Biological experiment reports

The design of b-glycosidases with planed substrate specificity for biotechnological application has received little attention. This is mostly a consequence of the lack of data on the molecular basis of the b-glycosidase specificity, namely data on the energy of the noncovalent interactions in the enzymetransition state complex. In an attempt to fill this gap, sitedirected mutagenesis and enzyme steady-state kinetic experiments with different substrates were conducted, using as model a digestive b-glycosidase (glycoside hydrolase family 1) from Spodoptera frugiperda (Lepidoptera) (Sfbgly50).

10p system191 01-06-2013 41 4   Download

The case of homologous monomeric c-type and oligomeric b-type crystallins has been described and analyzed in evolutionary terms. Data and hypotheses from molecular genetics and structural investigations converge and suggest a novel three-phase model for the evolutionary history of crystallin-type proteins. In the divergent cascades of monomeric and oligomeric crystallins, a pivotal role was played by alterations in the gene segments encoding the C-terminal extensions and the intermotif or interdomain linker peptides.

9p system191 01-06-2013 41 4   Download

Mitochondrial functional and structural impairment and generation of oxidative stress have been implicated in aging, various diseases and chemotherapies. This study analyzed azidothymidine (AZT)-caused failures in mitochondrial functions, in redox regulation and activation of the HIV-1 gene expression. We monitored intracellular concentrations of ATP and glutathione (GSH) as the indicators of energy production and redox conditions, respectively, during the time-course experiments with U937 and MOLT4 human lymphoid cells in the presence of AZT (0.05 mgÆmL)1) or H2O2 (0.01 mM) for 15–25 days.

7p system191 01-06-2013 42 3   Download

Frog p26olf is a novel S100-like Ca21-binding protein found in olfactory cilia. It consists of two S100-like domains aligned sequentially, and has a total of four Ca21-binding sites (known as EF-hands). In this study, to elucidate the mechanism of Ca21-binding to each EF-hand (named EF-A, -B, -C and -D from the N-terminus of p26olf), we examined Ca21-binding in wild-type p26olf and also in its mutants in which a glutamate at the –z coordinate position within each Ca21-binding loop was substituted for a glutamine.

8p system191 01-06-2013 29 4   Download

Human aromatase is responsible for estrogen biosynthesis and is implicated, in particular, in reproduction and estro-gen-dependent tumor proliferation. Themolecular structure model is largelyderived fromtheX-ray structure of bacterial cytochromes sharing only 15±20% identities with hP-450arom. In the present study, site directed mutagenesis experiments were performed to examine the role of K119, C124, I125, K130, E302, F320, D309, H475, D476, S470, I471 and I474 of aromatase in catalysis and for substrate binding....

13p research12 29-04-2013 33 3   Download

Ikkai&Ooi [Ikkai,T.&Ooi,T. (1966)Biochemistry5, 1551± 1560] made a thorough study of the e€ect of pressure on G- and F-actins. However, all of the measurements in their study were made after the release of pressure. In the present experiment in situ observations were attempted by using eATP to obtain further detailed kinetic and thermodynamic information about the behaviour of actin under pressure.

8p research12 29-04-2013 41 7   Download

Mild acid degradation of the lipopolysaccharide ofCitro-bacter gilleniiO9a,9b released a polysaccharide (PS), which was found to consist of a single monosaccharide, 4-acetamido-4,6-dideoxy-D-mannose (D-Rha4NAc,N-acetyl-D-perosamine). PS was studied by methylation analysis and 1 H-NMR and 13 C-NMR spectroscopy, using two-dimen-sional 1 H, 1 H COSY, TOCSY, NOESY, and H-detected 1 H, 13 C heteronuclear correlation experiments.

7p research12 29-04-2013 33 4   Download

Small-angle neutron scattering experiments were performed on horse azidometmyoglobin (MbN3) at pressures up to 300 MPa.Other spectroscopic techniques have shown that a reorganization of the secondary structure and of the active site occur in this pressure range. The present measurements, performedusingvarious concentrations ofMbN3, showthat the compactness of the protein is not altered as the value of its radius of gyration remains constant up to 300 MPa.

7p research12 23-04-2013 54 3   Download

We have recently identified an Ets element controlling over 90%of the basal expression of the human presenilin 1 (PS1) gene. We have also shown that Ets1 and Ets2 act as trans-activators of the PS1 gene by cotransfection experiments in SK-N-SH neuronal cells. The PS1 gene is widely but dif-ferentially expressed across tissues and the expression in brain appears to be restricted to neurons. To gain further insight into the regulation of the gene we have examined the regulation of PS1 by 12-O-tetradecanoylphorbol 13-acetate (TPA). ...

7p tumor12 22-04-2013 29 4   Download

Antioxidant protein 2 (AOP2) is a member of a family of thiol-specific antioxidants, recently renamedperoxiredoxins, that evolvedas part of anelaborate systemtocounteract and control detrimental effects of oxygen radicals. AOP2 is found in endothelial cells, erythrocytes, monocytes, T andB cells, but not in granulocytes. AOP2 was found solely in the cytoplasm and was not associated with the nuclear or membrane fractions; neither was it detectable in plasma.

8p tumor12 20-04-2013 34 2   Download

We report experiments to investigate the role of the physio-logically relevant protein tyrosine kinase Lck in the ordered phosphorylation of the T-cell receptorfchain. Six synthetic peptides were designed based on the sequences of the immunoreceptor tyrosine-based activation motifs (ITAMs) of thefchain. Preliminary 1 H-NMRstudies of recombinant f chain suggested that it is essentially unstructured and therefore that peptide mimics would serve as useful models for investigating individual ITAM tyrosines....

8p fptmusic 16-04-2013 26 1   Download

The wealth of available genomic data has spawned a corresponding interest in computational methods that can impart biological meaning and context to these experiments. Traditional computational methods have drawn rela-tionships between pairs of proteins or genes based on notions of equality or similarity between their patterns of occurrence or behavior. For exam-ple, two genes displaying similar variation in expression, over a number of experiments, may be predicted to be functionally related.

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Staphylococcalnuclease is a single domain protein with 149 amino acids. It has no disulfide bonds, which makes it a simple model for the study of pro-tein folding. In this study, 20 mutants of this protein were generated each with a single base substitution of glycine for negatively charged glutamic acid or aspartic acid. Using differential scanning microcalorimetry in ther-mal denaturation experiments, we identified two mutants, E75G and E129G, having approximately 43% and 44%, respectively, lowerDHcal values than the wild-type protein....

8p fptmusic 11-04-2013 44 3   Download

The (ba)8-barrel is the most common enzyme fold and it is capable of cata-lyzing an enormous diversity of reactions. It follows that this scaffold should be an ideal starting point for engineering novel enzymes by directed evolution. However, experiments to date have utilized in vivo screens or selections and the compatibility of (ba)8-barrels with in vitro selection methods remains largely untested.

14p fptmusic 11-04-2013 41 3   Download

Cellular regulation is a result of complex interactions arising from DNA–protein and protein–protein binding, autoreg-ulation, and compartmentalization and shuttling of regula-tory proteins. Experiments in molecular biology have identified these mechanisms recruited by a regulatory net-work.Mathematicalmodelsmaybe used to complement the knowledge-base provided byin vitroexperimental methods. Interactions identified byin vitroexperiments can lead to the hypothesis of multiple candidate models explaining the in vivo mechanism. ...

11p awards 05-04-2013 52 3   Download

For 30 years, the field of medical device technology has struggled to identify the specifications for optimal biocompatibility. For the vast majority of devices, clinical experience has eventually confirmed that the best performance is achieved when biocom-patibility is equated with biological safety that is materials are selected on the basis of maximizing bioinertness and minimizing toxicity and irritation.

7p inspiron33 26-03-2013 52 5   Download

The 3¢-processing of viral DNA extremities is the first step in the integra-tion process catalysed by human immunodeficiency virus (HIV)-1 integrase (IN). This reaction is relatively inefficient and processed DNAs are usually detectedin vitro under conditions of excess enzyme. Despite such experi-mental conditions, steady-state Michaelis–Menten formalism is often applied to calculate characteristic equilibrium⁄kinetic constants of IN.

15p inspiron33 26-03-2013 39 5   Download

hSfi1, a human centrosomal protein with homologs in other eukaryotic organisms, includes 23 repeats, each of 23 amino acids, separated by 10 residue linkers. The main molecular partner in the centrosome is a small, calcium-binding EF-hand protein, the human centrin 2. Using isothermal titration calorimetry experiments, we characterized the centrin-binding capacity of three isolated hSfi1 repeats, two exhibiting the general consen-sus motif and the third being the unique Pro-containing human repeat....

12p inspiron33 25-03-2013 36 5   Download

DNA polymeraseeco-operates with polymerasesaanddin the replicative DNA synthesis of eukaryotic cells. We describe here a specific physical interaction between DNA polymerase eand RNA polymerase II, evidenced by reciprocal immunoprecipitation experiments. The interacting RNA polymerase II was the hyperphosphorylated IIO form implicated in tran-

15p inspiron33 23-03-2013 44 3   Download

Fluorescence recovery after photobleaching (FRAP) and related techniques using green fluorescent protein (GFP)-tagged proteins are widely used to study the subcellular trafficking of proteins. It was concluded from these experiments that the cytokine-induced nuclear import of tyrosine-phos-phorylated (activated) signal transducer and activator of transcription 1 (STAT1) was rapid, while the constitutive shuttling of unphosphorylated STAT1 was determined to be inefficient.

12p galaxyss3 21-03-2013 39 4   Download