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Biomedical subjects

R Sharma

Publications and source records attributed to R Sharma.

At least 73 records · Page 4Linked to original sources
PubMed2001

Leptin, insulin sensitivity and growth hormone binding protein in chronic heart failure with and without cardiac cachexia.

OBJECTIVE: Regulation of growth hormone (GH) receptor expression and hence tissue GH sensitivity may be important for the conflicting results found in treatment studies with recombinant growth hormone in chronic heart failure (CHF). Growth hormone-binding protein (GHBP) corresponds to the extracellular domain of the GH receptor and is closely related to measures of body composition and, specifically, to size of visceral fat tissue. Leptin, the adipocyte specific (ob) gene product, has been proposed as the signal linking adipose tissue and GHBP/GH-receptor expression. CHF has recently been shown to be a hyperleptinaemic and insulin-resistant state regardless of aetiology. This study aimed to examine the influence of leptin on GHBP in CHF patients with and without cardiac cachexia compared with healthy control subjects. METHODS: We studied 47 male patients with CHF (mean age 61+/-2 years, New York Heart Association (NYHA)-class 2.7+/-0.1, left ventricular ejection fraction (LVEF) 28+/-2%, peak oxygen consumption 16.8+/-0.9 ml/kg/min) and 21 male healthy controls of similar age. Of the CHF patients, 19 were cachectic (cCHF; non-oedematous weight loss >7.5% over at least 6 months) and 28 non-cachectic (ncCHF; similar for age and LVEF). Insulin sensitivity was assessed by an intravenous glucose tolerance test using the minimal model approach. RESULTS: Compared with healthy controls, patients had elevated levels of leptin (7.6+/-0.7 vs 4.8+/-0.7 ng/ml, P<0.05), insulin (76.2+/-8.9 vs 41.4+/-6.0 pmol/l, P<0.01), and reduced insulin sensitivity (2.43+/-0.2 vs 3.48+/-0.3 min(-1).microU.ml(-1).10(4), P<0.005) but similar GHBP levels (901+/-73 vs 903+/-95 pmol/l). Leptin levels were increased in ncCHF (9.11+/-1.0 ng/ml, P=0.001) but were not different from normal in cCHF (5.32+/-0.7 ng/ml, P>0.5). After correction for total body fat mass, both ncCHF and cCHF were hyperleptinaemic (41.8+/-3.8 and 37.9+/-0.38 vs 24.4+/-2.1 ng/ml/100 g, ANOVA P=0.001). In both patients and controls there was a direct correlation between leptin levels and GHBP (r=0.70 and r=0.71 respectively, both P<0.0001). This relationship was stronger than between GHBP and several parameters of body composition (body mass index (BMI), total and regional body fat mass or % body fat) and held true when sub-groups were tested individually (ncCHF r=0.62, P<0.001; cCHF r=0.79, P<0.0001). In multivariate regression analysis in all CHF patients, serum leptin levels emerged as the strongest predictor of GHBP, independent of age, BMI, total and regional fat mass or % body fat, fasting insulin level and insulin sensitivity. CONCLUSION: Fat mass corrected leptin levels are elevated in CHF patients with and without cachexia. Reduced total fat mass may account for lower leptin levels in cachectic CHF patients compared with non cachectic patients. Leptin strongly predicts GHBP levels in CHF regardless of its hyperleptinaemic state or severely altered body composition as in cardiac cachexia. Leptin could be the signalling link between adipose tissue and GHBP/GH receptor expression in CHF.

Adipose Tissue↗
PubMed2001

Reversal of hyperalgesia by transplantation in lateral hypothalamic lesioned rats.

Lateral hypothalamus (LHA) plays a very important role in the modulation of nociceptive behaviour. The stimulation of LHA is known to produce analgesia of both tonic and phasic pain. The present study reports hyperalgesia induced by lateral hypothalamic lesions and the effect of fetal (gestation day 16) hypothalamic transplant on the nociceptive response to phasic thermal noxious stimulation [tail flick latency (TFL)] in LHA lesioned rats. The TFL decreased significantly (12.91 +/- 3.91 sec to 10.51+/- 1.23 sec) following LHA lesion. However, after transplantation, the TFL did not change. This is the first report of a hypothalamic transplant inducing recovery of a nociceptive response.

Animals↗
PubMed2001

Early detection of radiation-induced glomerular injury by albumin permeability assay.

Renal irradiation leads predictably to glomerular vascular injury, cell lysis, matrix accumulation, sclerosis and loss of renal function. The immediate effects of renal irradiation that may be associated with glomerular pathology and proteinuria are not clear in the human disease or its rat model. We hypothesized that radiation-induced injury causes immediate and subtle alterations in glomerular physiology independent of the neurohumoral and hemodynamic regulatory mechanisms. We employed a sensitive in vitro functional assay of glomerular albumin permeability (P(alb)) to demonstrate radiation-induced damage to the glomerular filtration barrier immediately after total-body irradiation of rats. In blinded experiments, control rats were sham-treated, and experimental rats received 9.5 Gy X rays. Rats were killed humanely at 1 h to 9 weeks after irradiation and glomeruli were isolated. In parallel experiments, glomeruli were isolated from normal rats and irradiated in vitro. The change in glomerular capillary permeability due to an experimental oncotic gradient was determined using videomicroscopy and P(alb) was calculated. Results show that in vivo or in vitro irradiation of glomeruli caused an increased P(alb) at 1 h. Increased P(alb) was observed up to 3 weeks after irradiation. Glomeruli from mice irradiated with 9.5 or 19.0 Gy X rays did not show increased P(alb) at 1 h postirradiation. We conclude that glomerular protein permeability of irradiated rats increases in a dose-dependent manner immediately after irradiation and that it appears to be independent of hemodynamic or systemic influences.

Albumins↗
PubMed2001

A flow cell assay for evaluation of whole cell drug efflux kinetics: analysis of paclitaxel efflux in CCRF-CEM leukemia cells overexpressing P-glycoprotein.

P-glycoprotein (Pgp) mediates drug accumulation defects in malignant cells in vitro. It confers resistance to multiple drugs including paclitaxel, an agent useful in treating malignancies including acute leukemia. Pgp-mediated drug resistance appears to be due to primary active drug-transport as well as other effects on membrane permeability, but the relative contribution of each is unclear. Flow cells are useful for differentiating transport-mediated efflux from altered membrane permeability, but their utility is limited to attached cells. We developed a novel flow cell to study drug efflux kinetics in suspension culture cells and examined paclitaxel efflux in resistant CEM/VLB100 leukemia cells, which overexpress Pgp, compared with its sensitive CEM parent line. Paclitaxel efflux from both cell lines was described by bi-exponential kinetics. The predominant initial rapid component increased linearly with paclitaxel concentration, consistent with passive efflux, and was faster in CEM/VLB100 than CEM cells. The slow terminal component of efflux was also more rapid for CEM/VLB100 than CEM, and was saturable (V(max)= 9.1 +/- 1.1 versus 3.5 +/- 0.3 pmol/min/10(7) cells, respectively) at a lower paclitaxel concentration than the parental CEM cells (k(m) = 63 +/- 46 nM versus 144 +/- 56 nM, respectively). In CEM/VLB100 cells, this saturable component was inhibited by verapamil and was temperature-sensitive, consistent with Pgp-mediated transport. Verapamil also inhibited the rapid component of efflux, suggesting additional effects on membrane permeability. Our studies show that the present technique is useful for studying drug transport and that effects of Pgp on membrane permeability contribute significantly to the net drug-accumulation defect.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Lichen planus of the eyelids. A report of 5 cases.

Involvement of the eye and related structures is rarely reported in lichen planus. Only eleven cases with eyelid involvement are reported in the literature. Lesions on the eyelids may be the only manifestation of lichen planus or it may be a part of the disease process present on other parts of the body. When only eyelids are involved, clinical diagnosis may be difficult. Lichen planus should be considered in the differential diagnosis of erythematous papular lesions on the eyelids.

Administration, Topical↗
PubMed2001

Hydrocortisone and triiodothyronine regulation of malate-aspartate shuttle enzymes during postnatal development of chicken.

The normal endogenous level of malate-aspartate shuttle enzymes and its regulation by hydrocortisone and triiodothyronine were studied in the liver and kidney of 0-, 30- and 60-day old male Rhode Island Red (RIR) chicken. The endogenous activity of cytosolic malate dehydrogenase (c-MDH) was significantly higher in the liver of day 30 as compared to day 0 and 60. In contrast, mitochondrial malate dehydrogenase (m-MDH) activity decreased at day 60 in the liver. However, both c- and m-MDH had significantly lower activities at day 0, which increased sharply at day 30 and 60 in the kidney. On the other hand, activity of both cytosolic and mitochondrial aspartate aminotransferase (c- and m-AsAT) showed peak value at day 30 in both liver and kidney. Hydrocortisone administration induced c-MDH in the liver at all the ages studied, but did not influence the activity of the isoenzymes in the kidney whereas, it induced m-MDH in the liver at day 0 and in kidney at day 30. Administration of hydrocortisone, however, did not influence AsAT isoenzymes (c- and m-AsAT) in either of the tissues at any of the postnatal ages. Triiodothyronine induced c-MDH in the liver at all the ages whereas kidney isoenzyme was induced only at day 60. In contrast, m-MDH was induced by triiodothyronine in both liver and kidney at day 30 and 60. Administration of triiodothyronine did not influence c-AsAT of liver and kidney at either of the ages, whereas it induced m-AsAT of only liver at day 0 and 60. These findings indicated a tissue- and age-specific expression of the malate-aspartate shuttle enzymes in chicken and difference in the regulation exerted by hydrocortisone and triiodothyronine during postnatal development of chicken.

Animals↗
PubMed2001

Purification and functional reconstitution of intact ral-binding Gtpase activating protein, RLIP76, in artificial liposomes.

We have recently shown that RLIP76, a ral-binding GTPase activating protein, mediates ATP-dependent transport of glutathione-conjugates (GS-E) and doxorubicin (DOX) (S. Awasthi et al., Biochemistry 39,9327,2000). Transport function of RLIP76 was found to be intact despite considerable proteolytic fragmentation in preparations used for those studies, suggesting either that the residual intact RLIP76 was responsible for transport activity, or that the transport activity could be reconstituted by fragments of RLIP76. If the former were true, intact RLIP76 would have a much higher specific activity for ATP-hydrolysis than the fragmented protein. We have addressed this question by comparing transport properties of recombinant RLIP76 and human erythrocyte membrane RLIP76 purified in buffers treated with either 100 or 500 microM serine protease inhibitor, PMSF. The purity and identity of recombinant and human erythrocyte RLIP76 was established by SDS/PAGE and Western-blot analysis. These studies confirmed the origin of the 38 kDa protein, previously referred to as DNP-SG ATPase, from RLIP76. Higher PMSF concentration resulted in lower yield of the 38 kDa band and higher yield of intact RLIP76 from both human and recombinant source. In contrast, the substrate-stimulated ATPase activity in presence of DNP-SG, doxorubicin, daunorubicin, or colchicine were unaffected by increased PMSF; similarly, ATP-dependent transport of doxorubicin in proteoliposomes reconstituted with RLIP76 was unaffected by higher PMSF. These results indicated that limited proteolysis by serine proteases does not abrogate the transport function of RLIP76. Comparison of transport kinetics for daunorubicin between recombinant vs human erythrocyte RLIP76 revealed higher specific activity of transport for tissue purified RLIP76, indicating that additional factors present in tissue purified RLIP76 can modulate its transport activity.

ATP-Binding Cassette Transporters↗
PubMed2001

Heterogeneity of transport systems for L-glutamine in mouse mammary gland.

The characteristics of the transport systems of L-glutamine in lactating mouse mammary gland have been studied. L-glutamine uptake was mediated by three Na+-dependent and one Na+-independent systems. The 2-(methylamino)isobutyric acid-sensitive component of Na+-dependent uptake exhibited the usual characteristics of system A. The other two Na+-dependent systems, which we have named BCI(-)-dependent and BCl(-)-independent, are the new systems identified. These are broad specificity systems and were discriminated on the basis of inhibition analysis, Cl- dependency and the effect of preloading mammary tissue with amino acids. While L-aspargine inhibited the uptake of L-glutamine via both these broad specificity systems, L-homoserine inhibited the uptake of L-glutamine via only BCl(-)-dependent system. The uptake of L-glutamine via the BCl(-)-independent system was upregulated by preloading mammary tissue with L-serine, while BCl(-)-dependent system was unaffected. The Na+-independent uptake of L-glutamine was inhibited by 2-aminobicyclo-(2,2,1)heptane carboxylic acid and other neutral amino acids, and identified as the system L.

Amino Acids, Cyclic↗
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↗