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B Sharma

Publications and source records attributed to B Sharma.

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

Zero background yeast reporter plasmids.

UAS-less reporter plasmids are widespread and powerful tools for the identification and analysis of binding sites for transcriptional activators. The common reporter plasmids for the yeast Saccharomyces cerevisiae are multicopy (2mu) vectors with the CYC1 core promoter upstream of the lacZ gene. Insertion of putative or known activator binding sites upstream of the core promoter puts lacZ (beta-galactosidase) expression under the control of the corresponding activator. Although these constructs have proved to work well for most purposes, they have certain limitations: (1) they give significant and carbon-source-dependent lacZ background expression; (2) unlike most other yeast promoters, the CYC1 upstream region has a partially open chromatin structure with an accessible TATA box; (3) they use only a single, moderately sensitive reporter; and (4) the use of multicopy vectors can result in activator titration. Here, we introduce novel reporter plasmids based on the yeast MEL1 (alpha-galactosidase) gene that can overcome all of these limitations. It is also shown that background expression is due to fortuitous activator binding sites within the plasmid backbones that are insufficiently shielded from the core promoters in the common CYC1 reporter plasmids.

Binding Sites↗
PubMed2000

Effect of tumor necrosis factor on hepatic oxidative stress and antioxidant defense indices in normal and Plasmodium yeolii nigeriensis infected mice.

BACKGROUND: Plasmodium yoelii nigeriensis (P. y. nigeriensis) produces lethal malaria infection in Swiss albino mice. Reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide along with endogenously produced tumor necrosis factor (TNF) have been implicated in the pathogenesis of malaria. OBJECTIVE: Study the effect of TNF on hepatic oxidative stress and antioxidant defense indices in normal and P. y. nigeriensis infected mice. METHODS: Mice were divided into four groups. Normal group, TNF treated group, P. y. nigeriensis infected group, and P. y. nigeriensis infected mice treated with TNF group (250 microg/kg body weight, i.p.). RESULTS: TNF treatment of normal mice caused a highly significant decrease in hepatic superoxide dismutase (SOD) while changes in other oxidative stress and antioxidant defense indices were nonsignificant. On the other hand, TNF treatment of P. y. nigeriensis infected mice caused a highly significant increase in hepatic xanthine oxidase, lipid peroxidation and a significant decrease in hepatic SOD with respect to infected mice. CONCLUSION: These results suggest that exogenous TNF acts synergistically with P. y. nigeriensis infection to generate oxidative stress in the host and also causes an impairment of antioxidant defense enzyme such as superoxide dismutase.

Analysis of Variance↗
PubMed1999

Asp-89: a critical residue in maintaining the oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase.

Aspartate residues function as proton acceptors in catalysis and are involved in ionic interactions stabilizing subunit assembly. In an attempt to unravel the role of a conserved aspartate (D89) in sheep-liver tetrameric serine hydroxymethyltransferase (SHMT), it was converted into aspargine by site-directed mutagenesis. The purified D89N mutant enzyme had a lower specific activity compared with the wild-type enzyme. It was a mixture of dimers and tetramers with the proportion of tetramers increasing with an increase in the pyridoxal-5'-phosphate (PLP) concentration used during purification. The D89N mutant tetramer was as active as the wild-type enzyme and had similar kinetic and spectral properties in the presence of 500 microM PLP. The quinonoid spectral intermediate commonly seen in the case of SHMT was also seen in the case of D89N mutant tetramer, although the amount of intermediate formed was lower. Although the purified dimer exhibited visible absorbance at 425 nm, it had a negligible visible CD spectrum at 425 nm and was only 5% active. The apo-D89N mutant tetramer was a dimer unlike the apo-form of the wild-type enzyme which was present predominantly as a tetramer. Furthermore the apo mutant dimer could not be reconstituted to the holo-form by the addition of excess PLP, suggesting that dimer-dimer interactions are weak in this mutant. The recently published crystal structure of human liver cytosolic recombinant SHMT indicates that this residue (D90 in the human enzyme) is located at the N-terminal end of the fourth helix of one subunit and packs against K39 from the second N-terminal helix of the other symmetry related subunit forming the tight dimer. D89 is at the interface of tight dimers where the PLP 5'-phosphate is also bound. Mutation of D89 could lead to weakened ionic interactions in the tight dimer interface, resulting in decreased affinity of the enzyme for the cofactor.

Animals↗
PubMed1999

Effect of carbaryl on some biochemical constituents of the blood and liver of Clarias batrachus, a fresh-water teleost.

Carbaryl, a carbamate pesticide, (LC50 15.08 mg/l for 96 hr, i.e. lethal concentration with 50% mortality) induced perturbations in the levels of certain biochemical components including the activities of some enzymes in the blood and liver of the fresh-water catfish, Clarias batrachus exposed to sublethal concentrations (1, 2 and 4 mg/l) of the pesticide for 96 hr and 15 days. The pesticide caused a decrease in the levels of total protein and glucose with a concomitant increase in the levels of inorganic phosphate and lactic acid in fish serum. However, very little change was recorded in the serum cholesterol level. The treatment of the fish with carbaryl led to a marked increase in the activities of transaminases (GOT and GPT), phosphatases (acid and alkaline) and lactate dehydrogenase in the fish serum, the magnitude of the effect being dependent on the pesticide concentration and duration of exposure. The increase in lactic acid concentration with subsequent decrease in glucose concentration indicates an enhanced rate of glycolysis due to pesticide stress. Furthermore, the significant decrease in the activity of fish liver succinate dehydrogenase suggests that anaerobic metabolism was favored over aerobic oxidation of glucose through Kreb's cycle in order to mitigate the energy crisis for survival. The rise in the activities of transaminases and acid phosphatase due to pesticide intoxication suggest enhanced protein catabolism and probable hepatocellular damage in the organism.

Animals↗
PubMed1999

Apoptosis in bovine herpesvirus-1 infected bovine peripheral blood mononuclear cells.

Apoptosis is a process whereby cells die in a controlled manner in response to various stimuli like cytotoxins, viral antigens and normal physiological signals during differentiation and development. Virus induced immunosuppression has been reported for various viral diseases including Bovine Herpesvirus-1 (BHV-1). In the present study, BHV-1 was found to cause apoptosis in ConA stimulated bovine peripheral blood mononuclear cells (PBMCs). Apoptotic index quantified by fluorescent dyes revealed a significant (P < 0.001) increase in percent apoptotic cells at 2, 24 and 48 hr post infection as compared to their respective non-infected controls. Apoptosis specific internucleosomal laddering in DNA from BHV-1 infected PBMCs was seen in agarose gel electrophoresis. No DNA fragmentation was observed in control non-infected PBMCs.

Animals↗
PubMed1998

Random amplified polymorphic DNA for the specific detection of bubaline Echinococcus granulosus by hybridization assay.

The polymerase chain reaction (PCR) method to randomly amplify polymorphic DNA (RAPD) was used to differentiate the bubaline and bovine strains of Echinococcus granulosus and buffalo host DNA. Four random oligonucleotide primers of 10-11 mer were analyzed for their ability to direct the amplification of polymorphic DNA fragments from parasites and bovine DNA. Significant DNA polymorphism was observed between the E. granulosus isolates. A selectively amplified DNA fragment 0.9 kilobase (kb) from E. granulosus buffalo isolate by primer AP2 (5'-TGCCGAGCTG-3') was reamplified and used as a DIG-labelled DNA probe. Dot-blot hybridization of total genomic DNA differentiated buffalo E. granulosus isolate from bovine isolate and bubaline host DNA with no detectable cross-hybridization signal.

Animals↗
PubMed1998

Antisense targeting of perlecan blocks tumor growth and angiogenesis in vivo.

Perlecan, a ubiquitous heparan sulfate proteoglycan, possesses angiogenic and growth-promoting attributes primarily by acting as a coreceptor for basic fibroblast growth factor (FGF-2). In this report we blocked perlecan expression by using either constitutive CMV-driven or doxycycline- inducible antisense constructs. Growth of colon carcinoma cells was markedly attenuated upon obliteration of perlecan gene expression and these effects correlated with reduced responsiveness to and affinity for mitogenic keratinocyte growth factor (FGF-7). Exogenous perlecan effectively reconstituted the activity of FGF-7 in the perlecan-deficient cells. Moreover, soluble FGF-7 specifically bound immobilized perlecan in a heparan sulfate-independent manner. In both tumor xenografts induced by human colon carcinoma cells and tumor allografts induced by highly invasive mouse melanoma cells, perlecan suppression caused substantial inhibition of tumor growth and neovascularization. Thus, perlecan is a potent inducer of tumor growth and angiogenesis in vivo and therapeutic interventions targeting this key modulator of tumor progression may improve cancer treatment.

Animals↗
PubMed1998

Impact of torture on refugees displaced within the developing world: symptomatology among Bhutanese refugees in Nepal.

CONTEXT: Most of the world's refugees are displaced within the developing world. The impact of torture on such refugees is unknown. OBJECTIVE: To examine the impact of torture on Bhutanese refugees in Nepal. DESIGN: Case-control survey. Interviews were conducted by local physicians and included demographics, questions related to the torture experienced, a checklist of 40 medical complaints, and measures of posttraumatic stress disorder (PTSD), anxiety, and depression. SETTING: Bhutanese refugee community in the United Nations refugee camps in the Terai in eastern Nepal. PARTICIPANTS: A random sample of 526 tortured refugees and a control group of 526 nontortured refugees matched for age and sex. MAIN OUTCOME MEASURES: The Diagnostic and Statistical Manual of Mental Disorders, Revised Third Edition (DSM-III-R) criteria for PTSD and the Hopkins Symptom Checklist-25 (HSCL-25) for depression and anxiety. RESULTS: The 2 groups were similar on most demographic variables. The tortured refugees, as a group, suffered more on 15 of 17 DSM-III-RPTSD symptoms (P<.005) and had higher HSCL-25 anxiety and depression scores (P<.001) than nontortured refugees. Logistic regression analysis showed that history of torture predicted PTSD symptoms (odds ratio [OR], 4.6; 95% confidence interval [CI], 2.7-8.0), depression symptoms (OR, 1.9; 95% CI, 1.4-2.6), and anxiety symptoms (OR, 1.5; 95% CI, 1.1-1.9). Torture survivors who were Buddhist were less likely to be depressed (OR, 0.5; 95% CI, 0.3-0.9) or anxious (OR, 0.7; 95% CI, 0.4-1.0). Those who were male were less likely to experience anxiety (OR, 0.66; 95% CI, 0.44-1.00). Tortured refugees also presented more musculoskeletal system- and respiratory system-related complaints (P<.001 for both). CONCLUSION: Torture plays a significant role in the development of PTSD, depression, and anxiety symptoms among refugees from Bhutan living in the developing world.

Adult↗
PubMed1998

Transcriptional silencing of perlecan gene expression by interferon-gamma.

Perlecan, a heparan sulfate proteoglycan of basement membranes and cell surfaces, has been implicated in the control of tumor cell growth and metastasis because of its ability to bind and store growth factors and its activity as an inducer of angiogenesis. Because interferon-gamma (IFN-gamma), a cytokine with known antiproliferative and antitumoral activity, binds with high affinity to the heparan sulfate side chains of perlecan, we investigated the activity of IFN-gamma on perlecan gene expression and cell growth in colon carcinoma cells. We found that IFN-gamma rapidly and efficiently blocked perlecan gene expression with concurrent growth suppression, a phenomenon that was independent of a functional p21(WAF1/CIP1). These effects were transcriptionally mediated, did not require new protein synthesis, and were fully reversible. Moreover, we found these IFN-gamma-induced effects to be generalizable because they could be reproduced in a variety of cells with various histogenetic backgrounds. The transcriptional repression of the perlecan gene required intact Stat1 protein, and these effects were likely mediated by Stat1-binding sites in the distal promoter region. Thus, the IFN-gamma-mediated transcriptional repression of perlecan may represent a novel antitumoral effect of this cytokine through which it eliminates a powerful angiogenic stimulus.

Cell Cycle↗
PubMed1998

Use of prokaryotically expressed nucleocapsid protein as positive antigen in ELISA.

A cDNA library of Rinderpest vaccine virus was prepared in Zap Express vector (Stratagene). The Rinderpest 'N' gene specific clones were selected, characterized and thereafter expressed in E. coli XLOLR strain. The expressed protein was found to be immunogenic in western blot with hyperimmune sera. It reacted with rinderpest and 'N' protein specific monoclonal antibodies in Enzyme Linked Immunosorbent Assay (ELISA). Prokaryotically expressed 'N' protein also gave precipitin band in counter immunoelectrophoresis test (CIE). The expression of N protein was sufficient for its utility as positive antigen in CIE and ELISA used for rinderpest diagnosis.

Animals↗
PubMed1997

The role of His-134, -147, and -150 residues in subunit assembly, cofactor binding, and catalysis of sheep liver cytosolic serine hydroxymethyltransferase.

In an attempt to unravel the role of conserved histidine residues in the structure-function of sheep liver cytosolic serine hydroxymethyltransferase (SHMT), three site-specific mutants (H134N, H147N, and H150N) were constructed and expressed. H134N and H147N SHMTs had Km values for L-serine, L-allo-threonine and beta-phenylserine similar to that of wild type enzyme, although the kcat values were markedly decreased. H134N SHMT was obtained in a dimeric form with only 6% of bound pyridoxal 5'-phosphate (PLP) compared with the wild type enzyme. Increasing concentrations of PLP (up to 500 microM) enhanced the enzyme activity without changing its oligomeric structure, indicating that His-134 may be involved in dimer-dimer interactions. H147N SHMT was obtained in a tetrameric form but with very little PLP (3%) bound to it, suggesting that this residue was probably involved in cofactor binding. Unlike the wild type enzyme, the cofactor could be easily removed by dialysis from H147N SHMT, and the apoenzyme thus formed was present predominantly in the dimeric form, indicating that PLP binding is at the dimer-dimer interface. H150N SHMT was obtained in a tetrameric form with bound PLP. However, the mutant had very little enzyme activity (<2%). The kcat/Km values for L-serine, L-allo-threonine and beta-phenylserine were 80-, 56-, and 33-fold less compared with wild type enzyme. Unlike the wild type enzyme, it failed to form the characteristic quinonoid intermediate and was unable to carry out the exchange of 2-S proton from glycine in the presence of H4-folate. However, it could form an external aldimine with serine and glycine. The wild type and the mutant enzyme had similar Kd values for serine and glycine. These results suggest that His-150 may be the base that abstracts the alpha-proton of the substrate, leading to formation of the quinonoid intermediate in the reaction catalyzed by SHMT.

Amino Acid Sequence↗
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↗