Biomedical subjects

V Narayanaswami

Publications and source records attributed to V Narayanaswami.

25 records · Page 2Linked to original sources

PubMed1994

Metabolic relevance of selenium in the insect Corcyra cephalonica. Uptake of 75Se and subcellular distribution.

Requirement, uptake, and subcellular distribution of Na2(75)SeO3 in the larvae of the insect C. cephalonica was investigated. That Se is well tolerated by C. cephalonica upto an added level of 2 ppm in the diet is suggested by the observed increase in body weight, total protein, and succinate dehydrogenase levels. Significant increases in the State 3 respiration ensued with Se supplementation up to 2 ppm in the mitochondrial oxidation of D-glycerol 1-phosphate, succinate and NADH, along with concomitant unaltered State 4 respiration, leading to enhanced RCR values. Maximal uptake of 75Se was registered in the larvae maintained on basal diet when subjected to short-term exposure to 0.5 ppm 75Se level. When exposure level was further increased up to 20 ppm, the observed decrease in the uptake of 75Se suggested that Se status of larvae itself controlled the tissue uptake. Subcellular distribution pattern revealed maximal incorporation of 75Se (cpm/g tissue) in the supernatant fraction, whereas, maximal specific 75Se activity (cpm/mg protein) was associated with the mitochondrial fraction. Autoradiography of the soluble fractions indicated the presence of single selenoprotein in the larval group with short term 2 ppm 75Se exposure. Inherent Se controls both the extent and the nature of distribution of mitochondrial 75Se incorporation. Uptake of 45Ca by the insect mitochondria was enhanced by dietary Se up to 2 ppm but was unaffected by addition of in vitro 75Se in the medium. A more fundamental role for Se in the mitochondrial energy metabolism emerges from these studies.

Animals↗

PubMed1993

Protein-lipid interactions and Torpedo californica nicotinic acetylcholine receptor function. 1. Spatial disposition of cysteine residues in the gamma subunit analyzed by fluorescence-quenching and energy-transfer measurements.

The nicotinic acetylcholine receptor from Torpedo californica was labeled with a fluorescent, lipophilic probe, N-(1-pyrenyl)maleimide, specific for sulfhydryls in a hydrophobic environment, and was found to alkylate Cys 416, Cys 420 and Cys 451 in the gamma subunit [Li, L., Schuchard, M., Palma, A., Pradier, L., & McNamee, M.G. (1990) Biochemistry 29, 5428-5436]. The spatial disposition of the acetylcholine receptor-bound pyrene with respect to the membrane bilayer was assessed by a combination of fluorescence-quenching and resonance energy transfer measurements, under conditions of selective labeling of the gamma subunit. Quenching of pyrene fluorescence by spin-labeled fatty acids with the doxyl group at positions C-5 and C-12 revealed that the former was more effective, with a Stern-Volmer quenching constant of 0.187 compared to 0.072 for the latter, suggesting that the fluorophore(s) are located closer to the membrane-water interface rather than the hydrophobic interior. Energy transfer was found to occur from tryptophan in the acetylcholine receptor to cysteine-bound pyrene with a distance of separation of approximately 18 A. However, there was no energy transfer when pyrene-labeled AChR was reconstituted into membranes containing brominated phospholipids and cholesterol, suggesting that the fluorophore(s) responsible for energy transfer are located in the membrane domain. Thus, the N-(1-pyrenyl)maleimide can be used to monitor lipid-protein interactions of the AChR.

Animals↗

PubMed1993

Protein-lipid interactions and Torpedo californica nicotinic acetylcholine receptor function. 2. Membrane fluidity and ligand-mediated alteration in the accessibility of gamma subunit cysteine residues to cholesterol.

Fluorescence-quenching and energy-transfer measurements were carried out to further characterize lipid-protein interactions involving the nicotinic acetylcholine receptor (AChR) from Torpedo californica in reconstituted membranes. To assess the fluidity of the receptor microenvironment, cis- and trans-parinaric acids were used to take advantage of the preferential partitioning behavior of the trans isomer for the gel phase. A relatively higher extent of energy transfer from the intrinsic tryptophan fluorescence of AChR in dielaidoylphosphatidylcholine bilayers to cis-parinaric acid in both the gel and the fluid phase suggests that the AChR is surrounded by a relatively fluid annulus of lipids. The ability of AChR to accommodate and interact with specific lipids such as cholesterol and fatty acids in the vicinity of pyrene-labeled cysteine residues in the membranous domain and/or the membrane-water interface region of the gamma subunit was assessed. Pyrene-labeled AChR prepared in (6,7-dibromostearoyl)phosphatidylcholine showed a 25% decrease in fluorescence as sites accessible to phospholipids were occupied; subsequent addition of dibromocholesterol hemisuccinate (DiBrCHS) caused further quenching by about 25%. This result is consistent with the presence of sites accessible to cholesterol, but not accessible to phospholipids, in the vicinity of the cysteine-bound pyrene in the membranous domain of the AChR. Quenching by DiBrCHS was sensitive to the presence of an AChR activator (carbamylcholine) but not a competitive antagonist (alpha-bungarotoxin). The Stern-Volmer quenching constant was 0.123 in the absence of added ligands and 0.167 and 0.134 in the presence of carbamylcholine and alpha-bungarotoxin, respectively, corresponding to accessibilities of 65%, 90%, and 70%.

Animals↗

PubMed1991

ATP-dependent S-(2,4-dinitrophenyl)glutathione transport in canalicular plasma membrane vesicles from rat liver.

Uptake of the thioether S-(2,4-dinitrophenyl)glutathione (DNPSG) in canalicular plasma membrane vesicles from rat liver is enhanced in the presence of ATP and exhibits an overshoot with a transient 5.5-fold accumulation of DNPSG. Stimulation by ATP is not caused by the generation of a membrane potential, based on responses of the indicator dye oxonol V. ATP-dependent uptake has an apparent Km of 71 microM for DNPSG and a Vmax of 0.34 nmol.min-1.mg of vesicle protein-1. Protein thiol groups are essential for transport activity as indicated by the sensitivity of DNPSG transport to sulfhydryl reagents. There is competitive inhibition with other thioethers, S-hexylglutathione (Ki = 66 microM), the photoaffinity label S-(4-azidophenacyl)glutathione (Ki = 56 microM), as well as with glutathione disulfide (Ki = 0.44 mM) and with the bile acid taurocholate (Ki = 0.61 mM). GSH (2 mM) or cholate (0.4 mM) does not inhibit. Both glutathione disulfide and taurocholate show ATP-dependent transport in the canalicular membrane vesicles which is inhibited by DNPSG. No ATP-dependent transport is found for GSH. Transport of DNPSG is also inhibited competitively by alpha-naphthyl-beta-D-glucuronide (Ki = 0.42 mM) but not by alpha-naphthylsulfate (2 mM), and there is substantial inhibition with the glucuronides from ebselen and p-nitrophenol. The results indicate that the canalicular transport system for DNPSG is directly driven by ATP and that the biliary transport of other classes of compounds may also proceed via this system.

Adenosine Triphosphate↗

PubMed1990

Oxidative damage to mitochondria and protection by ebselen and other antioxidants.

Iron/ascorbate induced lipid peroxidation in liver mitochondria isolated from normal and glutathione-depleted rats was monitored by low-level chemiluminescence and by accumulation of thiobarbituric acid-reactive substances (TBARS). Antioxidant capacity was assessed by the duration of the lag phase preceding the onset of active peroxidation. The lag phases in state 4 and in the presence of uncouplers were similar, but shorter in the presence of ADP (state 3). In glutathione-depleted rats the lag periods were less than those in normal mitochondria. A biphasic pattern of loss of membrane alpha-tocopherol was typical in state 4 with about 55% remaining after 40 min, while in presence of ADP there was a steady and rapid loss to about 30% of the initial level. Synthetic antioxidants such as ebselen or its glutathione adduct protected mitochondrial membranes against peroxidative reactions. There was a 5-fold increase in the lag phase with 1 microM ebselen in state 4 (lag doubling concentration, 0.4 microM) and a significantly lower rate of loss of alpha-tocopherol with about 90% of the initial level still remaining after 40 min. Likewise, the lag doubling concentrations were 0.04 microM for diethyldithiocarbamate, 0.3 microM for 5-hydroxyindole, 10 microM for dihydroxyphenylalanine and serotonin, and about 40 microM for epinephrine and norepinephrine.

Animals↗

PubMed1990

Reactivity of ebselen and related selenoorganic compounds with 1,2-dichloroethane radical cations and halogenated peroxyl radicals.

The reactivity of ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)one, and structurally related analogues was studied by pulse radiolysis. The rate constant for the reaction of ebselen with trichloromethylperoxyl radicals was determined to be 2.9 X 10(8) M-1 s-1, while its sulfur analogue, 2-phenyl-1,2-benzisothiazol-3(2H)one, was oxidized at much lower rates, k less than or equal to 10(7) M-1 s-1. Among several derivatives studied, the only other compound that exhibited a high rate constant was 2-(methylseleno)-benzoic acid-N-phenylamide. Oxidation of ebselen by other halogenated peroxyl radicals was also carried out and revealed a direct relationship between rate constant and the degree of halogenation of the oxidant. The transient radicals generated during oxidation of ebselen and the analogues were characterized by optical absorption and conductivity measurements and were attributed to one-electron-oxidized radical cations. The oxidation potentials were determined by cyclic voltammetry. Comparative evaluation of the in vitro behavior during microsomal lipid peroxidation revealed ebselen to be the most potent antioxidant of the compounds investigated, 2-(Methylseleno)-benzoic acid-N-phenylamide, despite its high rate constant for oxidation by halogenated peroxyl radicals, was found to be a poor antioxidant. The rate constant of oxidation of ebselen by trichloromethylperoxyl radicals is comparable to that of alpha-tocopherol under similar conditions, underscoring the potential pharmacological interest of ebselen as an antioxidant.

Antioxidants↗

PubMed1990

Antioxidant activity of ebselen and related selenoorganic compounds in microsomal lipid peroxidation.

Ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)one, and its derivatives were compared for their ability to protect microsomal membranes against iron/ADP/ascorbate-induced lipid peroxidation, measured as low-level chemiluminescence and accumulation of thiobarbituric acid-reactive substances (TBARS). The concentrations of the compounds required to double the lag time of the control with no added antioxidants were 0.13 microM for ebselen, 0.5 microM for the N-pyridyl analog, 0.3-0.7 microM for the selenylsulfides, about 1.0 microM for the selenoxide derivative and 2.0 microM for the sulfur analog of ebselen. The open-chain seleno- and thioether derivatives, on the other hand, exhibited comparatively low abilities to protect the membrane, the lag doubling concentrations for these compounds being 100-1,000 fold higher than that for ebselen. The rate of loss of alpha-tocopherol in the microsomal membrane during peroxidation was significantly diminished in the presence of 0.1-0.5 microM ebselen, while the glutathione adduct of ebselen was equally effective in protecting the loss of alpha-tocopherol. The sulphur analogue and, the benzylated and methylated derivatives of ebselen did not afford protection. Ebselen was without effect in microsomes from vitamin E-deficient rats up to 20 microM, indicative of the dependence of its protective ability upon alpha-tocopherol.

Adenosine Diphosphate↗