PubMed Health⌕ Search

Biomedical subjects

R Sharma

Publications and source records attributed to R Sharma.

At least 163 records · Page 9Linked to original sources
PubMed2000

Up-regulation of Na,K-ATPase beta 1 transcription by hyperoxia is mediated by SP1/SP3 binding.

The sodium pump, Na,K-ATPase, is an important protein for maintaining intracellular ion concentration, cellular volume, and ion transport and is regulated both transcriptionally and post-transcriptionally. We previously demonstrated that hyperoxia increased Na,K-ATPase beta(1) gene expression in Madin-Darby canine kidney (MDCK) cells. In this study, we identify a DNA element necessary for up-regulation of the Na,K-ATPase beta(1) transcription by hyperoxia and evaluate the nuclear proteins responsible for this up-regulation. Transient transfection experiments in MDCK cells using sequential 5'-deletions of the rat Na,K-ATPase beta(1) promoter-luciferase fusion gene demonstrated promoter activation by hyperoxia between -102 and +151. The hyperoxia response was localized to a 7-base pair region between -62 and -55, which contained a GC-rich region consistent with a consensus sequence for the SP1 family, that was sufficient for up-regulation by hyperoxia. This GC element exhibited both basal and hyperoxia-induced promoter activity and bound both transcription factors SP1 and SP3 in electrophoretic mobility shift assays. In addition, electrophoretic mobility shift assays demonstrated increased binding of SP1/SP3 in cells exposed to hyperoxia while mutation of this element eliminated protein binding. Other GC sites within the proximal promoter also demonstrated up-regulation of transcription by hyperoxia, however, the site at -55 had higher affinity for SP proteins.

Animals↗
PubMed2000

Age-dependent effects of Aroclor 1254R on calcium uptake by subcellular organelles in selected brain regions of rats.

Earlier reports from our laboratory have indicated that polychlorinated biphenyls (PCBs) affect signal transduction mechanisms in brain, including Ca2+ homeostasis, phosphoinositol hydrolysis, and protein kinase C (PKC) translocation in mature neurons and adult brain homogenate preparations. Present studies were designed to investigate whether there were any brain region-, gender-, or age-dependent effects of PCBs on 45Ca2+-uptake by two subcellular organelles, microsomes and mitochondria. We have studied in vitro effects of a widely studied commercial PCB mixture, Aroclor 1254R, on 45Ca2+-uptake by microsomes and mitochondria in cerebellum, frontal cortex and hippocampus of postnatal day (PND) 7, 21, and 90-120 (adult) male and female Long-Evans (LE)-rats. In general, microsomal and mitochondrial 45Ca2+-uptake in selected brain regions increased with age; PND 7<PND 21< or =adults. Among three brain regions, hippocampus had relatively lower microsomal 45Ca2+-uptake than cerebellum and frontal cortex throughout the development. Mitochondrial 45Ca2+-uptake was comparable in three brain regions of PND 7 and adult animals, but in PND 21 rats, the cerebellum had much higher activity than frontal cortex and hippocampus. No gender-related differences were seen in 45Ca2+-uptake by either microsomes or mitochondria in selected brain regions throughout development. Inhibition of 45Ca2+-uptake by Aroclor 1254 in a concentration-dependent manner was observed throughout the study. However, the degree of inhibition of microsomal 45Ca2+-uptake in these brain regions by Aroclor 1254 increased with age, PND 7<PND 21< or =adults (IC50s=21-34, 8-20 and 10-14 microM, respectively). Brain region-specific differential sensitivity to Aroclor 1254 on the inhibition of microsomal 45Ca2+-uptake was not seen in PND 7 and adult animals but in PND 21 rats, hippocampus was more sensitive than the other selected brain regions. There were no age-, gender- or brain region-specific differential effects of Aroclor 1254 on mitochondrial 45Ca2+-uptake. These results indicate that a commercial PCB mixture, Aroclor 1254, inhibited 45Ca2+-uptake by both microsomes and mitochondria uniformly in selected brain regions of males and females during development. However, the inhibition of microsomal 45Ca2+-uptake by Aroclor 1254 increased with age. The age- and gender-related differential sensitivity to Aroclor 1254 may be attributed to the changes in calcium homeostasis in various brain regions during development.

Age Factors↗
PubMed2000

Transgenic expression of cecropin B, an antibacterial peptide from Bombyx mori, confers enhanced resistance to bacterial leaf blight in rice.

The short persistence of cecropin B peptide in plants, due to post-translational degradation, is a serious impediment in its effective utilization for developing bacterial resistance transgenic plants. Two DNA constructs encoding the full-length precursor of cecropin B peptide and the mature sequence of cecropin B peptide preceded by a signal peptide derived from rice chitinase gene were transformed in rice. The differences in the transcriptional levels in independent transgenic lines showed moderate to high expression of cecropin B gene that correlated well with the differences in cecropin B accumulation observed by Western blot analysis. The development of lesions resulting from infection by Xanthomonas oryzae pv. oryzae was significantly confined in the infected leaflet of transgenic lines, when compared with the control plants.

Animals↗
PubMed2000

Streptozotocin-induced diabetes and glucocorticoid receptor regulation: tissue- and age-specific variation.

Streptozotocin (STZ) -induced diabetic effects were analyzed for glucocorticoid receptor (GR) level and for in vitro activation of GR by specific binding analysis, using [3H]dexamethasone, a synthetic glucocorticoid, and by DNA cellulose and nuclear binding assay, in the liver and kidney of 15- (immature) and 120-day-old (mature) male mice. Comparison of GR level (fmol/mg protein) among the control mice reveals decreased (22-33%) specific binding in the liver and kidney of mature mice compared with immature ones. Scatchard analyses, however, reveal no change in the affinity (K(d)) of receptor at these two ages of mice. STZ-induced diabetes did not alter the level of GR in either of the tissues at both the ages studied. The GR from both the tissues underwent thermal activation, albeit the extent of activation was more pronounced in mature liver compared to immature, with no such difference of activation in the kidney. In diabetic mice, the activation of hepatic GR exhibits reduced DNA cellulose ( approximately 20-23%) and nuclear (24-30%) binding compared to control mice. In contrast, thermal activation of kidney GR does not show marked differences in diabetic mice at either of the ages studied. Cross-mixing experiments (i.e. binding of activated GR from diabetic mice to nuclei of control and vice-versa) performed on the mature liver, indicate receptor specificity. These findings reveal tissue- and age- specific variations in the level of GR that is not influenced under diabetic conditions. However, the activation of hepatic GR is reduced during STZ-induced diabetes that might play a role in controlling glucose homeostasis in diabetic animals.

Aging↗
PubMed2000

Molecular cloning, genomic organization, and biochemical characterization of myristoyl-CoA:protein N-myristoyltransferase from Arabidopsis thaliana.

Myristoyl-CoA:protein N-myristoyltransferase (NMT, EC 2.3.1.97) catalyzes the co-translational addition of myristic acid to the amino-terminal glycine residue of a number of important proteins of diverse functions. We have isolated a full-length Arabidopsis thaliana cDNA encoding NMT (AtNMT1), the first described from a higher plant. This AtNMT1 cDNA clone has an open reading frame of 434 amino acids and a predicted molecular mass of 48,706 Da. The primary structure is 50% identical to the mammalian NMTs. Analyses of Southern blots, genomic clones, and database sequences suggested that the A. thaliana genome contains two copies of NMT gene, which are present on different chromosomes and have distinct genomic organizations. The recombinant AtNMT1 expressed in Escherichia coli exhibited a high catalytic efficiency for the peptides derived from putative plant myristoylated proteins AtCDPK6 and Fen kinase. The AtNMT was similar to the mammalian NMTs with respect to a relative specificity for myristoyl CoA among the acyl CoA donors and also inhibition by the bovine brain NMT inhibitor NIP(71). The AtNMT1 expression profile indicated ubiquity in roots, stem, leaves, flowers, and siliques (approximately 1.7 kb transcript and approximately 50 kDa immunoreactive polypeptide) but a greater level in the younger tissue, which are developmentally very active. NMT activity was also evident in all these tissues. Subcellular distribution studies indicated that, in leaf extracts, approximately 60% of AtNMT activity was associated with the ribosomal fractions, whereas approximately 30% of the activity was observed in the cytosolic fractions. The NMT is biologically important to plants, as noted from the stunted development when the AtNMT1 was down-regulated in transgenic Arabidopsis under the control of an enhanced CaMV 35S promoter. The results presented in this study provide the first direct molecular evidence for plant protein N-myristoylation and a mechanistic basis for understanding the role of this protein modification in plants.

Acyltransferases↗
PubMed2000

The ATP hydrolytic activity of purified ZntA, a Pb(II)/Cd(II)/Zn(II)-translocating ATPase from Escherichia coli.

ZntA, a soft metal-translocating P1-type ATPase from Escherichia coli, confers resistance to Pb(II), Cd(II), and Zn(II). ZntA was expressed as a histidyl-tagged protein, solubilized from membranes with Triton X-100, and purified to homogeneity. The soft metal-dependent ATP hydrolysis activity of purified ZntA was characterized. The activity was specific for Pb(II), Cd(II), Zn(II), and Hg(II), with the highest activity obtained when the metals were present as thiolate complexes of cysteine or glutathione. The maximal ATPase activity of ZntA was approximately 3 micromol/(mg x min) obtained with the Pb(II)-thiolate complex. In the absence of thiolates, Cd(II) inhibits ZntA above pH 6, whereas the Cd(II)-thiolate complexes stimulate activity, suggesting that a metal-thiolate complex is the true substrate in vivo. These results are consistent with the physiological role of ZntA as mediator of resistance to toxic concentrations of the divalent soft metals, Pb(II), Cd(II), and Zn(II), by ATP-dependent efflux. Our results confirm that ZntA is the first Pb(II)-dependent ATPase discovered to date.

Adenosine Triphosphatases↗