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Biomedical subjects

A Datta

Publications and source records attributed to A Datta.

At least 55 records · Page 3Linked to original sources
PubMed2000

Checkpoint-dependent activation of mutagenic repair in Saccharomyces cerevisiae pol3-01 mutants.

The Saccharomyces cerevisiae DNA polymerase delta proofreading exonuclease-defective mutation pol3-01 is known to cause high rates of accumulating mutations. The pol3-01 mutant was found to have abnormal cell cycle progression due to activation of the S phase checkpoint. Inactivation of the S phase checkpoint suppressed both the pol3-01 cell cycle progression defect and mutator phenotype, indicating that the pol3-01 mutator phenotype was dependent on the S phase damage checkpoint pathway. Epistasis analysis suggested that a portion of the pol3-01 mutator phenotype involves members of the RAD6 epistasis group that function in both error-free and error-prone repair. These results indicate that activation of a checkpoint in response to certain types of replicative defects can result in the accumulation of mutations.

Base Sequence↗
PubMed2000

Impact of zinc supplementation in malnourished children with acute watery diarrhoea.

A double-blind, randomized, controlled clinical trial was conducted on 80 malnourished children with acute dehydrating diarrhoea to evaluate the efficacy of oral supplementation of zinc as an adjunct therapy to oral rehydration solution (ORS). After decoding it was observed that 44 children received zinc sulphate (177 mg/kg/day in three divided doses equivalent to 40 mg elemental zinc) in a syrup form and 36 children received only syrup placebo. Clinical parameters and microbiological findings of stool samples were comparable in the two groups at the time of enrollment. All the children (100 per cent) in the zinc supplemented group and 32 (89 per cent) children in the placebo group recovered within 5 days of hospitalization (p = 0.04). The zinc supplemented group had a significantly shorter duration of diarrhoea (70.4 +/- 10.0 vs. 103.4 +/- 17.1 h; p = 0.0001), passed less liquid stool (1.5 +/- 0.7 vs. 2.4 +/- 0.7kg; p=0.0001), consumed less oral rehydration solution (2.5 +/- 1.0 vs. 3.6 +/- 0.8 litre; p = 0.0001) and other liquids (867.0 +/- 466.1 vs. 1354.7 +/- 675.6 ml; p = 0.0001) as compared to the placebo group. Our findings suggest that zinc supplementation as an adjunct therapy to ORS has beneficial effects on the clinical course of dehydrating acute diarrhoea.

Acute Disease↗
PubMed2000

In situ and interrupted-growth studies of the self-assembly of octadecyltrichlorosilane monolayers

We have examined the self-assembly process of octadecyltrichlorosilane on silicon using x-ray reflectivity. By comparing the commonly used "interrupted-growth" characterization technique with results obtained in situ, we have determined that quenching the growth and then rinsing and drying the sample introduces free area into the film, presumably by removal of non-cross-linked (physisorbed) molecules. Reintroduction of a quenched and rinsed film to solvent does not restore the thickness of the film to its previous value. We have also performed in situ growth studies over a range of concentrations. For all concentrations, we observe growth of islands of vertical molecules. The growth follows Langmuir kinetics, except at short times for low concentration solutions.

Journal Article↗
PubMed2000

Stimulation of mitotic recombination events by high levels of RNA polymerase II transcription in yeast.

The impact of high levels of RNA polymerase II transcription on mitotic recombination was examined using lys2 recombination substrates positioned on nonhomologous chromosomes. Substrates were used that could produce Lys(+) recombinants by either a simple (noncrossover) gene conversion event or a crossover-associated recombination event, by only a simple gene conversion event, or by only a crossover event. Transcription of the lys2 substrates was regulated by the highly inducible GAL1-10 promoter or the low-level LYS2 promoter, with GAL1-10 promoter activity being controlled by the presence or absence of the Gal80p negative regulatory protein. Transcription was found to stimulate recombination in all assays used, but the level of stimulation varied depending on whether only one or both substrates were highly transcribed. In addition, there was an asymmetry in the types of recombination events observed when one substrate versus the other was highly transcribed. Finally, the lys2 substrates were positioned as direct repeats on the same chromosome and were found to exhibit a different recombinational response to high levels of transcription from that exhibited by the repeats on nonhomologous chromosomes. The relevance of these results to the mechanisms of transcription-associated recombination are discussed.

Aldehyde Oxidoreductases↗
PubMed2000

Effect of pH on the fluorescence and absorption spectra of hypericin in reverse micelles.

The well-characterized, monodispersed nature of reverse micelles formed by sodium bis(2-ethylhexyl)sulfosuccinate/heptane and their usefulness in approximating a membrane-like environment have been exploited to investigate the effect of pH and water pool size on the photophysical properties of hypericin (Hyp). Our measurements reveal two titratable groups of pKa approximately 1.5 and approximately 12.5. These are assigned to the HypH+/Hyp equilibrium (the deprotonation of a carbonyl group) and the Hyp-/Hyp2- equilibrium (the deprotonation of a peri hydroxyl group). The low-energy absorbance maxima of HypH+, of Hyp and Hyp- and of Hyp2- are 583, 594 and 613 nm, respectively. Neither at pH 13 nor at 1 M HCl is the system entirely in the Hyp2- or the HypH+ forms. Ours is the first study of Hyp in reverse micelles as well as the first time-resolved study of Hyp as a function of pH.

Anthracenes↗
PubMed2000

Multidimensional reaction coordinate for the excited-state H-atom transfer in perylene quinones: importance of the 7-membered ring in hypocrellins A and B.

The excited-state intramolecular H-atom transfer reactions of hypocrellins B and A are compared by using time-resolved absorption and fluorescence upconversion techniques. The hypocrellin B photophysics are well described by a simple model involving one ground-state species and excited-state forward and reverse H-atom transfer with a nonfluorescent excited state. We suggest that excited-state conformational changes are coupled to the H-atom transfer in hypocrellin B just as gauche/anti changes are coupled to the H-atom transfer in hypocrellin A.

Kinetics↗
PubMed2000

5-Hydroxytryptamine-inhibiting property of Feverfew: role of parthenolide content.

AIM: To study the mechanism of antimigraine activity of Tanacetum parthenium (Feverfew), its extracts and parthenolide, a component of Feverfew, by observing their effect on 5-HT storage and release, and stimulation of 5-HT2B and 5-HT2A receptors. Also to standardize a dosage form of Feverfew with respect to its parthenolide content. METHODS: Isometric responses to 5-HT and an indirect acting serotonergic, d-fenfluramine, were obtained on rat fundus and ileum. In one set of experiments the effect of dichloromethane extract of Feverfew and parthenolide was observed on the above. The extract was then thermally degraded upto 10%, 23%, and 33% with respect to its parthenolide content by keeping at 60 degrees C and 75% relative humidity and the experiments were repeated. In another set of experiments rats were fed with 20 mg/kg Feverfew powder (equivalent to a human dose of 500 micrograms parthenolide per day) for 30 d or were i.p. injected with parthenolide (23.4 micrograms/day) for 7 d. In the same set of experiments one group of rats were fed with 15% and 77% degraded Feverfew powder in the same dose as mentioned above. After 30 days the effects of the above were observed on 5-HT and d-fenfluramine. Feverfew was specially formulated and tested for stability under accelerated conditions. RESULTS: Parthenolide (1 x 10(-5) mol/L) non-competitively antagonised the effects of d-fenfluramine but had no significant effect on 5-HT2B and 5-HT2A receptors in rat fundus and ileum at 30 min which turned significant on increasing the incubation time to 1.5 h, in rat fundus. Parthenolide (5 x 10(-5) mol/L) followed the same trend. However, Feverfew extract (1 x 10(-5) mol/L) potently and directly blocked 5-HT2B and 5-HT2A receptors and neuronally released 5-HT. At 5 x 10(-5) mol/L the extract potently and irreversibly blocked the above. Both parthenolide and Feverfew extract showed a time-dependency in their action. The extract when degraded thermally upto 10% could significantly block the 5-HT receptors and neuronal release of 5-HT, however, on further degradation it lost its inhibitory capacity markedly. Similar results were observed in rats fed orally with undergraded and degraded Feverfew powder and injected i.p. with parthenolide. Feverfew powder was more effective than any of its extracts or pure parthenolide. CONCLUSION: Feverfew powder is more potent than any of its extract or parthenolide alone in its antiserotonergic activity. Degraded Feverfew extracts show a marked decrease in their antiserotonergic activity. With thermally degraded Feverfew powder containing less contents of parthenolide no built-up antiserotonergic responses were observed after one month. This ascertains that Feverfew should be dispensed in a properly stabilized form wherein its parthenolide content is not degraded to less than 90% of the original content.

Drug Stability↗
PubMed1999

Adhesion and invasion of a mutant Shigella flexneri to an eukaryotic cell line in absence of the 220-kb virulence plasmid.

A Shigella flexneri strain, cured of the large 220-kb virulence plasmid, expresses adhering and invading ability in confluent monolayers of HeLa cells similar to its parent strain. Invasion by both the parent and the cured strains resulted in alteration of the monomeric actin (G) in the total actin pool of HeLa cells. Other indicators of invasive characteristics of virulent Shigella strains such as production of keratoconjunctivitis in guinea pig eye in vivo, Congo red binding and expression of contact hemolysin however, indicated loss of invasive properties in the plasmid cured strain. Further, pretreatment of bacterial cells with para-bromophenacyl bromide (p-BPB), a specific chemical inhibitor of phospholipase A, adversely affected adhesion to and invasion of HeLa cells in vitro, irrespective of the presence of the 220-kb plasmid indicating the possible involvement of the enzyme phospholipase A in the invasion process. Adherence of both the strains to guinea pig colonic epithelial cells (CECs) in vitro was reduced significantly on pretreatment of bacteria or CECs with p-BPB. Expression of exocellular enzymes viz. protease, elastase, phospholipase A and phospholipase C were not related to the large plasmid.

Acetophenones↗
PubMed1999

Comparative analysis of cytotoxin, hemolysin, hemagglutinin and exocellular enzymes among clinical and environmental isolates of vibrio cholerae O139 and non-O1, non-O139.

The presence of three major virulence genes toxR, tcpA and ctxA as well as expression of several putative virulence factors were compared in 12 Vibrio cholerae O139 and non-O1,non-O139 strains of clinical and environmental origin. All the strains possessed the gene encoding the regulatory protein TOXR. None of the non-O1, non-O139 strains as well as one of the O139 environmental strains carried the genes for ctxA and tcpA. Statistically significant differences in hemagglutinin and hemolysin production were observed amongst the strains depending on the source of their isolation. Expression of extracellular enzymes such as protease, elastase, neuraminidase, phospholipase A and phospholipase C, however, did not vary significantly from the groups of strains isolated from different sources.

Animals↗
PubMed1999

Purification of a mannose/glucose-specific hemagglutinin/lectin from a Vibrio cholerae O1 strain.

A cell-associated mannose/glucose-specific hemagglutinin (MSHA) has been purified from a strain of Vibrio cholerae O1 by chromatography on a chitin column followed by affinity purification on Sephadex G100. The purified protein gave a single stained band of 40 kDa by SDS-PAGE, exhibited high affinity towards D-mannose and D-glucose but was resistant to L-fucose and N-acetyl-D-glucosamine. The purified MSHA was revealed as a globular form of protein under electron microscope. In immunodiffusion tests the purified MSHA produced a single precipitin band against homologous antisera and antisera raised against the whole cell bacteria without any reactivity towards antisera raised against the purified N-acetyl-D-glucosamine-specific lectin of the same bacterial strain. Immunogold labelling confirmed the location of hemagglutinin on the surface of the bacteria. Purified MSHA reacted strongly with sera from convalescent cholera patients in immunodiffusion tests.

Animals↗
PubMed1998

Use of monoclonal antibodies to identify phospholipase C as the enterotoxic factor of the bifunctional hemolysin-phospholipase C molecule of Vibrio cholerae O139.

Two hybrid clones producing monoclonal antibodies (MAbs) raised against the purified enterotoxic hemolysin-phospholipase C (HlyPC) bifunctional molecule of a Vibrio cholerae O139 strain were used to study its enterotoxicity in relation to its hemolytic and enzymatic activities. Fab fragments of MAbs from ascites produced by the two hybrids neutralized the hemolytic activity of HlyPC, leaving the enzymatic activity unaffected. In ligated rabbit ileal loop and infant mouse intestine, the Fab fragments of the MAbs were not able to neutralize the enterotoxicity of HlyPC, suggesting that PC rather than Hly is the enterotoxic moiety of the molecule. The enterotoxicity of the purified PC molecule isolated from an Hly- spontaneous mutant of the HlyPC-producing parent strain further confirms this contention. The Hly molecule isolated from a PC- mutant was not diarrheagenic.

Animals↗
PubMed1997

Purification, characterization, and inhibition of peptide deformylase from Escherichia coli.

Peptide deformylase (EC 3.5.1.31) catalyzes the removal of a formyl group from the N-termini of nascent ribosome-synthesized polypeptides, an obligatory step during protein maturation in eubacteria. Since its discovery in crude Escherichia coli extracts 3 decades ago, the deformylase has resisted all attempts of purification or characterization due to its extraordinary lability. By placing the coding sequence (def gene) of Escherichia coli deformylase behind a bacteriophage T7 promoter, we have, however, been able to overexpress this deformylase in Escherichia coli. Overproduction has allowed the purification of > 50 mg of deformylase enzyme from each liter of cell culture. Purified deformylase is highly active toward N-formylated peptide substrates. A new, sensitive assay for the deformylase has been developed by measuring the amount of released formate using a formate dehydrogenase. This has allowed for the assessment of the catalytic properties of peptide deformylase using a series of synthetic N-formylated peptides as substrates. The deformylase exhibits strong preference for an L-methionine or the isosteric norleucine at the N-terminus of a substrate and has broad specificity for the rest of the residues. Small divalent metal chelators strongly inhibit the E. coli deformylase. In particular, certain 1,2- and 1,3-dithiol compounds act as potent, time-dependent inhibitors of the peptide deformylase.

Amidohydrolases↗
PubMed1997

Dual roles for DNA sequence identity and the mismatch repair system in the regulation of mitotic crossing-over in yeast.

Sequence divergence acts as a potent barrier to homologous recombination; much of this barrier derives from an antirecombination activity exerted by mismatch repair proteins. An inverted repeat assay system with recombination substrates ranging in identity from 74% to 100% has been used to define the relationship between sequence divergence and the rate of mitotic crossing-over in yeast. To elucidate the role of the mismatch repair machinery in regulating recombination between mismatched substrates, we performed experiments in both wild-type and mismatch repair defective strains. We find that a single mismatch is sufficient to inhibit recombination between otherwise identical sequences, and that this inhibition is dependent on the mismatch repair system. Additional mismatches have a cumulative negative effect on the recombination rate. With sequence divergence of up to approximately 10%, the inhibitory effect of mismatches results mainly from antirecombination activity of the mismatch repair system. With greater levels of divergence, recombination is inefficient even in the absence of mismatch repair activity. In both wild-type and mismatch repair defective strains, an approximate log-linear relationship is observed between the recombination rate and the level of sequence divergence.

Crossing Over, Genetic↗
PubMed1997

Importance of the amino terminus in maintenance of oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase.

The role of the amino and carboxyl-terminal regions of cytosolic serine hydroxymethyltransferase (SHMT) in subunit assembly and catalysis was studied using six amino-terminal (lacking the first 6, 14, 30, 49, 58, and 75 residues) and two carboxyl-terminal (lacking the last 49 and 185 residues) deletion mutants. These mutants were constructed from a full length cDNA clone using restriction enzyme/PCR-based methods and overexpressed in Escherichia coli. The overexpressed proteins, des-(A1-K6)-SHMT and des-(A1-W14)-SHMT were present in the soluble fraction and they were purified to homogeneity. The deletion clones, for des-(A1-V30)-SHMT and des-(A1-L49)-SHMT were expressed at very low levels, whereas des-(A1-R58)-SHMT, des-(A1-G75)-SHMT, des-(Q435-F483)-SHMT and des-(L299-F483)-SHMT mutant proteins were not soluble and formed inclusion bodies. Des-(A1-K6)-SHMT and des-(A1-W14)-SHMT catalyzed both the tetrahydrofolate-dependent and tetrahydrofolate-independent reactions, generating characteristic spectral intermediates with glycine and tetrahydrofolate. The two mutants had similar kinetic parameters to that of the recombinant SHMT (rSHMT). However, at 55 degrees C, the des-(A1-W14)-SHMT lost almost all the activity within 5 min, while at the same temperature rSHMT and des-(A1-K6)-SHMT retained 85% and 70% activity, respectively. Thermal denaturation studies showed that des-(A1-W14)-SHMT had a lower apparent melting temperature (52 degrees C) compared to rSHMT (56 degrees C) and des-(A1-K6)-SHMT (55 degrees C), suggesting that N-terminal deletion had resulted in a decrease in the thermal stability of the enzyme. Further, urea induced inactivation of the enzymes revealed that 50% inactivation occurred at a lower urea concentration (1.2+/-0.1 M) in the case of des-(A1-W14)-SHMT compared to rSHMT (1.8+/-0.1 M) and des-(A1-K6)-SHMT (1.7+/-0.1 M). The apoenzyme of des-(A1-W14)-SHMT was present predominantly in the dimer form, whereas the apoenzymes of rSHMT and des-(A1-K6)-SHMT were a mixture of tetramers (approximately 75% and approximately 65%, respectively) and dimers. While, rSHMT and des-(A1-K6)-SHMT apoenzymes could be reconstituted upon the addition of pyridoxal-5'-phosphate to 96% and 94% enzyme activity, respectively, des-(A1-W14)-SHMT apoenzyme could be reconstituted only up to 22%. The percentage activity regained correlated with the appearance of visible CD at 425 nm and with the amount of enzyme present in the tetrameric form upon reconstitution as monitored by gel filtration. These results demonstrate that, in addition to the cofactor, the N-terminal arm plays an important role in stabilizing the tetrameric structure of SHMT.

Amino Acid Sequence↗