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Biomedical subjects

S Khurana

Publications and source records attributed to S Khurana.

At least 19 recordsLinked to original sources
PubMed1999

Heparin-binding property of human prolactin: a novel aspect of prolactin biology.

Prolactin (PRL) shares several characteristics with growth factors and cytokines, many of which are known to bind to heparan sulfate proteoglycans. In this study we examined the heparin-binding properties of selected members of the PRL/GH family, using heparin affinity columns followed by gel electrophoresis/Western blotting. Purified human PRL and its cleaved 16K fragment, but not human GH or placental lactogen, were retained on the heparin column and were displaced by 0.5 M NaCl. Native PRL in human pituitary extracts and amniotic fluid showed a similar binding affinity to heparin as the purified hormone. None of the other hormones tested, e.g., rat, ovine and bovine PRL, glycosylated ovine PRL or rat GH, bound to heparin. Two consensus heparin-binding sequences are present in human PRL but not in the other hormones included in this study. We postulate that the heparin-binding capability of PRL affects its biological activity as a growth factor and the angiostatic actions of its 16K fragment.

Amniotic Fluid↗
PubMed1998

Crystal structure of 2,5-diketo-D-gluconic acid reductase A complexed with NADPH at 2.1-A resolution.

The three-dimensional structure of Corynebacterium 2, 5-diketo-D-gluconic acid reductase A (2,5-DKGR A; EC 1.1.1.-), in complex with cofactor NADPH, has been solved by using x-ray crystallographic data to 2.1-A resolution. This enzyme catalyzes stereospecific reduction of 2,5-diketo-D-gluconate (2,5-DKG) to 2-keto-L-gulonate. Thus the three-dimensional structure has now been solved for a prokaryotic example of the aldo-keto reductase superfamily. The details of the binding of the NADPH cofactor help to explain why 2,5-DKGR exhibits lower binding affinity for cofactor than the related human aldose reductase does. Furthermore, changes in the local loop structure near the cofactor suggest that 2,5-DKGR will not exhibit the biphasic cofactor binding characteristics observed in aldose reductase. Although the crystal structure does not include substrate, the two ordered water molecules present within the substrate-binding pocket are postulated to provide positional landmarks for the substrate 5-keto and 4-hydroxyl groups. The structural basis for several previously described active-site mutants of 2,5-DKGR A is also proposed. Recent research efforts have described a novel approach to the synthesis of L-ascorbate (vitamin C) by using a genetically engineered microorganism that is capable of synthesizing 2,5-DKG from glucose and subsequently is transformed with the gene for 2,5-DKGR. These modifications create a microorganism capable of direct production of 2-keto-L-gulonate from D-glucose, and the gulonate can subsequently be converted into vitamin C. In economic terms, vitamin C is the single most important specialty chemical manufactured in the world. Understanding the structural determinants of specificity, catalysis, and stability for 2,5-DKGR A is of substantial commercial interest.

Bacterial Proteins↗
PubMed1998

Subcellular redistribution is involved in acute regulation of the brush border Na+/H+ exchanger isoform 3 in human colon adenocarcinoma cell line Caco-2. Protein kinase C-mediated inhibition of the exchanger.

Na+/H+ exchanger isoform 3 (NHE3), an epithelial brush border isoform of the Na+/H+ exchanger gene family, plays an important role in reabsorption of Na+ in the small intestine, the colon, and the kidney. In several cell types, phorbol 12-myristate 13-acetate (PMA) acutely inhibits NHE3 activity by changes in Vmax, but the mechanism of this inhibition is unknown. We investigated the role of subcellular redistribution of NHE3 in the PMA-induced inhibition of endogenous brush border NHE3 in a model human colon adenocarcinoma cell line, Caco-2. Subcellular localization of NHE3 was examined by confocal morphometric analysis complemented with cell surface biotinylation and compared with NHE3 activity evaluated by fluorometric measurement of intracellular pH. PMA inhibited NHE3 activity by 28% (p < 0.01), which was associated with a decrease of the ratio of the brush border/subapical cytoplasmic compartment of NHE3 from approximately 4.3 to approximately 2.4. This translocation resulted in 10-15% of the total cell NHE3 being shifted from the brush border pool to the cytoplasmic pool. These effects were mediated by protein kinase C, since they were blocked by the protein kinase C inhibitor H7. We conclude that inhibition of NHE3 by protein kinase C in Caco-2 cells involves redistribution of the exchanger from brush border into a subapical cytoplasmic compartment, and that this mechanism contributes approximately 50% to the overall protein kinase C-induced inhibition of the exchanger.

Caco-2 Cells↗
PubMed1998

Quantitative contribution of NHE2 and NHE3 to rabbit ileal brush-border Na+/H+ exchange.

Intestinal neutral NaCl absorption, which is made up of brush-border (BB) Na+/H+ exchange linked to BB Cl-/HCO3- exchange, is up- and downregulated as part of digestion and diarrheal diseases. Glucocorticoids stimulate ileal NaCl absorption and BB Na+/H+ exchange. Intestinal BB contains two Na+/H+ exchanger isoforms, NHE2 and NHE3, but their relative roles in rabbit ileal BB Na+/H+ exchange has not been determined. A technique to separate the contribution of NHE2 and NHE3 to ileal BB Na+/H+ exchange activity was standardized by using an amiloride-related compound, HOE-694. Under basal conditions, both NHE2 and NHE3 contribute approximately 50% to ileal Na+/H+ exchange. Glucocorticoids (methylprednisolone) increase BB Na+/H+ exchange (2.5 times) but increase only ileal NHE3 activity (4.1 times), without an effect on NHE2 activity. Thus ileal BB Na+/H+ exchange in animals treated with glucocorticoids is 69% via NHE3. A quantitative Western analysis for NHE3 was developed, using as an internal standard a fusion protein of the COOH-terminal 85 amino acids of NHE3 and maltose binding protein. Glucocorticoid treatment increased the amount of BB NHE3. The quantitative Western analysis showed that NHE3 makes up 0.018% of ileal BB protein in control rabbits and 0.042% (2.3 times as much) in methylprednisolone-treated rabbits. Methylprednisolone treatment did not alter the amount of ileal BB NHE2 protein. NHE3 turnover number was estimated to be 458 cycles/s under basal conditions and 708 cycles/s in glucocorticoid-treated ileum. Thus methylprednisolone stimulates ileal BB Na+/H+ exchange activity only by an effect on NHE3 and not on NHE2; it does so primarily by increasing the amount of BB NHE3, although it also increases the NHE3 turnover number.

Animals↗
PubMed1998

G protein-coupled receptors in gastrointestinal physiology. III. Asymmetry in plasma membrane signal transduction: lessons from brush-border Na+/H+ exchangers.

Signal transduction in epithelial cells adds another level of complexity to the signaling that occurs in symmetrical cells, in the form of the need to coordinate and keep separate signals at the apical and basolateral membranes. Regulation by protein kinases of ileal NaCl absorption and its component brush-border Na+/H+ exchanger are used as an example of how signaling in epithelial cells must deal with spatial localization of signals, protein-protein interactions, signaling molecules, and the involvement of the transport protein being regulated in collecting and focusing the signals generated at the receptor and beyond.

Animals↗
PubMed1998

The role of Gardnerella vaginalis in nonspecific vaginitis in intra uterine contraceptive device users.

Two Hundred Forty patients who had Intra Uterine Contraceptive Device (IUCD) and manifested of nonspecific vaginitis were investigated for the presence of G. vaginalis. Pure growth of this organism was obtained in 14(5.8%) cases while 116(48.3%) cases showed this organism in association with other organisms e.g. Esch. coli (11.7%), Klebsiella (9.2%), Candida (9.2%), Strept. faecalis (7.3%), Proteus species (5.8%) and Staph. albus (5%).

Anti-Bacterial Agents↗
PubMed1998

Incidence of dengue fever in relation to climatic factors in Ludhiana, Punjab.

An outbreak of dengue fever occurred in Ludhiana in 1996 and 1997. A total of 505 patients who attended the hospital attached to Dayanand Medical College, Ludhiana were clinically diagnosed to have dengue fever. Of these, 460 cases were noticed between October and December 1996 while during 1997 only 45 dengue fever cases were observed. Serological examination using dengue IgG and IgM blot was performed with single serum samples of 189 patients. Of these, 129 serum samples were detected positive for anti dengue antibodies. Twenty eight patients died in the dengue epidemic, 12 of whom suffered from dengue haemorrhagic fever (DHF) and six with dengue shock syndrome (DSS). Male patients outnumbered the female patients. Seasonal and cyclic pattern of the disease incidence was observed.

Adolescent↗
PubMed1998

Incidence of HIV risk and prevention of infection in health care settings.

Tht HIV epidemic has created awareness about blood borne pathogens transmission in health care settings. Although, the risk of transmission of HIV and other blood borne pathogen may account for a very small proportion, yet the potential for such transmission always exists. Despite this, the use of proper and careful sterile procedures is sufficient to substantially reduce or even eliminate the risk of HIV infection.

Adult↗
PubMed1997

Ileal microvillar protein villin is tyrosine-phosphorylated and associates with PLC-gamma1. Role of cytoskeletal rearrangement in the carbachol-induced inhibition of ileal NaCl absorption.

In ileal absorptive cells, carbachol inhibits NaCl absorption and its component brush border Na+/H+ exchanger, acting via basolateral membrane receptors. This carbachol effect involves (i) activation of brush border phosphatidylinositol 4,5-bisphosphate-specific phospholipase C (PLC) activity and brush border but not basolateral membrane translocation of PLC-gamma1 (Khurana, S., Kreydiyyeh, S., Aronzon, A., Hoogerwerf, W. A., Rhee, S. G., Donowitz, M., and Cohen, M. E. (1996) Biochem. J. 313, 509-518); and (ii) brush border tyrosine kinase(s) because mucosal but not serosal addition of the tyrosine kinase inhibitor genistein prevents the carbachol-induced inhibition of NaCl absorption and brush border Na+/H+ exchange. In the present work we identify a pool of villin (a brush border actin-binding protein) in the microvillus membrane fraction of rabbit ileum; this pool of villin is tyrosine-phosphorylated and associates with brush border membrane PLC-gamma1. Villin is present both in the Triton X-100-soluble and -insoluble fractions of the brush border. The Triton X-100-soluble pool is approximately 4-fold smaller than the brush border pool of villin that is present in the Triton X-100-insoluble fraction. Only the villin present in the Triton X-100-soluble fraction of ileal villus brush border associates with PLC-gamma1 and is tyrosine-phosphorylated. Carbachol increases the tyrosine phosphorylation of villin rapidly (as early as 30 s) and transiently. Carbachol also increases the amount of tyrosine-phosphorylated villin that associates with PLC-gamma1. These studies demonstrate that carbachol effects on NaCl absorption are accompanied by an increase in brush border PLC-gamma1 association with villin and an increase in tyrosine phosphorylation of villin. To study the role of cytoskeletal rearrangement in carbachol-induced inhibition of NaCl absorption, we used the F-actin stabilizing drug jasplakinolide. Jasplakinolide prevents the carbachol inhibition of ileal NaCl absorption. This suggests that F-actin severing is necessary for carbachol to inhibit ileal villus NaCl absorption. Since villin is known to sever actin, these studies suggest a role for villin in the signaling cascade that begins at the basolateral membrane with carbachol binding to its receptor and ends at the apical membrane in inhibition of NaCl absorption.

Actins↗
PubMed1997

The variable domain glycosylation in a monoclonal antibody specific to GnRH modulates antigen binding.

Functionally important glycosylation has been identified in the antigen binding domain of an anti-GnRH monoclonal antibody. Presence of mannose and sialic acid residues is revealed from con A immunoblots and positive staining with a sialic acid detection kit, respectively. Desialylation of the antibody reduces GnRH binding, suggesting the role of terminal sialic acid residues in modulating antigen binding. The crystal structure of the Fab fragment shows electron density adjacent to the antigen binding site which may be attributed to the covalently attached carbohydrate moiety. Thus, the presence of sialic acid containing mannose-rich carbohydrate moiety near the antigen binding site of a monoclonal antibody Fab fragment is relevant for defining antibody specificity.

Animals↗
PubMed1997

Topological analysis of the functional mimicry between a peptide and a carbohydrate moiety.

The shared surface topology of two chemically dissimilar but functionally equivalent molecular structures has been analyzed. A carbohydrate moiety (alpha-D-mannopyranoside) and a peptide molecule (DVFYPYPYASGS) bind to concanavalin A at a common binding site. The cross-reactivity of the polyclonal antibodies (pAbs) was used for understanding the topological relationship between these two independent ligands. The anti-alpha-D-mannopyranoside pAbs recognized various peptide ligands of concanavalin A, and the anti-DVFYPYPYASGS pAbs recognized the carbohydrate ligands, providing direct evidence of molecular mimicry. On the basis of differential binding of various rationally designed peptide analogs to the anti-alpha-D-mannopyranoside pAbs, it was possible to identify different peptide residues critical for the mimicry. The comparison of circular dichroism profiles of the designed analogs suggests that the carbohydrate mimicking conformation of the peptide ligand incorporates a polyproline type II structural fold. The concanavalin A binding activity of these analogs was found to have a direct correlation with the topological relationship between peptide and carbohydrate ligands.

Animals↗
PubMed1997

Acute occlusion of a left internal mammary artery graft immediately after redo coronary artery bypass surgery: successful rescue PTCA.

A patient with a patent left internal mammary artery (LIMA) to the left anterior descending (LAD) artery required repeat bypass grafting to the distal right coronary artery (RCA). Intraoperative injury to the LIMA resulted in acute anterior myocardial infarction, which was managed by successful rescue percutaneous transluminal coronary angioplasty (PTCA).

Angioplasty, Balloon, Coronary↗
PubMed1997

Monitoring platelet glycoprotein IIb/IIIa-fibrin interaction with tissue factor-activated thromboelastography.

Computerized thromboelastography (TEG) was used to study platelet glycoprotein IIb/IIIa function, characterize the consequences of the interaction between polymerizing fibrin and activated platelets, and establish a quantitative assay of platelet function. The ability of platelets to augment the shear elastic modulus of blood clots was measured by TEG under conditions of maximal platelet activation during ex vivo clot formation accelerated by recombinant human tissue factor (TF). Under these conditions, platelets significantly enhance clot strength eightfold (relative to platelet-free fibrin clots). This effect, inhibited by cytochalasin D and c7E3 Fab, appears to be dependent on the transmission of platelet contractile force to fibrin, through glycoprotein IIb/IIIa receptors. The c7E3 Fab dose response of TF-TEG clot strength is identical to results with platelet aggregation induced by the thrombin receptor agonist peptide (50% inhibitory concentration (IC50 = 3.6 microg/ml); adenosine diphosphate-induced aggregation is easier to inhibit (IC50 = 1.2 microg/ml). The results obtained with this system are reproducible (coefficient of variation for clot strength 4%; intraclass correlation coefficient 0.96). As a clinical assay, TF-triggered computerized TEG is easy to perform at the bedside, provides on-line results within 30 minutes, and may offer advantages over conventional measures of platelet function.

Abciximab↗
PubMed1997

Heterogeneity in buffalo pituitary prolactin.

The Ellis procedure of serial extraction of gonadotropins and growth hormone (GH) followed by alkaline ethanol extraction was adopted to process freshly frozen buffalo pituitaries. The procedure after slight modification was found very useful as more than 2 mg of GH free immunoreactive prolactin (PRL) could be isolated from each gram of wet pituitary tissue. Further, the biochemical purity and immunobiological potency of the extracted PRL, designated as P-I, was comparable with that of the highly purified samples of homologous and heterologous PRLs. No non-PRL protein was detectable in P-I. Micro-heterogeneity with regard to size, charge, co- and post-translational modifications was also investigated under different conditions of extraction and at different stages of purification. Immunological and biological potencies were compared in homologous competitive enzyme linked immunosorbent assay (ELISA) developed for buffalo PRL and in rat Nb2 lymphoma proliferation assay respectively. Structural heterogeneity was observed in all the preparations checked including fresh pituitary homogenate and highly purified hormone. Nevertheless a 25 K species corresponding to the hormone monomer was always the only paramount form comprising more than 90% of the total PRL protein in all the samples including P-I. Similar size forms were observed in all preparations and were found to be equivalents of monomers, dimers, covalent-and non-covalent multimers, disulphide bridged forms and cleaved fragments. Other sibling species identified were glycosylated PRL, charge isoforms and forms that perhaps differed in their extractability from the pituitary tissue. Strong apparent size heterogeneity was displayed by the monomeric buffalo PRL. In light of these observations and the information on the structural and functional significance and the consequences of polymeric forms, the use of a heterogeneous PRL (P-I) as a reference hormone is recommended for a valid assay.

Animals↗
PubMed1997

Two distinct pituitary cell lines from mouse intermediate lobe tumors: a cell that produces prolactin-regulating factor and a melanotroph [seecomments].

The intermediate lobe (IL) of the pituitary produces a PRL-regulating factor (PRF). Targeted tumorigenesis, using the POMC promoter ligated to SV40 large T antigen (Tag), generated transgenic mice that develop IL tumors with PRF activity. Our goal was to establish and characterize a PRF-producing cell line. Two cell lines, which differ markedly in size and morphology, were independently developed from IL tumors and designated mIL5 and mIL39. These cells are transformed, as judged by rapid proliferation, low serum requirements, and generation of secondary tumors in nude mice. RT-PCR revealed that mIL39, but not mIL5 cells, express POMC and dopamine D2 receptors, typical of a melanotroph phenotype. Although mIL5 cells originated from an IL tumor, they do not express messenger RNA for SV40 Tag. The bioassay for PRF used GH3 cells stably transfected with the PRL promoter ligated to a luciferase reporter gene (GH3/luc). Coculture of mIL5 with GH3/luc cells induced cell-density dependent increases in PRL gene expression and release, whereas mIL39 cells showed negligible PRF activity. Incubation of GH3/luc cells with conditioned media from mIL5, but not mIL39 cells, stimulated PRL gene expression and release up to 10-fold. Coculture of mIL5 cells with primary rat anterior pituitary cells stimulated PRL, but not GH, release. Fractionation of mIL5 cell extracts by reverse phase HPLC resolved PRF activity into one major and one minor peak. In conclusion, we have developed two novel and distinct cell lines from mouse intermediate lobe tumors. The first reported melanotroph cell line, mIL39, could provide a valuable model for studying dopaminergic regulation of POMC gene expression and release. In contrast, the mIL5 cells do not express POMC, D2 receptors, or SV40 Tag and appear to have been immortalized by a spontaneous mutation(s). These cells produce and secrete a potent PRF and could be used for the purification and biochemical characterization of PRF.

Animals↗
PubMed1997

Family physicians' preferences for computerized decision-support hardware and software.

BACKGROUND: While computers are now widely used by family physicians for billing and patient registration purposes, their use as decision-support tools is still quite limited. The purpose of this study was to determine the current use of computer hardware and software by family physicians, and the characteristics these physicians desire in computerized decision-support hardware and software. METHODS: A cross-sectional survey of a random sample of 250 Michigan family physicians was undertaken in mid-1995. These physicians were asked about their current use of a variety of computer hardware and software. They were also asked to rate the value of different kinds of decision-support information potentially available by computer. The survey instrument also gathered the family physicians' preferences for design factors (both hardware and software), such as the size of a computer, the time needed to access information, and the frequency of updates. RESULTS: Word processing on desktop computers and hospital information systems are the most widely used computer applications by family physicians. Physicians are most interested in computer-based information on drugs, storage and generation of patient education materials, and accessing treatment recommendations. Most feel that semiannual or annual updates of information are adequate, and would like a uniform interface. A high percentage of physicians (84.5% of all physicians and 94.1% of younger physicians) stated that they would consider carrying a handheld computer. CONCLUSIONS: There is significant interest in several types of clinical decision-support software. Based on the results of this study, such software should have following characteristics: (1) be available for handheld as well as networked and desktop computers, (2) include drug information (particularly warnings, interactions, and side effects), (3) include overviews of treatment recommendations, (4) include patient education materials, and (5) have a uniform user interface and be updated at least annually.

Adult↗
PubMed1997

Infectious diarrhoea--an update.

Infectious diarrhoea is one of the most common illness affecting mankind. Recent advancement have led to detailed understanding of causative agents and the pathogenesis of the infections. Fluid and electrolyte replacement remains the most important aspect of treatment. The role of antimicrobial agents is discussed.

Adenoviridae↗