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A Bhatnagar

Publications and source records attributed to A Bhatnagar.

At least 145 records · Page 8Linked to original sources
PubMed1992

Adrenalectomy, corticosteroid replacement and their importance for drug-induced memory-enhancement in mice.

Adrenalectomy blocks the memory-improving effect of piracetam-like compounds in mice. If this blockade is due to the removal of endogenous corticosteroids, replacement therapy with exogenous corticosteroids should reinstate the effects on memory. The present experiments were designed to determine the appropriate replacement dose (concentration in the drinking fluid) for corticosterone and aldosterone, the main corticosteroids in mice. Based on the effects of corticosterone on thymus weight, replacement with 3 micrograms/ml corticosterone given in the drinking fluid (0.9% NaCl) for one week was found to be appropriate. The appropriate replacement dose for aldosterone was found by giving aldosterone to adrenalectomized (ADX) mice in the drinking fluid in combination with 3 micrograms/ml corticosterone. The combination of 3 micrograms/ml corticosterone + 30 ng/ml aldosterone resulted in a plasma ratio of corticosterone/aldosterone which most closely approximated the ratio seen in sham-ADX control animals. The physiologic adequacy of the corticosteroid replacement doses resulting from this study were clearly demonstrated in subsequent behavioral experiments where blockade of the memory-enhancing effects of piracetam by adrenalectomy were overcome by replacement with either 3 micrograms/ml corticosterone or 30 ng/ml aldosterone given in the drinking fluid.

Adrenalectomy↗
PubMed1992

Mechanism of inhibition of aldose reductase by menadione (vitamin K3).

Incubation of human placental aldose reductase (EC 1.1.1.21) with menadione (0.5-3.0 mM) resulted in time-dependent loss of the catalytic activity of the enzyme. Kinetic analysis of the data suggests that the inactivation process follows a single apparent rate constant that displays hyperbolic dependence on menadione concentration, indicating that menadione forms a kinetically significant, dissociable complex with the enzyme before the formation of an inactive enzyme-menadione complex. The inactivation of the enzyme with menadione was reversed upon dialysis of the inactivated enzyme against buffer containing 10 mM dithiothreitol suggesting that menadione reacts with enzyme sulfhydryl residue(s). Inactivation of the enzyme was significantly prevented by dithiothreitol (5 mM), NADPH (0.1 mM), and DL-glyceraldehyde (10 mM). Correlation of the fractional remaining activity with the extent of modification indicates that loss of catalytic activity corresponds to the modification of a single amino acid residue of the enzyme protein. Recombinant human aldose reductase, obtained by overexpression in Escherichia coli, and aldose reductase in which Cys-80 or Cys-303 was replaced by serine were also inactivated by menadione. However, enzyme in which Cys-298 was replaced by serine was insensitive to menadione. On the basis of these observations, it is suggested that menadione forms a thiodione-like adduct with Cys-298, leading to inactivation of the enzyme.

Aldehyde Reductase↗
PubMed1991

Structure-activity correlations in human kidney aldehyde reductase-catalyzed reduction of para-substituted benzaldehyde by 3-acetyl pyridine adenine dinucleotide phosphate.

Steady-state kinetic parameters of the human kidney aldehyde reductase-catalyzed reduction of para-substituted benzaldehydes by 3-acetyl pyridine dinucleotide phosphate (3-APADPH) were determined. The kcat of aldehyde reduction by 3-APADPH was 2- to 4-fold lower than by NADPH. The dissociation constant of 3-APADPH from the enzyme-coenzyme complex was higher (77 microM) than that of NADPH (5.3 microM). Primary deuterium kinetic isotope effects on both kcat and kcat/Km for para-substituted benzaldehyde reduction by 3-APADPH (with the exception of para-carboxybenzaldehyde) were equal and on average 2.82 +/- 0.21, suggesting that these reactions follow a rapid equilibrium-ordered reaction scheme in which the hydride transfer step is rate-limiting. Multiple regression analysis of the data suggests that benzaldehyde reduction depends upon electronic substituent effects, characterized by a rho value of 0.5. These data are consistent with a transition state in which the charge on the aldehyde carbonyl increases relative to the charge on this group in the ground state. A positive deviation of para-carboxybenzaldehyde from the linear correlation between other benzaldehydes and the substituent constant sigma + suggests a specific interaction of the carboxyl substituent of the substrate with the enzyme.

Alcohol Dehydrogenase↗
PubMed1991

Human liver aldehyde reductase: pH dependence of steady-state kinetic parameters.

The pH dependence of steady-state parameters for aldehyde reduction and alcohol oxidation were determined in the human liver aldehyde reductase reaction. The maximum velocity of aldehyde reduction with NADPH or 3-acetyl pyridine adenine dinucleotide phosphate (3-APADPH) was pH independent at low pH but decreased at high pH with a pK of 8.9-9.6. The V/K for both nucleotides decreased below a pK of 5.7-6.2, as did the pKi of competitive inhibitors NADP and ATP-ribose, suggesting that the 2'-phosphate of the nucleotide has to be deprotonated for binding to the enzyme. The pK of the 2'-phosphate of NADPH appears to be perturbed in the ternary complexes to 5.2-5.4. The V/K for NADPH, the V/K for 3-APADPH, and the pKi of ATP-ribose also decreased above a pK of 9-10, suggesting interaction of the 2'-phosphate of the nucleotide with a protonated base, perhaps lysine. Since protonation of a residue with a pK of 8 (evident in V/K for DL-glyceraldehyde and V/K for L-gulonate versus pH profiles) appears to be essential for aldehyde reduction, and deprotonation for alcohol oxidation, this residue appears to act as a general acid-base catalyst. An additional anion binding site with a pK of 9.94 facilitates the binding of carboxylic substrates such as D-glucuronate. With NADPH as the coenzyme the primary deuterium isotope effects on V and V/K for NADPH were close to unity and pH independent, suggesting that the hydride transfer step is not rate determining over the experimental pH range. With 3-APADPH as the coenzyme, the maximum velocity, relative to NADPH was three- to four-fold lower. Isotope effects on V, V/K for 3-APADPH, and V/K for D-glucuronate were pH independent and equal to 2.2-2.8, indicating that the chemical step of the reaction is relatively insensitive to pH. These data suggest that substrates bind to both the protonated and the deprotonated forms of the enzyme, though only the protonated enzyme catalyzes aldehyde reduction and the deprotonated enzyme catalyzes alcohol oxidation. On the basis of these results a scheme for the chemical mechanism of aldehyde reductase is postulated.

Adenosine Diphosphate Ribose↗
PubMed1991

Purification and characterization of aldose reductase and aldehyde reductase from human kidney.

Aldose reductase and aldehyde reductases have been purified to homogeneity from human kidney and have molecular weights of 32,000 and 40,000 and isoelectric pH 5.8 and 5.3, respectively. Aldose reductase, beside catalyzing the reduction of various aldehydes, reduces aldo-sugars, whereas aldehyde reductase, does not reduce aldo-sugars. Aldose reductase activity is expressed with either NADH or NADPH as cofactor, whereas aldehyde reductase utilizes only NADPH. Both enzymes are inhibited to varying degrees by aldose reductase inhibitors. Antibodies against bovine lens aldose reductase precipitated aldose reductase but not aldehyde reductase. The sequence of addition of the substrates to aldehyde reductase is ordered and to aldose reductase is random, whereas for both the enzymes the release of product is ordered with NADP released last.

Aldehyde Reductase↗
PubMed1990

Inhibition kinetics of human kidney aldose and aldehyde reductases by aldose reductase inhibitors.

Kinetic patterns of inhibition of homogenous human kidney aldose reductase (AR, EC 1.1.1.21) and aldehyde reductase II (AR II, EC 1.1.1.19) by statil, ICI 105552 [1-(3,4-dichlorobenzyl)-3-methyl-1,2-dihydro-2-oxoquinol-4-yl acetic acid], tolrestat, alrestatin, chromone carboxylic acid (CCA), quercetin, phenobarbital and sorbinil were studied. On the basis of the kinetic nature of inhibition, the inhibitors were classified into four distinct categories. For aldose reductase, sorbinil and phenobarbital were noncompetitive (NC; category I) and CCA and alrestatin were uncompetitive (UC; category II) to both the aldehyde substrate and NADPH. Quercetin and ICI 105552 were NC to the aldehyde and UC to NADPH (category III) and tolrestat and statil were UC to the aldehyde and NC to NADPH (category IV). For AR II, sorbinil and alrestatin were category I inhibitors, ICI 105552 and statil belong to category II, phenobarbital, tolrestat and CCA to category III, and quercetin to category IV. To determine the specificity of inhibition, the ratios of the inhibition constants (Kii) for AR and AR II were calculated. A lower ratio indicates greater specificity. With aldehyde as the varied substrate the specificity ratios were: statil less than ICI 105552 less than alrestatin less than tolrestat less than quercetin less than CCA less than sorbinil less than phenobarbital, and with NADPH as the varied substrate, ICI 105552 less than statil less than alrestatin less than tolrestat less than quercetin less than CCA less than sorbinil less than phenobarbital. For AR, double-inhibition plots generated for one inhibitor from each kinetic category versus sorbinil showed that AR inhibitors of categories I-III bind to the same site on the protein molecule as sorbinil. However, tolrestat seemed to bind to a site different from the sorbinil binding site. For AR II, inhibitors from all the four categories appeared to bind to the same inhibitor binding site.

Aldehyde Reductase↗
PubMed1990

Involvement of a steroidal component in the mechanism of action of piracetam-like nootropics.

Since adrenalectomy abolishes the memory-enhancing effects of piracetam and its derivatives, oxiracetam, aniracetam and pramiracetam, the question arises whether endogenous steroids play a role in their mechanism of action. We show that inhibition of steroid biosynthesis by aminoglutethimide and blockade of the aldosterone receptors by epoxymexrenone completely suppress the memory-improving effects of the nootropics. These results indicate that steroids, or, more precisely, activities mediated by the aldosterone receptors, might be involved in the mechanism of action of this class of nootropics. Blockade of aldosterone receptors, however, does not block the effects of cholinomimetics on memory, indicating the involvement of another mechanism of action.

Aminoglutethimide↗
PubMed1990

Oxidative stress alters specific membrane currents in isolated cardiac myocytes.

To evaluate the effects of oxidative stress on cardiac membrane currents, single cells from frog ventricle were exposed to tert-butyl hydroperoxide (t-BHP). Incubation of these cells with 2 mM t-BHP causes a rapid depletion of cellular glutathione, followed by a more gradual increase in the contents of malonaldialdehyde and conjugated dienes. Effects of this rapidly evolving oxidative stress were studied on sodium, calcium, and potassium currents of isolated ventricular cells. t-BHP caused a progressive decrease in the magnitude of sodium current obtained on depolarization from a holding potential of -85 mV, which was accompanied by a shift in the reversal potential toward more negative potentials. The voltage dependence of the steady-state parameters for activation and inactivation was shifted, such that in peroxide-exposed cells, there was a greater overlap of activation and inactivation parameters, which would be expected to result in an increased window current. In addition, in the presence of t-BHP, the time constant for activation was decreased at most depolarizing potentials, whereas the time constant for inactivation was increased. The resultant sodium current transients were, therefore, slower in the presence of the peroxide because of slower inactivation. Prolonged exposure of the cells to t-BHP led to a complete and selective inhibition of the Na+ current. However, even when all the Na+ current was inhibited, the K+ and Ca2+ currents remained essentially unaltered. Also, no large outward currents were observed at this stage, indicating that ATP concentration was not drastically decreased. The barrier properties of plasma membrane remained intact, as it was possible to form gigohm seals between the patch pipette and the plasma membrane of cells treated with 2-14 mM t-BHP for up to 30 minutes. These results account for the proarrhythmic effects of free radicals and oxidative stress on cardiac tissues.

Animals↗
PubMed1989

Functional cysteinyl residues in human placental aldose reductase.

Incubation of human placental aldose reductase (EC 1.1.1.21) with the sulfhydryl oxidizing reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM) results in a biexponential loss of catalytic activity. Inactivation by DTNB or NEM is prevented by saturating concentrations of NADPH. ATP-ribose offers partial protection against inactivation by DTNB, whereas NADP, nicotinamide mononucleotide (NMN), and the substrates glyceraldehyde and glucose offer little or no protection. The inactivation by DTNB was reversed by dithiothreitol and partially by 2-mercaptoethanol but not by KCN. When the release of 2-nitro-5-mercaptobenzoic acid was measured, 3 mol of sulfhydryl residues was found to be modified per mole of the enzyme by DTNB. Correlation of the fractional activity remaining with the extent of modification by the statistical method of C.-L. Tsou (1962, Sci. Sin. 11, 1535-1558) indicates that of the three reactive residues, one reacts at a faster rate than the other two, and that two residues are essential for the catalytic activity of the enzyme. Labeling of the total sulfhydryl by [14C]NEM and quantification of DTNB-reactive residues in the enzyme denatured by 6 M urea indicates that a total of seven sulfhydryl residues are present in the protein. The modification of the enzyme did not affect Km glyceraldehyde, but the modified enzyme had a lower Km NADPH. Kinetic analysis of the data suggests that a biexponential nature of inactivation could be due to the formation of a dissociable E:DTNB complex and the presence of a partially active enzyme species.

Aldehyde Reductase↗
PubMed1989

Activation of aldose reductase by nonenzymatic glycosylation.

Incubation of human erythrocyte with 50 mM glucose results in glycosylation of aldose reductase besides hemoglobin A and other proteins. The glycosylation of aldose reductase is established by adsorption of the enzyme on phenyl boronate (PBA-60) column. Furthermore, the enzyme was purified to an apparent homogeneity from the erythrocytes incubated with 50 mM glucose and the glycosylation was quantitated by reduction with tritiated sodium borohydride. The glycosylated aldose reductase exhibits lower Km for glucose and NADPH as compared to unglycosylated enzyme and is not inhibited by phosphorylated intermediates such as ADP, 1,3-DPG, 2,3-DPG and 3-PGA, whereas physiological concentrations of these intermediates almost completely inhibit the unglycosylated enzyme. In addition, the glycosylated enzyme is less susceptible to aldose reductase inhibitors such as sorbinil and alrestatin. In hyperglycemia, blood glucose higher than 11 mM, almost all the aldose reductase is glycosylated.

Aldehyde Reductase↗
PubMed1989

Spectral properties of human placental aldose reductase and aldehyde reductase II.

Circular dichroism and fluorescence spectra of aldose reductase (E.C.1.1.1.21) and aldehyde reductase II (E.C.1.1.1.19) purified to homogeneity from human placenta have been studied. The alpha helical content of aldose reductase and aldehyde reductase II was 51% and 56%, respectively, whereas no beta helical structure was found in either case. In the case of aldose reductase, the secondary structure was unaffected at alkaline pH (9.5), whereas a drastic alteration in the structure was observed at 58 degrees C. The secondary structure of aldehyde reductase II, on the other hand, remained unaffected at higher pH and temperature.

Aldehyde Reductase↗
PubMed1989

Involvement of sulfhydryl residues in aldose reductase-inhibitor interaction.

Purification and storage of aldose reductase isolated from human placenta or kidney in beta-mercaptoethanol-containing buffers causes a time-dependent decrease in its catalytic activity, as well as in its sensitivity to inhibition by aldose reductase inhibitors such as sorbinil. Dithiothreitol (DTT) slowly regenerated the enzyme activity, as well as reversed the alterations in the sensitivity of the enzyme to sorbinil. In contrast to sorbinil, the inhibition of aldose reductase by tolrestat was less affected by purification and/or storage in beta-mercaptoethanol-containing buffers. Kinetic analysis of the rate of increase in sensitivity of the enzyme to sorbinil on incubation with DTT reveals that the reaction follows two kinetically distinct rate constants. Also, sorbinil protected the enzyme from inactivation with sulfhydryl-modifying reagents 5,5'-dithiobis(2-nitrobenzoic acid) and glutathione disulfide. The enzyme stored in beta-mercaptoethanol migrates as two distinct bands, one corresponding to molecular weight 36,000 and the other to molecular weight 33,000, on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas under reducing conditions the protein migrates as a single discrete band corresponding to molecular weight 36,000. Moreover, the molecular weight 33,000 form of the enzyme could be converted to the molecular weight 36,000 form on reduction with DTT, indicating that the molecular weight 33,000 form of the enzyme is due to intramolecular disulfide bond(s) formed, which presumably cause the protein to assume a more folded conformation and migrate faster through the gel, and not due to proteolysis. These studies indicate that oxidation of sulfhydryl residues, including disulfide bond formation, during purification and storage in beta-mercaptoethanol-containing buffers alters the sensitivity of the enzyme to some inhibitors.

Aldehyde Reductase↗
PubMed1989

Characterization of a wide host range plasmid pANV-6 from Citrobacter diversus.

pANV-6 is a 5.85 kb, streptomycin resistant, high copy number plasmid isolated from a multi drug resistant clinical isolate of C. diversus by transformation in Escherichia coli C-600. The plasmid was very stable, noncurable and could not be amplified with chloramphenicol. It was non-conjugative among Enterobacteriaceae hosts. Plasmid was transferred by transformation to several Gram negative and Gram positive hosts. These included Esch. coli, Serratia marcescens, Salmonella typhimurium, Klebsiella pneumoniae, Staphylococcus aureus, Corynebacterium glutamicum. From Staph. aureus it was transferred by conjugation to other Staph. aureus, Staph. epidermidis and Bacillus subtilis. Plasmid was incompatible with standard plasmids belonging to incompatibility groups H1, H2 = S,J,P and T of coliform bacteria. Plasmid has one site for restriction enzyme EcoRI and Pst I, three for Bgl 1 and non for Bgl 1, Bam HI, Hind III and X ba I.

Citrobacter↗
PubMed1988

The kinetic mechanism of human placental aldose reductase and aldehyde reductase II.

The kinetic mechanism of NADPH-dependent aldehyde reductase II and aldose reductase, purified from human placenta, has been studied using L-glucuronate and DL-glyceraldehyde as their respective substrates. For aldehyde reductase II, the initial velocity and product inhibition studies (using NADP and gulonate) indicate that the enzyme reaction sequence is ordered with NADPH binding to the free enzyme and NADP being the last product to be released. Inhibition patterns using menadione (an analog of the aldehydic substrate) and ATP-ribose (an analog of NADPH) are also consistent with a compulsory ordered reaction sequence. Isotope effects of deuterium-substituted NADPH (NADPD) also corroborate the above reaction scheme and indicate that hydride transfer is not the sole rate-limiting step in the reaction sequence. For aldose reductase, initial velocity patterns, product, and dead-end inhibition studies indicate a random binding pattern of the substrates and an ordered release of product; the coenzyme is released last. A steady-state random mechanism is also consistent with deuterium isotope effects of NADPD on the reaction sequence catalyzed by this enzyme. However, the hydride transfer step seems to be more rate determining for aldose reductase than for aldehyde reductase II.

Aldehyde Reductase↗
PubMed1988

Elevated serum iron levels following administration of cisplatinum.

An increase in serum iron levels and a decrease in serum unsaturated iron binding capacity (uIBC) were noted following the administration of cisplatinum to 9 children with malignancies. The mean serum iron concentration increased from a pretreatment level of 75.7 +/- 30.5 micrograms/ml to a posttreatment level of 162.1 +/- 65.3 micrograms/ml with the first cisplatinum treatment course (p less than 0.004). The uIBC concomitantly decreased from 181.9 +/- 33.7 micrograms/ml to 86.4 +/- 44.6 micrograms/ml (p less than 0.0005). A cumulative effect was noted following subsequent courses. The levels returned to baseline values within 2-4 months following cessation of therapy in 6 children in whom follow-up data were available. It is possible that this reversal of the iron/uIBC ratio is the result of cisplatinum competition for iron binding sites to proteins.

Child↗
PubMed1988

Effects of sodium deoxycholate on the mechanical and electrical activities and ultrastructure of guinea pig atria.

The mechanism of cardio-inhibitory effects of sodium deoxycholate (DOC) was investigated by studying its effects on the contractility, action potentials (APs) and ultrastructure of guinea pig atrial preparations. DOC (10(-7)-10(-4) M) caused reversible negative ino- (NIE) and chrono-tropy in spontaneously beating (SBA) and NIE in electrically driven left (EDA) atria. At higher doses (greater than or equal to 1.10(-3) M) DOC caused irreversible inhibition of contractions. Atropine (10(-7)-10(-4) M) failed to inhibit both the reversible and irreversible effects of DOC. The NIE due to lower doses of DOC (less than or equal to 1.10(-4) M) was inhibited by higher [Ca2+]0, isoprenaline (10(-6)-10(-4) M), and noradrenaline (10(-6)-10(-5) M), which did not alter the dose of DOC required for the irreversible and complete NIE. In lower doses (10(-7)-10(-4) M) DOC caused a reversible inhibition of the AP durations at -20 and -40 mV (APD20 and APD40, respectively), but increased the AP duration at 90% repolarisation (APD90). At higher doses (greater than 5.10(-4) M) it caused an irreversible membrane depolarization, reduction in APD20 and APD40, and complete cessation of electrical activity. The ultrastructural changes in atria treated with 1.10(-4) M DOC were characterized by poorly delineated glycocalyx and at greater than 1.10(-3) M by disruption of sarcolemma and sarcoplasmic reticulum and swelling disruption of mitochondria. Taken together these observations show that DOC caused reversible and irreversible inhibition of atrial contractions at low (10(-7)-10(-4) M) and high (greater than 5.10(-4) M) concentrations, respectively, by different mechanisms. The former effect is due to inhibition of Ca2+ channel activity and the latter due to its detergent property causing removal of subcellular components.

Action Potentials↗