%, p < 0.01, respectively) as well as liver CO and COHb levels (196 ± 59% and 203 ± 59%, respec- tively). Serum unconjugated bilirubin increased from 0.28 to 8.87 lmol/l in BDL, p < 0.001. Addition of taurocholic acid 50 lM and more to liver homogenates significantly increased lipid peroxidation (p < 0.01). Serum peroxyl radical scavenging capacity was significantly higher in BDL rats (210 ± 12%, p < 0.0001) compared to controls. Lipid peroxidation triggered by high concentrations of bile acids is the main source of CO in cholestasis. Elevated serum bilirubin and CO levels mediate anti- oxidant protection in response to acute cholestatic injury despite decreased HO activity. Acknowledgement: Supported by grant IGAMZ Nr.9366-3.

P8–111 ERK inhibitor UO126 decreases p21 expression without affecting p53 status and apoptosis induction D. Muthna1, M. Rezacova1, J. Cmielova1, R. Havelek1 and J. Vavrova2 1Faculty of Medicine in Hradec Kra´love´, Department of Medical Biochemistry, Hradec Kra´love´, CZECH REPUBLIC, 2Faculty of Military Health Sciences Hradec Kralove, Department of Radiobi- ology, Hradec Kra´love´, CZECH REPUBLIC

Quinolones are a family of synthetic carboxylic acid derivatives that possess broad spectrum antibacterial activity. Enrofloxacin (EF) is a second generation fluorinated quinolone that has wide- spread applications in antibacterial therapy of mammalian and non-mammalian species. Its mechanism of action is not thor- oughly understood, but it is believed to act by inhibiting bacterial DNA-gyrase (a type-II topoisomerase), thereby preventing DNA supercoiling and DNA synthesis (1,2). Brown trout (Salmo trutta fario) is an important fish species in Turkey. Brown trout (Salmo trutta fario) is not only a dominant fish species in the streams of eastern Turkey but also a potential commercial species for Tur- key. This study was carried out in order to determine the in vivo effect of enrofloxacin on the paraoxonase (PON), catalase (CAT), peroxidase (POX) and carbonic anhydrase (CA) activities on brown trout (Salmo trutta fario). For these studies, groups of six brown trout’s (163 ± 21 g) were selected and in each one intravenous enrofloxacin (10 mg/kg and 40 hours) application was carried out. A group of six brown trouts (Salmo trutta fario) was included in the study for a control group, which was not subject to drug application (3–5). For enrofloxacin, a mean of the serum, heart, muscle and liver paraxonase activity, and erythrocytes, heart, muscle and liver catalase, and carbonic an- hydrase activities were determined and compared to the control groups. PON, CAT, POX and CA have a vital function in many kinds of tissues and play an important role in metabolism. In our experience results, the effects of enrofloxacin on PON, CAT, POX, and CA could be dramatic and systemic. References: 1. Brown SA. J Vet Pharmacol Ther 1996; 19: 1–14. 2. Papich MG, Riviere JE. Fluoroquinolone antimicrobial drugs. In: Veterinary Pharmacology and Therapeutics Chapter 45, 8th Edn. Adams HR, (ed), Ames, IA: Iowa State University Press, 2001; 898–917. 3. Demir Y, Nadaroðlu H, Demir N. Pharm Biol 2006; 44(5): 396–399. 4. Demir Y. Nadaroðlu H. Demir N. Biol Pharm Bull 2004; 27: 1730–1734. 5. Demir Y. Nadaroðlu H. Demir N. Pharm Biol 2008; 46: 393– 399. Aim: Our work consists in evaluating changes in protein expres- sion and apoptosis induction in MOLT-4 leukemia cells after treatment with UO126 (an inhibitor of ERK) and consequent irradiation with the dose of 1 resp. 3 Gy. Methods: Apoptosis has been measured by flow cytometry. Cell viability has been determined using Trypan blue exclusion tech- nique. Western blotting has been used for determination of changes in protein expression. Results: Treatment of MOLT-4 cells with UO126 has no signifi- cant effect on cell viability, percentage of apoptotic cells equals in case of cells only irradiated and both irradiated and UO126 treated. Level of protein p53 stays unchanged during the whole interval of treatment. Phosphorylation status at serine 15 and 392 do not vary either in only irradiated or UO126 treated and irradiated cells. On the contrary p21 induction seems to be ERK dependent, once the ERK phosphorylation is blocked, p21 induc- tion is weaker. Conclusion: UO126, an inhibitor of MAPK kinase ERK, does not increase radiation sensitivity of MOLT-4 cells either does not affect p53 status, but causes weaker p21 induction. Acknowledgement: This work was supported by The Ministry of Education of Czech Republic, project MSM 0021620820.

P8–113 Antimicrobial properties of photosensitizers methylene blue and neutral red encapsulated in liposomes F. Nakonechny1, M. Nisnevitch1 and Y. Nitzan2 1Ariel University Center of Samaria, Chemical Engineering and Biotechnology, Ariel, ISRAEL, 2Bar-Ilan University, Faculty of Life Sciences, Ramat-Gan, ISRAEL

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Background: It is critically important to avoid negative side- effects of medication on healthy human organs and tissues and to mitigate the side-effects of medical treatment. The develop-

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P8–115 Rapide PCR-RFLP screening for a novel BRCA2 mutation found in a north-eastern Romanian population L. Negura1, E. Carasevici1, A. Negura2, N. Uhrhammer3 and Y. J. Bignon3 1Gr.T.Popa University of Medicine and Pharmacy, Immunology, Iasi, ROMANIA, 2University ‘‘Alexandru Ioan Cuza’’, Biochemistry, Iasi, ROMANIA, 3‘‘Jean Perrin’’ Molecular Oncology Center, Molecular Genetics, Clermont-Ferrand, FRANCE

ment of bacterial resistance to antibiotics is also a serious prob- lem, caused by excessive and improper use of such drugs. The ideal solution to this problem is to target drugs via deployment of appropriate agents. Amongst such agents liposomes demon- strate great capacity to deliver drugs to organs or tissues and afford better control of the rate of drug release over time. In the present study the antibacterial efficacy of liposomal forms of photosensitizers (PS) methylene blue (MB) and neutral red (NR) was tested for the first time. The cytotoxic effect of PS is well known and is based on their ability to absorb the energy of light in creating a chemical reaction that produces reactive oxygen and free radicals, which destroy cells. Methods: In this study we tested the antimicrobial efficacy of MB and NR in free and liposome-included forms against repre- sentative species of Gram-positive and Gram-negative bacteria. In addition, we selected the conditions of liposome preparation and effective usage of encapsulated PS, including liposome com- position, lipid concentration, sonication time, PS location in lipo- some, illumination time and incubation temperature. Results: We found that both PS were able to mediate killing of the Gram-positive bacteria S. aureus and Sarcina lutea. In the case of the Gram-negative species, MB was cytotoxic against the Shigella flexneri, NR inactivated E. coli and Salmonella para B. In each case MB and NR encapsulated in liposomes gave a much stronger antimicrobial effect compared to their free forms. Conclusions: We conclude that encapsulation of PS into lipo- somes increases their efficacy against gram-positive and gram- negative bacteria.

P8–114 Alternative splicing of the human FOLH1 gene near its 5’ termini N. Vaclav1, V. Ales2, S. Pavel1 and K. Jan1 1Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences, Biochemistry, Prague 6, CZECH REPUBLIC, 2Charles University 2nd Faculty of Medicine, Pediatric Hematol- ogy and Oncology, Prague 5, CZECH REPUBLIC

Introduction: As very little information is available in Romania on hereditary breast and ovarian cancer, we started the first oncogenetic study, searching for mutations in the cancer predis- position genes BRCA1 and BRCA2 in hereditary breast and ovarian cancer (HBOC) families. Patients and methods: We have identified and recruited 15 high-risk families with at least three cases of epithelial breast or ovarian cancer within the same family line. All patients agreed by written informed consent. DNA was extracted from peripheral blood. The entire coding sequence of both genes was analysed using amplification and Sanger sequencing. RFLP-PCR was used to screen for specific mutations. Results: We identified a novel BRCA2 mutation in exon 21, c.8680C>T. The T for C substitution creates a stop codon at position 2894, truncating the last C-terminal 525 amino acids of BRCA2 protein. This domain is responsible for molecular inter- actions with Rad51, and is involved in nuclear localization of BRCA2. Its deletion can be considered responsible for functional loss of the BRCA2 tumour suppressor in cancer cases. We imag- ined a simple and rapid screening for c.8680C>T identification. This substitution creates an additional HpyCH4III restriction site, allowing a quick identification of the mutation by RFLP- PCR and agarose gel electrophoresis. Conclusions: This outcome, the first one in Romania, could open the way for a population study to determine the frequency of c.8680C>T in the Romanian population, and for an eventual founder effect. This could also develop in Romania the oncoge- netic approach and follow-up of BRCA mutations bearers.

the GCPII splice variants represent

P8–116 Antimicrobial peptides from larvae of the flesh fly Neobellieria bullata T. Neubauerova´ 1,3, M. Mackova´ 1,3, T. Macek1,3, M. Sanda1,2 and Z. Voburka3 1Institute of Chemical Technology, Biochemistry and Microbiology, Prague, CZECH REPUBLIC, 2Academy of Science, Mass Spec- trometry, Prague, CZECH REPUBLIC, 3Academy of Science, Joint laboratories of ICT Prague and Department of Organic chemistry and biochemistry, Prague, CZECH REPUBLIC

in lymph node prostate carcinoma cell

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

FOLH1 gene encodes glutamate carboxypeptidase II (GCPII), a type II integral membrane protein. GCPII is predominantly expressed in prostate and upregulated during the prostate cancer (hence its second name prostate specific membrane antigen, PSMA). Besides the wild type, several alternative splice variants near the 5’ end of the FOLH1 mRNA have been described. Vari- ants called PSM‘, PSM-C, PSM-D and PSM-F differ from each other by truncation or insertion of exons near the 5’end of the FOLH1 mRNA. Since the PSMA to PSM‘ ratio increases during the progression of prostate cancer, the analysis and quantification of important diagnostic approach to prostate cancer, and possibly also to other malig- nancies. In this study we examined the presence of alternative line splice variants (LNCaP). Total RNA isolation from culture grown cells was employed, followed by traditional RT PCR (using specific primer pairs covering the first three constitutive exons) and cloning of the amplified products with subsequent sequencing of the clones. Except PSM-C and PSM-D we cloned all previously described and several novel GCPII splice variants resulting from different combinations of already known and of a few novel exons. We obtained also presumably PSM-C and PSM-D clones, though differing in few nucleotides from reported sequences. Interestingly some of the variants might code for alternative proteins differing from the wild type in insertions between the transmembrane and extracellular domain whereas others for protein with intracellular and transmembrane parts substituted with a predicted signal pep- tide. In the last years the resistance of microbial pathogens had increased. There will be fewer possibilities to cure multiresistance infections by existing antibiotics in the future. This can become a great problem for public health. One of the potential solutions seems to be short cationic peptides synthesized as a part of innate immunity. In our project larvae of the flesh fly Neobellieria bulla- ta were chosen as a source of these peptides. The haemolymph of larvae was collected from non-induced and induced larvae by pinprick to the larvae body. It was gradually centrifuged and pre- cipitated by acidified methanol. Subsequently these fractions were separated by chromatographic methods (SPE column, RP-HPLC) to gain peptide fractions. These fractions were identified by chro- matographic methods (PAGE) and characterized by MALDI and N-terminal sequencing. Antimicrobial activity of fractions was

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Poster Presentations

inhibited by 24 mM of free MB for both types of excitation, and by 12 mM when enclosed into liposomes and activated by light or by encapsulated luminol. Analogous results were obtained with TB as the PS. As bacterial resistance to PS molecules has not been demonstrated, the use of CPAT may prove to be a novel and more effective form of antimicrobial therapy, particu- larly for internal infections not currently accessible to traditional PACT. screened by diffusion method. The tested model organisms were bacteria (Escherichia coli, Pseudomonas aeruginosa, Staphylococ- cus aureus, Bacillus megatherium and Listeria innocua) and fungi (Candida scotii, Aspergillus ochraceus and Mucor species). Some fractions isolated from both non-induced and induced larvae were active against B. megatherium and C. scotii. Tricine electro- phoresis showed a presence of small peptides in active fractions. Acknowledgement: Thanks to the grants GACˇ R 522/09/1693, MSM 6046137305 and Z 40550506.

P8–119 Testosterone-induced cellular proliferation and telomerase up-regulation in ovarian cancer M. Nourbakhsh1, M. Zahraei1, A. Golestani1, M. H. Modarresi2 and F. Karami-Tehrani3 1Tehran University of Medical Sciences, Biochemistry, Tehran, IRAN, 2Tehran University of Medical Sciences, Genetics, Tehran, IRAN, 3Tarbiat Modarres University, Biochemistry, Tehran, IRAN

P8–117 Salivary cytokines profiling by use of xMAP technology in oral squamous cell carcinoma M. I. Nicolescu1, L. Albulescu2, C. Tanase2, O. Dinca3, A. Bucur3 and C. Ursaciuc1 1‘‘Victor Babes’’ National Institute of Pathology, Immunology, Bucharest, ROMANIA, 2‘‘Victor Babes’’ National Institute of Pathology, Biochemistry, Bucharest, ROMANIA, 3‘‘Carol Davila’’ University of Medicine and Pharmacy, Oral and Maxillofacial Surgery, Bucharest, ROMANIA

The levels of a set of salivary cytokines were determined in patients with oral squamous cell carcinoma (OSCC) in order to establish a potential salivary biomarker. IL6, IL8, IL10 and TNFa were analyzed by xMAP technology, using whole unstimu- lated saliva samples from 10 OSCC patients and 10 healthy per- sons. The results showed a higher levels of IL6, IL8 and TNFa in OSCC patients than in healthy control group. IL10 variances showed no statistical significance. Smoking has not shown any influence on assayed salivary cytokines levels.

P8–118 Photodynamic antimicrobial chemotherapy with photosensitizers in liposomes under external and chemiluminescent excitation M. Nisnevitch1, F. Nakonechny1, M. Firer1 and Y. Nitzan2 1Ariel University Center of Samaria, Chemical Engineering and Biotechnology, Ariel, ISRAEL, 2Bar-Ilan University, The Mina and Everard Faculty of Life Sciences, Ramat-Gan, ISRAEL

Ovarian cancer is one of the most common and lethal types of cancer in women. Although its etiology is not fully understood, evidence bearing on the pathophysiology of ovarian cancer appears to support a role for androgens. In most cancers, includ- ing epithelial ovarian cancer, telomerase is upregulated which results in an immortal phenotype. The aim of this study was to investigate the effect of testosterone on proliferation of epithelial ovarian cancer cells, expression of telomerase catalytic subunit (hTERT) and the effect of PI3K/AKT inhibitors on this process. OVCAR-3 cell line were cultured and treated with different con- centrations of testosterone. MTT assay was used for evaluation of cellular proliferation in response to testosterone. hTERT gene expression was measured by real-time PCR carried out on synthesized from total RNA extracted cDNA, which was from the cells. The results showed that 48 hours treatment with 1 lM concentration of testosterone stimulated proliferation of OVCAR-3 cells and elevated the expression of hTERT signifi- cantly. On the other hand, PI3K/AKT inhibitor suppressed testosterone-induced up-regulation of hTERT expression. In con- clusion our results support the role of testosterone in ovarian cancer progression and the possible involvement of PI3K/AKT pathway.

P8–120 Role of vitamin C on mitotic activity of lymphocytes in thymus of streptozotocin- induced diabetic rats D. Okan, N. Ozsoy and S. Cebesoy Faculty of Science, Ankara University, Biology, Ankara, TUR- KEY

(n = 9), diabetes

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

The increasing resistance of bacteria to antibiotics is already a serious problem, caused by excessive and improper use of these drugs. One alternative to traditional antibiotics is Photodynamic Antimicrobial ChemoTherapy (PACT), which is based on the use of a photosensitizer (PS), activated by illumination with visible light. The poor penetration of visible light through the skin limits the application of PACT mainly to the treatment of skin infec- tions. To overcome this problem we report here the exploitation of light emitted in the chemiluminescent reaction of luminol to excite the PS and call this process Chemiluminescent Photody- namic Antimicrobial Therapy (CPAT). In order to increase intra- cellular delivery of both PS and luminol into microbial cells, we encapsulated them into the dipalmitoylphosphatidylcholine - based liposomes. We studied the effect of free and nanoparticle- enclosed PSs (methylene blue, MB and toluedene blue, TB) on Gram-positive Staph. aureus and Gram-negative E. coli under excitation by white luminescent light and by light emitted by free or liposome enclosed luminol. Conventional PACT and CPAT were similarly effective against both types of bacteria. With regard to CPAT, the strongest MIC reduction was achieved when both PS and luminol were liposome encapsulated. In the case of E. coli, the MIC value of free MB, activated by external light or by reaction of free luminol, was 190 mM whereas the MIC of MB in the liposomal form was as 50% lower under excitation with external light or by luminol in liposomes. Staph. aureus was Lymphocytes are chiefly cells of the thymus. The mitotic activity of thymic lymphocytes has been investigated by several research- ers. In the present study, we studied the effect of vitamin C (ascorbic acid) on the thymic lymphocytes of Streptozotocin (STZ)-induced diabetic rats, by examining their mitotic activities. Three experimental groups including 29 male adult Wistar Albino rats with weights 200 ± 20 g have been utilised in this research; control (n = 10), diabetes + vitamin C (n = 10). Rats were injected intraperitoneally a single dose of 45 mg/kg streptozotocin dissolved in citrate buffer to induce dia- betes. Body weights and fasting blood glucose were measured at the beginning and the end of the experiment. Diabetes + vitamin C groups of animals received intragastric AA 20 mg/kg daily for 21 days. In diabetes and control groups, tap water was given to the animals in the same way. The animals were then decapitated

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Abstracts

P8–122 Cyclamen as an ethnomedicine: is it safe? O. Ozgun, S. Arslan, A. Semiz, M. Kapdag, M. Oztas, R. Mammadov and A. Sen Pamukkale, Biology, Denizli, TURKEY

(EROD),

under ether anaesthesia. Thymus were dissected free and fixed in 2.5% phosphate-buffered glutaraldehyde, pH 7.2, and kept cold for 2 hours. After rinsing several times in cold phosphate buffer, the tissues were postfixed in 1% osmium tetroxide solution for 2 hours. Fixed tissues were dehydrated in a series of graded etha- nol, placed into propylene oxide and embedded in araldite. Semi- thin sections were stained toluidin blue and examined with light microscopy. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with transmission electron micros- copy. Light and electron microscope studies revealed normal ultrastructure in controls. Lymphocytes showed cell divisions. The first indication of the diabetes was the appearance of a large number of degenerating thymocytes in the cortex. There were pyknotic nuclei in degenerating thymocytes. In some diabetic rat groups, lipid–laden lymphocytes were found. Proliferative activity in the thymic lymphocytes was considerably lower in the diabetic rats. This may have been caused by the lowering of mitotic index due to diabetes. However, mitotic activity for control and STZ + vitamin C treated groups was essentially similar. The results suggest that vitamin C can be a protective stimulator against decreasing mitotic activity and degenerated lymphocytes in diabetics.

Cyclamen trochopterantum is one of the endemic species of cycla- mens in the southwestern Turkey that is widely used as an ethno- medicine for treatment of hemorrhoids and eczema and expelling digestive tract worms. Despite its wide usage, there is no avail- able information about their actions on xenobiotic metabolism. In this respect, the aim of this study is to determine the effect of water extracts of cyclamen’s tuberose on hepatic CYP1A1/2 and CYP2E1 enzymes that are mainly involved in carcinogen metabo- lism. Male Wistar rats were treated with four different doses of cyclamen tuberose water for 10 consecutive days. extract CYP2E1 associated aniline 4-hydroxylase (A4H), and CYP1A1/2 associated caffeine N-demethylase (CND), ethoxyresorufin O- and methoxyresorufin O-demethylase dethylase (MROD) activities were measured. A4H activity was increased in all groups between 1.2–2.2-fold. Similarly, CYP1A2 dependent CND and MROD activities were increased significantly in all groups. On the other hand, CYP1A1 dependent EROD activity was increased in the three groups. But, degree of induction was decreased with increasing dose. Moreover, this activity was signif- icantly inhibited (51%) in the group taken highest concentration of extract. In the light of the present study, it is expected that people using this plant as a herbal remedy may have increased risk of carcinogenesis due to co-exposure of chemicals such as ni- trosamines, aromatic amines and benzo(a)pyrene may be increased because of induction of CYP1A1/2 and CYP2E1 enzymes. Studies, such as Western blot and RT-PCR analysis, are still underway to determine the expression levels of these enzymes.

P8–121 The effects of thiamine supplementation on frontal cerebral cortex in a rat model of semistarvation N. Orhan1, O. Ekizoglu2, S. Korur2, N. Arican2, M. Kaya3, C. Gurses4, I. Elmas2 and B. Ahishali5 1Istanbul University Institute of Experimental Medicine, Neurosci- ence, Istanbul, TURKEY, 2Istanbul Faculty of Medicine, Forensic Medicine, Istanbul, TURKEY, 3Istanbul Faculty of Medicine, Physiology, Istanbul, TURKEY, 4Istanbul Faculty of Medicine, Neurology, Istanbul, TURKEY, 5Istanbul Faculty of Medicine, Histology, Istanbul, TURKEY

P8–123 Antioxidant properties and cytotoxic effects of essential oils from Salvia pisidica L. on hepatoma G2 cells A. Ozkan1 and A. Erdogan2 1Akdeniz Universit, Biology, Antalya, TURKEY, 2Science Institute, Biology, Antalya, TURKEY

The aim of this study was to evaluate the effect of daily thiamine intake on frontal cortex in a model of semistarvation based on the experience obtained from hunger strikes in Turkey. Sprague Dawley rats were randomly allocated into three groups. Group I was accepted as control and the rats were nourished with normal diet for 2 weeks while the rats in groups II and III were fed with special nutrition formulations (70% sucrose in 0.9% NaCl ent- eral diet). The rats in group III were also administered with 0.5 mg/kg/day thiamine (i.v). At the end of the second week, frontal cortex region of brains were obtained for measurement of the antioxidant enzyme activities [superoxide dismutase (SOD)], reduced glutathione and lipid peroxidation [malondialdehyde (MDA)]. MDA levels in frontal cortex regions of group II were found significantly higher than groups I and III (p < 0.05), while SOD activity in group II was significantly lower than the other groups (p < 0.05). When glutathione levels were evaluated, no depletion was observed in both experimental groups, instead, it showed significantly higher levels when compared to control (p < 0.05). In conclusion, high MDA and low SOD levels observed in frontal cortex of rat brains indicated the existence of oxidative stress in semistarvation model. The increased glutathi- one levels might reflect the dynamic mechanisms in the antioxi- dant activity. Oxidative stress levels were increased among semistarvation groups while thiamine had a preservative effect on neuronal tissue, which suggests a possible regulatory role for this vitamin in protection against oxidative stress.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

The aim of this work was to investigate cytotoxic effect of essen- tial oils on Hepatoma G2 cells (HepG2) and to investigate their possible protective (antioxidant) effects against hydrogen perox- ide-induced cytotoxicity. The antioxidant capacity of essential oils obtained from Salvia pisidica were exhibited by the scaveng- ing activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2- deoxyribose oxidation assay, reducing power assay and b-car- otene-linoleate model system. In DPPH assay, the IC50 value of essential oils was found 9.13 + 0, 65 lg/ml. Similarly, essential oils showed the relative antioxidant activity (RRA%) 63.20% for the inhibition of linoleic acid oxidation. Also, essential oils showed higher hydroxyl radical scavenging activity and reducing power capacity dependent on concentrations. Testing of cytotoxic activity was performed by the CellTiter-Blue(cid:3) Cell Viability assay (resazurin). The amount of cytotoxicity in HepG2 cell treated with the essential oils and its combinations with hydrogen perox- ide (H2O2) was measured this method. We found out that essen- tial oils showed cytotoxic effect on HepG2 cells and IC50 was found 128 lg/ml. As a very important we consider the finding that essential oils significantly reduced the cytototoxic effects induced in HepG2 cells by the strong oxidant H2O2. We assume that decreasing cytotoxic effect of essential oils can be accompa- nied by their antioxidant action (£50 lg/ml).

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P8–124 Wheatgrass extract affects lipid peroxidation levels in human promyelocytic leukemia cell line HL-60 T. Ozkan1, F. Bakar2, A. Z. Karabay2, A. Akinci2, B. Altinok1, A. Karadag3, S. Aydos3 and A. Sunguroglu3 1Ankara University Biotechnology Institute, Biotechnology, Ankara, TURKEY, 2Ankara University Faculty of Pharmacy, Bio- chemistry, Ankara, TURKEY, 3Ankara University Faculty of Medicine, Medical Biology, Ankara, TURKEY

P8–126 Effect of different environmental parameters upon the expression of resistance features in Escherichia coli aquatic strains E. Panus1, N. Rosoiu2, C. Bleotu3, C. Iordache3, C. Delcaru-Larion3, O. Drecea3, M. Bucur3, L. Marutescu3, L. M. Ditu3, M. M. Mitache4, V. Lazar3 and M. C. Chifiriuc3 1Institute of Public Health, Microbiology, Constanta, ROMANIA, 2Faculty of Medicine, Ovidius University, Constanta, ROMANIA, 3University of Bucharest, Faculty of Biology, Bucharest, ROMA- NIA, 4Public Health Authority, Microbiology, Bucharest, ROMA- NIA

Wheatgrass, the young grass of Triticum aestivum, is a rich source of vitamins, amino acids, chlorophyll and minerals in a bioavailable form. Improving the digestive system, preventing cancer and promoting detoxification are among the controversial health benefits of wheatgrass extract. Recently, there has been increased interest in research of the role of oxidative stress in car- cinogenesis. The objective of this study was to determine how wheatgrass extract effects HL-60 cells in terms of lipid peroxida- tion levels. Human promyelocytic leukemia cell line HL-60 was cultured in RPMI 1640 medium supplemented with fetal bovine serum, streptomycin, penicillin and glutamine at 37(cid:2)C in a humidified atmosphere containing 5% CO2. Cells (8 · 104) were seeded to 96-well plates for incubation in the absence or presence of different concentrations of wheatgrass extract (0.5%; 1.5%; 2.5%; 3.5%, 5%, 7 5%, 10% v/v) for 24 hours. After incubation, amounts of MDA (malondialdehyde) were measured spectropho- tometrically. Our results show that wheatgrass extract increases lipid peroxidation levels significantly in HL-60 cells in a dose dependent manner. Its significant effective doses are 1.5%; 2.5%; 3.5%, 5%, 7.5%, 10% v/v (p < 0,001). Our earlier findings have shown that wheatgrass extract decreases proliferation of different cancer cell lines. We conclude that the antiproliferative effect of wheatgrass extract might be related to its effects on oxidative sta- tus of cells.

P8–125 Protective effect of vitamin C treatment on diabetes-induced ultrastructural changes in the thymic capillaries of rats N. Ozsoy, S. Cebesoy and D. Okan Department of Biology, Ankara University Faculty of Science, Ankara, TURKEY

Purpose: To investigate the relationships among the expression of different bacterial resistance features, different incubation tem- peratures and chemical composition of the culture media in E. coli aquatic strains. Material and methods: Twenty strains of E. coli isolated from Black Sea were investigated for the expression of resistance to different bivalent metals (Cu, Co, Mn, Zn, Ni) compounds. The experiments were performed comparatively at different incuba- tion temperatures (22(cid:2)C, 37(cid:2)C and 44(cid:2)C) in aerobic and anaero- bic conditions, NaCl concentrations (from 0% to 10%), glucose (1.5% and 3%) and pH (4, 7 and 9.6). Results: The metals resistance patterns varied with the tested parameter and the bivalent metal compounds. The temperature growth induced an increase in susceptibility of the tested strains to Zn (from 85% at 22(cid:2)C, 20% at 37(cid:2)C and 100% at 44(cid:2)C), Mn (from 50% at 22(cid:2)C, 15% at 37(cid:2)C and 75% at 44(cid:2)C), Cu (from 10% at 22(cid:2)C, 0% at 37(cid:2)C and 55% at 44(cid:2)C), (from 10% at 22(cid:2)C, 0% at 37(cid:2)C and 30% at 44(cid:2)C), Ni (from 0% at 22(cid:2)C, 0% at 37(cid:2)C and 5% at 44(cid:2)C). Concerning the influence of salinity, the highest 10% NaCl induced susceptibility to all tested metals, followed by 2%, 6% and 7% NaCl with susceptibility to four of five metals. The highest susceptibility levels to Zn and Mn were expressed in inverse order to 3%, 4%, 6%, 7%, 0%, 2% and 0.5 % NaCl, while to Cu at 3%, 2% and 7% NaCl. The tested strains were very resistant to Ni and Co at the majority of tested salinities. Concerning the relationship between the chemical com- position of the culture medium and the susceptibility levels to metals, the higher glucose concentration of 3% and the alkaline pH induced higher rates of susceptibility to Mn, Zn and Ni. Conclusion: The expression of resistance features of the E. coli strains is strongly influenced by the incubation temperature and salinity, demonstrating the role of these parameters in the selec- tion of resistance genes in the aquatic strains.

P8–127 New case: a female patient with a mosaicism in the SLC16A2 gene encoding the specific thyroid hormone transporter monocarboxylate transporter 8 (MCT8) P. Ferro-Gallego1, R. Lago-Leston1, I. Pinal-Fernandez1, M. Castro-Gago2 and L. Dominguez-Gerpe1 1IDIS - Universidad de Santiago de Compostela, Departamento de Medicina, Santiago de Compostela, SPAIN, 2Complejo Hospitala- rio Universitario de Santiago, Servicio de Neuropediatria, Santiago de Compostela, SPAIN

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Microangiopathy may represent one of the most critical mecha- nisms involved in diabetes mellitus. The development of microan- giopathy accounts for many of the complications seen in the diabetics. Previous research indicated that a low plasma ascorbic acid level might promote microangiopathy in diabetes. Therefore, the fine structure of the capillaries is of considerable interest. In the present study, we examined the ultrastructural changes in the thymic capillary of male Wistar rats having Streptozotocin- induced diabetes mellitus (STZ), and treated with vitamin C. Dia- betes was induced by a single intraperitoneally injected dose of STZ (45 mg/kg). Diabetic rats were treated with intragastrically administered vitamin C (ascorbic acid) 20 mg/kg daily for 21 days. Endothelial injury and closed capillaries were seen in diabetic rats compared with control groups. Endothelial prolifer- ation was noted in the thymus capillaries of the STZ treated rats. Their mitochondria were swollen and there was loss of cristae. Thickening of basal lamina and capillary proliferation occurred in diabetic rats. There was less capillary damage in the vitamin C treated diabetic groups. These findings and results must be con- sidered as an indication that the vitamin C has a positive effect on the diabetic rats. Introduction: Monocarboxylate transporter 8 (MCT8), encoded by the SLC16A2 gene in the X chromosome, is a specific thyroid hormone transporter. Mutations in this gene cause a severe neu- rological syndrome in males. The aim of this work was to per- form a genetic study in a 8 years old female (Patient 1), which manifested a serious disorder of behavior and learning associated

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P8–129 Modulation of adenosine A2A receptor cell surface levels by a DNA demethylating agent S. Pe´ rez-Buira1, J. Albasanz2, G. Dentesano1, M. Martı´ n2, I. Ferrer1 and M. Barrachina1 1Institut d’Investigacio´ Biomedica de Bellvitge, Institute of Neuro- pathology, L’Hospitalet (BCN), SPAIN, 2Universidad de Castilla- La Mancha, Dept. Quı´mica Inorga´nica Orga´nica y Bioquı´mica (Facultad de Quı´micas), Ciudad Real, SPAIN

with epilepsy. Characteristic thyroid hormone serum patterns were also found: elevated F-T3 and low F-T4 levels. The TSH concentrations were normal. Methods: SLC16A2 and TRb genes were sequenced from gDNA from Patient 1 and Patient 2 (non-genetic pathology) using ABI technology. Additionally, PCR purified products of SLC16A2 exon 1 and exon 2 from each patient were cloned into the pGEMT-easy vector system. Approximately two hundred col- onies from exon 1, and 50 colonies from exon 2 were sequenced and analyzed. Results and Conclusions: Patient 1 exhibited the germ-line SLC16A2 S107P SNP in heterozygosis. In addition, six colonies containing the cloned products from exon 1 showed five point mutations: E114K, E142G, V148M, P0153S and P194S, and one deletion del14G. The TRb gene showed no mutations. The results suggest the presence of a mosaicism in the SLC16A2 gene in Patient 1.

Adenosine A2A receptors (A2ARs) appear to play important roles in inflammation and certain diseases of the nervous system. Phar- macological modulation of A2ARs is particularly useful in Par- kinson’s disease and has been tested in schizophrenia. However, little is known about the regulation of A2AR gene (ADORA2A). A bioinformatic analysis of the entire 5¢ UTR region of human ADORA2A (nearly 15 Kb) revealed the presence of three CpG islands. The percentage of DNA methylation in the different CpG islands was analyzed in two human cell lines, HeLa and U87-MG, using the SEQUENOM Mass Array platform. To test whether DNA methylation regulates endogenous A2ARs expres- sion levels, HeLa and U87-MG cells were treated with the DNA demethylating agent 5-azacytidine, 5 lM for 48 hours. Reduced DNA methylation and increased A2AR mRNA expression as revealed by TaqMan assay, and by increased A2AR radioligand binding of [3H]ZM241385, were demonstrated in HeLa cells when compared with non-treated cells. No modifications were seen in U87-MG cells. These results show for the first time that DNA methylation plays a role in ADORA2A transcription and, subse- quently, in constitutive A2AR cell surface levels. Increased under- standing of epigenetic regulation of ADORA2A may have therapeutic implications in inflammation and in certain neurolog- ical diseases.

P8–128 Metabolomic profiles of hydrophilic and lipophilic thyroid cell line extracts from normal and pathological tissues by 1H NMR spectroscopy I. Pinal-Fernandez1, M. Martin-Pastor2, S. Bravo3, P. Ferro-Gallego1, R. Lago-Leston1, C. Alvarez3 and L. Dominguez-Gerpe1 1IDIS - Universidad de Santiago de Compostela, Departamento de Medicina, Santiago de Compostela, SPAIN, 2Unidad de Resonan- cia Magne´tica Nuclear, RIAIDT. Universidad de Santiago de Compostela, Santiago de Compostela, SPAIN, 3Facultad de Medi- cina, Departamento de Fisiologia, Santiago de Compostela, SPAIN

P8–130 Insights in the regulation of gender-specific LRP-2 expression in the kidney J. A. Plieschnig1, T. M. Bajari2, W. J. Schneider1 and M. Hermann1 1Medical University Vienna - Max. F. Perutz Laboratories, Medi- cal Biochemistry, Vienna, AUSTRIA, 2Medical University Vienna, Universita¨tsklinik fu¨r Innere Medizin III, Vienna, AUSTRIA

Introduction: Thyroid nodules diagnosis using FNA is not always conclusive due to the similarity among certain types of benign and malign tumours. Particularly, in some cases of follicu- lar carcinomas and adenomas, the diagnostics is possible only after surgery. To avoid unnecessary surgical procedures the study of new methods to improve diagnosis is desirable. Our aim was to study the metabolomic profiles by 1H NMR spectroscopy of cell line extracts from different thyroid pathologies and its possi- ble application in diagnosis. Methods: Normal, benign and malign thyroid follicular cell lines were processed to separate hydrophilic and lipophilic metabolites. 1H NMR spectra were obtained for each type of extracts in a 17.6 T Varian INOVA NMR spectrometer operating at a 1H fre- quency of 750 MHz. A 5 mm triple resonance 1H/13C/31P probe with three orthogonal shielded gradients of maximum strength 66.7, 28.3 and 25.4 G/cm in z-, x- and y-directions, respectively, was used. The spectrometer control software was VNMR/ VNMRJ (1.1d). The NMR data were processed and analyzed with MestRe-C v3.x software (Mestrelab Inc.) Results and conclusions: The peak assignment and quantifica- tion showed differences between cells from normal and pathologi- cal tissues. Moreover, differences in specific peaks were also found among different pathologies. The results are very promis- ing and pointing to a possible use of 1H NMR spectroscopic profiles for diagnostic purposes.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

LRP-2 (Megalin, gp330), a member of the LDL receptor family, was originally identified as the auto-antigen in a rat model dis- ease known as Heymann nephritis. This receptor is highly expressed on the apical surface of polarized epithelia, which show high endocytic activity. LRP-2 is not only mediating endocytosis, but is also involved in signal transduction, vitamin homeostasis and hormone regulation, which are important processes in the course of embryonic development. In our studies, avian and rodent species are used to identify the underlying mechanisms of steroid hormone regulation of LRP-2 expression. Additionally, human cell systems will be included in our studies. We were able to identify and clone potential estrogen responsive elements (ERE) in the promoter region of the chicken and human LRP-2 gene, indicating a possible hormone regulation. Luciferase assays should help to demonstrate the functionality of the thus far iden- tified EREs. Furthermore, quantitative PCR experiments indicate a difference in male, female, and estrogen-treated animals. We are able to show that after a steady increase of LRP-2 expression in the developing chicken kidney, a gender-specific difference arises when the animals become sexually mature. High protein levels are maintained until the animals grow older and sex hor- mones start to affect the organism. Protein levels in the female kidney remain high, in contrast to low LRP-2 levels in the male

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organ. After estrogen administration for different time periods, the receptor levels reach those observed in hens. The underlying mechanisms of a gender-specific LRP-2 expression will be eluci- dated in future studies.

P8–131 Characterization of three plant nucleases: properties and anticancer potential T. Podzimek1, T. Podzimek2, J. Matousek1, P. Lipovova2 and B. Kralova2 1Biology Centre ASCR v.v.i., Institute of Plant Molecular Biology, Ceske Budejovice, CZECH REPUBLIC, 2Institute of Chemical Technology Prague, Department of Biochemistry and Microbiol- ogy, Prague 6, CZECH REPUBLIC

Czech Ministry and of anticancer activity of ellipticine to neuroblastomas was synergi- cally increased by VPA and TSA. Combination therapy more effectively inhibited the growth of both neuroblastoma cells than single-agent (ellipticine) treatment, decreasing values of IC50 for ellipticine in a dose-dependent manner. Whereas VPA at concen- trations of 2 mM is able to increase the cytotoxicity of ellipticine up to 10-fold, the only concentration of 100 nM TSA produced up to 2-fold higher cytotoxicity in neuroblastoma cells. A higher sensitivity of neuroblastoma cells to ellipticine correlated with an increase in formation of covalent ellipticine-derived DNA ad- ducts that was found to be one of the most important DNA- damaging mechanisms of ellipticine action in neuroblastomas. No effect of VPA was detected on expression of CYP3A4, the major enzyme responsible for ellipticine activation. Stimulation effects of VPA and TSA on toxicity of ellipticine to neuroblas- toma cells follow from their ability to make cell DNA more accessible to the ellipticine-mediated damage and not from their effects on expression and activities of enzymes activating this drug. Acknowledgement: Supported by GACR (303/09/0472), IGA (NR9522-3/2007) Education (MSM0021620813).

P8–133 Protein profiles of seminal plasma and spermatozoa in patients with normal and pathological spermiograms P. Manaskova-Postlerova, V. Jonakova and J. Peknicova Institute of Biotechnology AS CR, Laboratory of Diagnostics for Reproductive Medicine, Prague 4, CZECH REPUBLIC

supported by MSMT: In the recent years, the properties and biological functions of plant nucleases are studied with increasing intensity. In medical research, efforts are carried out to find new anticancerogenic agents, preferably with no significant side effects. Plant nucleases seem to contribute to these efforts. Our work is aimed on charac- terization of three plant nucleases, TBN1 from tomato leaves, HBN1 from hop and ABN1 from Arabis brassica. The amino acid sequence homology of these enzymes is quite high. Perhaps, this is the reason for their similar catalytical properties. These enzymes are sugar non-specific, therefore are able to cleave both DNA and RNA. Moreover, they can also cleave double-stranded and single-stranded substrates, including also dsRNA. In addi- tion, these enzymes are capable of cleavage an artificial substrate polyA with more efficiency than RNA substrate and possess the 3’- nucleotidase activity. Their temperature optima are about 60(cid:2)C, and pH optima between 5.1 and 6.3. The simple addition of divalent cations, such as Zn2+, Mg2+, Mn2+ and Ca2+ into the reaction mixture inhibits the reaction rather than stimulate – only dsDNA activity of ABN1 is increased by Mn2+ nearly twice. Aside from the biochemical properties characterization, the antitumorogenic effect of the nucleases as well as possible nega- tive side effects were examined. TBN1 possesses cytotoxic effect on melanoma or neuroblastoma cells. In comparison with well studied animal BS-RNase, TBN1 has significantly lower negative side effects, such as embryotoxicity, aspermatogenicity or immu- nogenicity. Acknowledgement: This work is MSM6046137305 and GACR 521/09/1214.

P8–132 Ellipticine combined with histone deactylase inhibitors, valproic acid and trichostatin A, is an effective DNA damage strategy in neuroblastoma cells J. Poljakova1, J. Hrebackova2, J. Hrabeta2, T. Eckschlager2, E. Frei3 and M. Stiborova1 1Charles University Faculty of Science, Biochemistry, Prague, CZECH REPUBLIC, 2Charles University 2nd Faculty of Medicine, Pediatric Hematology and Oncology, Prague, CZECH REPUBLIC, 3German Cancer Research Center, Molecular Toxicology, Heidelberg, GERMANY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

and 523/08/HD64 GACR, Recently, the number of pairs with a reproductive problem has been growing up. In men, many causes of infertility are encoun- tered, and are represented by the reduced number of spermatozoa in the ejaculate, decreased sperm motility or morphological defects of sperm cells. We characterized proteins of seminal plasma and spermatozoa of patients with different spermiograms by electrophoretic methods (SDS-PAGE, 2D-PAGE). Protein profiles of sperm from men with normal and pathological spermi- ograms were compared. Further characterization was carried out by detection of glycoproteins. Most variances in protein profiles were noted in seminal plasma of patients with azoospermia and in the sperm extract of men with oligozoospermia. Visible differ- ences detected by glycoprotein determination were found in sam- ples of seminal plasma and sperm in patients with pathological spermiograms in high-molecular-weight proteins. Additionally, we monitored activities of proteolytic and glycolytic enzymes using substrate zymography. We detected a decreased activity of glycolytic enzyme hyaluronidase in samples of seminal plasma in men with oligozoospermia. Hyaluronidase present on the sperm surface and inside the sperm acrosome participates in the sperm penetration through the matrix of the ovum envelopes. Quantita- tive changes or decreased activity of sperm can impact their fer- tilizing ability. Further studies will be focused on protein and enzymatic differences in pathological samples. Protein identifica- tion or determination of decreasing enzymatic activity in seminal plasma and sperm might help explain the background of fertiliz- ing failure in men with pathological spermiograms. Acknowledgement: This work was supported by grants 303/ 09/1285 1M0601 MSMT and 50520701 AVOZ. Valproic acid (VPA) and trichostatin A (TSA) exert antitumour activity as histone deacetylase inhibitors, whereas ellipticine action is based mainly on DNA intercalation, inhibition of topo- isomerase II and formation of covalent DNA adducts mediated by cytochromes P450 (CYPs) and peroxidases. We evaluated the effects of both two types of antitumor compounds cytotoxic to human neuroblastoma UKF-NB-3 and UKF-NB-4 cell lines in combination therapy on cell growth. The results show that the

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P8–134 Experimental gene therapy intervention and radiation-induced late pulmonary fibrosis development M. Przybyszewska1, J. Miloszewska1, S. Rzonca1, H. Trembacz1, K. Pysniak2 and M. Zalewska3 1Cancer Center Institute of Oncology, Cell Biology Department, Warsaw, POLAND, 2Cancer Center Institute of Oncology, Department of Genetics and Animal Breeding, Warsaw, POLAND, 3Cancer Center Institute of Oncology, Medical Physics Depart- ment, Warsaw, POLAND

GSPE (250 mg/kg) was orally administrated. Blood was obtained from the tail vein after 1, 3, 5 and 7 hours. Our results indicated that a single oral administration of GSPE blocked the increase of plasma TG (26%), Chylomicrons (24%) Very Low Density Lipo- protein (24%) and free fatty acid (14%), induced by the lard ingestion in control animals. Under the same conditions, GSPE did not affect the level of plasma b-hydroxybutyrate. These results indicate that proanthocyanidins gently improve the status of circulating lipoproteins in the postprandrial state, suggesting a decrease of lipid absorption together with a reduction of TG syn- thesis, both in liver and intestine. These findings support the idea that proanthocyanidins are powerful agents for preventing and treating lipid altered metabolic states. Acknowledgement: This work was supported by the Spanish Government (grant AGL2005-04889).

P8–136 Comparison of two DNA binding dyes for applications of high-resolution melting analysis J. Radvansky, G. Minarik and L. Kadasi Department of Molecular Biology, Comenius University, Bratislava, SLOVAK REPUBLIC

Late lung fibrosis following radiotherapy is a serious medical problem. Ionizing radiation of the thoracic region results in an inflammatory reaction involving a deregulation of several cyto- kines and leading to cell damage, ECM deposition and an irre- versible pulmonary fibrosis. The role of TNFa in lung fibrotic desease has been discussed in a number of studies. We aimed to investigate the antifibrotic effect of diminishing the levels of TNFa in the lung tissue by the elevated serum sTNFR-II concen- trations. Using the pcDNA 3.1 expressing vector we constructed plasmid encoding the mouse extracellular domain of TNFR-II. Cell transfection efficiency of the plasmid, and gene and protein expression were assessed. An animal model of fibrosis-sensitive, inbred mouse C57Bl/6J strain, with well documented, radiation- induced, elevated TNFa level in the lung was used. Three days following a single pTNFR-IIs or pcDNA plasmid transfection (100 mg DNA/mouse), mice were exposed to ionizing radiation (20 or 15 Gy) and subsequent intramuscular plasmids injections were performed 2, 4 or 8 times at 2-week intervals. The intramus- cular TNFR-II gene transfection resulted in the increase of serum TNFR-II levels. The transfection was safe and a very low level of plasmid DNA was found at the distant sites of the recipients’ organisms. Animals that received pTNFR-IIs injections devel- oped radiation-induced lung fibrosis a little earlier than the con- trol mice. This could result from too low in vivo gene transfer efficiency, short half-life of the TNFR-II protein and/or from the the postulated anti-fibrotic activity of TNFa. In conclusion, pTNFR-IIs plasmid is a useful vector for the intramuscular TNFR-II gene introduction resulting in an increase of serum TNFR II levels. However, the increased levels of TNFR II before and after irradiation, do not prevent long-term experimental post-radiation lung fibrosis in mice.

High-resolution melting analysis of PCR products is a relatively newly introduced method for rapid and simple genotyping and mutation scanning. The melting profile of a PCR amplicon is generated most commonly by monitoring the fluorescence of a DNA binding fluorescent dye. The aims of our study were to compare the possibility of using a new fluorescent dye EvaGreen for genotyping of two different types of sequence alterations by high-resolution melting analysis. We genotyped a single nucleo- tide substitution (G to A at the W24X locus of the GJB2 gene, causing non-syndromic hearing loss) and the 1 kb Alu insertion/ deletion polymorphism (at the DMPK locus, associated with myotonic dystrophy type 1). We also studied the effect of the amplicon length to the genotyping possibilities in both cases by different combinations of primers. For HRM analyses we used the 96-well Light Scanner instrument. In the case of W24X muta- tion the lengths of amplicons, we studied, were 169, 97 and 55 bp, respectively. The three genotypes were successfully identi- fied in the amplicons of all investigated length with both dyes. The Alu insertion/deletion polymorphism was detected using the three-primer amplification protocol where the insertion allele is represented by a 1007 bp and the deletion allele by a 493 bp product. Alternatively, with modified primer combination the insertion and deletion allele is represented by a 381 and 493 bp amplicon, respectively. The genotypes were determinable with both primer combinations and both dyes. Our results suggest that EvaGreen may represent a suitable dye for genotyping using high-resolution melting analysis.

P8–135 Oral administration of proanthocyanidins reduces postprandial hypertriglyceridemia in rats fed with lard oil H. Quesada, S. Dı´ az, D. Pajuelo, A. Ferna´ ndez, M. J. Salvado´ , L. Arola and C. Blade´ Rovira i Virgili University, Biochemistry and Biotechnology, Tarragona, SPAIN

P8–137 Hypoxic regulation of HIF and NF-jB signaling in prostate and mesothelioma cell lines L. Ravenna1, A. Verdina2, L. Principessa3, C. Fagliarone2, L. Salvatori3, F. Caporuscio3, M. Russo3 and E. Petrangeli3 1Patologia Molecolare, Istituto di Biologia e Patologia Molecolari, Roma, ITALY, 2Sviluppo e programmi terapeutici, Istituti Fisiote- rapici Ospitalieri, Roma, ITALY, 3Medicina Sperimentale, Univer- sita di Roma Sapienza, Roma, ITALY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Inflammation and hypoxia have an acknowledged pathogenetic role in prostate and mesothelioma cancers. The purpose of this study is to investigate the effects of hypoxia in the modulation of Dietary fat is one of the most important environmental factors associated with the incidence of cardiovascular diseases (CVD). Regular consumption of proanthocyanidins, a type of flavonoids, has been associated with a reduced risk to develop CVD. In a previous study, it has been demonstrated that an oral gavage of grape seed proanthocyanidins (GSPE) lowers plasma apolipopro- tein B (apo B) and triglyceride (TG) levels in normolipidemic rats fed with standard diet 5 hours after the treatment. The aim of this study was to determine the effects of GSPE on plasma trigly- cerides and lipoproteins kinetics during the lipid absorption. To this end, male Wistar rats were deprived of food for 14 hours before the experiment. A lard oil (2.5 ml/kg) with or without

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Poster Presentations

playing a major role in mycobacteria detection through TLR2 and initiation of an anti-mycobacterial response.

P8–139 Modulation of COMT activity and expression by mineral nutrients L. Ribeiro1, P. Bastos2, J. Araujo1 and M. J. Martins1 1Biochemistry, Faculty of Medicine, University of Porto, Porto, PORTUGAL, 2Biology School of Sciences, University of Minho, Braga, PORTUGAL

pro-inflammatory factors in these tumors. We selected three tumorigenic prostate cell lines, LNCaP (androgen-dependent), PC3 and DU145 (androgen-independent) and two mesothelioma cell lines, MPP89 (non-tumorigenic) and MSTO-211H (tumori- genic). After hypoxic stimulation, we compared the kinetics of activation of HIF1alpha and 2alpha and of NF-jB (p65 and p50), the transcription factors more involved in the response to hypoxic and inflammatory stimuli. Activation of HIF1alpha, HIF2alpha and NF-jB followed different kinetics but compara- ble in all the prostatic cell lines. In particular, nuclear accumula- tion of HIF1alpha started early after oxygen deprivation (10¢), reached the top after 4 hours and declined to the base level by 24 hours. Expression of HIF1alpha mRNA did not change for 4 hours, then abruptly decreased, remaining stable under the base level up to 72 hours. HIF 2 alpha did not seem to respond to reduced oxygen both at mRNA and protein level. Nuclear trans- location of p65 and p50 NF-jB subunits preceded the HIF1alpha activation peak. Differently, in mesothelioma, nuclear transloca- tion of NF-jB subunits started 6 hours after oxygen withdrawal and followed HIF1alpha maximum activation. In this cancer model HIF2alpha did not appear to be modulated by hypoxic conditions either. Our data show that in prostate and mesotheli- oma, the cross-talk between HIF1alpha and NF-jB, described by some authors employs different mechanisms. Therefore also the genes controlled by the two transcription factors must have dif- ferent patterns of activation. By silencing HIF1alpha and p65 we intend to clarify their individual contributions to the activation of an inflammatory reparative phenotype and to deepen their role in cancer progression.

P8–138 Characterization of lipomannan as a major ligand of Toll-like receptor 2 promoting anti-mycobacterial responses A. Ray, G. Puzo and J. Nigou Mecanismes moleculaires des infections mycobacteriennes, Institut de pharmacologie et de biologie structurale, Toulouse, FRANCE

Magnesium, whose dietary intake in much of the Western World population is less than the recommended dietary allowances, is essential for several cell reactions and varied metabolic and phys- iological functions. COMT (catechol-O-methyltransferase) is a magnesium dependent enzyme that catalyzes the methylation of catechol substrates using S-adenosyl-L-methionine as a methyl donor. Although COMT is involved in the metabolism of various compounds, including estrogens and polyphenols, it plays a most important role in the metabolism of catecholamines, much more adrenaline than of noradrenaline. These amines play key roles in the regulation of numerous physiological processes and are being implicated in an ever-expanding array of neurological, psychiat- ric, endocrine, and cardiovascular disorders. Recently, much attention has been devoted to the impact of COMT polymor- phisms on cardiovascular health. The aim of this study was to test a) the hypothesis that a relative insufficiency of magnesium leads to lower COMT activity, and a higher magnesium intake favors the enzyme activity and b) the effect of these different diets on COMT expression. Adult Wistar rats were treated for 7 weeks with three different diets (group 1: standard; group 2: high sodium, low magnesium; group 3: high magnesium). At the end of treatment COMT expression was evaluated in the liver and adrenal glands through RT-PCR, and COMT activity, in the same tissues, through exposure to adrenaline and measurement of metanephrine by HPLC. The COMT mRNA expression in adrenal glands significantly decreased with high magnesium intake, being about 46% lower in this group compared with the standard diet group (n = 4–6, p < 0.05). In liver, the expression of COMT mRNA remained unchanged in response to the three different diets (n = 6). These preliminary findings show that high magnesium intake differently affect COMT mRNA expression in liver and adrenal gland. Results obtained from COMT activity, currently in preparation, will provide a more complete picture of the influence of magnesium diet disposal in COMT function in both organs.

P8–140 Hormonal responses to exhausting physical exercise L. Ribeiro1, M. Moz1, A. Ascensao2, M. Jose2 and M. Mendanha1 1Biochemistry, Faculty of Medicine, University of Porto, Porto, PORTUGAL, 2Sport Biology, FCDEF, University of Porto, Porto, PORTUGAL

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

An adequate balance between catabolic processes (mobilization of energy), induced by stress hormones such as adrenaline (AD), noradrenaline (NA) and cortisol, and anabolic processes (repair, healing, growth), induced by steroid sex (such as testosterone) and growth hormones, is vital for health and survival. Regular moderate physical exercise is known to contribute to such ana- bolic effects and to an earlier homeostasis restoration. However, excessive physical exercise may lead to an inadequate endocrine response, eventually contributing as a risk factor to disease initia- Innate immune system relies on evolutionarily conserved pattern recognition receptors (PRR), among them Toll-like receptors (TLR), that recognize a set of molecular structures conserved among microbes and termed ‘Microbe-associated molecular pat- terns’ (MAMPs). Toll-like receptor 2 is a major PRR allowing the detection by human host cells of Mycobacterium tuberculosis, the etiologic agent of tuberculosis. Beside the well characterized lipoproteins, others and us have identified another class of TLR2 ligands restricted to mycobacteria and related actinomycetes, namely lipoglycans. However, their real contribution in the stim- ulation of the TLR2-mediated pro-inflammatory response initi- ated by the detection of mycobacteria remained elusive. Using various Mycobacterium smegmatis knock-out mutants defi- cient for the production of the different lipoglycans, we have found that one of them, lipomannan, indeed plays a key role in the TLR2-dependent pro-inflammatory cytokine production induced by the whole mycobacteria. The lipomannan-deficient M. smegmatis mutant, which is impaired in NF-jB activation, also shows an increased ability to survive in activated THP-1 macrophage cell line, as compared to its wild-type parent strain. Unfortunately, so far lipomannan-deficient strains cannot be obtained by genetic manipulation in the pathogenic species M. tuberculosis. However, we have identified some M. tuberculosis clinical isolates that naturally do not produce lipoglycans. Inter- estingly, the lipomannan-deficient bacteria are greatly impaired in their ability to induce NF-jB activation and cytokine production. Altogether these data define lipomannans as bona fide MAMPs

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opportunity for improved cultivation technique in tissue engi- neering. Acknowledgements: Supported by Grant GAAS CR KAN 2001 00801 and 126 08 9005.

in cortisol plasmatic increases

P8–142 Hexavalent chromium and lung cancer: new perspectives C. F. D. Rodrigues1, A. Almeida2, I. M. Carreira3, E. Matoso4, A. M. Urbano5, M. F. Botelho3, L. Carvalho3, M. Alves6, C. Monteiro6 and M. C. Alpoim7 1Department of Zoology CNBC and CIMAGO, University of Coimbra, Coimbra, PORTUGAL, 2Centro de Histocompatibilidade do Centro, Ministe´rio da Sau´de, Coimbra, PORTUGAL, 3Faculty of Medicine and CIMAGO, University of Coimbra, Coimbra, PORTUGAL, 4Faculty of Medicine, University of Coimbra, Coimbra, PORTUGAL, 5Department of Biochemistry and CIMAGO, University of Coimbra, Coimbra, PORTUGAL, 6Faculty of Pharmacy, University of Lisbon, Lisbon, PORTUGAL, 7Department of Biochemistry CNBC and CIMAGO, University of Coimbra, Coimbra, PORTUGAL

tion and/or exacerbation. The aim of our work was to examine the effect of exposure to an intense and extremely stressful train- ing on several plasma stress hormone levels in healthy male indi- viduals. This study was conducted establishing two groups of subjects: one group conditioned by a previous 3 months rigorous training (Group A; n = 7) and another one not submitted to this training (Group B; n = 7). After this period, all the individuals were submitted to an intense and extremely stressful physical pro- gram training (which consisted of running, walking, gymnastics, climbing, while loaded or unloaded with backpacks, etc.) during 72 hours. Blood samples were collected before (basal), immedi- ately after 72 hours of training, and 48 hours (rest period) after this period. AD and NA plasmatic levels were quantified by HPLC with electrochemical detection, whereas plasmatic levels of either cortisol or testosterone were measured by using commer- cial radioimmunoassay kits. Immediately after exercise, signifi- cant levels were observed (p < 0.05) in group A, while in group B there was only a ten- dency to an increase. However, in this last group, contrary to group A, after the rest period cortisol levels remained abnormally high. In both groups, although there were no differences in tes- tosterone plasmatic levels following exercise, a significant increase was observed after rest (p < 0.05). In relation to CA, the most relevant finding was that individuals without previous exercise (group B) presented higher basal NA plasmatic levels than the individuals previously trained (p < 0.05). Our results suggest that in order to adequately respond to an intense and extremely stressful exercise it is necessary to be in a good fitness condition. Acknowledgements: Supported by Reitoria da Universidade do Porto and Caixa Geral de Depo´ sitos (IRIC/61).

genes

P8–141 Cell adhesion on artificial materials modified for tissue engineering S. Rimpelova´ 1, N. Kasa´ lkova´ 2, V. Svorcik2 and T. Ruml1 1Department of Biochemistry and Microbiology, Institute of Chemical Technology Prague, Prague 6, CZECH REPUBLIC, 2Department of Polymers, Institute of Chemical Technology Prague, Prague 6, CZECH REPUBLIC

Hexavalent chromium [Cr(VI)] is an ionic form of the element chromium avowed as a human lung carcinogen. Despite the vast knowledge on the (bio)chemical mechanisms underlying Cr(VI)-induced cell damage, the molecular pathways conducing to malignancy still remain obscure. To make clear Cr(VI)- induced carcinogenic process, an in vitro cell model was estab- lished by chronically exposing BEAS-2B cells to 1 lM Cr(VI). Besides the changes in cells’ ploidy and a decrease in cloning efficiency documented soon after Cr(VI)-exposure, behind 12 passages the culture started to became heterogeneous presenting morphological abnormal cells in between the normal epithelial diamond-shaped cells. Abnormal clones were isolated from those heterogeneous cultures and cultivated at a very low cellu- lar density. The colonies obtained were isolated and expanded, allowing the establishment of sub-clonal cell lines. One of those sub-clonal cell lines, RenG2, showed several malignant features such as abnormal growth pattern and morphology, an aneu- ploid karyotype and over-expression of commonly involved in malignant transformation (c-MYC, EGFR, HIF-1a, and LDH-A), as well as the ability to induce tumors when injected in nude mice. RenG2 sub-clonal cell line also revealed no microsatellite instability (MSI) and a functional MLH1 pro- tein, which points to a functional mismatch repair system. Our results suggest that prolonged exposure to Cr(VI) leads to the emergence of altered cells resistant to cytotoxicity, which paved the way to the accumulation of mutations through the selection of the most resistant variants. Thus, our findings are suggestive for a role of the resistance phenotype pathway in the origin of the so called chromate lung cancer.

P8–143 Glutathione role on proliferation and differentiation processes of osteoblastic cells C. Romagnoli1, F. Favilli1, G. Marcucci2, I. Tognarini2, S. Sabina2, S. Catarzi1, T. Iantomasi1, M. L. Brandi2 and M. T. Vincenzini1 1Biochemical Sciences, University, Firenze, ITALY, 2Internal Medicine, University, Firenze, ITALY

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Tissue engineering, one of modern interdisciplinary scientific fields, is of growing importance, especially for variety of clinical applications including the treatment of burns, chronic venous ulcers etc. Cell-material interface plays a key role in the interac- tion of cells with artificial materials designed for construction of body implants. Physical (wettability, roughness, polarity, electri- cal charge, elasticity, rigidity, etc.) and chemical properties (vari- ety of chemical functional groups) of the material surface strictly influence the cell adhesion. To determine the material surface and enhance its biocompatibility, it was modified by Ar+ plasma irradiation. These modifications lead to the formation of radicals and double bonds, which improve spontaneous adsorption of various biomolecules (cell adhesion-molecules). The minimal ligand specifically recognized by integrins (cell adhesion recep- tors) is a tripeptide arginine-glycine-aspartate (RGD). As a matrix high density polyethylene (HDPE) was used, whose bind- ing capacity for specific ligand (RGD) was enhanced by Ar+ plasma irradiation. The efficiency of this modification for increased adhesion and growth of epidermal cells was tested in vi- tro by using cell line of mouse embryonal fibroblasts (NIH 3T3). In comparison with glycine modified matrix serving as a control (smaller than minimal ligand recognized by integrins), growth of our used cells was better on RGD. This result indicates that using of cell adhesion molecules fragments provides a great Introduction: Glutathione (GSH) is the main intracellular anti- oxidant and the ratio GSH/oxidized GSH (GSSG) is used to measure cellular redox status that, recently, has been related to bone metabolism and osteoporosis. This study reports some data

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supported by CNMP extracellular matrix from conjunctive, cartilaginous and bone tis- sue. Experimental data demonstrate that the extracts rich in gly- cosaminoglycans obtained from small sea fish can be conditioned in various pharmaceutical forms with valuable therapeutic actions. Acknowledgement: This work was PNCDI 2 research program 4, Project 6.1.-043/2007-2010.

P8–145 Cytotoxic activity of genistein-8-C-glucoside and genistein against human SK-OV-3 ovarian carcinoma cells A. Rucinska1, S. Rozalska2, S. Kirko3 and T. Gabryelak1 1Department of General Biophysics, University of Lodz, Lodz, POLAND, 2Department of Industrial Microbiology and Biotech- nology, University of Lodz, Lodz, POLAND, 3Institute of Pharma- cology and Biochemistry, National Academy of Sciences, Grodno, BELARUS

on the relationship between changes of GSH levels and processes of proliferation and differentiation in osteoblastic cells. Methods: Human SaOS-2 cell line was cultured and cell growth, viability and osteogenic differentiation were performed as previ- ously described (1). Proliferation and differentiation were evalu- ated in GSH depleted cells by buthionine sulfoximine, a specific inhibitor of GSH synthesis, and in cells treated with GSH or N- acetyl cysteine. GSH/GSSG ratio was measured by HPLC method. Results: GSH/GSSG ratio levels during the proliferation and differentiation of SaOS-2 cells were determined and the results showed a significant increase of this ratio during cell differentia- tion in comparison with levels measured in proliferating cells. Variations of intracellular oxidative status, obtained by the mod- ulation of the GSH/GSSG ratio, showed that the proliferation was not affected by an increase of oxidative status, whereas, when this decreased the cell growth was significantly reduced. Differently, the differentiation process was activated in GSH treated cells and decreased in cells with low levels of GSH/GSSG ratio; this effect seems to be prevalently related to the early phase of the differentiation. Conclusions:These data can be useful to clarify metabolic pro- therapeutical cesses of bone regeneration and suggest novel approaches both in osteoporosis treatment and diseases of bone tissue. Acknowledgements: MIUR. Reference: 1. I.Tognarini et al., Biomaterials 2008; 29: 809–824.

P8–144 In vitro and in vivo investigation of some bioactive extracts rich in glycosaminoglycans obtained from small sea fish N. Rosoiu1, R. Nita2, D. Ene2, M. Constantinovici2 and L. Olariu2 1Department of Biochemistry, Academy of Romanian Scientists, Ovidius University Constanta, ROMANIA, 2Research Department, S.C. Biotehnos S.A., Otopeni, ROMANIA

Genistein and genistein-8-C-glucoside (G8CG) belong to isoflav- ones, which are a subclass of flavonoids, a large group of poly- phenolic compounds widely distributed in plants. Numerous in vitro studies suggest that isoflavones, particularly genistein have both chemopreventive and chemotherapeutic potential in multiple tumor types. In the current investigation we used geni- stein and glycosylated genistein (genistein-8-C-glucoside) isolated from flowers of lupine (Lupinus luteus L.). We studied the effects of G8CG and combination genistein-G8CG on human ovarian carcinoma cell line SK-OV-3. The cells were exposed to various concentrations of isoflavones (ranging from 1–90 lM) for 24 and 48 hours. The colorimetric MTT assay was used to assess cyto- toxicity and acridine orange (AO)/ethidium bromide (EB) stain- ing technique was employed for the detection of apoptotic and necrotic cell death. The morphological changes of SK-OV-3 cells were examined by a confocal laser scanning microscope (CLSM). The level of reactive oxygen species (ROS) was estimated by DCFH-DA assay. Mitochondrial membrane potential changes were monitored using a fluorescent probe rhodamine 123 by a CLSM and flow cytometry. The results revealed that G8CG and the mixture of isoflavones genistein-G8CG exerted multiple sup- pressive effects on ovarian cancer cells, including proliferation inhibition, induction of apoptotic cell death and loss of mito- chondrial membrane potential. These data suggest that genistein- 8-C-glucoside from Lupinus luteus and combination of genistein- G8CG can be identified as a future, potential chemotherapeutic agent in the treatment of chemoresistant ovarian cancer.

P8–146 Genistein derivatives potentiate inhibition of cancer cells growth by irradiation A. Rusin1, A. Gogler1, A. Gruca1, D. Bochenek1, J. Zawisza2, W. Szeja2, G. Grynkiewicz3 and Z. Krawczyk1 1Department of Tumor Biology, Maria Sklodowska-Curie Memo- rial Cancer Center and Institute of Oncology Gliwice, Gliwice, POLAND, 2Department of Organic Chemistry Bioorganic Chemis- try and Biotechnology, Silesian University of Technology, Gliwice, POLAND, 3Chemistry Department, Pharmaceutical Research Institute, Warsaw, POLAND

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Genistein, a natural isoflavone product, has been shown to inhi- bit cell proliferation and induce apoptotic cell death. It has also been shown to augment the effect of irradiation against human cancer cell lines in vitro and tumors in animal models in vivo. Recently, we found in screening studies of genistein glycoconju- gates some compounds significantly more cytotoxic than a parent New data regarding the in vitro and in vivo therapeutic effects of some extracts rich in glycosaminoglycans (GAG’s) obtained through a patented technology from small sea fish (Sprattus sprattus sprattus, Odontogadus merlangus euxinus and Engraulis encrassicolus ponticus species) are shown. The results show that GAG’s extracts modulate the assembly process of collagen fibers in a dose-effect manner. The significant decrease of hyaluroni- dase, collagenase and elastase activities emphasize the positive intervention of the GAG’s extracts in the enzymes status involved in extracellular matrix degradation. The anti-inflammatory activ- ity of the GAG’s extract was investigated in vivo on Wistar rats with carragenaan-induced paw edema. Results were similar to those of diclofenac, emphasizing the strong anti-inflammatory effect of GAG’s. Antioxidant activity has been investigated in acellular system using the DPPH (1,1-diphenyl-2-picrylhydrazyl) method and in cellular system by flow cytometry (using vascular endothelial cells, HUVEC). The results show a strong antioxidant activity depending on the bioextract concentration. Also, their high content in sulfated glycosaminoglycans (44%) make the bio- active extracts from small see fish useful to prevent the unsettle of the macromolecular structure and keep the functionality of the

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P8–148 Kinetic characterization of new selective collagenase 3 (MMP-13) inhibitors as potential therapeutic agents in osteoarthritis S. Santamaria1, E. Nuti1, F. Casalini1, S. Avramova1, G. Cercignani2, E. Orlandini1, S. Nencetti1, N. Han Lim3, R. Visse3, H. Nagase3 and A. Rossello1 1Dipartimento di Scienze Farmaceutiche, University of Pisa, Pisa, ITALY, 2Dipartimento di Biologia, University of Pisa, Pisa, ITALY, 3Department of Matrix Biology, Imperial College, Lon- don, UK

compound, genistein. The aim of our work was to assess the effect of novel genistein derivatives used in combination with irradiation on cancer cell growth inhibition. Two cell lines were used in the study, HCT 116 and Du145. Cells were pretreated for 24 hours with the tested compounds and then irradiated by clini- cal linear accelerator. Cellular responses to the experimental treatment were assessed by clonogenic survival, cell cycle arrest- ment, micro- and multinucleation, centrosome splitting and apop- totic cells score. The activity of tyrosine kinases was assessed in K-lisa test and EGFR phosphorylation was assessed in Western blots. We found that some genistein derivatives are more potent than genistein for enhancement of the effect of irradiation in col- ony formation assay. The tested compounds were able to inhibit cell cycle in G2/M phase and to inhibit activity of protein kinas- es, among them EGFR. We also observed increased index of abnormal mitoses and mitotic death in cells treated with novel genistein derivatives. The mechanism of the synergistic inhibition of cells proliferation is not described yet, however, the results indicate that different pathways contribute to lethality caused by combined treatment with genistein derivatives and irradiation.

Osteoarthritis (OA) is a debilitating disease in which the protec- tive cushion of cartilage is destroyed, resulting in pain and reduced mobility. The major constituents of cartilage are collagen fibrils and aggrecan, a large aggregating proteoglycan. Matrix metalloproteinases (MMPs) are a class of zinc-dependent endo- peptidases known to play key roles in the remodeling and turn- over of extracellular matrix (ECM). MMP-13 (collagenase-3) catalyses the cleavage of typeII collagen, the main structural pro- tein in articular cartilage. This enzyme is not present in normal adult tissues but is expressed in the joint and articular cartilage of OA patients. Since aberrant MMP-13 activity is regarded as the main cause of irreversible cartilage damage in OA, MMP-13 synthetic inhibitors (MMPI) are sought as potential OA drugs. Here we report the in vitro evaluation and kinetic characteriza- tion of a new series of arylsulfonamido hydroxamates, belonging to the new NO-alkylsulfonamido based MMPIs, as potential selective MMP-13 inhibitors. Among these MMPIs a new potent (nanomolar) and highly selective slow-binding MMP-13-inhibitor was discovered by enzymatic assay and was successively tested in vitro on collagen and on cartilage explants. Further studies on this class of compounds are going on in order to evaluate their pharmacokinetic properties and in vivo efficacy in OA models.

P8–147 Switchable fluorescent orthotopic model of brain tumour for investigation the role of GCPII expression P. Sacha1, J. Starkova1, K. Sramkova1, S. Vaculin2, M. Franek2, J. Zamecnik3 and J. Konvalinka1 1Biochemistry, Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague 6, CZECH REPUBLIC, 2Department of Normal Pathological and Clinical Physiology, Third Faculty of Medicine, Charles University, Prague 2, CZECH REPUBLIC, 3Department of Pathology and Molecular Medicine, Second Medical Faculty and University Hospital Motol, Charles University, Prague 5, CZECH REPUBLIC

P8–149 Hemorphin demonstrates anticancer properties by affecting tumor DNA structure F. Sarukhanyan1, H. Stepanyan2, J. Garibyan2, I. Grigoryan3, Y. Dalyan3 and N. Barkhudaryan1 1Neuropeptides Biochemistry, H.Buniatian Inst. of Biochemistry NAS RA, Yerevan, ARMENIA, 2Cancer Chemotherapy, Institute of Fine Organic Chemistry NAS RA, Yerevan, ARMENIA, 3Molecular Physics, Yerevan State University, Yerevan, ARMENIA

GCPII is well established marker of prostate adenocarcinoma. It is also expressed in majority of solid tumour vasculature. The role of GCPII in tumours remains controversial since there are reports suggesting both positive and negative impact of GCPII expression/activity on tumor growth. In our previous work we found that GCPII is also expressed in several astrocytomas, abundant brain tumours. GCPII is known to cleave NAAG to yield L-glutamate. High concentration of L-glutamate is neuro- toxic to neurons and might thus promote tumour expansion. To address the influence of GCPII expression on brain tumour growth, we have developed a switchable orthotopic fluorescent in immunodeficient mouse. This approach xenograft model enables the visualization of intracranial tumour growth in vivo in time course with gene of interest switched by doxycycline either on or off using the same cell clone. Astrocytoma cell line U373MG was chosen as a suitable, easy to transfect cell line, lacking constitutive GCPII expression. After tumourigenicity test- ing, stable transfectants of regulation protein coded by pTet-off- Advanced were selected. The tumourigenic and highly regulative clones were stable transfected by mKate (a novel monomeric very bright far-red fluorescent protein marker) to visualize tumour growth. After transfection by GCPII, this cell line enables to ana- lyze the role of GCPII in tumour growth in vivo. This method might represent a general approach for the analysis of the possi- ble role of exogenous genes/gene products on glioblastoma growth using U373MG cell line. Acknowledgement: Supported grants: Centre for New Antivi- rals and Antineoplastics, project No. 1M0508 and Research Goal 21620816.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

LVVYPW, a member of hemorphins family, was shown to inhi- bit the tumor cells growth in rats, inoculated with sarcoma-45 (S-45). It has been found that intraperitoneal administration of rats, bearing S-45, with LVVYPW inhibits DNA methylation. Inhibition of DNA methylation is a specific characteristic of anti- tumour drugs and it is well known that methylation of DNA often correlates with the lack of transcriptional activity. Earlier we found that hemorphins modulated the activity of calcineurin, which controls production of some regulatory proteins on gene transcription level via activation of NFAT (nuclear factor of acti- vated T cell) transcription factors. It is suggested that our hemor- phin can also affect genes transcription by modulation of NFAT binding to DNA via calcineurin/NFAT pathway. However, it is not excluded that hemorphin may directly bind to DNA by hydrophobic interaction and form DNA-LVVYPW complex with transcriptional pro- changed conformation, which will affect cesses. By using calorimetric approaches the thermodynamic investigation of DNA, isolated from S-45 bearing rats that received LVVYPW treatment, revealed the increased value of enthalpy (16.2 kcal/mol) in comparison with enthalpy of S-45

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DNA (9.23 kcal/mol). It is suggested that ability of LVVYPW to affect DNA structure is one of the mechanisms underlying anti- cancer effect of hemorphin.

P8–150 Transformation by retroviral vectors of bone marrow-derived mesenchymal cells causes mitochondria-dependent cAMP-sensitive oxidative stress R. Scrima, C. Piccoli, M. Ripoli, A. D’Aprile, G. Quarato, O. Cela, D. Boffoli and N. Capitanio Biomedical Sciences, Medical School, University of Foggia, Foggia, ITALY

tubules. Renal hypouricemia predispose the individuals to exer- cise-induced acute renal failure and nephrolithiasis and is charac- terized by hypouricemia and increased excretion fraction (EF) of uric acid. Most of the described patients are Japanese, who have loss-of-function mutations in hURAT1 gene. Hypouricemia is sometimes overlooked, therefore we have set up the complete investigations for the unexplained hypouricemia. The evaluation of clinical history with attention to renal failure, urolithiasis, sei- zures and immunodeficiency is important. From the group of 569 hypouricemic patients the cases (with repetitive serum uric acid less than 60 lmol/L and urate EF more than 43%) were selected for analysis of the SLC22A12 gene. Sequence analysis of the 17 Czech patients revealed three sequence variations in the promotor and 5¢-UTR region and five variations in exonic regions. Three transition and four deletions, yet unpublished, were found in six patients. The function and imunohistochemistry analysis in Xenopus oocytes showed significantly decreased urate transport activity of these variants. Sequence analysis of SLC2A9 of the four subjects, where we did not find sequence variation in the SLC22A12, is in the process. In conclusion – we have diagnosed first patients of non-Asian origin with the defects in hURAT1 gene. This genetic defect is still unrecognized condition and prob- ably not widespread in Japan and Korea only. Acknowledgement: Supported by grant – MSM 0021620806 Czech Republic.

P8–152 O-Glycosylation-mediated heterogeneity of 67– 76 region of endogenous pro-Brain Natriuretic Peptide (proBNP) and its processing A. Semenov1, N. Karpova2, A. Postnikov1, K. Seferian1, N. Tamm1, D. Serebryanaya2 and A. Katrukha1 1R&D, HyTest Ltd, Turku, FINLAND, 2Biochemistry, Moscow State University, Moscow, RUSSIA

Background: Retroviral vectors have been used in human gene therapy trials because they can stably introduce therapeutic genes in patient’s cells. Their applicability is frustrated by the limited viability of transformed cells and by risks linked to selection of oncogene-mutated clones. In this study we analysed the mito- chondrial oxidative metabolism in IL7-stromal cells to reveal pos- sible alterations influencing cell survival. Methods: A respirometric and spectrophotometric analysis of the OXPHOS complexes activity and related ROS production by confocal microscopy was carried out in IL 7 engineered- and con- trol stromal cells. Results: We showed that LXSN-NeoR/IL-7-engineered mesen- chymal cells exhibited a marked enhancement of ROS production compared to untransfected cells. Surprisingly, this effect appeared to be independent on the gene product carried by the retroviral vehicle because it was reproducible in cells transfected with the plasmid alone. Stable transfection of mesenchymal cells with pBABE-puro, a different vector, caused similar redox unbalance suggesting a more general effect. The enhanced production of ROS was attributable to mitochondrial dysfunction and brought back to altered activity of the Complex I of the respiratory chain. The oxidative stress in transfected mesenchymal stem cells was completely reversed by treatment with cAMP-analogue thus pointing to alteration in the PKA-dependent signalling pathway of the host cell. Conclusions: Engineered stromal cells show a reduced complex I activity responsible of ROS production completely reversed by bcAMP treatment; this could explain the partial unsuccess of the transplantation protocol and provides insights to improve the efficiency of the engineered stromal cell therapy in regenerative medicine.

P8–151 Defects of renal urate transporters and their clinical consequences I. Sebesta1, B. Stiburkova2 and J. Bartl2 1First Faculty of Medicine, Institute of Clinical Biochemistry and Laboratory Diagnostics Inst.Inher.Metabolic Disorders, Charles University Prague, Prague 2, CZECH REPUBLIC, 2First Faculty of Medicine, Institute of Inherited Metabolic Disorders, Charles University in Prague, Prague 2, CZECH REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Recent studies have led to significant advances in the understand- ing of the pathogenesis of hyperuricemia and gout. These find- ings include genetic variation within the regulators of urate homeostasis. Moreover, the identification of the urate transport- ers in the kidney (hURAT1 encoded by SLC22A12 gene, and URATv1 encoded by SLC2A9 gene) led to the molecular eluci- dation of idiopathic renal hypouricemia. hURAT1 is the major urate-anion exchanger in the kidney and plays the central role in the proximal reabsorption of urate on apical membrane of Brain natriuretic peptide (BNP) is a peptide hormone secreted into the circulation by cardiomyocytes. Active BNP-32 hormone (32 amino acid residues, a.a.r.) is formed from the precursor mol- ecule, proBNP (108 a.a.r.), by convertase-mediated cleavage. BNP concentration is markedly elevated in blood of patients with heart failure (HF). However, the major form of BNP in circula- tion is unprocessed proBNP displaying impaired hormonal activ- ity. Recently we have demonstrated that processing of proBNP expressed in HEK 293 cells is suppressed by O-glycosylation of the region (67–76 a.a.r.) located close to the cleavage site (Seme- nov et al. 2009). The aim of the present study was to analyze gly- cosylation status of 67–76 region of proBNP from plasma of HF patients and the occurrence of its cleavage. Using monoclonal antibodies with precise epitope specificity, we demonstrated that endogenous proBNP is heterogeneous in O-glycosylation status of 67–76 region. By means of affinity chromatography we extracted two different fractions of endogenous proBNP: non- glycosylated (325%) and glycosylated (375%) at 67–76 region. Incubation of non-glycosylated fraction with recombinant pro- hormone convertase furin resulted in the effective proBNP pro- cessing. MALDI-MS analysis revealed that processing led to BNP-32 formation. Glycosylated proBNP fraction was unsuscep- tible to convertase treatment. These results are in good agreement with the results obtained for recombinant proBNP expressed in eukaryotic cells. Summing: for the first time we report here the presence of non-glycosylated at 67–76 region fraction of endoge- nous proBNP. For unknown reason this fraction is not processed in vivo, but is susceptible to the convertase-mediated processing in vitro.

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oligodeoxyribonucleotide with high yields. Oligonucleotide conju- gates were obtained by treatment of anthracene containing oligo- nucleotide derivative with peptides with maleimide residue. All used peptides have demonstrated high ribonuclease activity on model RNA substrates. Thereby a series of peptide-oligonucleo- tide conjugates was synthesized. The ribonuclease activity of these conjugates will be investigated. Acknowledgement: This work was supported by RFBR 07-04- 00990-a, 08-04-90038-Bel-a.

P8–153 Simultaneous advanced glycation ends products (AGEs) and Aminoguanidine (AG) exposure on cultured human skin fibroblasts A. I. Serban1, C.M. Munteanu2, N.R. Gorgan2, M. Costache2 and A. Dinischiotu2 1Faculty of Veterinary Medicine Bucharest, Biochemistry and Molecular Biology, Bucharest, ROMANIA, 2University of Bucha- rest Faculty of Biology Bucharest, Biochemistry and Molecular Biology, Bucharest, ROMANIA

P8–155 Fluorogenic tagging of protein 3-nitrotyrosine with 4-(aminomethyl)benzenesulfonic acid for quantitative analysis and visualization of protein tyrosine nitration V. Sharov, E. Dremina, X. Li, J. Stobaugh and C. Schoeneich Pharmaceutical Chemistry, University of Kansas, Lawrence KS, USA

AGEs affect several gene expressions and the signal transduction pathways of target tissues. AG was used in diabetes treatment. Our goal was to study the effects of AG on the expression of RAGE, TGF-b, collagen I and III on AGE-BSA-treated fibro- blast cells at transcriptional and translational levels. The cells were treated for 12 and 24 hours with AGE-BSA in the range in the presence and absence of 10–100 lM AG. 50–200 lg/ml The mRNA expression of proteins was analyzed by qPCR. The collagens from conditioned media and RAGE from membranes were investigated by Western immunoblot. The TGF-b level was determined by ELISA. Parallel experiments with unglycated BSA were performed. The concentration of 50 lg/ml AGE-BSA up- regulated the mRNA and protein expression of RAGE, TGF-b and both procollagens, but at higher levels this effect was dimin- ished. Therefore the treatment with AG was performed at this concentration. After 12 hours of exposure, the relative expression ratio (R) of RAGE and TGF-b increased by 6.6 respectively 5.16 fold in the presence of 10 lM AG. At higher AG concentrations, R values decreased, but at 100 lM AG they were 4.5 respectively 3.86. The procollagen a2(I) and a1(III) R enhanced with the increase of the AG level. After 24 hours, R for all investigated genes downregulated with the increase of drug concentration compared to control. It seems that TGF-b is regulated by RAGE-AGE interaction and plays a pivotal role in mediating collagen deposits in diabetes, and AG is efficient only at higher concentration.

P8–154 Peptide-oligonucleotide conjugates as potential ribonucleases I. Serpokrylova, L. Koroleva and V. Silnikov Laboratory of organic chemistry, Institute of chemical biology and fundamental medicine, Novosibirsk, RUSSIA

Protein 3-nitrotyrosine (3-NT) has been recognized as an impor- tant biomarker of oxidative/nitrosative stress associated with inflammatory and degenerative diseases, and biological aging. Analysis of protein-bound 3-NT in vivo continues to represent a challenge since it frequently does not accumulate in high yields and/or targets low abundant proteins. Here, we describe an approach for fluorescent tagging and sequence-specific quantita- tion of peptide-bound 3-NT residues based on the reduction to 3-aminotyrosine followed by selective reaction with 4-(amino- methyl)benzenesulfonic acid (ABS) in the presence of K3Fe(CN)6 to form a highly fluorescent 2-phenylbenzoxasole product. Syn- thetic 3-NT peptide (0.005–1 lM) upon reduction with 10 mM sodium dithionite and tagging with 2 mM ABS and 5 lM K3Fe(CN)6 in 0.1 M Na2HPO4 buffer (pH 9.0) was converted with > 95% yields to a single fluorescent product incorporating 2 ABS molecules per 3-NT residue with fluorescence excitation and emission maxima at 360 and 480 nm, respectively, and a flu- orescence quantum yield of 0.77, based on reverse-phase HPLC with UV and fluorescence detection, fluorescence spectroscopy and LC-MS/MS analyses. This derivatization was successfully tested for quantitative analysis of in vitro Tyr nitration in tryptic digests of a model protein, rabbit muscle phosphorylase b, and in a complex mixture of proteins from cultured C2C12 cells either spiked with nitropeptides or exposed to peroxynitrite, with detec- tion limit of ca. 1 pmol 3-NT by fluorescence spectrometry. LC- MS/MS analysis of ABS tagged peptides revealed that the fluo- rescent derivatives undergo efficient backbone fragmentations, allowing for sequence-specific characterization of protein Tyr nitration. Fluorescent derivatization of protein 3-NT with ABS is useful for the visualization and quantitation of nitrated proteins separated by HPLC and gel electrophoresis, and for fluorescence microscopy analysis of nitrated proteins in tissue sections.

P8–156 Effect of fibrillar Ab1-42 on antioxidant enzymes and intracellular ROS in SK-N-MC cells H. Shaykhalishahi and Y. Razieh Biochemistry, Instiute of Biochemistry and Biophysics, Tehran, IRAN

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

The main purpose of our studies is the design of efficient artificial ribonucleases capable of cleaving RNA substrates and further development of specific RNA cleaving therapeutics for modula- tion of gene expression and inactivation of viral genomes. Syn- thetic ribonuclease consists of two parts. The first one is an oligonucleotide delivery, which is complementary to a segment of RNA target. And the second one is a functional group that is playing an important role in catalytic center of natural enzymes. Oligonucleotide conjugates with peptides and peptide-like mole- cules have a wide application in molecular biology in aim of cor- rection of biochemical processes. Several strategies are used for the preparation of oligonucleotide conjugates with peptides or other compounds containing functional groups. The considerable quantity of ways of conjugation is offered but only small part has found real application in practical synthesis. We decided to try a new strategy of construction of peptide-oligonucleotide con- jugate. We have therefore designed suitable phosphoramidite reagent that can be used during standard step in oligonucleotide assembly by phosphoramidite methods. For this purpose the new amidophosphite with anthracene residue was synthesized. This phosphoramidite was introduced in different positions of 18-mer Ab1-42 is the main constituent of senile plaques characterized in Alzheimer’s disease (AD). It is repeatedly demonstrated that aggregation of Ab is heavily involved in the pathogenesis of AD. On the other hand, it has been shown that oxidative stress is clo- sely linked to Ab cytotoxicity. In this study, we studied the cyto- toxic effect of Ab1-42 on the neuroblastoma SK-N-MC cell line at

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level of intracellular oxidative stress and antioxidant enzymes. To prepare the fibrillar Ab1-42, the peptide was dissolved in PBS and kept at 37(cid:2)C for 10 days. Treatment of SK-N-MC cells with Ab1- 42 (10 lM) led to nearly 50% reduction in cell viability deter- mined by MTT assay. We also measured the intracellular level of ROS in the cells treated with 10 lM Ab1-42 by using DCF fluo- rescence assay. Our data indicated that treatment of the cells with Ab1-42 (10 lM) caused an increase in the extent of intracellular peroxide radicals by 90%, relative to untreated control cells. Fur- treatment of SK-N-MC cells with 10 lM Ab1-42 thermore, increased the activity of superoxide dismutase enzyme by about 70% and decreased the activity of catalase by 36%, respectively, relative to untreated control cells. Numerous studies suggest that amyloid beta aggregates with different conformations might have dissimilar effects on the different neuroblastoma cell lines. How- ever, we postulate that collecting and comparing those data might be useful to explore, in more detail, mechanisms involved in Ab cytotoxicity.

of L-carnitine biosynthesis and uptake by mildronate results in facilitated glucose oxidation. The aim of the present study was to investigate, whether the long-term mildronate treatment could influence the blood glucose concentration, as well as prevent dia- betic complications in experimental model of type 2 diabetic Goto-Kakizaki (G-K) rats. G-K rats were treated with mildro- nate at doses of 100 and 200 mg/kg p.o. daily for 28 days. Plasma glucose level, lipid profile and insulin concentration were determined. Experimental isolated rat heart infarction model and isolated aortic rings were used to investigate effects of mildronate administration on the cardiovascular system. Nociceptive sensi- tivity was measured with a tail-flick test. Mildronate treatment decreased the L-carnitine concentration in rat plasma and gradu- ally decreased the blood glucose concentration in both the fasted and fed state. In isolated rat heart infarction experiment, in mildronate treated hearts the necrotic zone was reduced for about 30% as compared to untreated rat hearts. In addition, mildronate at a dose of 200 mg/kg ameliorated contractile responsiveness of G-K rat aortic rings to phenylephrine and pre- vented the loss of nociceptive sensitivity. In conclusion, results show for the first time that mildronate is protective in experimen- tal diabetic G-K rat model. These results indicate that mildronate treatment could be beneficial in diabetic patients.

P8–157 Use of cell permeable RANK receptor inhibitor for suppression of bone loss J. H. Shin, H. K. Choi, K. H. Kim, J. Y. Huh, S. A. Lee and S. Y. Lee College of Natural Sciences Life Science, Ewha Womans University, Seoul, SOUTH KOREA

P8–159 The analysis of the products of lipid peroxidation in erythrocytes as possible markers of Alzheimer disease A. Skoumalova1, E. Topinkova2 and J. Wilhelm1 1Department of Medical Chemistry and Biochemistry, Charles Uni- versity 2nd Faculty of Medicine, Prague, CZECH REPUBLIC, 2Department of Geriatrics, Charles University 1st Faculty of Medi- cine, Prague, CZECH REPUBLIC

Regulation of osteoclast (OC) formation and function is a key to understanding the pathogenesis of skeletal disorders and to devel- oping pharmaceutical agents that arrest the progression of disor- ders such as osteoporosis. Gene targeting studies have shown that RANK plays a critical role in OC differentiation and func- tion and blocking RANK is a potential strategy for preventing bone destruction. Here we developed a cell-permeable inhibitor, termed the RANK receptor inhibitor (RRI), which targets a spe- cific motif of RANK. The RRI peptide blocked the RANKL- induced OC formation and resorptive function and caused OC apoptosis. The inhibitory effects of the peptide on OCs were due to a disruption of the actin cytoskeleton, which resulted from impaired downstream signaling of RANK linked to Vav3, Rac1, and Cdc42. On the other hand, the RRI peptide did not affected CD40 and IL-1b downstream signaling that are closely related to the formation of OC and the important components in the immune system. Thus, this study reveals a novel RANK-initiated regulatory pathway, which leads to OC maturation and mainte- nance of cell survival. These results could be potentially used to develop selective therapeutic agents for the treatment of osteopo- rosis and other bone diseases without jeopardizing the immune system.

P8–158 The beneficial effects of inhibitor of L-carnitine biosynthesis in experimental rat model of type 2 diabetes E. Skapare1, R. Vilskersts2, B. Svalbe3 and M. Dambrova3 1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Riga Stradins University, Riga, LATVIA, 2Faculty of Medicine, University of Latvia, Riga, LATVIA, 3Laboratory of Pharmaceuti- cal Pharmacology, Latvian Institute of Organic Synthesis, Riga, LATVIA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Alzheimer disease (AD) is the most common form of dementia. Free radicals play an important role in its pathogenesis. Even though the source of increased free radical production is still unclear, the toxic effect of amyloid b (Ab) has been discussed. Ab is formed from the membrane-bound precursor. In AD patients Ab-amyloidosis has been found in senile plaques and in the brain blood vessels and is accompanied with increased free radical production. Erythrocytes in the brain blood vessels inter- act with Ab and can bind it. Free radicals initiate the lipid perox- idation process in the erythrocyte membrane leading to the production of peroxidation products, lipofuscin like pigments (LFP). The fluorescent characteristics of these pigments differ in various pathologies. In our previous study, we found the specific LFP in erythrocytes of demented dogs that are used as a model for AD. In this project, we have characterized the fluorescent products in erythrocyte membranes of AD patients. The erythro- cytes of the patients and the age-matched controls were analyzed by means of tridimentional fluorescence spectroscopy. Since we found an increased production of LFP in the group of patients, we analyzed the synchronous spectra of the LFP. Synchronous spectra make it possible to compare the changes in the composi- tion of LFP. Our results indicate that the presence of the specific products of lipid peroxidation in erythrocytes accompany the oxi- dative stress in Alzheimer disease. It could be used for the diag- nosis and monitoring of the therapy. Acknowledgement: This work was supported by GACR (grant 220 032). Type 2 diabetes is a widespread disease characterized by elevated blood glucose level and insulin resistance, increased risk of car- diovascular pathologies and predisposition to peripheral neuropa- thy. It has been shown recently that inhibition of the pathways

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P8–160 Hepatic CYP3A and PXR expression after long- term treatment with GnRH analogs R. Skowronek, P. Czekaj, A. Suszka-Switek, A. Bryzek, D. Plewka, E. Czech and R. Wiaderkiewicz Department of Histology, Medical University of Silesia, Katowice, POLAND

effects on calcium-induced MPT. Long-term preincubation of mitochondria with glucose metabolites had no effect. Conclusion: We have confirmed the potency of methylglyoxal as MPT inhibitor. High concentrations of glucose and glucose 1-phosphate can also inhibit MPT, which was not described pre- viously. It is possible that the inhibition of MPT by these sub- strates results in accumulation of ROS within mitochondria and contributes to the development of diabetic complications. The ROS production in mitochondria under these conditions is cur- rently investigated.

P8–162 Cholesterol, lanosterol, and ergosterol attenuate the membrane association of LL-37(W27F) and temporin L R. Sood and P. Kinnunen Medical Biochemistry, University of Helsinki, Helsinki, FINLAND

In the present study, we addressed the effects of cholesterol, ergosterol, and lanosterol on the membrane association of two structurally and functionally diverse AMPs viz. LL-37(F27W) and temporin L (TemL) using fluorescence spectroscopy. Sterol concentration dependent effects on the membrane association of these peptides were observed. At XSterol = 0.5 cholesterol was most effective in reducing the membrane intercalation of both LL-37(F27W) and TemL, the corresponding efficiencies of the three sterols decreasing as cholesterol > lanosterol ‡ ergosterol and cholesterol > lanosterol > ergosterol. It is conceivable that part of the selection pressure for the chemical evolution of cho- lesterol may have derived from the ability to protect the AMP- secreting host cell from the membrane damaging action of the antimicrobial peptides.

Long-term therapy by synthetic analogs of GnRH leads to desen- sitization of hypophysis, and results in repression of hypotha- lamic-pituitary-gonadal axis. The changes in levels of pituitary and sex hormones can modify expression of hormone-dependent cytochromes P450 and PXR. The aim of the study was to investi- gate the neuroendocrine regulation of the expression of hormone- dependent CYP3A subfamily and to indicate, whether, a long- term administration of GnRH receptor agonist and antagonist to adult female rats influences CYP3A and PXR expression and localization within the liver, and also how quick and in what degree these changes are reversible. The study was performed on Sprague–Dawley adult rat females administered intraperitoneally with dalarelin (a new GnRH receptor agonist) and cetrorelix (antagonist) (6 lg/kg of b.w., respectively). The liver was taken after 1, 2, 3 months of treatment and 1, 2, and 4 weeks after fin- ishing administration. Localization of CYP3A isoforms and PXR receptor within the liver acinus was analyzed immunohistochemi- cally. Then, CYP3A1, CYP3A2, CYP3A9 and PXR mRNA (RT-PCR) and proteins (Western blotting) were identified and quantified by densitometry. Our results showed that dalarelin and cetrorelix, in different way change expression pattern, but not localization of the CYP3A subfamily members, influencing the GnRH receptors and hypothalamic-pituitary-gonadal axis. It can be potentially associated with changes in the metabolism rate of drugs administered parallely with GnRH analogs, activation of some xenobiotics and toxic effects in organs. Thus, the choice of a type of GnRH analogs for individualized therapy should be carefully considered.

P8–163 5-Fluoro-2¢-deoxyuridine induces fetal hemoglobin production in human erythroid precursor cells from healthy and thalassemic donors P. Spyrou and M. Kleanthous Nephrology & Genetics, Thalassemia, Nicosia, CYPRUS

P8–161 The influence of glucose metabolites on mitochondrial permeability transition J. Skrha Jr., J. Gall, R. Buchal, E. Sedlackova and J. Platenik Institute of Medical Biochemistry, First Faculty of Medicine Charles University Prague, Prague, CZECH REPUBLIC

that could agents increase c-globin

Compounds with nucleoside analog chemical structure were found to affect DNA synthesis and transcription due to their structure similarity with DNA bases. Compounds like 5-azacyti- dine were found to activate the expression of the human c-globin gene by causing hypomethylation or down regulate it. The pur- pose of this study focuses on finding nucleoside analogs and hy- pomethylating gene expression for therapeutic purposes for the treatment of b-thalas- semia. Among the compounds screened, 5-fluoro-2¢deoxyuridine which is an anticancer drug showed a promising effect on HbF induction. 5-Fluoro-2¢deoxyuridine increased the c-globin pro- moter activity in MEL transfected cells and HbF production in erythroid liquid progenitor cultures in normal and thalassemic donors measured by HPLC. HbF induction was also combined with an increase of the percentage of F cells and c-mRNA levels. The increase of these parameters clearly shows its potent HbF inducing activity that makes it a candidate target for further study.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Introduction: Mitochondrial production of reactive oxygen spe- cies (ROS) due to upregulated glucose oxidation supposedly plays a crucial role in pathogenesis of long-term diabetic compli- cations. We investigate the effect of commercially available glu- cose metabolites on the mitochondrial permeability transition pore (MPT) and mitochondrial ROS production. Methods: Mitochondria were isolated from rat liver. The inner membrane potential was measured by fluorescent probe JC-1, and mitochondrial swelling (MPT) was detected as light scatter. Mitochondria were energized by succinate with rotenone (com- plex I inhibitor). The effects of glucose, glucose 6-phosphate or glucose+ATP+Mg, fructose 6-phosphate, glucose 1-phosphate (each 5 and 30 mM), glyceraldehyde and methylglyoxal (each 1 and 6 mM) on calcium-induced MPT were measured. Further- more, mitochondria were preincubated with the glucose metabo- lites for 5 hours and processed in the same way. Results: Lack of effect of glucose 6-phosphate or glu- cose + ATP + Mg indicated absence of glucokinase (known to affect the pore) in our mitochondrial preparations. From all the glucose metabolites tested, only 30 mM glucose, 30 mM glucose 1-phosphate and 6 mM methylglyoxal had certain inhibitory

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Poster Presentations

P8–164 Specificity of secreted proteomes from cardiac stem cells and neonatal myocytes M. Stastna1,2, I. Chimenti3, E. Marban4 and J. Van Eyk1 1Johns Hopkins University, Medicine, Baltimore, MD, USA, 2Institute of Analytical Chemistry of the ASCR, Brno, CZECH REPUBLIC, 3Medicine, University La Sapienza, Rome, ITALY, 4Cardiology, Cedar-sinai Medical Center, Los Angeles, CA, USA

release kinetics in vitro. The nanoemulsion was prepared using 5% Aerosol OT in eucalyptus oil. This system was optically transparent and allowed a solubilization of water, polar organic compounds, peptides, proteins, etc. Normally, after addition of an aqueous peptide solution and vigorous shaking for several sec- onds, a transparent DSIP-containing nanoemulsion was obtained. The sizes of nanocontainers loaded with the peptide were deter- mined using photon correlation spectroscopy with He-Ne laser. The mean diameter of nanoemulsion particles was found to be in the range of 2–35 nm depending on water content in the system. To mimic the system stability in vivo, the release kinetics of the peptides encapsulated in nanocontainers in vitro using a dialysis of the peptide samples through semipermeable membrane was studied. Peptide concentration in dialysates was determined by HPLC. As found, DSIP release from nanoemulsion system was significantly slower compared to that one from aqueous solution. An introduction of polymer compounds (chitosan, DEAE-dex- tran) provided an additional prolongation of the peptide release. DSIP entrapment into nanoemulsion significantly increased its storage stability. Thus, the method for DSIP entrapment in reverse nanoemulsion was developed and the peptide slowdown release using in vitro model was shown.

P8–166 A new approach in the use of GlyT1 inhibitors for treatment of schizophrenia G. Szabo´ Molecular Pharmacology, EGIS PLC, Budapest, HUNGARY

factors were (i)

Adult heart has a limited capability for endogenous renewal of damaged tissue after events such as heart attack or myocardial infarction. Stem cell-based therapy is emerging efficient approach for myocardial regeneration. Cardiac stem cells create a small population of resident stem cells in a heart from which cardiac cells can be derived in case of injury. There are increasing proofs that stem cells secrete proteins into the vicinity of injury that can modulate regeneration in paracrine/autocrine manner. We ana- lyzed proteins in media of cardiac stem cells (CSCs) isolated from cardiac tissue specimens of 12 to 16 weeks old rats and compared them to secretome of neonatal rat ventricular myocytes (NRVMs). Media conditions were optimized to allow minimize serum addition without inducing cell death. The media were col- lected after 48 hours, concentrated and proteins were separated by reversed phase HPLC prior to mass spectrometry. Over 80 non redundant proteins in total were identified. Of those, 48.2% and 22.4% of proteins were NRVM-specific and CSC-specific, respectively and majority of proteins were integral plasma mem- brane or known secreted proteins. Our selection criteria for can- didate paracrine/autocrine robust protein identification, (ii) cell specific and not in control media, (iii) known to be secreted. 10 proteins met these criteria; adrenomed- ullin and CTGF were previously suggested to be potential stem cell paracrine factors, but others are novel. They were further tested to determine their effect on CSC proliferation. 3 proteins, myocyte-specific ANP and CTGF and stem cell-specific ST2, were found to affect CSC proliferation.

P8–165 Delta-sleep inducing peptide entrapped into reverse nanoemulsions: preparation and study of in vitro release T. Sukhanova1, L. Filatova2, E. Efremov3, I. Prudchenko3, S. Uglanova2, N. Klyachko2 and E. Markvicheva1 1Laboratory Polymers for Biology, Institute of Bioorganic Chemis- try, Moscow, RUSSIA, 2Chemical Department, Lomonosov Mos- cow State University, Moscow, RUSSIA, 3Laboratory of Peptide Chemistry, Institute of Bioorganic Chemistry, Moscow, RUSSIA

Schizophrenia is one of the most prevailing diseases in the devel- oped countries. According to the latest hypothesis it is caused by the hypofunction a glutamatergic system. In this respect the most important receptors are the NMDA receptors (NMDARs), the increased activity of NMDARs can ameliorate some schizo- phrenic symptoms. NMDARs have two amino acid co-agonists: the glutamate and the glycine. Local elevation of glycine level can enhance the effect of glutamatergic neurotransmission. One of the function of glycine transporter type 1 (GlyT1) is the elimi- nation of glycin from the synaptic cleft, so the inhibition of GlyT1 can ameliorate the hypofunctioned glutamatergic neuro- transmission. Moreover, the direct increase of glycine level by glycine administration has beneficial effect in schizophrenic patients. However, GlyT1 occurs not only in the CNS but in some peripheral tissues as well; the erytrocytes seem to be the most important of them. Our hypothesis is that the inhibition of the erytrocyte GlyT1 results in a serum glycine level increase, the glycin enters the brain and strengthens the glutamatergic neuro- transmission. We experimentally proved that NFPS (a GlyT1 inhibitor) increases the serum glycine level both in vitro and in vivo. On the base of the results we suggest the use of GlyT1 inhibitors that do not enter the brain, so decreasing the CNS side effects.

P8–167 Detection optimization and analysis of cell-free fetal nucleic acids in maternal peripheral blood for non-invasive prenatal diagnostics T. Szemes1, G. Minarik2, B. Vlkova2, P. Celec3 and J. Turna1 1Department of Molecular Biology, Comenius University – Faculty of Natural Sciences, Bratislava, SLOVAK REPUBLIC, 2Geneton s.r.o, Bratislava, SLOVAK REPUBLIC, 3Department of Patho- logical Physiology, Comenius University, Faculty of Medicine, Bra- tislava, SLOVAK REPUBLIC

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Delta-sleep inducing peptide (DSIP, TrpAlaGlyGlyAspAlaSer- GlyGlu) occurs in various organs, tissues and body fluids show- ing a wide spectrum of functions, such as pain modulation, stress response, cardiac cycle normalization, antiepileptic activity. DSIP also exhibits antioxidant properties and considerably inhibits a stress-induced elevation of lipid peroxidation intensity as well as free radicals content in a brain and other tissues. Peripheral administration of DSIP in a small dose significantly increases ani- mal resistance to acute emotional stress preventing cardiovascular disturbances. Among DSIP disadvantages there are its low chem- ical stability and fast biodegradation in a human body. Entrap- ment of bioactive molecules in various vehicles (nanoparticles, nano- and microcapsules, micelles etc) protects them from bio- degradation providing a sustained drug release. That is why micellar systems and nanoemulsions can be considered as promis- ing vehicles for various proteins and peptides. The aim of the study was to develop a technique for DSIP entrapment in reverse nanoemulsion (water/oil), to study the peptide stability and its Prenatal diagnosis of genetic disorders is currently performed on fetal cell samples collected by chorionic villus sampling or amnio-

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Abstracts

P8–169 Kinetics of the reduction of phenoxazine and phenothiazine dyes by NADH in aqueous solution O. Tacal and I. Ozer Department of Biochemistry, Hacettepe University School of Pharmacy, Ankara, TURKEY

centhesis. These invasive approaches to sampling of fetal genetic material bear a small but not negligible risk to both subjected mother and child. Non-invasive procedure based on analysis of extracellular (cell-free) nucleic acids present in maternal periph- eral blood appears to be a very promising strategy for analysis of fetal genetic material allowing early testing and determination of its genetic status while eliminating the risk. In general, the intro- duction of non-invasive analysis into routine clinical practice is preceded by rigorous optimization of relevant methods accompa- nied with robust evaluation studies. Currently, we focus on opti- mization of fetal gender rapid and reliable method for determination. For this purpose, we collected blood samples of pregnant women at all three trimesters after their informed con- sent, and extracted cell-free nucleid acids from maternal plasma fraction. We used real-time PCR based assay for detection of Y-chromosome specific sequences present in multiple copies per genome. Furthermore, we evaluate a real-time PCR assay for determination of fetal rhesus D (RhD) status of RhD negative pregnant women in order to perform non-invasive fetal RhD genotyping.

P8–168 Derivatives of tunicamycin as effective inhibitors of classical swine fever virus B. Szewczyk1, E. Krol1, I. Wandzik2, W. Szeja2 and G. Grynkiewicz3 1Molecular Virology, University of Gdansk, Gdansk, POLAND, 2Organic Chemistry, Silesian Technical University, Gliwice, POLAND, 3Organic Chemistry, Pharmaceutical Institute, Warsaw, POLAND

The kinetics of the reduction of phenoxazine and phenothiazine dyes [meldola blue (MB), nile blue (NB), toluidine blue O (TBO), methylene blue (MetB), thionine (TH)] by NADH in aqueous solution were studied spectrophotometrically. The reactions involved intermediate dye-NADH complex formation. In the case of MB and TH the formation of the complex was accompanied by a change in dye absorbance, pointing to charge transfer inter- action between donor and acceptor species. The reduction of TBO, MetB and NB proceeded without intermediate changes in absorptivity. The dissociation and turnover rate constants (Kd and k) for the dye-NADH complexes were 0.27 ± 0.040 mM and 66 ± 3.0 min-1 (for MB), 2.0 ± 0.45 mM and 5.4 ± 1.4 min-1 (for TH), 0.63 ± 0.18 mM and 0.77 ± 0.15 min-1 (for 0.42 ± 0.05 mM and 0.16 ± 0.03 min-1(for MetB), TBO), 0.52 ± 0.08 mM and 0.10 ± 0.02 min-1 (for NB). Overall, there was a linear correlation between the redox potentials of the dyes and the reduction rate constants. Dye reduction in solution was characterized by up to 94,000-fold lower turnover rate constants than reported for electron transfer between NADH and surface- immobilized dyes in electrocatalytic NADH oxidation. Compari- son of the redox behavior of free and immobilized dyes suggested that electron transfer on electrode surfaces must be rate-limited by physical phenomena.

P8–170 Inhibition of microtubule polymerisation to block epithelial-mesenchymal transition in an in vitro model of posterior capsular opacification F. Tholozan1, B. Lou2 and R. Quinlan1 1School of Biological and Biomedical Sciences, Durham University, Durham, UK, 2Zhongshan Ophthalmic Centre, Sun Yat-Sen Uni- versity, Guangzhou, CHINA

Classical swine fever virus (CSFV) is often used as a surrogate model to elucidate the role of envelope glycoproteins of hepatitis C virus (HCV). These two viruses are homologous in genomic organization, replication and protein function. Glycoproteins E2, E0 (Erns) and E1 of CSFV play a major role in the initial stages of viral infection. They are detected on the external part of viral particles. It has been found that some glycosylation inhibitors, such as tunicamycin, which act at the early stages of glycan chain processing, can influence not only glycosylation, but also the sta- bility of E2 and E0 glycoproteins, effectively inhibiting the for- mation of glycoprotein complexes and the yield of the virus. Because tunicamycin is relatively toxic to the cells, we have syn- thesized a number of inhibitors mimicking tunicamycin structure or a part of this structure. The main aim of this work was to study the influence of tunicamycin derivatives on penetration and propagation of CSF virus, and on maturation of viral envelope glycoproteins. To this end we have investigated the formation of glycoprotein dimers by immunoperoxidase monolayer assay and by immunoblotting (Western blotting). Some of inhibitors effec- tively arrested viral growth without significant toxicity for mam- malian cells. These inhibitors were further studied in order to elucidate the molecular mechanism of their antiviral effect using different mammalian and insect cell lines and it has been found that most of them inhibit N-glycosylation at the stage of glycan modification characteristic for mammalian cells. These results for CSFV were used in the initial characterization of the effect of the inhibitors on recombinant HCV glycoproteins.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Combretastatin-A-4-phosphate (CA4P) is an inhibitor of micro- tubule polymerisation that has been shown to disrupt vasculature within tumors (Quan et al., Int.J.Cancer 2008; 122:1730–1737). We tested CA4P as a potential inhibitor of lens epithelial cells epithelial-mesenchymal transition (EMT) during posterior cap- sular opacification (PCO), a common complication of cataract surgery. Using alpha-tubulin immunofluorescence and immuno- blotting, a 30 minutes exposure to 1mM CA4P was sufficient to effectively depolymerise microtubule networks in both in vitro cultured immortalised bovine lens epithelial cells and primary bovine lens epithelial cells grown in vitro on their natural base- ment membrane, the lens capsule. This effect persisted up to 48 hours after CA4P exposure. Lens epithelial cell proliferation was also affected, as assayed by Ki67 immunofluorescence, MTT-based cell viability assays and cell cycle analysis by flow cytometry. Hallmarks of EMT, namely loss of cell polarity and migratory behaviour, were also investigated in primary bovine lens epithelial cells grown on the lens capsule and were decreased by CA4P exposure, although cell multilayering seemed unaf- fected. These results identify a potential novel therapeutic appli- an already well-characterised compound, with cation of

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Poster Presentations

significant effectiveness being achieved for up to two days after a single 30 minutes exposure.

P8–171 New non-synthetic molecules are able to inhibit apoptosis M. V. Traversa1, S. Carelli1, G. Giuliani2, A. M. Di Giulio3 and A. Gorio3 1Dept. Medicine Surgery and Dentistry, University of Milan, Milan, ITALY, 2Research and Development, Giuliani Pharmaceuti- cals, Milan, ITALY, 3Dept. Medicine Surgery and Dentistry, University of Milan, Milan, ITALY

ERB-hcAb, a human, ‘compact’ antibody, in which two Erbicin molecules are fused to the Fc of a human IgG1. Both ERB-hRN- ase and ERB-hcAb severely inhibit the growth of ErbB2-positive tumours and target an ErbB2 epitope different from that recog- nized by Herceptin. Preliminary studies suggest that they are not immunogenic and do not display cardiotoxic effects. To define and implement the antitumor potential of Erbicin-Derived-Immu- noAgents (EDIA), we have extensively characterized for the first time the interactions of EDIA and Herceptin with the extracellu- lar domain (ECD) of ErbB2 by three different methods. We found that EDIA bind soluble ECD with a lower affinity than that measured for the native antigen on tumor cells. Herceptin instead shows a higher affinity for soluble ErbB2-ECD. Accord- ingly, ErbB2-ECD abolished the in vitro antitumor activity of Herceptin with no effects on that of EDIA. These results suggest that the fraction of immunoagent neutralized by free ECD shed into the bloodstream is much higher for Herceptin than for EDIA, which thus could be used at lower therapeutic doses com- pared to those employed with Herceptin. The localization through epitope mapping of the antigenic peptide segments rec- ognized by EDIA, different from those recognized by Herceptin, could lead to the identification of novel epitopes on ErbB2 to be used as potential therapeutic targets to mitigate anti-ErbB2-asso- ciated cardiotoxicity. Overall, EDIA are immunoagents of high potential interest for immunotherapy of ErbB2-positive carcino- mas. Acknowledgements: The support to our research of Biotecnol SA, Lisbon, and AIRC are gratefully acknowledged

Interestingly, combined treatment shape.

P8–173 Assessments of enhancers and coating substrate on the efficiency of substrate- mediated transfection delivered by branched polyethylenimine W. C. Tseng, K. W. Chang, C. H. Tang and L. Y. Su Chemical Engineering Department, National Taiwan University of Science and Technology, Taipei, TAIWAN

Hair exerts a range of functions including thermoregulation, physical protection, sensory activity. Mature and actively grow- ing hair follicles become anchored in the subcutis, and regenerate by spontaneous repetitive cycles of growth (anagen), apoptosis- driven regression (catagen), and quiescence (telogen). We focused our study on investigating the effect of new therapeutic molecules capable of counteracting the regression phase. We have tested our working hypothesis on HFDPC cells, grown in culture in Follicle Dermal Papilla Growth Medium, and apoptosis was induced by 24 hours incubation with 1 lM staurosporin. This treatment resulted in a marked activation of caspase-3 accompa- nied by cytoskeletal degradation, nuclear blebbing, and cellular fragmentation. The addition of spermidin or rutine in the micro- molar range concentration reduced staurosporin-induced caspase activity by over 50%, when the two agents were added simulta- neously, equal level of caspase-3 inhibition was achieved with concentration 10-fold lower. Zeaxantine alone was ineffective, however, when added to the combined spermidin and rutine treatment, the staurosporin-induced caspase activity was almost totally counteracted and the enzymatic activity was significantly reduced. This combined treatment was also effective in prevent- ing staurosporin-mediated cellular damage. The extent of cell loss was greatly reduced and there was a total preservation and nor- mal distribution of actin-tubulin cytoskeleton with normal cellu- lar also our counteracted caspase-3 over-expression induced by staurosporin. In conclusion, these experimental molecules can counteract HFDPC cells apoptosis and may represent an effective prevent- ing treatment for the catagen phase of the hair bulb life cycle.

P8–172 Binding of human immunoagents and herceptin to the ErbB2 receptor F. Troise1, V. Cafaro2, C. Giancola3, G. D’Alessio2 and C. De Lorenzo2 1Biotecnologie mediche, CEINGE, Naples, ITALY, 2Department of Structural and Functional Biology, University of Naples ‘Federico II’, Naples, ITALY, 3Department of Chemistry, University of Naples ‘Federico II’, Naples, ITALY

resistant is

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Gene delivery is an important step in the preparation of cell arrays. A highly efficient delivery system is required to modulate the cellular functions for analysis. In this study, branched poly- ethylenimine (bPEI), a very potent and cost-effective polymer for transfection, was used as a primary carrier for gene delivery. The percentage of cells expressing green fluorescent protein was used to indicate the transfection efficiency. Various substrates were coated on slides for the examination of cell growth and transfec- tion efficiency. Different enhancers, such as polyethylene glycol, dextran and gelatin, were combined with the transfection reagent in an attempt to increase the transfection efficiency. For the slides coated with gelatin typeB that could provide better cell growth, the transfection efficiency was observed to strongly depend on the amount of plasmid contained within the transfec- tion reagent in a dosage response. In the absence of any enhanc- ers, the efficiency reached 20% whereas in the presence of polyethylene glycol of MW 2000 or gelatin typeA, the efficiency could be further enhanced to around 70%. When bPEI was replaced with dextran-grafted bPEI, a more efficient carrier for transfection in cell culture, very minimal levels of GFP positive cells were detected, suggesting that different factors might exist in transfecting cells in culture and those in microarrys for PEI-based vectors to achieve high efficiency. This study showed that using bPEI combined with an enhancer as a gene delivery system could efficiently modulate cellular functions for the preparation of cell arrays. Over-expression of ErbB2 (Her2/neu) receptor is associated with progression of malignancy of breast cancer, and is a sign of a poor prognosis. Herceptin, a humanized anti-ErbB2 antibody, has proved to be effective in the immunotherapy of breast carci- it can engender cardiotoxicity and a high noma. However, fraction of breast cancer patients to Herceptin treatment. We have engineered novel fully human anti-ErbB2 immunoagents: Erbicin, a human scFv; ERB-hRNase, a human immunoRNase made up of Erbicin fused to a human RNase;

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three-dimensional structures of the E. coli and newly determined human protein were used to interpret structure/function relation- ships for the mutations in the human isoform. Acknowledgement: Supported by Grants: GAUK 257540 54007, MSM0021620806, 1M6837805002.

P8–174 Effects of Thymus sipyleus and taurine on liver antioxidant status in Ehrlich ascites solid tumor bearing mice G. Turna, N. Kilic, S. Sari and Z. Yildirim Medical Biochemistry, Gazi University Faculty of Medicine, Ankara, TURKEY

P8–176 Chemotherapy of Duchenne’s muscular dystrophy Y. Urade, M. Hayashi, T. Maruyama, S. Kamauchi, I. Mohri and K. Aritake Molecular Behavioral Biology, Osaka Biosciense Institute, Suita, JAPAN

on based novel H-PGDS

In this study, taurine (200 mg/kg/day) and the methanol extract of Thymus sipyleus Boiss. subsp. sipyleus var. Sipyleus (an ende- mic species in Ayas region in Ankara,Turkey) (1.5 g/kg/day) were injected to Swiss albino mice. The effects of these substances on liver advanced oxidation protein product (AOPP), malondialde- hyde (MDA) and glutathione (GSH) levels and the superoxide dismutase (SOD) activities were studied. Thirty-six (36) animals were divided into six equal groups as healthy control, tumor con- trol, thyme treatment, thyme protective, taurine protective and taurine treatment. The protective groups received these sub- stances for 17 days starting from the 1st day after the tumor injection. The treatment groups received these substances for 7 days starting from the 11th day after tumor injection. AOPP level in the taurine treatment group decreased significantly when compared to the tumor control group (p < 0.05). MDA level in the taurine protective group decreased significantly when com- pared to the tumor control group (p < 0.05). GSH level in the taurine protective group was insignificantly higher than the tumor control group (p > 0.05). SOD activity was significantly higher in the thyme treatment group than the tumor control group (p < 0.05). In conclusion, taurine treatment significantly decreased the increased MDA and AOPP levels due to the oxida- tive stress. However, GSH levels and SOD activities were not affected significantly. The methanol extract of Thymus sipyleus increased the SOD activities. However MDA, AOPP and GSH levels were not changed.

Duchenne muscular dystrophy (DMD) is an X-linked muscular abnormality caused by the loss of dystrophin and is one of the most gravely genetic disorders. We have recently found that hematopoietic prostaglandin (PG) D synthase (H-PGDS) was induced in grouped necrotic muscle fibers in DMD patients (Oki- naga T. et al., Acta Neuropathol. 2002; 104: 377–384). We devel- oped the X-ray inhibitors crystallographic analysis of human H-PGDS complexed with its prototype inhibitor, HQL-79 (Aritake K. et al., J. Biol. Chem. 2006: 281: 15277–15286). In this study, we developed a novel therapy for DMD by inhibition of H-PGDS. H-PGDS was local- ized in the necrotic muscle fibers and accumulated macrophages in mdx mice. Oral administration of H-PGDS inhibitors for 5 days prevented the expansion of muscular necrosis in an mdx mouse model, as measured by X-ray computed tomography (CT) imaging enhanced by non-ionic contrast media. The treatment with H-PGDS inhibitors also decreased the expression of mRNAs of pro-inflammatory cytokines. These results indicate that PGD2 produced by H-PGDS plays important pathological roles on the expansion of muscle necrosis. H-PGDS inhibitor also accelerated the accumulation and activation of macrophages in the necrotic area. These results indicate that PGD2 produced by H-PGDS is involved in the expansion of muscle necrosis in DMD and that inhibition of H-PGDS is a novel therapy for DMD.

P8–175 Impact of mutations found in the hydroxymethylbilane synthase gene on the biochemical and enzymatic protein properties D. Ulbrichova1, M. Hrdinka1, V. Saudek2 and P. Martasek1 1Pediatrics, Charles University First Faculty of Medicine, Prague 2, CZECH REPUBLIC, 2Laboratory of Molecular Pathology Institute of Inherited Metabolic Disorders, Charles University First Faculty of Medicine, Prague 2, CZECH REPUBLIC

P8–177 Polymeric carriers covalently bound with 5¢-triphosphates of nucleoside analogs for reduced cytotoxicity and enhanced drug activity S. Vasileva, D. Konevetz, O. Zakharova, N. Lukyanchuk and V. Silnikov Laboratory of organic synthesis, Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, RUSSIA

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Nucleoside analogs remain the first-line antiviral chemotherapeu- tic agents. Nucleoside analogs treatment is associated with severe toxicity and acquirement of viral and metabolic resistance. Administration of active phosphorylated drugs will result in bypassing the first step of intracellular activation and faster intracellular accumulation of NTP. Therefore, we face an unmet medical need for the improvement of the drug efficacy and bio- distribution through delivery of activated-form nucleoside ana- logs to the site of treatment. Different polymeric drug delivery systems have been introduced and evaluated in recent years. Nanoparticulate drug carriers (microgels, nanogels) were devel- oped for the delivery of nucleoside analogs in their active phos- phorylated form [1]. ionic complexes are In these systems formed. However, known successful applications of NTP only include covalent phospholipid conjugates of anti-HIV drug, 3¢- Acute intermittent porphyria (AIP) is an inborn autosomal domi- nant disorder. The underlying cause is a defect in the gene coding for hydroxymethylbilane synthase (HMBS). Diagnosis of AIP is crucial for preventing life-threatening, acute attacks among both symptomatic and asymptomatic carriers. HMBS is the third the heme biosynthetic pathway. Two isoenzymes, enzyme of 42 kDa housekeeping and 40 kDa erythroid-specific, are indepen- dently expressed. HMBS isoforms from several different species have been studied and their enzymatic and kinetic properties have been described. The crystallographic structures of HMBS from E. coli and human have been determined. The aims of this study were to identify the gene defects in each of the newly diag- nosed AIP patients and their families and to establish their struc- ture/function consequences. We report seven mutations, four previously described and three novel mutations. To establish the effects of mutations on enzyme function, biochemical character- ization of the expressed normal recombinant and mutated pro- teins was performed. Prokaryotic expression of the HMBS mutant alleles revealed that, with the exception of one, all muta- tions produced little, if any, enzymatic activity. Moreover, the

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versidad Michoacana de San Nicola´s de Hidalgo, Morelia, MEXICO

carriers with covalently

azidothymidine (AZT) demonstrating a higher bioavailability and lower toxicity than AZT [2]. In this work we propose drug deliv- ery systems on the base of linear polyethylenimine (MW 25 kDa) with various percentages of cholesterol modifications (0.5–5%) and others polymeric and nanoparticulate carriers covalently bound with NTP. We have synthesized polymeric carriers cova- lently bound with fluorescein-labelled UTP, AZT-TP, and fluo- bound AZT-TP. rescein-labelled Intracellular delivery of compounds was shown by fluorescent microscopy. Antiviral effect of obtained conjugates is under study. Acknowledgement: This work was supported by Russian Fund for Fundamental Research, Grant1. 07-04-00990-a` , 08-04-90038. References: 1. E. Kohli, H. Y. Han, A. D. Zeman and S. V. Vinogradov. J Control Release 2007; 121: 19–27. 2. S. L. Morris-Natschke, K. S. Ishaq and L. S. Kucera. Curr Pharm Des. 2003; 9: 1441–1451.

Different bacterial strains from the Enterobacteriaceae family are responsible for diarrheal diseases. Such diseases represent a public health problem in Mexico and are associated to the consumption of contaminated food. In this work, enterobacteria found in cow’s raw milk, manure and soil from small scale dairy farms were iden- tified in order to establish the epidemiologic risks associated to milk consumption from these farms. Three hundred and ten iso- lates were analyzed and 185 were positive to Escherichia coli, 5 to Klebsiella oxytoca, 11 to Enterobacter genus (E. cloacae, E. sak- azakii and E. hormaechi) and 9 to the genus Shigella (S. sonnei and S. dysenteriae). The rest of the isolates were identified as entero- bacteria non-associated to public health problems. However, these bacteria were identified for the first time in these types of samples. Fifty-eight isolates were identified as Brenneria rubrifaciens, and 38 belonged to the genus Pectobacterium (P. carotovorum, P. wasa- biae and P. cypripedii), all of them are phytopathogenic bacteria. On the other hand, four isolates were identified as Photorhabdus luminescens, which is an insect pathogen. Analysis by RAPD-PCR of the E. coli population revealed that it is composed by four clones which are distributed indistinctively among the milk, soil and manure samples. These results show that milk obtained from small scale dairy farms in Mexico is contaminated by pathogens that can be found in manure and soil. Therefore, such milk repre- sents an epidemiologic risk that must be carefully evaluated in order to avoid gastrointestinal diseases.

P8–178 Typing bacterial isolates from feces of healthy Mexican volunteers and analysis of the genetic bases for tetracycline resistance G. Va´ zquez-Marrufo1, I. Tafolla-Munoz2, V. A. Robinson-Fuentes2 and M. S. Va´ zquez-Garciduenas2 1Fac. de Medicina Veterinaria y Zootecnia, Universidad Michoa- cana de San Nicola´s de Hidalgo, Morelia, MEXICO, 2Fac. de Ciencias Me´dicas y Biolo´gicas, Universidad Michoacana de San Nicola´s de Hidalgo, Morelia, MEXICO

P8–180 Modulatory effect of adenosine on oxidative stress parameters in hepatic ischemia/ reperfusion (I/R) injury A. Veljkovic1, G. Kocic1, T. Cvetkovic1, J. Nikolic1, J. Basic1, T. Jevtovic1 and M. Radojkovic2 1Medical Faculty, Institute of Biochemistry, Nis, SERBIA, 2Clinics of Surgery, Clinical Centre, Nis, SERBIA

From 87 bacterial isolates from feces of healthy volunteers, 85 were identified as E. coli and two as Morganella morganii by par- tial sequence of the 16S rRNA gene. The presence of virulence genes was also investigated by PCR and we found that 18 E. coli strains belonged to the enterotoxigenic (ETEC) pathotype, 14 of which contained the labile (LT) toxin gene and four contained both, the LT and stable toxin (ST) genes. The rest of E. coli strains were considered commensal because it was not found any virulence gene of the four E. coli pathotypes: ETEC, EIEC, EPEC, EHEC. Surprisingly, the LT gene was found in one M. morganii isolate and in the other one both, the LT and ST, genes were present. Tetracycline is still broadly used in Mexico to treat diarrheal diseases. Therefore, we searched by PCR in forty two E. coli and the two M. morganii strains for genetic determinants to tetracycline resistance. Some ETEC (6) and commensal (7) strains contained the two tetA and tetB genes. The tetB gene was found in 12 commensal and three ETEC strains; whereas the tetA gene was found in nine and one strains, respectively. The tetC gene was only found in two commensal strains and 12 did not contained any of the three resistance genes. The two M. morganii strains contained both tetA and tetB genes. These results clearly show high variations in the genetic determinants associated to tetracycline resistance among commensal and diarrheagenic strains from Mexico.

Hepatic surgery may be complicated by liver failure. There are many reports on the role of reactive oxygen species (ROS) in the pathogenesis of ischemia/reperfusion (I/R) injury. Complex mani- festations such as rapid depletion in cellular ATP, increase in cytosolic calcium, activation of phospholipases and proteases, infiltration of neutrophils as well as arachidonic acid metabolism may reflect changes in the steady-state concentration of pro/an- tioxidants resulting in oxidative stress. The aim of this study was to examine the effect of adenosine preconditioning in protecting against a hepatic I/R injury. We have studied the lipid peroxida- tion levels (as MDA), protein carbonyl content, glutathione (GSH) levels, catalase (CAT) and glutathione peroxidase (GPx) activity in liver homogenate. Male rabbits, 1 month old, were used for the study, allocated into two groups. The first group I/R animals were subjected to 45 minutes of right-lobe hepatic ische- mia, followed by 48 hours of reperfusion. The second group received intraportally infusion of adenosine in physiological solu- tion in dose of 1 mg/kg 20 minutes before I/R damage. The ani- mals were killed 48 hours afterwards, livers were quickly removed, frozen and homogenised on ice. Obtained results dem- onstrated that MDA was significantly decreased in second group compared to I/R group (p < 0.001). The levels of GSH as well as CAT activities were increased in adenosine treated group. The results also showed statistically significant decrease in activity of GPx and gGT (p < 0.001 vs. I/R group). These results suggest that intraportally infused adenosine attenuates reperfusion injury of the liver, presumably by suppressing the activation of neu- trophils and oxidative stress.

P8–179 Enterobacteria associated to small scale dairy farms from Mexico G. Va´ zquez-Marrufo1, A. Rosales-Castillo1, V. A. Robinson-Fuentes2, O. Chassin-Noria3 and M. S. Va´ zquez-Garciduenas2 1Fac. de Medicina Veterinaria y Zootecnia, Universidad Michoa- cana de San Nicola´s de Hidalgo, Morelia, MEXICO, 2Fac. de Ciencias Me´dicas y Biolo´gicas, Universidad Michoacana de San Nicola´s de Hidalgo, Morelia, MEXICO, 3Fac. de Biologı´a, Uni-

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P8–181 Mildronate orotate: its cardioprotective and antiarrhythmic effects and mechanism of action R. Vilskersts1, J. Kuka2, B. Svalbe3, E. Liepinsh3 and M. Dambrova3 1Faculty of Medicine, University of Latvia, Riga, LATVIA, 2Faculty of Pharmacy, Rigas Stradins University, Riga, LATVIA, 3Laboratory of Pharmaceutical Pharmacology, Latvian Institute of Organic Synthesis, Riga, LATVIA

decreased (p = 0.008) and correlated with CRP (r = -0.65, p < 0.05) and erythrocyte GST activity was increased (p = 0.04) in patients with high inflammatory markers. The concentration of vitamin C was lower (p = 0.008) in patients with severe foot ulcers and also (p = 0.001) in those with CRP > 1 mg/dL. In the group treated with insulin the levels of CRP (p = 0.01), HbA1c (p = 0.03), catalase (p = 0.02) and GPx (p = 0.05) were lower and uric acid was increased (p = 0.03) versus group with- out insulin. In conclusion, plasma vitamin C and thiols levels were reduced in diabetic patients with severe foot ulcers and high inflammatory markers. Type 2 diabetic patients treated with insu- lin have a better blood glucose control, a lower CRP level and reduced oxidative stress. is of the one strategies therapy treatment of

P8–183 Characterization of recombinant cysteine synthase in Caenorhabditis elegans R. Vozdek1, A. Hnizda1, J. Krijt1, M. Kodicek2 and V. Kozich1 1Institute of Inherited Metabolic Diseases, Charles University First Faculty of Medicine, Prague 2, CZECH REPUBLIC, 2Depart- ment of Biochemistry and Microbiology, Institute of Chemical Technology Faculty of Food and Biochemical Technology, Prague 6, CZECH REPUBLIC

Ischemic heart disease is characterized by altered energy metabo- lism, which results in decreased cardiac functionality and arrhyth- mia. Metabolic for pharmacological ischemia-related pathologies. Mildronate and orotic acid act as cardioprotective agents by influencing L-carnitine dependent energy metabolism pathways and regulating nucleoside metabolism, respectively. This study was performed to compare the cardioprotective effects of mildro- nate, orotic acid and mildronate orotate in experimental models of isolated rat heart infarction and angina pectoris. In addition, we tested the effect of mildronate and mildronate orotate in experimental models of arrhythmia induced by ischemia-reperfu- sion, calcium chloride and aconitine. Treatment with mildronate and orotic acid for 14 days reduced necrotic area for 25%, whereas mildronate orotate treatment decreased the infarct size for 50%. Moreover, we found that the administration of mildro- nate and its orotate salt significantly decreased the duration and incidence of arrhythmias in experimental models of arrhythmia. The results of the present study provide evidence that the combi- nation of orotic acid and mildronate possesses additive pharma- cological effects and that mildronate orotate might be considered as a powerful therapeutic agent facilitating chronic recovery from ischemia in cardiac tissues. We hypothesize that the possible mechanism of interaction between regulatory mechanisms of mildronate and orotate could involve activation of UTP and gly- cogen synthesis that protects myocardium during ischemia by maintaining higher ATP levels and improving activity of mito- chondrial complexes.

Nematode Caenorhabditis elegans could be a suitable model to study metabolic and cellular consequences of homocystinuria due to cystathionine b-synthase (CBS) deficiency. However, metabo- lism of sulfur amino acids in C. elegans is as yet unknown, namely the steps in cysteine biosynthetic pathways. Cysteine can be synthesized either via the transsulfuration pathway which uti- lizes homocysteine by CBS or via the assimilation pathway which uses sulfide by cysteine synthase (CS). In silico analysis of C. elegans database identified four orthologs of human CBS, namely ZC373.1, C17G1.7, K10H10.2 and R08E5.2. The aim of this study was to express the gene C17G1.7 (predicted CS) in pro- karyotic system, to purify and further characterize this recombi- nant protein. Molecular weight of polypeptide chain was determined to be 37.2 kDa by MALDI-TOF MS. Blue Native electrophoresis revealed a molecular weight of 70 kDa suggesting that recombinant CS is a dimer. Purified protein contains pyri- doxal 5’-phosphate (PLP) as determined by UV/VIS absorption spectrometry; circular dichroism showed characteristic PLP maxi- mum confirming its localization in a centre of organized globular protein. We determined that purified enzyme has very specific en- zymic activity for CS reaction; other possible activities were not detected. Recombinant CS exhibited KM values for O-acetyl-L - serine and sulfide of 5.54 and 4.23 mM, respectively, and a turn- over number of 139 and 134/seconds, respectively. These data show that C17G1.7 could play an important role in cysteine bio- synthesis since C. elegans genome contains also a CBS gene, we hypothesize that nematode utilizes both cysteine biosythesis path- ways – sulfur assimilation and transsulfuration pathway.

P8–182 Antioxidants, inflammation, glucose blood control and severity of diabetic foot ulcers B. Virgolici1, M. Mohora1, I. Stoian1, L. Gaman1, D. Lixandru1, A. Coman2, V. Gruia1, J. Vertommen3 and B. Manuel-Y-Keenoy3 1Biochemistry, University of Medicine and Pharmacy ‘‘Carol Davila’’, Bucharest, ROMANIA, 2Biochemistry, ‘‘N. Paulescu’’ Institute of Diabetes Nutrition and Metabolic Diseases, Bucharest, ROMANIA, 3Biochemistry, Antwerp Metabolic Research Unit University of Antwerp, Antwerpen, BELGIUM

P8–184 Hemodialysis causes an increase in Annexin V binding to blood platelets B. Walkowiak1, A. Sobol2, M. Kaminska1, M. Stasiak3 and J. Syzmanski3 1Department of Biophysics, Technical University of Lodz, Lodz, POLAND, 2University Hospital No. 1, Medical University of Lodz, Lodz, POLAND, 3Department of Molecular and Medical Biophysics, Medical University of Lodz, Lodz, POLAND

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Blood platelet proteome of hemodialyzed uremic patients exhibits significant difference in comparison to the blood platelet prote- Oxidative stress, inflammation and obesity are involved in type 2 diabetes. Insulin is itself an inhibitor of acute-phase protein syn- thesis. The aim of this study is to assess the relationship between plasma inflammation and antioxidant markers in diabetic foot patients, treated with and without insulin. Forty type 2 diabetic patients, with different stages of foot ulceration were divided according the severity of foot lesions, plasma C-reactive protein values and therapy (with and without insulin). Twenty healthy controls were involved. The activity of enzymatic (GPx, SOD, catalase in RBC) and the level of nonenzymatic (retinol, vitamin C, vitamin E and uric acid) antioxidants did not correlate with any of the inflammation markers (C-reactive protein, ceruloplas- min, haptoglobin) in the studied groups. Plasma thiols were

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Acknowledgement: This work was supported by the Scientific and Technical Research Council of Turkey (TUBITAK), with a project number of 107 T 783.

P8–186 Antiproliferative effects and influence on liver condition after per os administration of novel cytostatic maleimide derivate S. Yablonska, O. Lynchak, O. Filinska, G. Ostrovska, N. Shkira, I. Kharchuk, K. Nizheradze and V. Rybalchenko Biological, Taras Shevchenko Kyiv National University, Kiev, UKRAINE

ome of healthy subjects. This alteration is manifested by the pres- ence of high concentrations of low molecular peptides within the whole range of pI. Increased platelet apoptosis has been put for- ward as a possible cause of this phenomenon (1). The aim of the present research was to assess whether blood platelet populations from hemodialyzed uremic patients exhibit greater binding of Annexin V (a marker of apoptosis) than control samples from healthy donors. Blood was obtained from uremic patients imme- diately after hemodialysis. At the same time samples from control healthy donors were also collected. Blood was anticoagulated with sodium citrate and was immediately exposed to propidium iodide, fluorescent labeled Annexin V and CD61 antibodies. The samples were incubated for 10 minutes in the dark and next the labeled samples were processed in a BectonDickinson FACScan flow cytofluorimeter. Our preliminary study was performed for six patients and six controls. It was found that the blood platelet populations of hemodialyzed patients exhibited significantly higher levels of fluorescence intensity attributed to Annexin V. This intensity was independent of patient sex and age. The results support the hypothesis that blood platelet contact with artificial surfaces during the process of hemodialysys may be partially responsible for triggering of blood platelet apoptosis. Acknowledgement: The project was supported by the grant number N N401 227434 Reference: 1. Walkowiak B, Kaminska M, Okroj W, Tanski W, Sobol A, Zbrog Z. Przybyszewska-Doros I. Blood platelet proteome is changed in uremic patients. Platelets 2007; 18(5): 386–388.

P8–185 Beta amyloid fibril destabilizing activities of novel monoamine oxidase (MAO) inhibitors S. Yabanoglu1, A. Ercan1, N. Kelekci-Gokhan2, B. Salih3 and G. Ucar1 1Faculty of Pharmacy Department of Biochemistry, Hacettepe University, Ankara, TURKEY, 2Faculty of Pharmacy Department of Pharmaceutical Chemistry, Hacettepe University, Ankara, TURKEY, 3Department of Chemistry, Hacettepe University, Ankara, TURKEY

Introduction: Maleimide derivate 1-(4-Cl-benzyl)-3-Cl-4-(CF3- phenylamino)-1H-pyrrol-2.5-dione (MI-1) is novel protein kinases inhibitor that has been synthesized in ChemBioCenter (Kyiv, Uk- raine). It exhibits cytostatic effects on cancer cell lines but after oral administration this compound might cause unpredictable effects. Hepatic chemicals metabolism, often with an imbalance between generations of reactive oxygen species and its degrada- tion, can cause liver damage. Results: MI-1 caused cell growth suppression in cultures of transformed and tumor cell lines (HEK297, MCF-7, HeLa) with IC50 in micromolar concentration range. Maximal suppression (up to 80–90%) was registered in 10–100 lM. Primary cell cul- tures of normal human myocytes, fibroblasts and endotheliocytes are resistant enough to the inhibitory action. In 100 lM concen- tration of MI-1 cell growth was inhibited only to 20–30%. At the same time cell morphology and monolayer structure was not altered. Per os administration of MI-1 in vivo (health rat) in dose 2.7 mg/kg/day (approx. 100 lM in blood) for 30 days has not provoked physiological dysfunctions. There were no significant histological changes in the small and large intestines and in the liver. But the proliferation index in mucous coat of small intes- tine reduced up to 35%. 10 days intragastric MI-1 injection in dose 5 mg/kg/day did not cause changes in lipid and protein per- oxidation. MI-1 did not cause changes of catalase activity but produced decrease of superoxide dismutase activity (SOD) in twice. The influence of MI-1 on SOD activity is not connected with oxidant-antioxidant balance considering the above men- tioned effects. No significant changes of glutathione peroxidase activity and glutathione content under the influence of MI-1 have been detected. But glutathione-S-transferase (GST) activity increase up to 30%. Elevation of GST activity might suggest about increasing of the cell resistance to injected compound. Conclusion: The novel cytostatic maleimide derivate MI-1, which suppress tumor cell growth, does not cause morpho-physi- ological changes and dose not perturb oxidation-antioxidant balance after per os administration. This compound may be con- sidered as potential low toxic cytostatic drug.

P8–187 Pyrophosphate analogs as inhibitors of phosphorolytic activity of HIV-1 reverse transcriptase D. Yanvarev, O. Smirnova, A. Ivanov, A. Tatarintsev and M. Kukhanova Laboratory of chemical and biological analysis of biopolymers and cells, Engelhardt Institute of Molecular Biology RAS, Moscow, RUSSIA

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

The HIV resistance to 3’-azido-3’-deoxythymidine (AZT) is real- ized via an increased rate of phosphorolytic excision of AZT- monophosphate (AZTMP) from the growing chain of viral DNA. This process is Ca-dependent and catalyzed by viral Alzheimer’s Disease (AD) is a common progressive neurodegen- erative disease characterized by intraneuronal neurofibrillary tan- gles and the extracellular deposition of the amyloid b-peptide. Ab peptide is shown to be a proteolytic product of amyloid pre- cursor protein (APP), and polymerization of Ab (1–40) and Ab (1–42) peptides, leads to the formation of insoluble b-amyloid fibrils (fAb) in AD brains. Destabilization of preformed fAb is suggested to be an attractive therapeutic target for the treatment of AD. Increased brain and platelet monoamine oxidase-B (MAO-B) activities in AD patients suggested that oxidants formed by MAO catalysis may cause the neurodegeneration, and MAO inhibitors have some neuroprotective effects. Recent stud- ies indicating that MAO-B inhibitors prevent the Ab-fibril aggre- gation and senile plaque formation in AD are promising. On the basis of our preliminary studies showing that some N-substituted thiocarbamoyl derivatives had potent monoamine oxidase inhibi- tory activities, in the present study we synthesized some N-substi- tuted thiocarbamoyl-2-pyrazoline derivatives as MAO inhibitors and evaluated their b-amyloid fibril destabilizing activities using spectroscopy, fluorescence spectroscopy, electron microscopy and MALDI Mass Spectroscopy. Our results showed that pyrazoline fAb in vitro. derivatives directly inhibited the deposition of Although exact mechanism of the anti-amyloidogenic activity of these derivatives is unclear, pyrazolines appear as the future promising molecules for the development of new anti-Alzheimer drugs.

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two orders lower. We

P8–189 Chaperone-like activity of the dimeric beta- caseins: a first study towards development of Gemini-like protein surfactant R. Yousefi1, R. Yousefi2, N. Gheibi2, A. Sharifizadeh3, A. A. Moosavi-Movahedi3, A. A. Saboury4, J. M. Chobert5 and T. Haertle6 1Department of Biology, Science and Research Branch Azad Uni- versity Punak square, Tehran, IRAN, 2Department of Biochemistry and Biophysics, Qazvin University of Medical Sciences, Qazvin, IRAN, 3Institute of Biochemistry and Biophysics (IBB), The Uni- versity of Tehran, Tehran, IRAN, 4Institute of Biochemistry and Biophysics (IBB), The University of Tehran, Tehran, IRAN, 5BIA-FIPL, Institut National de la Recherche Agronomique (INRA), Nantes, FRANCE, 6BIA-FIPL, Institut National de la Recherche Agronomique (INRA), Nantes, FRANCE

the synthesized compounds in MT-4 cell

reverse transcriptase (RT), an enzyme also involved in the syn- thesis of viral DNA. The substrates of HIV RT in the excision reaction are inorganic pyrophosphate (PPi) or nucleoside triphos- phates, such as ATP and GTP. Recently it was shown that methylenediphosphonic analogs of PPi inhibited HIV-1 RT and RT-catalyzed AZT excision in the presence of ATP. The most active compounds contained a phenyl or biphenyl substituents adjacent to the bridging carbon (PC(X)P) (Bioorg. Med. Chem. 2008(16):8959–67). The activities of inhibitors lacking chelating properties towards Mg2+ because of modified phosphate residues several meth- were synthesized ylenediphosphonates Ph(CH2)n–C(X)(H2PO3)2 (X = H,OH or NH2) bearing phenyl groups joined to the PC(X)P backbone with linkers of varied lengths. We evaluated the dependence of their inhibitory properties from the distance between the aromatic fragment and the chelating group (PC[X]P) and studied the impact of structure of the chelating group on the inhibitory potential of the tested compounds. The results of in vitro inhibi- tion of HIV RT-catalyzed elongation DNA and inhibition of AZTMP excision induced by ATP and PPi are presented. The cy- totoxicities of line (human lymphatic cells) and Huh-7 (human hepatocytes) were also studied. Acknowledgement: The work was supported by the RFBR projects no. 09-04-01221 and no. 08-04-00552.

P8–188 How to affect resveratrol treatment on oxidant/antioxidant status and mRNA expressions of COX-1, COX-2 and NF-kB in diabetic cardiomyopathy S. Menevse1, A. S. Yar1, E. Alp1, D. Sahin2 and N. Altan2 1Medical Biology and Genetics, Gazi University Faculty of Medi- cine, Ankara, TURKEY, 2Medical Biochemistry, Gazi University Faculty of Medicine, Ankara, TURKEY

As a member of intrinsically unstructured protein (IUP) family, beta-CN (casein) contains relatively high amount of prolyl resi- dues, adopts non-compact and flexible structure and exhibits anti-aggregation (chaperone-like) ability in vitro. Like many other chaperones native beta-CN lacks cysteinyl residue and possesses distinct hydrophilic and hydrophobic domains, enhancing solubil- ity in aqueous media and allowing binding lipophilic molecules, respectively. In this study the chaperone-like activity of different molecular forms of beta-CN was examined based on suppression of aggregation of insulin and alcohol dehydrogenase (ADH). The two recombinant beta-CNs as C4 beta-CN (with cysteinyl residue in position 4) and C208 beta-CN ( with cysteinyl residue in posi- tion 208), expressed and purified from E. coli, consequently lack the phosphoryl residues, exhibited significantly lower chaperone- like activity than native beta-CN. To mimic Gemini (Bis) surfac- tants, which have considerably greater solubilizing capabilities than conventional surfactants the dimeric ‘quasi palindromic’ forms of C4 beta-CN and C208 beta-CN connected via disulfide bridge were produced and their chaperone-like activities were compared with those of monomeric forms. While C4 beta-CND (dimer with two distal hydrophobic domains) similar to the monomeric form of recombinant beta-CNs, exhibited poor chap- erone-like activity, a significant chaperone-like activity was observed in case of C208 beta-CND, which possesses two distal hydrophilic domains. The obtained results demonstrate the signif- icant role played by the polar contributions of phosphoryl resi- dues and N-terminal hydrophilic domain of native beta-CN as important functional elements in enhancing of chaperone-like activity of this protein. Moreover the current study might be con- sidered as an initial study towards designing/development of Gemini-like protein/peptide surfactants.

P8–190 MECP2 and CDKL5 mutation analysis in patients with Rett syndrome D. Zahorakova1, A. Puchmajerova1, A. Baxova2 and P. Martasek1 1Department of Pediatrics, First Faculty of Medicine Charles University in Prague and General University Hospital, Prague, CZECH REPUBLIC, 2Institute of Biology and Clinical Genetics, General University Hospital, Prague, CZECH REPUBLIC

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Rett syndrome (RTT) is a severe X-linked dominant neurodevel- opmental disorder primarily caused by de novo mutations in the MECP2 gene. The MeCP2 protein binds to methylated DNA and plays an important role in chromatin remodeling, silencing of many tissue-specific and imprinted genes, and also in RNA splicing. Mutations in another X-linked gene, CDKL5, have been reported in several atypical Rett patients with early onset seizures Hyperglycemia is a serious health problem for those with diabe- tes. Exposure to high levels of glucose induces cardiomyopathy associated with the production of reactive oxygen species (ROS) and inflammation. Nuclear factor jB (NF-jB) is a nuclear tran- scription factor that regulates expression of target genes including growth factors and inflammatory mediators such as COX-2. Cy- clooxygenase (COX), which have the isoforms of COX-1 and COX-2, plays a role in the formation of important biological mediators called prostanoids. Resveratrol (RSV), a natural flavo- noid antioxidant, suppresses COX-2 expression via inhibition of NF-kB activation and reduces oxidative stress in diabetic rats. The present study was designed in order to elucidate the effects of RSV on COX-1, COX-2 and NF-kB mRNA levels in diabetic cardiomyopathy. We also aimed to demonstrate the levels of lipid peroxidation breakdown products such as malondialdehyde (MDA), ferrous oxidation xylenol orange (FOX) and activity of superoxide dismutase (SOD). Three months-old, 44 Wistar albino male rats were used for the study. After the induction of Strepto- zotocin (55 mg/kg), every day 10 mg/kg RSV was administered intraperitoneally for 4 weeks. mRNA levels were measured by real time PCR assay and FOX, MDA levels and SOD activity were determined in heart homogenates. In this study, there were no statistically significant alteration in COX-1, COX-2 and NF- (p > 0.05). SOD activity kB mRNA levels among groups decreased in RSV treated sodium citrat buffer (sham) control and RSV treated DM group (p < 0.05). Higher doses or differ- ent periods of RSV treatment may alter mRNA expressions of these genes and antioxidant capacity.

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3.8±0.3 M and maximal inhibition of 80% similar to that obtained with liposomes. Presumably, zinc ions bind to a group that is hardly accessible in the fully oxidized cytochrome oxidase, but becomes exposed to the cation in one of the partially reduced states of the enzyme. Reduction of CuB is tentatively proposed to be responsible for the effect.

P8–192 Protective effects of elevated intracellular bilirubin: systemic hyperbilirubinemia versus local induction of heme oxygenase-1 J. Zelenka1, L. Muchova2, L. Zdrahalova2 and L. Vitek2 1Institute of Physiology AS CR v.v.i., Prague 4, CZECH REPUB- LIC, 2Institute of Biochemistry and Laboratory Diagnostics, Charles University, Prague 2, CZECH REPUBLIC

as well as in the patients with early infantile epileptic encephalop- athy 2 (EIEE2). It has been suggested that CDKL5 and MeCP2 may belong to the same molecular pathway. We report the muta- tion analysis of the MECP2 gene in 114 RTT patients and CDKL5 gene in 12 atypical RTT patients (previously negative for MECP2 mutations) and 7 patients with suspected EIEE2. The patients were screened for mutations by DNA sequencing of the coding sequence and the exon/intron boundaries of the MECP2 and CDKL5 gene. Mutation-negative cases were subsequently examined by multiplex ligation-dependent probe amplification (MLPA) to identify large deletions/duplications. We optimized the high-resolution melting (HRM) assay as the screening method to precede the sequencing analysis. Altogether, we identi- fied the causal MECP2 mutations in 76 RTT patients including 2 large deletions detected by MLPA. The analysis of the CDKL5 gene did not reveal any mutation in our patients. Furthermore, we established the HRM as a rapid and sensitive screening method to detect sequence variations in most amplicons of both genes. Acknowledgement: Supported by grants IGA NR9215 and GA UK 257927 92707.

P8–191 Zn2+ added from P-side block with high affinity cytochrome c oxidase in phospholipid vesicles or in intact mitochondria W. Zakirzianova1, T. Vygodina2 and A. Konstantinov2 1School of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, RUSSIA, 2A. N. Belozersky Institute of Physico-Chemical Biology, M. V. Lomonosov Moscow State University, Bioenergetics, Moscow, RUSSIA

Objective: Unconjugated bilirubin (UCB) is an endogenous metabolite with promising therapeutic potential due to its antiox- idant, cytoprotective and antiinflammatory properties. However, processes determining its intracellular and tissue distribution remain unknown. The aim of our study was to characterize rele- vant sources and sinks of UCB on the cellular level. Methods: Human hepatoblastoma HepG2 cells were grown in media with defined UCB levels. Heme oxygenase-1 (HO-1) was induced with heme or arsenic oxide and inhibited with taurocho- late. Oxidative stress was enhanced with sodium azide. Intracellu- lar levels of UCB, heme and malondialdehyde were determined using HPLC. HO-1 activity and expression were estimated using GC and Western blot, respectively. Results: Intracellular UCB correlated with UCB/albumin ratio in media (R2 = 0.998). Heme and arsenic oxide caused increase in HO-1 activity and expression leading to significant elevation of intracellular UCB compared to controls (p = 0.01). In contrast, oxidative stress dramatically decreased cellular UCB levels (p < 0.0001). Depletion of UCB was, however, significantly reduced under hyperbilirubinemic conditions (p = 0.01) and even induction of HO-1 completely abolished by simultaneous (p < 0.0001). Conclusion: Local induction of HO-1 and systemic hyperbiliru- binemia protect cells against oxidative stress-associated depletion of UCB, a natural cytoprotectant. Acknowledgement: The work was supported by grant IGA MZ NR9366-3 given by the Czech Ministry of Health.

P8–193 Ionic mechanisms of chloroform induced arrhythmia Y. Zhou1, T. Wong1 and G. Li2 1Physiology, The University of Hong Kong, Hong Kong City, HONG KONG, 2Medicine, The University of Hong Kong, Hong Kong City, HONG KONG

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Chloroform, an organic solvent widely used in industrial produc- tion, is found to cause intoxication of lethal arrhythmias. How- ever, the ionic mechanisms of its arrhythmogenic effects are still unknown. The present study was designed to investigate the elec- trophysiological basis involved. Methods: In isolated rat heat model, ECGs were recorded and analyzed to investigate the arrhythmogenic effects of chloroform. Whole-cell patch clamp was employed to study its effects on pacemaker channel (HCN2), Nav1.5 channel, human cardiac ether-a-go-go related (hERG) K+ channel or inward rectifier K+ channel (Kir2.1), which were stably expressed in HEK 239 cells respectively. Zn2+ has been shown to block enzymatic activity of the mito- chondrial and bacterial cytochrome c oxidase (COX). Crystal structure of COX reveal several Zn2+-binding sites located at the opposing parts of the protein exposed to the P- and N- aqueous phases separated by the membrane. Inhibitory effect of micromo- lar Zn2+ on the isolated COX was assigned to binding of the cat- ion at the orifices of the D- and K-input proton channels which was confirmed by crystal structure. The data on the inhibitory effect of the external Zn2+ are rather controversial. Originally described by Nicholls and Singh inhibition of the COX turnover in proteoliposomes by external Zn2+ according to later report from Ferguson-Miller’s group is highly effective (Ki 5 lM) only if there is membrane potential across the membrane while it requires mM Zn2+ in de-energized state. At the same time, inhi- bition of the COX-reconstituted vesicles by micromolar Zn2+ in the presence of the uncouplers was described by our group. In this work we characterize the effect quantitatively. It was shown that turnover of COX in liposomes as well as in the whole mito- chondria is rapidly inhibited by external Zn2+. The effect is observed in the de-energized vesicles but does require slow elec- tron flux into the oxidase promoted by aerobic preincubation of the vesicles with Zn2+ and weak reductans (tris-bipyridyl com- plex of ruthenium(II)+aniline). Under such conditions the inhibi- tory effect of Zn2+ binding with the oxidase at the P-side is fully reversible and titrates in liposome-bound bovine COX with Ki of 1±0.3 M and maximal inhibition of 80–90%. A close Ki value of 0.64±0.03 M has been determined with liposome-reconstituted COX from Rhodobacter sphaeroides. Similar pattern of the inhi- bition by external Zn2+ was observed with the whole uncoupled mitochondria from rat liver, heart, kidney and skeletal muscle except that no addition of reductants was required because of the electron flux from endogenous substrates. Accordingly Zn2+- induced inhibition was strongly suppressed by rotenone and mal- onate. Inhibition was fully reversible and titrates with Ki of

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P8–195 Adenylosuccinate lyase deficiency; genotype– phenotype correlation M. Zikanova, V. Skopova, H. Hulkova, J. Krijt and S. Kmoch Institute of Inherited Metabolic Disorders, Charles University First Faculty of Medicine, Prague 2, CZECH REPUBLIC

inactivation shifted curve and it

treatments of acute effective for Results: In Langendoff-perfused rat hearts, ECG recording showed that chloroform (5 mM) slowed the heart rate. In addi- tion, chloroform inhibited HCN2, Nav1.5 and hERG channels in a concentration-dependent manner; however, it had no effect on Kir2.1 channel. Specifically, chloroform inhibited HCN2 channel with IC50 at 3.4 mM. In addition, it shifted the activation curve of HCN2 channel toward hyperpolarized potential. Similarly, chloroform suppressed Nav1.5 currents to 75.5%, 52.4%, and 17.2% of basal levels at 5, 10 and 15 mM, respectively. Further- more, it slowed down the recovery of Nav1.5 channel from inac- tivation, toward the hyperpolarized potential, leaving no difference on the activation curve. For hERG channel, chloroform inhibited it with IC50 at 4.3 mM. Conclusion: These results demonstrate that chloroform blocks multiple cardiac ion channels including HCN2 channel, Nav1.5 channel and hERG channels, which might contribute to the chlo- roform-induced lethal arrhythmias. These findings provide poten- tial chloroform targets intoxication.

P8–194 Major Urinary Protein-I increases energy expenditure and improves glucose intolerance through enhancing mitochondrial functions in skeletal muscle in diabetic mice W. Zhu1, X. Hui2, K. S. Lam1, Y. Wang3, J. Zhang1 and A. Xu1 1Medicine, The University of Hong Kong, Hong Kong City, HONG KONG, 2Shanghai Information Center for Life Science, Chinese Academy of Sciences, Shanghai, CHINA, 3Phamacology, The University of Hong Kong, Hong Kong City, HONG KONG

Adenylosuccinate lyase deficiency (ADSL) is a rare autosomal recessive disease of purine metabolism affecting predominantly central nervous system. Although spectrum and severity of clini- cal symptoms overlaps, three forms of ADSL deficiency – severe neonatal, severe childhood and mild myopathic – can be distin- guished clinically based on onset and severity of symptoms and biochemically on diverse ratios of accumulating succinylpurines concentration in body fluids (SAdo/SAICAr ratio). The patho- genic mechanisms leading to the development of symptoms and underlying the phenotypic and biochemical heterogeneity remain unknown. So far we analysed a cohort of 22 patients from 18 families and identified 16 ADSL mutations. We expressed and characterized catalytic properties of corresponding recombinant wt and individual mutant ADSL proteins. We found evidence that residual enzyme activity, calculated as a mean of homoallelic activities, correlates with severity of phenotype, but no ground for the varied SAdo/SAICAr ratio was found. We coexpressed mutant proteins corresponding with patients’ genotypes as a reflection of the fact that two probably structurally different mutant enzymes are involved in tetrameric structure building in vivo. First experiments showed that enzyme activities of coex- pressed proteins differ from calculated activities. We also investi- gated expression patterns of ADSL in available tissues from three patients. Western blot analysis showed severely reduced amounts of ADSL in patients’ tissues compared to controls. Immunochemistry in brain confirmed these results. Based on these observations we hypothesize, that selective affection of cen- tral nervous system can be attributed rather to the toxic effects of accumulating succinylpurines than tissue specific involvement of ADSL function.

P8–196 Effect of melatonin and metabolites on advanced glycation end products-modified hemoglobin -mediated oxidation of low density lipoprotein J. Zuwala-Jagiello1, E. Murawska-Cialowicz2 and J. Gorka-Dynysiewicz1 1Department of Pharmaceutical Biochemistry, University of Medi- cine Wroclaw, Wroclaw, POLAND, 2Department of Physiology and Biochemistry, University of Physical Education Wroclaw, Wroclaw, POLAND

Major urinary protein-I (MUP-I) is a low molecular weight secreted protein predominantly produced from liver. It structur- ally belongs to the lipocalin family that possesses the property to carry small hydrophobic ligands such as pheromones. However, the physiological function of MUP-I remains poorly understood. Here we provide evidence demonstrating that MUP-I is an important player in regulating energy expenditure and metabo- lism in mice. Both microarray and real time PCR analysis dem- onstrated that the MUP-I mRNA abundance in the liver tissue of db/db obese mice was reduced by approximately 34 folds com- pared to their lean littermates, whereas this change was partially reversed by treatment with the insulin sensitizing drug rosiglitaz- one. Accordingly, MUP-I protein concentrations in both plasma and urine in db/db mice mirrored the changes in its mRNA lev- els. Chronic elevation of circulating MUP-I in db/db mice using osmotic pump-based protein delivery system decreased glucose intolerance and insulin resistance. Indirect calorimetry analysis showed replenishment of recombinant MUP-I in this mouse model enhanced energy expenditure, increased locomotor activity and raised core body temperature. At the molecular level, MUP- I-mediated improvement in metabolic profiles was accompanied by increased expression of genes involved in mitochondrial bio- genesis, elevated mitochondrial oxidative capacity, decreased tri- accumulation and enhanced insulin-evoked Akt glyceride signaling in skeletal muscle, but not in the liver. Altogether, these findings raise the possibility that MUP-I deficiency might contrib- ute to the metabolic dysregulation in obese/diabetic mice, and suggest the beneficial metabolic effects of MUP-I are attributed in part to its ability in increasing mitochondrial functions in skel- etal muscle.

FEBS Journal 276 (Suppl. 1) 95–356 (2009) ª 2009 The Authors Journal compilation ª 2009 Federation of European Biochemical Societies

Since oxidation of LDL is initiated by advanced glycation end products-modified hemoglobin (AGEs-Hb), a predictor for dia- betic vascular complications, the present study was undertaken to investigate the influence of melatonin as well as its metabolites, i.e., the main breakdown product 6-hydroxymelatonin and the precursor 5-hydroxytryptamine on this reaction. Vitamin C, a nutritive antioxidant, was also tested, for comparison reasons. The effects of melatonin and its physiological metabolites on oxi- dation of human LDL (0.2 mg of protein/mL) by AGEs-Hb or (10 lM, each) AGEsHb–haptoglobin complex (AGEsHb-Hp) were investigated in an in vitro system. The metabolites investi- gated were the precursor 5-hydroxytryptamine and the main breakdown product 6-hydroxymelatonin. Melatonin (10 lM) inhibited AGEsHb-Hp-dependent, but facilitated AGEs-Hb- dependent, oxidation of LDL in a dose-dependent manner.

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5-hydroxytryptamine (10 lM) and 6-hydroxymelatonin (10 lM) similarly inhibited AGEsHb-Hp-dependent, while facilitating AGEsHb-dependent, oxidation of LDL. However, the effective- these metabolites (1.0 lM each) at mediating either ness of AGEsHb-Hp- or AGEsHb-dependent LDL oxidation was not equivalent. Thus, AGEsHb-Hp-dependent oxidation of LDL was most effectively inhibited by 5-hydroxytryptamine, an intermedi- ate effect was observed with 6-hydroxymelatonin, and vitamin C as least effective. In contrast, a reversal of this pattern was observed for facilitation of AGEsHb-dependent oxidation of LDL, with vitamin C being most effective and intermediate 5- hydroxytryptamine being least effective. The present results indi- cate that the melatonin may have some beneficial effects in con- trolling diabetic vascular complications. Its main breakdown product 6-hydroxymelatonin, however, inhibits AGEsHb-Hp- dependent LDL-oxidation comparably to vitamin C. 5-hydroxy- tryptamine may also play a role in the inhibition of AGEsHb- Hp-dependent LDL oxidation in vivo.

organs including bone structure. Estradiol has two hydroxyl groups which allowed chemical reactions. Thionyl chloride is che- mical which allowed the binding of hydroxyl groups to carboxyl groups. Small molecules such as pesticides, drugs, etc. are usually nonimmunogenic and hence do not elicit an immune response unless coupled with some macromolecules such as proteins (1) and polyelectrolytes (PE). The immunogenic properties of 17b- estradiol, immobilized in negatively charged polymer gels, were investigated, and the specificity of antibodies produced was ana- lyzed. The polymer gels developed were composed of a hydro- phobic estradiol core surrounded by hydrophilic polyanions as corona. As an immunogen, it was conceived to function via a dual mode, that is as a hapten-delivery system (prolongation effect) and as a polyelectrolyte adjuvant. Polymer gels containing estradiol appeared to possess a high estradiol-specific immuno- genicity even without the addition of traditional adjuvants (2). In this present study we prepared 17b-estradiol polyelectrolyte con- jugates by using thionyl chloride and 17b-estradiol-BSA-PE com- plexes via Metal (Cu2+) ions. This conjugates were characterized by using different chromatographic methods (GPC, HPLC) and Spectroscopic methods (FT-IR, UV and Fluorescence). References 1. Singh KV, Kaur J, Varshney GC, Raje M & Suri CR. Synth- esis and characterization of hapten-protein conjugates for anti- body production against small molecules. Bioconjugate Chem 2004; 15: 168–173.

P8-197 Covalent conjugation of ß-estradiol with polymers by using thionyil chloride methods S. Derman, K. Kizilbey and Z. Akdeste Yildiz Technical University, Bioengineering, Istanbul, TURKEY

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2. Basalp A, Mustafaeva Z, Mustafaev M, Bermek E. Immune involved in polymer gels: antigen response to 17b-estradiol specificity and affinity of hybridoma clones. Hybridoma 2000; 19: 495–499. Estradiol (Estra-1,3,5(10)-triene-3,17ß-diol) is a sex hormone with a molecular weight 273,4 and it is also present in males; it repre- sents the major estrogen in humans. Estradiol has not only a cri- tical impact on reproductive functioning, but also affects other

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