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Biomedical subjects

V Ravichandran

Publications and source records attributed to V Ravichandran.

At least 19 recordsLinked to original sources
PubMed2004

Spectrophotometric analysis of raloxifene hydrochloride in pure and pharmaceutical formulations.

Two simple and sensitive spectrophotometric methods (A and B) for the determination of raloxifene hydrochloride in bulk samples and pharmaceutical formulations are described. Method A is based on the oxidation of the drug with ferric chloride and coupling with potassium ferric cyanide. Method B is based on reduction of the drug with Fehling's reagent. Bluish green color formed in method A absorbs at 735 nm and brown color produced in method B absorbs at 430 nm.

Chemistry, Pharmaceutical↗
PubMed2002

The GTP binding proteins Gem and Rad are negative regulators of the Rho-Rho kinase pathway.

The cytoskeletal changes that alter cellular morphogenesis and motility depend upon a complex interplay among molecules that regulate actin, myosin, and other cytoskeletal components. The Rho family of GTP binding proteins are important upstream mediators of cytoskeletal organization. Gem and Rad are members of another family of small GTP binding proteins (the Rad, Gem, and Kir family) for which biochemical functions have been mostly unknown. Here we show that Gem and Rad interface with the Rho pathway through association with the Rho effectors, Rho kinase (ROK) alpha and beta. Gem binds ROKbeta independently of RhoA in the ROKbeta coiled-coil region adjacent to the Rho binding domain. Expression of Gem inhibited ROKbeta-mediated phosphorylation of myosin light chain and myosin phosphatase, but not LIM kinase, suggesting that Gem acts by modifying the substrate specificity of ROKbeta. Gem or Rad expression led to cell flattening and neurite extension in N1E-115 neuroblastoma cells. In interference assays, Gem opposed ROKbeta- and Rad opposed ROKalpha-mediated cell rounding and neurite retraction. Gem did not oppose cell rounding initiated by ROKbeta containing a deletion of the Gem binding region, demonstrating that Gem binding to ROKbeta is required for the effects observed. In epithelial or fibroblastic cells, Gem or Rad expression resulted in stress fiber and focal adhesion disassembly. In addition, Gem reverted the anchorage-independent growth and invasiveness of Dbl-transformed fibroblasts. These results identify physiological roles for Gem and Rad in cytoskeletal regulation mediated by ROK.

3T3 Cells↗
PubMed2001

The PDB data uniformity project.

The Protein Data Bank (PDB; http://www.rcsb.org/pdb/) is the single worldwide archive of structural data of biological macromolecules. This paper describes the data uniformity project that is underway to address the inconsistency in PDB data.

Databases, Factual↗
PubMed1999

Phosphorylation of SNAP-23 by the novel kinase SNAK regulates t-SNARE complex assembly.

The docking and fusion of cargo-containing vesicles with target membranes of eukaryotic cells is mediated by the interaction of SNARE proteins present on both vesicle and target membranes. In many cases, the target membrane SNARE, or t-SNARE, exists as a complex of syntaxin with a member of the SNAP-25 family of palmitoylated proteins. We have identified a novel human kinase SNAK (SNARE kinase) that specifically phosphorylates the nonneuronal t-SNARE SNAP-23 in vivo. Interestingly, only SNAP-23 that is not assembled into t-SNARE complexes is phosphorylated by SNAK, and phosphorylated SNAP-23 resides exclusively in the cytosol. Coexpression with SNAK significantly enhances the stability of unassembled SNAP-23, and as a consequence, the assembly of newly synthesized SNAP-23 with syntaxin is augmented. These data demonstrate that phosphorylation of SNAP-23 by SNAK enhances the kinetics of t-SNARE assembly in vivo.

Carrier Proteins↗
PubMed1997

Cloning and identification of human syntaxin 5 as a synaptobrevin/VAMP binding protein.

Syntaxins are transmembrane proteins that function in regulating transport vesicle docking and fusion with target membranes in neuronal and nonneuronal cells. Vesicle docking is thought to be regulated in part by the specific interactions of syntaxin with a vesicle-associated membrane protein termed synaptobrevin/VAMP. We have cloned a 1557-bp cDNA that encodes the human syntaxin 5 isoform, using a combination of PCR and colony-screening methods. The deduced 301 amino-acid sequence of human syntaxin 5 shares 96% identity with rat syntaxin 5. Like rat syntaxin 1A, human syntaxin 5 binds to synaptobrevin/VAMP in vitro. The identification of human syntaxin 5 as a synaptobrevin/VAMP-binding protein supports the hypothesis that syntaxin 5 regulates protein transport by binding to vesicle-associated membrane proteins.

Animals↗
PubMed1996

Identification of a novel syntaxin- and synaptobrevin/VAMP-binding protein, SNAP-23, expressed in non-neuronal tissues.

The specificity of vesicular transport is regulated, in part, by the interaction of a vesicle-associated membrane protein termed synaptobrevin/VAMP with a target compartment membrane protein termed syntaxin. These proteins, together with SNAP-25 (synaptosome-associated protein of 25 kDa), form a complex which serves as a binding site for the general membrane fusion machinery. Synaptobrevin/VAMP and syntaxin are ubiquitously expressed proteins and are believed to be involved in vesicular transport in most (if not all) cells. However, SNAP-25 is present almost exclusively in the brain, suggesting that a ubiquitously expressed homolog of SNAP-25 exists to facilitate transport vesicle/target membrane fusion in other tissues. Using the yeast two-hybrid system, we have identified a 23-kDa protein from human B lymphocytes (termed SNAP-23) that binds tightly to multiple syntaxins and synaptobrevins/VAMPs in vitro. SNAP-23 is 59% identical with SNAP-25. Unlike SNAP-25, SNAP-23 was expressed in all tissues examined. These findings suggest that SNAP-23 is an essential component of the high affinity receptor for the general membrane fusion machinery and an important regulator of transport vesicle docking and fusion in all mammalian cells.

Amino Acid Sequence↗
PubMed1996

Protein S-thiolation and dethiolation during the respiratory burst in human monocytes. A reversible post-translational modification with potential for buffering the effects of oxidant stress.

Stimulation of the respiratory burst in mouse macrophages or human neutrophils results in the formation of disulfide bonds between low m.w. thiols and sulfhydryl groups on specific cytosolic proteins (S-thiolation). S-thiolation is reversible in certain chemical systems. The aim of the present study was to analyze the dynamic nature of this process in human monocytes under physiologic conditions. We report here that the extent of S-thiolation and the rate of respiratory burst stimulated by opsonized zymosan or phorbol diester increased for 10 to 20 min and then declined (dethiolation) in close association. Individual proteins underwent S-thiolation and dethiolation at different rates. H2O--appeared particularly effective in mediating S-thiolation, based on inhibition of S-thiolation by added catalase and accentuation by azide, which inhibits cellular catalase. S-thiolation did not occur in stimulated monocytes from patients with chronic granulomatous disease. The addition of H2O2 to monocytes or lymphocytes induced rapid S-thiolation (1 to 3 min); a subsequent dethiolation returned most of the proteins to baseline by 15 to 30 min. At 0 degrees C and after addition of 1,3-bis-(2-chloroethyl)-1-nitrosourea, there was effective S-thiolation on exposure to H2O2, but dethiolation was inhibited, suggesting a possible role for glutathione (GSH)/thioredoxin reductase systems in this process. GSH was determined to be the most abundant low m.w. thiol bound to S-thiolated proteins, but gamma-glutamylcysteine and cysteine were also bound. The time of maximal reduction in cytosolic GSH during the respiratory burst (10 min) coincided with the time at which protein-bound GSH was highest. S-thiolation-dethiolation represents a reversible post-translational modification that could protect cellular proteins from irreversible oxidative damage.

Blood Proteins↗
PubMed1996

Diamide primes neutrophils for enhanced release of superoxide anion: relationship to S-thiolation of cellular proteins.

Stimulation of the respiratory burst in phagocytes induces the formation of mixed disulfides between sulfhydryl groups of proteins and low-molecular-weight thiols. We hypothesized that this process (S-thiolation) might be involved in turning off the respiratory burst. However, induction of S-thiolation by pretreatment of neutrophils with diamide, a direct thiol oxidizing agent, actually primed the cells for a two- to fivefold increase in total release and fourfold increase in rate of release of 02- on stimulation by f-Met-Leu-Phe. Generation of intracellular oxidants (hydroethidine fluorescence) was increased ninefold. Priming and S-thiolation were apparent at 1 min of incubation and peaked at 5-10 min. Diamide pretreatment also reduced the lag time between addition of phorbol diester and release of 02- by a mean of 23 s (41%). Dithioerythritol, a sulfhydryl-reducing agent, abolished both the S-thiolation and priming mediated by diamide. H202 also induced priming and S-thiolation; and these were eliminated by dithioerythritol. In contrast to the effect of endotoxin, diamide priming did not affect Ca2+ homeostasis of the neutrophils. Diamide did not significantly alter NADPH oxidase activity in a cell-free system. These findings suggest that sulfhydryl groups on one or more proteins play an important role in modulating the respiratory burst.

Blood Proteins↗
PubMed1994

S-thiolation of glyceraldehyde-3-phosphate dehydrogenase induced by the phagocytosis-associated respiratory burst in blood monocytes.

Chemical oxidants can induce the covalent binding of low molecular weight thiols to reactive sulfhydryls on proteins (S-thiolation). We found that stimulation of the respiratory burst of human blood monocytes resulted in S-thiolation of several proteins, most prominently one of 38 kDa. This purified protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by enzyme activity, immunoblotting, and amino acid analysis. After stimulation of the respiratory burst, S-thiolation of GAPDH gradually increased, and cytosol GAPDH activity decreased; so that at 60 min, GAPDH activity was reduced by approximately 40%. Activity was restored by the addition of the sulfhydryl-reducing agent dithioerythritol. H2O2 appeared to be particularly important in mediating S-thiolation during the respiratory burst. Exposure of monocytes to H2O2 induced concentration-dependent S-thiolation of GAPDH and a concomitant decrease in enzyme activity. The addition of respiratory burst stimuli to lymphocytes, which lack a full respiratory burst, had no effect on GAPDH S-thiolation or activity; but H2O2 induced S-thiolation of lymphocyte GAPDH and inhibition of enzyme activity. Stimulation of monocytes from three patients with chronic granulomatous disease resulted in no respiratory burst, S-thiolation of GAPDH, or inactivation of GAPDH activity. The thiols covalently bound to purified S-thiolated GAPDH were removed by dithioerythritol and were identified as glutathione and cysteine; glutathione was predominant. These results indicate that during the respiratory burst in monocytes, low molecular weight thiols can bind to specific cytosolic proteins, including GAPDH. It is possible that S-thiolation of cytosolic proteins serves to modulate cellular metabolic events during phagocytosis.

Ethylmaleimide↗
PubMed1993

Restoration of tissue antioxidants and prevention of renal stone deposition in vitamin B6 deficient rats fed with vitamin E or methionine.

Observed loss in body weight gain, increased lipid peroxidation reaction, decreased concentrations of antioxidants, ascorbic acid, alpha-tocopherol and reduced glutathione and antioxidant enzymes, glutathione peroxidase and catalase and increased concentration of hydroperoxides and hydroxyl radicals in vitamin B6 deficient rat liver [J Nutri Biochem, 2 (1991) 245] and kidney [Biochem International, 21 (1991) 599] were nearly normalized on feeding with vitamin E or methionine. Accumulation of oxalate and calcium during vitamin B6 deficiency was abolished by feeding vitamin E or methionine. Calcium oxalate deposition observed in vitamin B6 deficient kidney was completely prevented when fed along with vitamin E or methionine. However the hyperoxaluria and hypercalciuria persisted even after feeding with vitamin E or methionine.

Animals↗
PubMed1991

Starch as a renewable finish to improve the pesticide-protective properties of conventional workclothes.

Because many pesticide handlers persist in wearing and reusing conventional workclothes, a renewable functional finish that enhances the pesticide-protective qualities of fabrics would be useful. This study investigated the ability of starch to act as a pesticide trap, preventing transfer and increasing removal by laundering, and the effect of carboxymethyl cellulose on release of pesticide in laundry. The retention and distribution of methyl parathion (MeP) on 65% polyester/35% cotton fabric was studied with four finishes: starch and carboxymethyl cellulose (CMC) as nondurable finishes; durable press resin (DP) and durable press/carboxymethyl cellulose (DP/CMC) as durable finishes. Starching with an add-on of 8% (w/w) effectively reduced the area of contamination and enhanced the removal of methyl parathion from polyester/cotton fabrics. Residual pesticide values for CMC, DP, and DP/CMC finishes were similar to that of the unfinished fabric. While distribution profiles of methyl parathion throughout the yarn and fiber structures were similar for all the finishes, lower concentrations of pesticide were observed on the cotton fibers from the starched fabric. Starch reduced the pesticide transferring by rubbing from both 100% cotton and 65% polyester/cotton fabrics. These studies support the intriguing theory that starch can act as a pesticide trap on the fabric surface to decrease pesticide transfer and to enhance pesticide removal. Extensive penetration studies, field studies, and additional investigation of fiber, yarn, and fabric parameters are needed to further quantify the effects of starch.

Carboxymethylcellulose Sodium↗
PubMed1991

Increased plasma lipidperoxidation in vitamin B-6 deficient rats.

Lipidperoxidation in plasma of rats fed with vitamin B-6 deficient diet for a period of 12 weeks was studied with pair-fed controls. Plasma pyridoxal 5'-phosphate, alanine amino transferase and aspartate amino transferase, the markers of vitamin B-6 status, were significantly low in vitamin B-6 deficient rats. Plasma malondialdehyde level, conjugated dienes and lipofuscin like pigments were increased in vitamin B-6 deficiency. Increased levels of plasma lipids, calcium, iron and copper were observed in vitamin B-6 deficiency. Plasma susceptibility to lipidperoxidation was maximal in vitamin B-6 deficiency, upon stimulation by the promotors, Fe2+, Fe3+, Cu2+, ascorbate, t-butyl hydroperoxide and hydrogen peroxide.

Animals↗
PubMed1991

Effect of oral methionine and vitamin E on blood lipid peroxidation in vitamin B6 deficient rats.

Lipid peroxidation in blood of vitamin B6 deficient rats was significantly increased when compared to pair-fed controls. The observed increased lipid peroxidation in vitamin B6 deficiency was correlated with high levels of lipids, metal ions and low levels of antioxidants, alpha-tocopherol, ascorbic acid and reduced GSH. Supplementation of methionine or vitamin E along with the vitamin B6 deficient diet restored the levels of antioxidants to near normal and also protected against oxidative stress. However plasma TBARS level as well as total lipids were still elevated in M-B6 diet fed rats and normalized in E-B6-d rats.

Animals↗
PubMed1990

Increased lipid peroxidation in kidney of vitamin B-6 deficient rats.

Lipid peroxidation in kidney of rats fed with vitamin B-6 deficient diet for a period of 12 weeks was studied with pair-fed controls. The basal lipid peroxide level as well as the degree of susceptibility to lipid peroxidation in presence of promotors such as NADPH, ascorbate, t-butyl hydroperoxide, Fe2+, Cu2+ and oxalate, were increased in vitamin B-6 deficient kidney. The observed increased lipid peroxidation in vitamin B-6 deficient kidney was correlated with high levels of lipids, copper, iron, calcium and oxalate, low levels of antioxidants and antioxidant enzymes and increased levels of hydroperoxides and hydroxyl radicals.

Animals↗
PubMed1988

Conformation of methylated amino acids: structure of 3,4-dimethoxy-alpha-methyl-DL-phenylalanine sesquihydrate.

C12H17NO4.1.5H2O, Mr = 266.3, triclinic, P1, a = 5.872 (1), b = 11.437 (2), c = 20.434 (1) A, alpha = 95.74 (1), beta = 96.91 (1), gamma = 89.18 (1) degrees, V = 1355.5 A3, Z = 4, Dm = 1.29, D chi = 1.305 g cm-3, lambda(Cu K alpha) = 1.5418 A, mu = 8.3 cm-1, F(000) = 572, T = 294 K, R = 0.038 for 4006 reflections, I greater than or equal to 3 sigma(I). Both the molecules A and B in the asymmetric unit exist as zwitterions. With respect to the D enantiomer, the torsion angles psi 1 and psi 2 are +47.2 and -134.4 degrees in molecule A and +33.3 and -147.5 degrees in molecule B respectively. The torsion angles of the alpha-methyl group, NH3+ and COO- groups with respect to Cv are in molecules A and B respectively +67.2, +66.8, -174.3, -175.6, and -59.2 and -59.5 degrees. The hydrogen-bonding environment of water OW1 is trigonal nonplanar; OW2 is trigonal planar and OW3 is tetrahedral. The crystal structure is stabilized by a number of hydrogen bonds involving the amino and carboxylate groups of both molecules A and B and the water molecules.

Computer Simulation↗
PubMed1987

Ion pairs in alpha helices.

A survey of 47 globular proteins was made to determine the probability of occurrence of ion pairs separated by 1,2,3,... and 8 residues in the alpha helices. As a control, the probability of occurrence of like charged pairs was also determined. The survey showed that ion pairs of the type i,i +/- 3 and i, i +/- 4 are the most predominant. Such a preference was not observed for like charged pairs. The observed frequency of ion pairs is significantly greater than their expected frequency. The normalized frequencies of occurrence of the ion pairs were also found to increase generally with the helix length. These results indicate that the ion pairs may contribute to the stability of solvent-exposed alpha helices. Since the stabilization of protein secondary structure enhances the stability of protein tertiary structure, these results may throw light on the mechanism of protein folding.

Amino Acids↗