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Biomedical subjects

S Ghosh

Publications and source records attributed to S Ghosh.

At least 505 records · Page 28Linked to original sources
PubMed1996

Association of trans fatty acids (vegetable ghee) and clarified butter (Indian ghee) intake with higher risk of coronary artery disease in rural and urban populations with low fat consumption.

These cross-sectional surveys included 1769 rural (894 men and 875 women) and 1806 urban (904 men and 902 women) randomly selected subjects between 25-64 years of age from Moradabad in North India. The total prevalence of coronary artery disease based on clinical history and electrocardiogram was significantly higher in urban compared to rural men (11.0 vs. 3.9%) and women (6.9 vs. 2.6%), respectively. Food consumption patterns showed that important differences in relation to coronary artery disease were higher intake of total visible fat, milk and milk products, meat, eggs, sugar and jaggery in urban compared to rural subjects. Prevalence of coronary artery disease in relation to visible fat intake showed a higher prevalence rate with higher visible fat intake in both sexes and the trend was significant for total prevalence rates both for rural and urban men and women. Subgroup analysis among urban (694 men and 694 women) and rural (442 men and 435 women) subjects consuming moderate to high fat diets showed that subjects eating trans fatty acids plus clarified butter or those consuming clarified butter as total visible fat had a significantly higher prevalence of coronary artery disease compared to those consuming clarified butter plus vegetable oils in both rural (9.8, 7.1 vs. 3.0%) and urban (16.2, 13.5 vs. 11.0%) men as well as in rural (9.2, 4.5 vs. 1.5%) and urban (10.7, 8.8 vs. 6.4%) women. Univariate and multivariate regression analysis with adjustment for age showed that sedentariness in women, body mass index in urban men and women, milk and clarified butter plus trans fatty acids in both rural and urban in both sexes were significantly associated with coronary artery disease. It is possible that lower intake of total visible fat (20 g/day), decreased intake of milk, increased physical activity and cessation of smoking may benefit some populations in the prevention of coronary artery disease.

Adult↗
PubMed1996

A multifunctional vector system for heterologous expression of proteins in Escherichia coli. Expression of native and hexahistidyl fusion proteins, rapid purification of the fusion proteins, and removal of fusion peptide by Kex2 protease.

Vectors have been constructed for the general purpose of expressing foreign proteins in E. coli. These vectors allow the production in high yield of either native proteins or of fusion proteins which contain, at their amino terminus, the peptide Met Gly His6 Ser Gly Leu Phe Lys Arg/, where Leu Phe Lys Arg/ is the recognition site for Kex2 protease which cleaves at the site indicated by /. The His6 sequence is used as a ligand for the one-step affinity purification of the expressed proteins on columns containing Ni or Zn ions chelated to iminodiacetic acid-agarose. After affinity chromatography, the purification peptide is cleaved off with Kex2 protease from Saccharomyces cerevisiae. The vectors also allow site-directed mutagenesis and sequencing of the cloned gene to be expressed without any intermediate subcloning. For practical examples of over-expression, affinity purification, and removal of the purification peptide, we chose a high-molecular-weight protein, phospholipase C gamma 1 (PLC gamma 1, M(r) 148,000) and a low-molecular-weight protein, Hit-1 (M(r) 16,000). Both were obtained pure and in high yield. PLC gamma 1 was fully active; the function of Hit-1 is not known. A set of companion vectors for co-expression of additional proteins has also been developed. These allow expression of proteins which enhance the production or activity of the protein of primary interest and of proteins which exhibit trans-interactions.

Bacteriophage T7↗
PubMed1996

Biological activity of 4-(4-bromophenyl)-thiosemicarbazide.

The two molecules (A and B) in the asymmetric unit of the title compound, C7H8BrN3S, display different conformation. In both molecules, the S atom is trans to the NH2 group. The Br atoms of the two molecules approach each other at a distance of 3.573(2) A. The crystal structure of the bromine compound is isomorphous with that of its chlorine analogue. In the crystal structure, intramolecular N-H...N and intermolecular N-H...S hydrogen bonds help stabilize the molecular packing. The increased antibacterial activity of the title compound compared to that of its chlorine analogue may be attributed to the increase in electron density on the hydrazinic end of the thiosemicarbazide chain.

Anti-Bacterial Agents↗
PubMed1996

Sequence heterogeneity of Nef transcripts in HIV-1-infected subjects at different stages of disease.

Nef transcripts were analyzed from peripheral blood mononuclear cells of 10 HIV-1-infected subjects with 9-822 CD4+ lymphocytes/cu mm, including 4 individuals with a probable common source infection. There was no relationship between the phylogenetic position of the various nef sequences and the disease state of the person from whom they were derived. The nef open reading frame was disrupted in all three clones from only 1 subject. Functional analyses of a representative clone from each of the remaining 9 subjects showed that all nef alleles were capable of CD4 cell surface down-regulation, but only three nef alleles suppressed the induction of IL-2 transcription.

Amino Acid Sequence↗
PubMed1996

Human platelet signaling defect characterized by impaired production of inositol-1,4,5-triphosphate and phosphatidic acid and diminished Pleckstrin phosphorylation: evidence for defective phospholipase C activation.

Signal transduction on platelet activation involves phosphoinositide-specific phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositides and formation of inositol-1,4,5-triphosphate [I(1,4,5)P3], which mediates Ca2+ mobilization, and diacylglycerol (DG), which activates protein kinase C (PKC) to phosphorylate a 47-kD protein (Pleckstrin). We studied these events in two related patients previously reported (Blood 74:664, 1989) to have abnormal aggregation and 14C-serotonin secretion, and impaired intracellular Ca2+ mobilization in response to several agonists. Thrombin-induced I(1,4,5)P3 and phosphatidic acid formation were diminished. Pleckstrin phosphorylation was impaired on activation with thrombin, platelet-activating factor, and ionophore A23187, but was normal with PKC activator 1,2-dioctonyl-sn-glycerol (DiC8). Ca2+ mobilization induced by guanosine triphosphate (GTP) analog guanosine 5'-0-(3 thiotriphosphate) (GTP gamma S) was diminished. Pretreatment with either A23187 or DiC8 did not correct the impaired adenine diphosphate-induced secretion; however, upon stimulation with A23187 plus DiC8, pleckstrin phosphorylation and secretion were normal, indicating that both PKC activation and Ca2+ mobilization are essential for normal secretion. We conclude that these patients have a unique inherited platelet defect in formation of two key intracellular mediators [I(1,4,5)P3 and DG] and in the responses mediated by them due to a defect in postreceptor mechanisms of PLC activation.

Adenosine Diphosphate↗
PubMed1996

Okadaic acid overrides the S-phase check point and accelerates progression of G2-phase to induce premature mitosis in HeLa cells.

Okadaic acid has been shown to induce premature mitosis in HeLa cells belonging to both S-phase and G2-phase. The response is dependent on concentration and perhaps also on the differential susceptibility of cells to okadaic acid (OA). OA resembles caffeine in its ability to override the DNA-replication check point but differs in being capable of inducing premature mitosis in cells in G2-phase, thereby accelerating mitosis. Chromosomes from mitotic cells induced in G2-phase show a typical banding pattern similar to G-banding. From immunoblot experiments and survey of the relevant literature it has been argued that OA may modulate both a tyrosine kinase and a tyrosine phosphatase activity to override the S-phase checkpoint and accelerate the activation of MPF in cells in G2-phase, respectively.

Cell Cycle↗
PubMed1996

Catalytic properties of the purified rat hepatic cytosolic cholesteryl ester hydrolase.

The purified enzyme hydrolyzed cholesteryl oleate, cholesteryl linoleate, and triolein at similar rates over a broad range of concentrations. Hydrolytic activity was relatively low with p-nitrophenyl acetate, but much higher with PNP-esters of the more lipophilic C4-C18 fatty acids, in sharp contrast to microsomal esterases which hydrolyze PNP-acetate more efficiently. Zn2+, Cu2+, Cd2+, Hg2+, and phenylmethylsulfonyl fluoride inhibited, whereas N-ethyl maleimide and iodoacetamide stimulated activity of the pure enzyme. Limited trypsin digestion selectively inhibited cholesteryl esterase activity with retention of activity toward PNP-octanoate, suggesting involvement of a trypsin-labile loop in the lipophilic substrate binding pocket.

Animals↗
PubMed1996

The solution structure of the Raf-1 cysteine-rich domain: a novel ras and phospholipid binding site.

The Raf-1 protein kinase is the best-characterized downstream effector of activated Ras. Interaction with Ras leads to Raf-1 activation and results in transduction of cell growth and differentiation signals. The details of Raf-1 activation are unclear, but our characterization of a second Ras-binding site in the cysteine-rich domain (CRD) and the involvement of both Ras-binding sites in effective Raf-1-mediated transformation provides insight into the molecular aspects and consequences of Ras-Raf interactions. The Raf-1 CRD is a member of an emerging family of domains, many of which are found within signal transducing proteins. Several contain binding sites for diacylglycerol (or phorbol esters) and phosphatidylserine and are believed to play a role in membrane translocation and enzyme activation. The CRD from Raf-1 does not bind diacylglycerol but interacts with Ras and phosphatidylserine. To investigate the ligand-binding specificities associated with CRDs, we have determined the solution structure of the Raf-1 CRD using heteronuclear multidimensional NMR. We show that there are differences between this structure and the structures of two related domains from protein kinase C (PKC). The differences are confined to regions of the CRDs involved in binding phorbol ester in the PKC domains. Since phosphatidylserine is a common ligand, we expect its binding site to be located in regions where the structures of the Raf-1 and PKC domains are similar. The structure of the Raf-1 CRD represents an example of this family of domains that does not bind diacylglycerol and provides a framework for investigating its interactions with other molecules.

Amino Acid Sequence↗
PubMed1996

Age-related changes in mRNA, protein and catalytic activity of hepatic neutral cholesterol ester hydrolase in male rats: evidence for transcriptional regulation.

Messenger RNA, protein mass and catalytic activity of hepatic neutral cholesteryl ester hydrolase (CEH) were measured in male Sprague-Dawley rats, aged 6, 8, 9.5, 12 and 24 weeks (wks). CEH mRNA increased 101% from 6 to 9.5 wks, corresponding to onset of puberty, and declined by 52% from 12 to 24 wks. CEH mass was highly correlated with mRNA levels at all ages, increasing 170% from 6 to 9.5 wks and declining 61% from 12 to 24 wks. CEH activity was highly correlated with mass and mRNA from 8-24 wks, but was greater at 6 wks than the activity predicted by the measured mass. In all age groups, activity was consistently increased by activation of endogenous protein kinase A and consistently inhibited by alkaline phosphatase, suggesting that age-related differences in catalytic activity were not due to differences in the level of enzyme phosphorylation. These data suggest transcriptional regulation and indicate an important role for CEH in cholesterol homeostasis in the developing rat.

Aging↗
PubMed1996

The effect of age on calcium dynamics in rat brain in vivo.

Different biochemical changes accompany development and ageing. However, the time frame for a particular biochemical change may be different than that for another. In the ageing brain, neurological deficits related to ageing have been suggested to be due to a breakdown of calcium (Ca++) homeostasis, and an increase in intracellular Ca+2. To understand the dynamics of calcium in CNS in relation to normal ageing, we have determined 45Ca++ uptake by the different regions of brain in young (3 months) and aged (15 months) rats after a single i.v. injection of 90 microCi/kg 45Ca++. In the a-ed group, the initial plasma contents of the tracer were higher than in the young rats (3 months). The mean values of uptake (as fraction of injected dose/g x 10(5)) of 45Ca at different sites were higher in the 15-month-old animals than in the young rats (viz. cortex: 50 vs. 26; hippocampus: 90 vs. 46 and putamen: 54 vs. 30 at 120 min). Computer simulation of the uptake data indicated that the values of most of the fractional transport rates were similar or lower in the aged compared to the controls. However, the steady state ratio of intra/extracellular Ca++ was higher in the 15-month-old animals, suggestive of intracellular accumulation of Ca++.

Aging↗
PubMed1996

A sustained reduction in IkappaB-beta may contribute to persistent NF-kappaB activation in human endothelial cells.

The responses of vascular endothelial cells (EC) to tumor necrosis factor-alpha (TNF), interleukin-1alpha (IL-1), and phorbol myristate acetate (PMA) were compared with respect to the kinetics of (i) NF-kappaB activation, (ii) IkappaB-alpha and IkappaB-beta degradation, and (iii) NF-kappaB-dependent cell surface molecule expression. TNF rapidly (</=20 min) and persistently (>20 h) activates NF-kappaB; IL-1 rapidly activates NF-kappaB, but activity declines by 3 h and further by 20 h; PMA slowly and transiently activates NF-kappaB. Untreated EC contain the inhibitory proteins IkappaB-alpha and IkappaB-beta. The onset of NF-kappaB activation correlates with degradation of IkappaB-alpha, but IkappaB-alpha reappears by 4 h without resequestration of NF-kappaB. TNF causes a rapid but partial (50%) reduction in IkappaB-beta, which does not recover by 22 h; IL-1 and PMA cause slower and less sustained reductions in IkappaB-beta. All three agonists induce de novo expression of E-selectin (CD62E) and vascular cell adhesion molecule-1 (CD106) and increase expression of intercellular adhesion molecule-1 (CD54) at 4 h. TNF induces sustained increases in vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 and increases human leukocyte antigen class I molecules at 24 h. We conclude that TNF causes persistent activation of NF-kappaB in human EC and that this may result from sustained reductions in IkappaB-beta levels.

Base Sequence↗
PubMed1996

Interaction of a rat intestinal brush border membrane glycoprotein with type-1 fimbriae of Salmonella typhimurium.

Type-1 fimbriated Salmonella typhimurium was found to adhere to rat intestinal brush border membrane in a mannose sensitive manner. The maximum binding of the purified fimbriae observed with the rat illeal enterocytes was inhibited by 69.2% in presence of D-mannose. Brush border membrane from rat illeum was isolated, delipidified, solubilised and fractionated by affinity chromatography on type-1 fimbriae coupled Sepharose CL 4B column. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of the material eluted from the column with D-mannose revealed a single band of molecular weight 60 kDa. The direct binding of this affinity eluted glycoprotein to the purified type-1 fimbriae was demonstrated by a modified Western blot experiment. Our findings suggest that the 60 kDa glycoprotein may serve as a receptor for the type-1 fimbriae in the rat intestinal brush border membrane and thereby may help in mediating bacterial adherence to the host epithelium.

Animals↗
PubMed1996

Synergistic activation of transcription by the mutant and wild-type minimal transcriptional activation domain of VP16.

VP16 activates transcription by stimulating initiation, and for this function the aromatic residue at position 442 within its activation domain is critical. Recent studies have suggested that VP16 also stimulates transcriptional elongation. It has been shown that VP16 can activate transcription tethered downstream of the transcription start site to RNA. Here, we analyze the synergistic activation features of hybrid VP16 fusion proteins when tethered simultaneously to RNA downstream of the start site and to DNA upstream of a promoter in order to investigate its role in postinitiation control of transcription. Upon targeting the VP16 activation domain simultaneously to both DNA and RNA, high levels of transcriptional synergism is observed. Importantly, a transcription-defective VP16 minimal activation domain (amino acids 413-453) mutated at critical residue 442 (phenylalanine --> proline) maintained synergism, when bound to RNA, with the DNA-bound wild-type VP16 minimal activation domain. Targeting of this "functionally defective" VP16 minimal activation domain via RNA and an intact activation domain via DNA allowed us to uncover a postinitiation activity for VP16 not previously detected in DNA targeting studies. We suggest that, in addition to stimulating initiation, VP16 also acts at a postinitiation step involving residues other than the critical residue at position 442 within the same 41-amino acid minimal activation domain (amino acids 413-453) required for initiation.

Amino Acid Sequence↗
PubMed1996

Raf-1 kinase possesses distinct binding domains for phosphatidylserine and phosphatidic acid. Phosphatidic acid regulates the translocation of Raf-1 in 12-O-tetradecanoylphorbol-13-acetate-stimulated Madin-Darby canine kidney cells.

Previous studies demonstrated that the cysteine-rich amino-terminal domain of Raf-1 kinase interacts selectively with phosphatidylserine (Ghosh, S., Xie, W. Q., Quest, A. F. G., Mabrouk, G. M., Strum, J. C., and Bell, R. M. (1994) J. Biol. Chem. 269, 10000-10007). Further analysis showed that full-length Raf-1 bound to both phosphatidylserine and phosphatidic acid (PA). Specifically, a carboxyl-terminal domain of Raf-1 kinase (RafC; residues 295 648 of human Raf-1) interacted strongly with phosphatidic acid. The binding of RafC to PA displayed positive cooperativity with Hill numbers between 3.3 and 6.2; the apparent Kd ranged from 4.9 +/- 0.6 to 7.8 +/- 0.9 mol % PA. The interaction of RafC with PA displayed a pH dependence distinct from the interaction between the cysteine-rich domain of Raf-1 and PA. Also, the RafC-PA interaction was unaffected at high ionic strength. Of all the lipids tested, only PA and cardiolipin exhibited high affinity binding; other acidic lipids were either ineffective or weakly effective. By deletion mutagenesis, the PA binding site within RafC was narrowed down to a 35-amino acid segment between residues 389 and 423. RafC did not bind phosphatidyl alcohols; also, inhibition of PA formation in Madin-Darby canine kidney cells by treatment with 1% ethanol significantly reduced the translocation of Raf-1 from the cytosol to the membrane following stimulation with 12-O-tetradecanoylphorbol-13-acetate. These results suggest a potential role of the lipid second messenger, PA, in the regulation of translocation and subsequent activation of Raf-1 in vivo.

Amino Acid Sequence↗
PubMed1996

Role of cardiac renin-angiotensin system in the development of pressure-overload left ventricular hypertrophy in rats with abdominal aortic constriction.

Possible involvement of cardiac renin-angiotensin system (RAS) in pressure overload induced left ventricular hypertrophy (LVH) was investigated. Rats were subjected to abdominal aortic constriction (AAC) and examined the effects of 4 weeks treatments with an angiotensin converting enzyme (ACE) inhibitor, captopril and a vasodilator, hydralazine on haemodynamics and ventricular RNA, DNA, protein and myosin isoform pattern in sham and hypertrophied rats. AAC increased the mean arterial pressure (MAP) and systolic blood pressure (SBP), and resulted in increased left ventricle/body weight ratio, LV thickness, RNA and protein content, however total DNA was not changed. The expression of fetal isogene, beta-myosin heavy chain (beta-MHC), was markedly enhanced where as alpha-MHC was reduced. High-dose captopril (100 mg/kg p.o.,) significantly prevented the increase in haemodynamics, development of LVH, LV remodeling, increase in total protein, RNA and antithetical expression of myosin isoforms. Hydralazine (15 mg/kg p.o.,), did not modulate hypertrophic changes and low-dose captopril (1.5 mg/kg p.o.,) which has not produced any marked fall in MAP and SBP also modulated favourably the development of LVH and its biochemical markers. Thus, the prevention of the development of LVH and induction of beta-MHC by non-hypotensive doses of captopril may be related to the blockade of intracardiac production of angiotensin II rather than circulating system. These results suggest that cardiac RAS may play an important role in pressure overload induced LVH.

Angiotensin-Converting Enzyme Inhibitors↗