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Biomedical subjects

A Bhatnagar

Publications and source records attributed to A Bhatnagar.

At least 127 records · Page 7Linked to original sources
PubMed1994

Biochemical mechanism of irreversible cell injury caused by free radical-initiated reactions.

Effects of oxidative stress on isolated rat ventricular myocytes were studied. Myocyte viability was determined by the ability of these cells to retain rod-shaped morphology and to exclude trypan blue. The mean life time of myocytes was quantitated using the Weibull distribution function. Superfusion with 200 microM tert-butyl hydroperoxide (t-BHP) led to a time-dependent loss of cell viability, generation of the products of lipid peroxidation, oxidation of protein and non-protein thiols, a decrease in [ATP]i and in the cellular energy charge. Dithiothreitol (DTT, 5 mM) prolonged survival of myocytes exposed to t-BHP, attenuated oxidation of protein and non-protein thiols, and preserved the energy charge. Exposure to DTT did not affect the concentration of t-BHP-generated lipid peroxidation products. Promethazine (1 microM) prevented t-BHP-induced increase in the concentration of lipid peroxidation products, but did not prevent either loss of thiols or loss of cell viability. Superfusion with N-ethylmaleimide (NEM, 5 microM) also led to loss of cell viability, with accompanying decreases in protein and non-protein thiols, ATP and energy charge without the accumulation of the products of lipid peroxidation. Superfusion with FeSO4 (400 microM) and ascorbate (1 mM), (Fe-Asc) did not result in loss of cell viability or a decrease protein thiols or the energy charge. Superfusion with Fe-Asc, did, however, lead to a slight decrease in the concentration of non-protein thiols and ATP and a large increase in the concentration of lipid peroxidation products. Accumulation of lipid peroxidation products induced by Fe-Asc was prevented by promethazine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Human placental aldose reductase: role of Cys-298 in substrate and inhibitor binding.

Steady-state kinetic and inhibition properties of human placental aldose reductase carboxymethylated at Cys-298 were investigated. A comparison of the primary deuterium kinetic isotope effect on the reduced and the carboxymethylated enzymes suggests that carboxymethylation did not affect the reaction sequence of substrate binding and release. Values of DV/KD-glyceraldehyde greater than DV suggest that steps in the reaction scheme subsequent to hydride transfer, particularly the release of NADP may be rate limiting. Carboxymethylation of Cys-298 was also found to affect NADPH and aldehyde binding to the enzyme. Carboxymethylation had little effect on the secondary structure of the enzyme, but a comparison of the circular dichroic spectra of the reduced and carboxymethylated enzyme, suggests a weakened interaction between the nicotinamide and 2'-monophosphoadenosine 5'-diphosphoribose of NADPH, and the carboxymethylated enzyme. Interaction between Cys-298 and NADPH appears to determine the rate of isomerization of the E:NADP binary complex and carboxymethylation-induced decrease in kcat may be due to slower isomerization of the E:NADP binary complex. The carboxymethylated enzyme was less sensitive than the reduced enzyme to most aldose reductase inhibitors including sorbinil (d-6-fluoro-spiro[chroman-4,4'-imidazolidine]-2',5'-dione), except tolrestat (N-methyl-N-[(5-trifluromethyl-6-methoxy-1-naphthalenyl)- thiomethyl]glycine) and quercetin. On the basis of these observations it is suggested that Cys-298 may form a part of the 'S'-inhibitor binding site of the enzyme and may be responsible for tight binding of NADPH.

Aldehyde Reductase↗
PubMed1994

Differentiation of malignant and degenerative bone lesions using dexamethasone interventional 3- and 24-hour bone scintigraphy.

Seventy-seven adult patients with suspected skeletal metastases were divided into two groups. In group A (n = 30), following intravenous administration of 20 mCi (740 MBq) of technetium-99m methylene diphosphonate (99mTc-MDP), 3- and 24-h scintigraphy of bone lesions was performed. The 24/3 h lesion to bone background radiouptake ratio (RUR) was calculated for each lesion. In group B (n = 47), the same procedure was followed with dexamethasone intervention (10 mg in 24 h) following the 3-h acquisition. In group A, after determination of the critical point, malignant and degenerative bone lesions could be separated with a sensitivity, specificity and accuracy of 0.76, 0.72 and 0.73, respectively. The mean RUR of the malignant lesions was 1.20 +/- 0.23, and that of the benign lesions, 0.95 +/- 0.15. In group B cases, significantly increased sensitivity, specificity and accuracy of 0.87, 0.94 and 0.92, respectively, were found (P < 0.001). The mean RUR of the malignant lesions was 1.48 +/- 0.34, and that of degenerative lesions, 0.88 +/- 0.19. Dexamethasone interventional bone scintigraphy seems to be a new cost-effective method for differentiating malignant from degenerative bone lesions using the RUR.

Bone Diseases↗
PubMed1994

A novel method for labelling human immunoglobulin-G with 99Tcm suitable for inflammation scintigraphy.

An amount of 1.0 mg human immunoglobulin G (hIgG) treated with ascorbic acid at a molar ratio of 1:5000 for 16 h at 4-7 degrees C was mixed with 250 micrograms GHA and 5 micrograms stannous chloride dihydrate in normal saline. Radiolabelling of hIgG (> 98%) was achieved instantly when mixed with 99Tcm-pertechnetate. The preparation was sufficiently stable in serum at 37 degrees C. The competitive binding assay and gel electrophoresis of the native and reduced hIgG did not show any measurable loss in immunoreactivity and intactness due to its reduction. There was no significant decrease in the radiolabel of labelled hIgG when incubated with diethylenetriaminepentaacetate (DTPA) (50-fold), in contrast with about 9% loss of the radiolabel by treating it with a similar concentration of cysteine. Blood clearance of labelled hIgG in rabbits was biphasic with about 78 min and 8 h as T1/2 of the fast and slow phases. Biodistribution of the radiotracer in mice at 4 h showed its uptake by liver (10.2%), kidneys (5.39%), intestines (7.33%) and muscles (3.9%), which altered to 5.63, 3.20, 4.03 and 6.73%, respectively, at 24 h. The radiotracer was excreted through both renal and hepatobiliary routes. High accumulation of the radiolabelled hIgG in inflammatory lesions of the patients confirmed the clinical usefulness of the method developed.

Animals↗
PubMed1994

Evaluation of the healing process in fractures using early and delayed bone scanning.

Fifty-two fracture sites were evaluated by bone scintigraphy after acquiring similar views of the lesions at 4 and 24 h following technetium-99m methylene diphosphonate administration. After calculating 24/4 h radio-uptake ratio (RUR) and using 1.15 as the critical point of separation, fresh healing lesions could be separated from old healed fractures persisting as 'hot spots' with a sensitivity of 100% and specificity of 82%. The 24/4 h RUR technique is a new approach in measuring the osteoblastic activity at the lesion site. The technique appears to have clinical significance in the detection and follow up of the healing status of occult fractures where radiological procedures are of limited value.

Adolescent↗
PubMed1994

Trolox protects hyperglycemia-induced cataractogenesis in cultured rat lens.

Hyperglycemia-induced cataractogenesis has been studied in rat lenses cultured in 50 mM glucose using an inverted microscope connected with a Universal C-mount and a CCD camera. Digital images were acquired and the opacity was determined by quantitating the transmitted light. Antioxidants, butylated hydroxy toluene (BHT) and 6-hydroxy-2,5,7,8-tetramethenyl-chroman-2-carboxylic acid (Trolox) provided good protection against 50 mM glucose-induced cataractogenesis in rat lenses for upto 8 days. Sorbitol levels in the 50 mM glucose+antioxidant groups were approximately 1.5 mM fold higher than in 50 mM glucose. The results, besides further demonstrating that oxidative damage is the major mechanism of sugar-induced cataractogenesis, show that Trolox or related amphipathic compounds could be of therapeutic use in the prevention of diabetic cataracts.

Animals↗
PubMed1994

Energetics of cyanophage N-1 multiplication in the diazotrophic cyanobacterium Nostoc muscorum.

Cyanophage N-1 multiplication was investigated during the latent period of the virus, when 14CO2 fixation was inhibited whereas respiratory O2 uptake increased approximately 67% at 4 h after infection. A simultaneous decrease (70%) in the glycogen content of infected cells indicated its catabolic involvement. A chloramphenicol-sensitive rise in glucose-6-phosphate dehydrogenase activity as a result of N-1 infection partly explained the increase in aerobic respiration. The total ATP pool declined to 53% of the control while Ca(2+)-dependent ATPase activity also declined (25%). In contrast, Mg(2+)-dependent ATPase activity increased (80%) in comparison with uninfected cells. Results suggest that oxidative phosphorylation was more crucial in the control of cyanophage N-1 development than photophosphorylation under photoautotrophic growth conditions.

Adenosine Triphosphatases↗
PubMed1993

Bovine lens aldose reductase. pH-dependence of steady-state kinetic parameters and nucleotide binding.

The pH-dependence of nucleotide binding and steady-state kinetic parameters of aldehyde reduction and alcohol oxidation catalyzed by bovine lens aldose reductase were studied. The maximal velocity of aldehyde reduction with NADPH and p-chlorobenzaldehyde was pH independent at low pH but decreased at high pH with a pK of 7.6. The V/K of NADPH displayed a bell-shaped dependence on pH and decreased with a pKa of 5.3 and a pKb of 7.5. The dissociation constant of NADPH and 3-acetylpyridine adenine dinucleotide phosphate (3-APADP) increased at low pH with a pK of 5.6-5.8 and at high pH with a pK of 9.4-9.7. The pKi of NADP and NADPH decreased below a pH of 5 and 6.7 and above a pH of 8.5 and 9.7, respectively. The pK of 8.5-9.7 appears to be due to the interaction of the 2'-phosphate of the nucleotide with a protonated base, possibly a lysine residue. The maximal velocity of alcohol oxidation was pH independent at high pH but decreased at low pH with a pK of 6.5-7.0, when p-chlorobenzyl alcohol or benzyl alcohol and 3-APADP were used. The amino acid residue for alcohol binding has a pK of 7.5-8.2 and also appears in pKi profiles of sorbinil, a competitive inhibitor versus the alcohol. Large (3-3.5) isotope effects on maximal velocity obtained with benzyl alcohol and 3-APADP suggest that with these substrates the hydride transfer step is rate-limiting and a pK of 6.5-7.0 may be the true pK of the acid-base catalyst, possibly a histidine.

Aldehyde Reductase↗
PubMed1993

Identification of the reactive cysteine residue in human placenta aldose reductase.

Modification of human placental aldose reductase by iodoacetate (IAA) led to a mol/mol binding of IAA, a 40% decrease in the kcat, a 3-5-fold increase in the Km,NADPH and Km,glyceraldehyde and a 600-fold increase in the Ki,sorbinil; determined at pH 6.0. NADPH and 2'-monophosphoadenosine 5'-diphosphoribose but neither glyceraldehyde nor sorbinil, prevented carboxymethylation-induced changes. Cleavage of [14C]IAA-modified enzyme by trypsin resulted in two radiolabeled peptides: Val-297 to Lys-307 and Val-297 to Phe-315. In both these peptides Cys-298 was the only radiolabeled residue. It is suggested that Cys-298 regulates the kinetic and inhibition properties of the enzyme, but does not participate in catalysis.

Adenosine Diphosphate Ribose↗
PubMed1993

Aromatase inhibitors: synthesis, biological activity, and binding mode of azole-type compounds.

The enantiomers of the potent nonsteroidal inhibitor of aromatase fadrozole hydrochloride 3 have been separated and their absolute configuration determined by X-ray crystallography. On the basis of a molecular modeling comparison of the active enantiomer 4 and one of the most potent steroidal inhibitors reported to date, (19R)-10-thiiranylestr-4-ene-3,17-dione, 7, a model describing the relative binding modes of the azole-type and steroidal inhibitors of aromatase at the active site of the enzyme is proposed. It is suggested that the cyanophenyl moiety present in the most active azole inhibitors partially mimics the steroid backbone of the natural substrate for aromatase, androst-4-ene-3,17-dione, 1. The synthesis and biological testing of novel analogues of 3 used to define the accessible and nonaccessible volumes to ligands in the model of the active site of aromatase are reported.

Aromatase Inhibitors↗
PubMed1993

Digital image analysis of cultured rat lens during oxidative stress-induced cataractogenesis.

Isolated rat lenses were exposed to oxidative stress generated by 100 microM H2O2, 2 mM ADP and 100 microM ferrous ammonium sulfate. Oxidation-induced cataract formation was followed by measuring loss of transmitted light intensity using quantitative digital image analysis, which offers distinct advantages over conventional photography. In the presence of oxidants, total and average light transmitted by the lens decreased exponentially as a function of time; the cortex showing a greater rate of decline in transmitted light intensity than the nucleus, which led to a change in the distribution pattern of light intensity. Lenses developing oxidative cataracts also showed a significant increase in diameter and an increase in the total wet weight. Maximal increase in lens diameter preceded maximal decrease in light intensity. These studies demonstrate the utility of quantitative image analysis in studying changes in lens geometry and transparency, and suggest that cataract formation is a single rate process.

Animals↗
PubMed1993

Effect of extracellular ions and modulators of calcium transport on survival of tert-butyl hydroperoxide exposed cardiac myocytes.

OBJECTIVE: The aim was to investigate the effects of extracellular ions and agents that modify calcium translocating pathways on oxidative stress induced cell injury. METHODS: Survival of cardiac myocytes exposed to tert-butyl hydroperoxide (t-BHP, 0.2 to 2 mM) was estimated by their ability to maintain rod shaped morphology and exclude trypan blue, and by the release of lactate dehydrogenase (LDH). Mean life time (mu) of a myocyte was analysed in accordance with the Weibull distribution. RESULTS: In pCa 3.0 Ringer's solution, t-BHP exposed myocytes underwent time and concentration dependent hypercontracture. The fraction of rod shaped cells excluding trypan blue correlated with LDH release. The lifetime of a cell was increased upon reduction of [Ca]o. Myocytes exposed to t-BHP in pCa 7.0 or 8.0 Ringer's solution attained a rigor state and became permeable to trypan blue. t-BHP-induced hypercontracture was delayed on increasing [K]o or [Na]o. Reduction of [Na]o to 67.5 mM, but not to 13.5 mM, accelerated t-BHP induced hypercontracture. Early reduction of [Ca]o to a pCa of 8.0 had no protective effect if 1 mM calcium was reintroduced after half the duration of exposure. Verapamil (10 microM), nifedipine (1 microM), butanedione monoxamine (20 mM), and caffeine (10 mM) accelerated t-BHP-induced loss of rod shaped morphology. Ryanodine (1 microM), Trolox-C (1 microM), and butylated hydroxytoluene (BHT, 500 microM) had no effect on survival of t-BHP exposed myocytes. Survival was prolonged upon preincubation of the myocytes with BAPTA-AM or desferrioxamine or when the cells were exposed to t-BHP in the presence of La (1 mM) or N-propyl gallate (10 microM). CONCLUSIONS: Ferrous catalysed free radical reactions initiated by t-BHP lead to hypercontracture or rigor shortening of myocytes, depending upon [Ca]o. It is unlikely that t-BHP-induced hypercontracture is mediated by Ca influx through L- or T-type Ca channels or due to release of Ca from intracellular stores. Activation of Ca influx via Na-Ca exchange and/or increase in the passive membrane permeability to Ca is consistent with the observations reported. Myocyte rigor observed upon attenuation of Ca overload suggests contribution of Ca-independent processes.

Animals↗
PubMed1993

24 hour/3 hour radio-uptake technique for differentiating degenerative and malignant bony lesions in bone scanning.

Differentiating bony metastases from degenerative lesions is of great importance to the oncologist. Routine bone scanning using technetium-99m methylene diphosphonate is the investigation of choice for detecting bony lesions, but its specificity is low. Using the difference in radio-uptake behaviour of metastatic and degenerative lesions as the criterion, a 24/3 h radio-uptake, lesion to non-lesion ratio was used to separate out the two types of lesions. Radio-uptake ratio (RUR) distribution curves of malignant and degenerative lesions were found to be significantly different (P < 0.001). Taking 1.12 as the critical point, RUR of malignant lesions was found to be more than the critical point, and that of degenerative lesions was found to be less than the critical point. Sensitivity, specificity and accuracy were found to be 68%, 80% and 74%, respectively.

Bone Diseases↗
PubMed1992

Does sorbinil bind to the substrate binding site of aldose reductase?

With benzyl alcohol as the varied substrate, sorbinil was found to be a competitive inhibitor of aldose reductase, an enzyme implicated in the etiology of secondary diabetic complications. The K(is sorbinil) and the Vmax/Km (V/K) benzyl alcohol decreased at low pH with a pK of 7.5 and 7.7, respectively. These observations suggest that both sorbinil and benzyl alcohol bind to the same site on the enzyme. Active site inhibition by sorbinil is consistent with non-competitive inhibition patterns of sorbinil with nucleotide coenzyme or aldehyde as the varied substrate in the direction of aldehyde reduction.

Aldehyde Reductase↗
PubMed1992

Involvement of cysteine residues in catalysis and inhibition of human aldose reductase. Site-directed mutagenesis of Cys-80, -298, and -303.

In order to study the potential role of cysteinyl residues in catalysis and inhibition of human aldose reductase, mutants containing cysteine to serine substitution at positions 80 (ALR2:C80S), 298 (ALR2:C298S), and 303 (ALR2:C303S) were constructed. Mutation of Cys298 resulted in the most profound changes, as ALR2:C298S displayed 4- to 5-fold elevation in K'm(NADPH), K'm(DL-glyceraldehyde), and kcat(DL-glyceraldehyde) relative to wild type aldose reductase as well as a 10-fold higher Ki for the aldose reductase inhibitor sorbinil. Wild type and mutant reductases were equally sensitive to tolrestat, a structurally different reductase inhibitor. Carboxymethylation of the wild type enzyme or the C80S and C303S mutants led to a modest decrease in kcat as well as an increase in K'm(DL-glyceraldehyde) and Ki(sorbinil). These parameters were not significantly changed when ALR2:C298S was subjected to carboxymethylation. Lithium sulfate caused activation of ALR2:WT, C80S, and C303S but did not significantly affect the activity of ALR2:C298S. The differential sensitivity of wild type and mutant reductases to inhibition by sorbinil and tolrestat, before and after carboxymethylation, indicates that these inhibitors bind at different sites. These results suggest that Cys-298 is present near the active site and constitutes a regulatory group which controls the catalytic activity and inhibitor sensitivity of the enzyme.

Aldehyde Reductase↗
PubMed1992

Carboxymethylation-induced activation of bovine lens aldose reductase.

Carboxymethylation of bovine lens aldose reductase with 10 mM iodoacetate for 1 h at 25 degrees C led to a more than 4-fold increase in kcat. Carboxymethylation led to a 3- to 5-fold increase in Km NADPH and Km D-glyceraldehyde, whereas Km L-glyceraldehyde increased approx. 30-fold. Activation of the enzyme on carboxymethylation was accompanied by a decrease in the sensitivity of the enzyme to inhibition by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), sorbinil (Kii increased from 0.4 to 109 microM) and NADP (Kis increased from 0.01 to 0.03 mM), but not tolrestat. Activation of the enzyme was almost completely prevented by NADPH and to a lesser extent by DL-glyceraldehyde. Carboxymethylation of the enzyme did not result in the generation of several partially oxidized enzyme species, indicating the absence of partially carboxymethylated forms. Primary deuterium isotope effects on the reduced enzyme were consistent with a preferred ordered kinetic reaction scheme, in which hydride transfer is not rate limiting. The hydride transfer step does not seem to be significantly affected by carboxymethylation, nor do changes in the substrate binding steps seem to contribute to the observed rate enhancement. Increase in the turnover number of the enzyme on carboxymethylation appears to be due to facilitation of the isomerization of the E:NADP binary complex. The differential effect of carboxymethylation on sorbinil and tolrestat suggests distinct inhibitor sites on the enzyme, an S-site that binds sorbinil and a T-site that binds tolrestat.

Aldehyde Reductase↗