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Biomedical subjects

M K Jain

Publications and source records attributed to M K Jain.

At least 109 records · Page 6Linked to original sources
PubMed1995

Impaired pancreatic bicarbonate secretion in chronic malnutrition.

Twenty three children with recurrent episodes of diarrhea and chronic malnutrition were studied for pancreatic duct function. Those children were subjected to pancreatic stimulation with pancreozymin and secretin. Grade I malnourished children, as per Gomez classification, formed the control group. The water output from pancreas increased in malnourished children (p < 0.05). It correlated significantly to cationic transport (p < 0.01). Sodium and potassium together accounted for significant proportion of water output in pancreatic fluid. Potassium transport increased with increasing severity of malnutrition and may be responsible for the hypokalemia observed in malnourished children. Pancreatic secretion of bicarbonate decreased in severe malnutrition inspite of increased flow rate of pancreatic secretion. This is probably due to defective bicarbonate secretion likely to be located at pancreatic duct epithelial cell membrane.

Bicarbonates↗
PubMed1994

Phospholipase A2 engineering. Structural and functional roles of the highly conserved active site residue aspartate-49.

Site-directed mutagenesis and high-resolution two-dimensional (2D) proton nuclear magnetic resonance (NMR) were used to probe the structural and functional roles of a highly conserved residue, Asp-49, in the interfacial catalysis by bovine pancreatic phospholipase A2 (PLA2, overexpressed in Escherichia coli). According to crystal structures, the side chain carboxylate of Asp-49, along with the carbonyl oxygens of Tyr-28, Gly-30, and Gly-32, and two water molecules, provides the necessary ligands for Ca2+ which is essential for the enzymatic activity. The Asp-49 of PLA2 was changed to Asn, Glu, Gln, Lys, and Ala; the resulting mutants are named D49N, D49E, D49Q, D49K, and D49A, respectively. The conformational stabilities of all five mutants are similar to that of WT as judged by guanidine hydrochloride-induced denaturation. The structural analyses by NMR indicated no global perturbations upon substitutions, although localized conformational perturbations can be observed for less conserved replacements. Direct Ca2+ binding studies showed no specific binding for D49A, D49N, D49Q, and D49K; however, D49E retains a 12-fold weaker calcium binding affinity (Kd,Ca = 23 mM). The specific activities of all five mutant enzymes decrease significantly, ranging from 5.4 x 10(2)- to 5.8 x 10(5)-fold in comparison with that of the wild-type enzyme. The observed activities of mutants require the presence of Ca2+. This demonstrates the functional importance of Asp-49 in the catalytic mechanism of PLA2, presumably by helping to bind and properly orient Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Purification and characterization of a calcium-independent acidic phospholipase A2 from rat lung.

Several phospholipase A2 (PLA2) activities have been identified in rat lung homogenate and shown to be important in metabolism of lung phospholipids. One PLA2 activity is Ca(2+)-independent, active in vitro at pH 4, and inhibited by a substrate analogue, 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33). Purification of this rat lung PLA2 by approx. 550-fold was carried out by sequential column chromatographies using DE-52, Sephacryl-100, heparin-Sepharose, and phenyl-Sepharose columns. The purified activity had an acidic pH optimum, was Ca(2+)-independent, was inhibited by MJ33 in a dose-dependent manner (50% inhibition at 3 mol%), was unaffected by treatment with p-bromophenacyl bromide or mercaptoethanol, and had a unique N-terminal amino acid sequence. The apparent molecular mass was 15 kDa on gel electrophoresis and activity was recovered in part by renaturation of protein from the gel. The properties of this enzyme suggest a new class of PLA2.

Amino Acid Sequence↗
PubMed1994

Tight binding inhibitors of 85-kDa phospholipase A2 but not 14-kDa phospholipase A2 inhibit release of free arachidonate in thrombin-stimulated human platelets.

An analogue of arachidonic acid in which the COOH group is replaced by a trifluoromethyl ketone group (COCF3) has recently been shown to be a tight binding inhibitor of the 85-kDa cytosolic phospholipase A2 that is found in platelets and other cells (Street, I. P., Lin, H.-K., Laliberté, F., Ghomashchi, F. G., Wang, Z., Perrier, H., Tremblay, N. M., Huang, Z., Weech, P. K., and Gelb, M. H. (1993) Biochemistry 32, 5935-5940). This trifluoromethyl ketone inhibits most of the arachidonate release from the phospholipid pool in thrombin-stimulated human platelets at concentrations of 0-40 microM with 4 x 10(8) platelets/ml. A structure-function analysis of related compounds reveals a good correlation between the inhibition of the purified phospholipase A2 and the blockage of arachidonate release in platelets. A number of recently described potent inhibitors of the 14-kDa phospholipase A2 that is secreted from activated platelets have no effect on the level of free arachidonate production. Furthermore, the addition of a large amount of recombinant 14-kDa phospholipase A2 to platelets does not produce free arachidonate, and it does not alter the amount of arachidonate released following platelet activation with thrombin. These studies provide strong pharmacological evidence for the role of the cytosolic phospholipase A2 in producing most, if not all, of the liberated arachidonate in thrombin-stimulated human platelets, and they show that tight binding membrane-residing inhibitors of the cytosolic phospholipase A2 can block the eicosanoid cascade in living cells.

Arachidonic Acids↗
PubMed1994

Characterization of interfacial catalysis by Aeromonas hydrophila lipase/acyltransferase in the highly processive scooting mode.

A glycerophospholipid:cholesterol acyltransferase (GCAT) that also has lipase activity is secreted by the bacterium Aeromonas hydrophila. Hydrolysis of the sn-2-ester bond of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DMPM) vesicles by this enzyme is shown to occur in a highly processive scooting mode in which the enzyme, substrate, and the products of hydrolysis remain bound to the vesicle interface. This conclusion is based on the following observations. (a) When there is an excess of vesicles over enzyme, the hydrolysis of the sn-2-acyl group ceases after only a fraction of the total available substrate is hydrolyzed. Addition of more enzyme, but not of more substrate, leads to a new round of hydrolysis. (b) The extent of hydrolysis of vesicles per enzyme increases with the size of the vesicles, and it corresponds to the total hydrolysis of the outer monolayer of one vesicle by one enzyme. (c) The enzyme bound to vesicles composed of reaction products or of the non-hydrolyzable phospholipid 1,2-ditetradecyl-sn-glycero-3-phosphomethanol (DTPM) is not able to undergo intervesicle exchange. Instead, intervesicle transfer of the substrate or the bound enzyme due to vesicle fusion promotes hydrolysis of all of the vesicles present in the reaction mixture. (d) Addition of DTPM vesicles to a reaction mixture containing DMPM substrate vesicles and the enzyme has no noticeable effect on the course of hydrolysis. Substrate specificity studies in the scooting mode on DMPM vesicles reveal that GCAT displays essentially no selectivity in the hydrolysis of phospholipids with different polar head groups. Treatment of GCAT with trypsin, which removes a small peptide, results in an enzyme that displays comparable catalytic activity but increased affinity for the interface. Alkyltrifluoromethyl ketones are shown to be tight-binding competitive inhibitors of GCAT. The scooting mode analysis, which has previously been shown to provide a simplified approach for analyzing the steady-state kinetics of interfacial catalysis by secreted phospholipase A2, is also useful for analyzing the interfacial kinetic behavior of lipases.

Acyltransferases↗
PubMed1994

Functional significance of the conformational dynamics of the N-terminal segment of secreted phospholipase A2 at the interface.

The kinetic and fluorescence properties of several pig pancreatic phospholipase A2 (PLA2) with substitutions and deletions in the N-terminal region and of tyrosines 52 and 73 are characterized. The substitutions Ala-1-D-Ala or -Gly, Trp-3-Phe, Gln-4-Nle, Arg-6-Glu, Tyr-52-Phe, and Tyr-73-Phe had at the most only a modest effect on the interfacial catalytic activity on the anionic interface to which they bind with high affinity. The observed rate of hydrolysis in the scooting mode by deletion mutants lacking one or more successive residues from the N-terminal region was lower by 50-95%. Detailed kinetic analysis of the deletion mutant lacking Ala-1 (des-1-AMPA) showed that the 50% decrease in the rate is due to a 5-fold increase in the interfacial Michaelis-Menten parameter, KM*, without a significant change in kcat. These results and direct measurements show that the primary effect of Ala-1 deletion is to lower the affinity for the active site directed ligands. Although the affinity of these mutants for anionic interface remains the same as for the wild type, the affinity for zwitterionic neutral diluents is considerably lower. Significant differences in the fluorescence quantum yields and the heterogeneity in the frequency-domain fluorescence intensity decays of these enzymes suggest that both in solution and at the interface the N-terminal region is an ensemble of conformations rather than a discrete state. Additional results suggest that the interfacial microenvironment of Trp-3 in des-1-AMPA is more polar and Trp-3 is more accessible to quenching by acrylamide.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Phosphatidylinositol-specific phospholipase C from Bacillus cereus at the lipid-water interface: interfacial binding, catalysis, and activation.

Binding characteristics of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus binding to the phospholipid-water interface were determined by spectroscopic methods and correlated with PI-PLC's catalytic properties. Binding of the enzyme to micelles and bilayers of zwitterionic phosphocholines is accompanied by an increase in the fluorescence emission from tryptophan, whereas a decrease in the emission is observed with synthetic anionic lipids containing a phosphomethanol head group. A similar decrease in the tryptophan emission is observed with phosphatidylinositol (PI) analogues containing the phospho-D-1-myo-inositol head group, but not with the enantiomeric L-1-myo-inositol. In covesicles of PI and phosphatidylcholine (PC), the rate of cleavage of PI is reduced because, as a neutral diluent, PC effectively reduces the surface concentration of PI that the bound enzyme "sees" in the interface. This permits determination of the interfacial Michaelis constant (KM*) as 0.26 mol fraction for PI as substrate. On the other hand, ditetradecylglycerophosphomethanol (DTPM) acts as a kinetic competitive inhibitor in the covesicles. The spectroscopic and catalytic activity data taken together show that PI-PLC binds to the interface of aqueous dispersions of phospholipids with an apparent Kd (in terms of the lipid monomers) of about 10-50 microM. However, only lipids with an anionic head group, such as phosphomethanol and phospho-D-1-myo-inositol, are able to bind as single molecules into the active site of the enzyme at the interface. Enantiomeric phospho-L-1-myo-inositol or the zwitterionic phosphocholine head group has little affinity for the enzyme at the interface. Thus, PI-PLC appears to obey the two-stage, Michaelis-Menten adaptation of interfacial catalysis, according to which the binding of the enzyme to the interface precedes the steps of the catalytic turnover at the interface. Limit estimates suggest that on PI or PI/PC vesicles the catalysis occurs in the "scooting" mode with a moderate processivity. DTPM vesicles also inhibit the activity of PI-PLC toward the synthetic water-soluble substrate myo-inositol 1-(4-nitrophenyl phosphate), but the activity is enhanced severalfold in the presence of vesicles of zwitterionic phosphatidylcholine. Several possible explanations of this interfacial activation are considered within the general context of the kinetic scheme for interfacial catalysis. The kinetic results for the action of PI-PLC bound to vesicles are consistent with a model in which the interface acts as an "allosteric" effector of the catalytic rate constant, kcat, without affecting the substrate binding.

Bacillus cereus↗
PubMed1994

Inhibition of phospholipase A2.

Phospholipases A2 are involved in inflammatory processes such as the liberation of free arachidonic acid from the membrane pool for the biosynthesis of eicosanoids. Inhibitors of these enzymes are proving useful in determining the biological roles of phospholipases A2 in complex cellular processes and may also have therapeutic potential. Inhibition of these lipolytic enzymes is more difficult to characterize as the enzymatic reaction occurs at a lipid/water interface. This review focuses on the description of a number of classes of rationally designed phospholipase A2 inhibitors. The development of a theoretical framework for the proper analysis of inhibitors is presented. Structural studies of phospholipase A2-inhibitor complexes suggest how the lipolysis reaction is catalyzed. Finally, some recent results on the use of phospholipase A2 inhibitors in living cells and tissues are revealed.

Animals↗
PubMed1994

Anaerobic degradation of normal- and branched-chain Fatty acids with four or more carbons to methane by a syntrophic methanogenic triculture.

Syntrophic degradation of normal- and branched-chain fatty acids with 4 to 9 carbons was investigated with a mesophilic syntrophic isobutyrate-butyrate-degrading triculture consisting of the non-spore-forming, syntrophic, fatty acid-degrading, gram-positive rod-shaped strain IB, Methanobacterium formicicum T1N, and Methanosarcina mazei T18. This triculture converted butyrate and isobutyrate to methane and converted valerate and 2-methylbutyrate to propionate and methane. This triculture also degraded caproate, 4-methylvalerate, heptanoate, 2-methylhexanoate, caprylate, and pelargoate. During the syntrophic conversion of isobutyrate and butyrate, a reversible isomerization between butyrate and isobutyrate occurred; isobutyrate and butyrate were isomerized to the other isomeric form to reach nearly equal concentrations and then their concentrations decreased at the same rates. Butyrate was an intermediate of syntrophic isobutyrate degradation. When butyrate was degraded in the presence of propionate, 2-methylbutyrate was synthesized from propionate and isobutyrate formed from butyrate. During the syntrophic degradation of valerate, isobutyrate, butyrate, and 2-methylbutyrate were formed and then degraded. During syntrophic degradation of 2-methylbutyrate, isobutyrate and butyrate were formed and then degraded.

Journal Article↗
PubMed1994

Jejunal disaccharidases in protein energy malnutrition and recovery.

The jejunal disaccharidases, sucrase, maltase and lactase, were determined in jejunal biopsies obtained from 43 malnourished children and 10 controls. In the study group, 63% were girls and 93% had severe malnutrition. Lactase activity was significantly reduced in third and fourth degree malnutrition (p < 0.05 and p < 0.005, respectively), but maltase activity was significantly reduced only in the fourth degree malnutrition (p < 0.01). After recovery, maltase and sucrase activities showed a marginally significant increase (p = 0.06), where lactase showed no significant increase (p > 0.05). We conclude that jejunal disaccharidase activity decreases significantly with increasing severity of malnutrition, lactase being the most severely affected and the last to recover.

Biopsy↗
PubMed1994

A review of human immunodeficiency virus infection in India.

By the turn of the century 20-50 million adults are projected to be HIV positive in Asia, and India will have the largest burden of newly infected patients in a single country. To determine the present status of the epidemic, a systematic review of available data in India has been carried out. Regional differences in reported HIV seroprevalence were found, with high levels in western, northeastern, and southern India and lower levels in northern, central, and eastern India. While differences in the temporal introduction of HIV may be partly responsible, the more likely explanation is incomplete data and reporting. In the past 4 years HIV has rapidly increased among commercial sex workers and patients coming to sexually transmitted diseases clinics in western (Bombay) and southern (Madras and Vellore) India. Assessing HIV seroprevalence in the general population is difficult because of limited data, especially from rural areas, where 60% of Indians reside. Heterosexual contact with commercial sex workers is the major reported risk factor, except in eastern India, where intravenous drug use is common. The impact of transfusions or contaminated needles is uncertain, but between 25 and 75% of blood donations are not screened for HIV, and 15% of infected patients had received transfusions. By October 1993, 494 cases of AIDS had been reported, but this number grossly underestimates the real situation since HIV is often unrecognized. Tuberculosis is the major HIV-associated infection. In its efforts to control HIV, India needs to institute a standardized surveillance system to provide data needed to design and implement appropriate interventions.

Blood Transfusion↗
PubMed1993

Spectroscopic properties of the states of pig pancreatic phospholipase A2 at interfaces and their possible molecular origin.

The near-UV absorption and fluorescence spectroscopic properties of Trp-3 of pig pancreatic phospholipase A2 (PLA2) in aqueous solution (E form) or at the interface without (E* form) or with a ligand at the active site (E*L form) are characterized. In the E form, the single tryptophan residue is exposed on the protein surface to the aqueous environment, as it is freely accessible to aqueous quenchers such as succinimide and acrylamide. The fluorescence quantum yield of E is about one-third that of N-acetyl-tryptophanamide, indicating significant intramolecular quenching processes including charge-transfer reactions, as seen by the D2O effect. Upon binding of PLA2 to micelles of 1-hexadecylpropanediol-3-phosphocholine (E*), a positive difference spectrum with a shoulder at 284 nm (delta epsilon = 370 M-1 cm-1) is observed. Similar difference spectra are also observed upon binding of sulfate ion to the E form. The fluorescence emission of E* is blue-shifted by about 10 nm to 336 nm, with a 2-fold higher quantum yield. Trp-3 in E* is significantly shielded from aqueous quenchers, and the D2O effect on the quantum yield is still present. The UV difference spectrum for the E*-to-E*L transition is of large amplitude with peaks at 292 (delta epsilon = 2540 M-1 cm-1) and 284 nm (delta epsilon = 2100 M-1 cm-1), which suggests transfer of tryptophan from an aqueous to a less polar environment. Upon conversion to the E*L form, there is a further blue shift to 333 nm, with about a 20% increase in the fluorescence quantum yield.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗