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Biomedical subjects

A Bhatnagar

Publications and source records attributed to A Bhatnagar.

At least 91 records · Page 5Linked to original sources
PubMed1998

Technetium-99m-sestamibi parathyroid scintigraphy: effect of P-glycoprotein, histology and tumor size on detectability.

UNLABELLED: The purpose of this study was to evaluate the effect of P-glycoprotein (P-gp) levels, predominant histology and tumor size on the detectability of parathyroid adenomas with 99mTc-sestamibi scans. Although previous studies have shown that positivity correlates with tumor size, false-negative studies have been reported with large tumors and true-positives reported with very small tumors. Recent investigations have reported rapid washout of sestamibi in malignant tumors because of high levels of P-gp, similar to that seen with multidrug chemotherapy resistance. Therefore, we postulated that this mechanism might account for false-negative studies in parathyroid tumors. Preliminary reports have suggested that the predominant histological subtype influences positivity on 99mTc-sestamibi parathyroid scans. METHODS: We studied 25 patients with surgically confirmed parathyroid adenomas with 99mTc-sestamibi parathyroid scans. The results of the imaging study were correlated with tissue P-gp levels, predominant histological subtype and size of the surgically removed glands. RESULTS: There were 21 true-positive and 4 false-negative results. The size of the adenomas ranged from 0.12 to 8.64 ml. We found no correlation between the results of the dual-phase 99mTc-sestamibi study and either the predominant cell type or the level of P-gp. Positivity did correlate with the size of the adenoma (p=0.73, p < 0.0001). We cannot exclude the possibility that P-gp and cell type may still play a role in individual cases, but we suspect that other yet to be determined factors may influence 99mTc-sestamibi detectability in addition to tumor size.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Modification of aldose reductase by S-nitrosoglutathione.

Kinetic and structural changes in recombinant human aldose reductase (AR) due to modification by S-nitrosoglutathione (GSNO) were investigated. Incubation of the enzyme with 10-50 microM GSNO led to a time- and concentration-dependent inactivation of the enzyme, with a second-order rate constant of 0.087 +/- 0.009 M-1 min-1. However, upon exhaustive modification, 30-40% of the enzyme activity was retained. The non-inactivated enzyme displayed a 2-3-fold change in Km for NADPH and Km fordl-glyceraldehyde, whereas the Km for the lipid peroxidation product, 4-hydroxy-2-trans nonenal (HNE), was comparable to that of the untreated enzyme. The residual activity of the enzyme after GSNO treatment was less sensitive to inhibition by the active site inhibitor sorbinil or to activation by sulfate. Significantly higher catalytic activity was retained when the enzyme was modified in the presence of NADPH, suggesting relatively low reactivity of the E-NADPH complex with GSNO. The modification site was identified using site-directed mutants in which each of the solvent-exposed cysteines of the enzyme was replaced individually by serine. The mutant C298S was insensitive to GSNO, whereas the sensitivity of the mutants C303S and C80S was comparable to that of the wild-type enzyme. Electrospray ionization mass spectroscopy of the GSNO-modified enzyme revealed a major modified species (70% of the protein) with a molecular mass that was 306 Da higher than that of the untreated enzyme, which is consistent with the addition of a single glutathione molecule to the enzyme. The remaining 30% of the protein displayed a molecular mass that was not significantly different from that of the native enzyme. No nitrosated forms of the enzyme were observed. These results suggest that inactivation of AR by GSNO is due to the selective formation of a single mixed disulfide between glutathione and Cys-298 located at the NADP(H)-binding site of the enzyme.

Aldehyde Reductase↗
PubMed1997

Identification of residues in the class II-associated Ii peptide (CLIP) region of invariant chain that affect efficiency of MHC class II-mediated antigen presentation in an allele-dependent manner.

Invariant chain (Ii) associates with class II MHC molecules and is crucial for Ag presentation by class II molecules. A general explanation for how invariant chain (Ii) associates with polymorphic MHC class II molecules has been suggested by the crystallographic structure of CLIP (class II-associated Ii peptide) complexed with an HLA class II molecule, HLA-DR3. We show here that methionine residues at positions 93 and 99 in Ii are important in MHC class II-mediated Ag presentation, but function in an allele-dependent manner. Introduction of a Met-->Ala mutation at position 99 in Ii (M99AIi) impaired presentation of peptides derived from exogenous proteins by I-Ad and I-Au class II molecules. Mutating Met-->Ala in Ii at position 93 (M93AIi) abrogated presentation by I-Au molecules, but not by I-Ad. Impaired Ag presentation was associated with conformationally altered expression of I-A molecules on the surface of cells expressing mutated Ii. Cell surface CLIP staining and immunoprecipitation studies showed that both I-Ad and I-Au molecules were associated with an increased abundance of Ii peptides, CLIP, in cells expressing mutated Ii. These results show that methionine 93 and methionine 99 play an important physiologic role in Ii association with class II molecules by regulating release of CLIP from class II in the endocytic compartments to allow binding of cognate peptides.

Alanine↗
PubMed1997

Active site modification of aldose reductase by nitric oxide donors.

Nitric oxide (NO) donors sodium nitrosoprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), and 3-morpholinosydnonemine (SIN-1) caused a time- and concentration-dependent loss of catalytic activity of recombinant human placental aldose reductase. Modification of the enzyme was prevented by NADPH and NADP and reversed partially by dithiothreitol (DTT) and sodium borohydride. The protection by NADPH was lost in the presence of both substrates (NADPH and glyceraldehyde), indicating that the enzyme becomes sensitive to inhibition by SNP during catalysis. Site-directed mutant form of the enzyme, in which active site cys-298 was substituted with serine (C298S) was not inactivated by NO donors, whereas, ARC80S and ARC303 were as sensitive as the wild type enzyme, indicating that inactivation of aldose reductase is due to modification of the active site at cys298. These results suggest that NO may be an endogenous regulator of aldose reductase, and consequently the polyol pathway of glucose metabolism; which has been implicated in the pathogenesis of secondary diabetic complications.

Aldehyde Reductase↗
PubMed1997

Human immunodeficiency virus type-1 p24 sequence from an Indian strain: expression in Escherichia coli and implications in diagnostics.

A 637-bp fragment, corresponding to the p24 human immunodeficiency virus (HIV) core protein from the gag ORF, was PCR amplified from DNA isolated from peripheral blood mononuclear lymphocytes (PBML) of an asymptomatic HIV-1 seropositive human subject from Bombay and cloned into PCRScript SK(+). The nucleotide sequence revealed highest homology (98.6%) with the consensus sequence of the HIV-1 B subtype. The 637-bp KpnI-HindIII fragment was cloned downstream from a His6 tag in the pQE30 vector under the control of phage T5 promoter leading to production of a 6XHis-p24 fusion protein in Escherichia coli. It showed an approx. 24-kDa band by SDS-PAGE. The recombinant p24 reacted with serum samples from HIV-infected subjects when tested by Western blot and ELISA.

Amino Acid Sequence↗
PubMed1997

Contribution of osmotic changes to disintegrative globulization of single cortical fibers isolated from rat lens.

In this study the contribution of osmotic changes to disintegrative globulization of lens cortical fibers was examined. Single fiber cells were isolated by trypsinization of adult rat lens cortex, and morphological changes elicited by exposure to different external solutions were monitored optically. The survival of the fiber-shaped cells was analysed in accordance with the Weibull distribution. Changes in [Ca2+]i were measured using the fluorescent calcium-sensitive dye-Fluo-3. Exposure of isolated fiber cells to Ringer's solution (containing 2 mm Ca2+) led to an exponential increase in [Ca2+]i with a time constant of 10.2+/-0.8 min, and caused disintegrative globulization in 25+/-4 min (=Tg). The process of globulization as well as the rate of increase in [Ca2+]i was delayed by removing Cl- ions from the external media. Globulization was also delayed by adding 20% bovine serum albumin (Tg=107+/-3 min) or chloride channel inhibitors 5, nitro-2-(3-phenylpropylamino) benzoate (NPPB), dideoxyforskolin, niflumic acid, and tamoxifen. When the fiber cells were suspended in isotonic (280 mm sucrose) HEPES-sucrose (HS) or HEPES-EDTA-sucrose (HES) solution, no globulization was observed for an observation time of 120 min. However, exposure to hypotonic (180 mm) HES solution led to disintegration of fiber cells in 75+/-7 min. Disintegration of the fiber induced by hypotonic HES solution could be delayed by either 0. 05 mm leupeptin (Tg=97+/-6 min) or by pre-loading the fibers with BAPTA (Tg=100+/-4 min). Inhibition of membrane calcium transport by 0.5 mm La3+ had no effect on Tg in hypotonic HES. Addition of 2 mm Ca2+ to HES solution accelerated globulization, and Tg was 57+/-4, 69+/-5 and 102+/-6 min for hypo-, iso- and hyper- tonic solutions, respectively. Transient exposure to calcium also accelerated disintegrative globulization of fiber cells exposed subsequently to HES solution. These results suggest that in ionic media, part of the calcium influx in isolated fiber cells is mediated by the influx of chloride ions. In the absence of other ions, the fiber cells still accumulate calcium, although this calcium influx was independent of medium tonicity. Globulization-induced by hypotonic sucrose solution appears to be mediated by the activation of intracellular proteases and by cell swelling-induced release of calcium from internal stores. Such swelling-mediated disintegrative globulization of fiber cells may be of significance in understanding the cellular basis of diabetic cataracts.

Animals↗
PubMed1997

Renal imaging with 99Tc(m)-dextran.

Significant uptake and retention of 99Tc(m)-dextran (molecular weight: 81,000) in renal parenchyma was discovered during evaluation of its intravascular use. Renal SPET images confirmed this. This study was designed to evaluate 99Tc(m)-dextran as a renal cortex imaging agent. Stability of parenchymal retention was shown by insignificant outflow at 24 h and by frusemide intervention. Evaluation of the renal parameters of intravenous 99Tc(m)-dextran (n = 71 normal kidneys) and its comparison with 99Tc(m)-DTPA n = 10) and 99Tc(m)-DMSA(III) (n = 23) was undertaken. The early glomerular extraction phase of the renograms of 99Tc(m)-DTPA and 99Tc(m)-dextran appeared identical; parenchymal uptake of 99Tc(m)-dextran continued to increase and reached a near-plateau by 40-60 min. The mean cortex-to-background and cortex-to-liver ratios at 2 h with 99Tc(m)-dextran and 99Tc(m)-DMSA(III) were 14.9 and 9.2, and 16.0 and 8.9, respectively. The target-to-nontarget ratios were similar despite different absolute renal uptake values (12 vs 20% at 2 h) because of faster background clearance of 99Tc(m)-dextran. The mechanism of parenchymal retention of 99Tc(m)-dextran appears to be trapping at the endothelial-epithelial interphase of the glomerulus. Our initial experience suggests 99Tc(m)-dextran is a viable renal parenchyma imaging agent.

Adolescent↗
PubMed1997

Relevance of biophysical concepts in the scintigraphic detection of gastrointestinal leaks.

The application of some physical concepts is described to explain the inherent insensitivity of 99Tc(m)-red blood cell (RBC) and 99Tc(m)-human serum albumin (HSA) in the detection of gastrointestinal bleeding and protein-losing enteropathy (PLE), respectively. Inferences drawn from the laws of radioactive decay, partition physics, hydrodynamics and electrostatic forces have been used to suggest that the clearance pattern, size and charge of radiopharmaceuticals, and the viscosity of the medium, affect the sensitivity of these tests. As the outflow from gastrointestinal lesions is non-specific, other labelled macromolecules can be used instead of RBCs or HSA. Based on biophysical concepts, we suggest that chargeless macromolecules in the size range 5-50 nm are superior to the conventional agents used for this purpose.

Animals↗
PubMed1997

Contribution of ATP to oxidative stress-induced changes in action potential of isolated cardiac myocytes.

The role of ATP in oxidative stress-mediated changes in the cardiac action potential was investigated in isolated adult rat cardiac myocytes. Superfusion with H2O2 led to a decrease in the energy charge and depletion of nonprotein thiols and elicited hypercontracture of the myocytes. Treatment with 3-aminobenzamide (3-AB), an inhibitor of protein ribosylation, increased the lifetime of H2O2-exposed myocytes and attenuated depletion of ATP and nonprotein thiols. H2O2-mediated DNA strand breaks were increased in the presence of 3-AB. On exposure to H2O2, myocytes patch clamped with 1 mM ATP in the pipette initially displayed prolonged action potential durations (APD), which were later markedly abbreviated and accompanied by the activation of ATP-sensitive K+ currents (I(K,ATP)). The late decrease in APD was inhibited by glibenclamide (which inhibits I(K,ATP)), but the initial prolongation of the action potential was exacerbated. Treatment with 3-AB or recordings with 10 mM ATP in the patch pipette caused an initial delay in the expression of H2O2-induced changes, but later caused a more pronounced and sustained increase in APD. These interventions delayed the activation of I(K,ATP). Thus enhanced ribosylation (presumably due to activation of DNA repair) appears to be a significant source of ATP depletion under oxidative stress that, via activation of I(K,ATP), mediates oxidative modifications in the action potential.

Action Potentials↗
PubMed1997

Alterations in the light transmission through single lens fibers during calcium-mediated disintegrative globulization.

PURPOSE: The purpose of this study was to examine changes in the light transmission through single cortical fibers isolated from the rat lens during the process of disintegrative globulization. METHODS: Single cortical fibers were isolated from adult rat lens by treatment with trypsin in a solution containing 10 mM HEPES, 10 mM EDTA, and 280 mM sucrose (pH 7.4, 300 to 310 mOsm) HEPES-EDTA-sucrose (HES) solution. The isolated fibers were illuminated by a white light source, and the light transmission through the fibers was collected by a charge-coupled device camera and quantified by digital image analysis. In some experiments, thin sections of fixed lens cells were examined using transmission electron microscopy. RESULTS: Enzymatic dissociation of the lens yielded elongated fibers, which, in the presence of Ringer's solution (containing 2 mM Ca2+), underwent disintegrative globulization. Measurements of light transmission through elongated fibers suspended in HES solution showed maximal transmission at the center of the fiber. Exposure of the cortical fibers to Ringer's solution led to biphasic changes in the intensity of the transmitted light. Within 5 to 10 minutes of exposure to Ringer's solution, a general decrease in the light transmission across the long axis of the fiber was observed. Extended superfusion led to a local, apparent increase in light transmission corresponding to the formation of membrane blebs and globules. Images of disingerated globules focused above their equator showed bright halos with dark central zones. In electron micrographs, the single fibers showed uniform electron density. No significant inhomogeneities or precipitation of intracellular crystallins was observed in globules generated from fiber cells exposed to Ringer's solution; in addition, no high molecular weight protein aggregates were found in the globules. CONCLUSIONS: Exposure to calcium alters the light-transmitting properties of isolated cortical fibers. The initial decrease in the average light transmittance of the fiber appears to be secondary to cell swelling and may relate to protein-based opacification. An apparent increase in light transmission through calcium-generated globules is likely because of the Becke line generated by a mismatch between the refractive index of the medium and the globule cytoplasm and accentuated by the transition from rod-shaped to spheroidal morphology.

Animals↗
PubMed1997

Calcium homeostasis of isolated single cortical fibers of rat lens.

PURPOSE: To investigate the calcium homeostasis in single fiber cells isolated from rat ocular lens cortex and to quantify the changes in the concentration of free intracellular calcium [Ca2+]i during the process of disintegrative globulization. METHODS: Individual fiber cells from the cortex of the adult rat lens were isolated by treatment with trypsin in ion-free buffered sucrose. The isolated fiber cells were loaded with the acetoxymethyl esters of Fluo-3 or Calcium Green-2, or with Fluo-3 and Fura Red, and changes in [Ca2+]i of single cortical fibers were measured using a microfluorometer. The time course of increase of [Ca2+]i in fiber cells exposed to Ringer's solution was measured, and the effects on the increase of [Ca2+]i of calcium channel blocker, verapamil, Na-Ca exchange inhibitors Ni2+ and Zn2+, and protease inhibitor, leupeptin, Na+-free and K+-free media and Ca2+-containing isotonic sucrose solution, were investigated. RESULTS: In Hepes sucrose solution (containing approximately 1.5 microM Ca2+), the isolated fiber cells maintained stable values of [Ca2+]i at 99.6+/-10 nM (n = 32). Exposure of the isolated fibers to Ringer's solution (containing 2 mM Ca2+) led to a monoexponential increase of [Ca2+]i at a rate of 0.12 min(-1). This increase in [Ca2+]i was accompanied by disintegration of the isolated fibers into discrete but resealed globules. Changes in [Ca2+]i, monitored by using a two-dye ratiometric method using Fura Red and fluo-3, showed a progressive increase in [Ca2+]i in fibers exposed to Ringer's solution, preceding globulization. The [Ca2+]i in the globules in Ringer's solution, determined using Calcium Green-2, was 3.6+/-0.7 microM (n = 23). Compared with that in fibers in Ringer's solution, the rate of increase of [Ca2+]i in fibers was much slower in the presence of 50 microM verapamil (0.047 min[-1]), in Na+-free (0.086 min[-1]) and in K+-free (0.062 min[-1]) Ringer's solution, or when the fibers were suspended in Hepes-sucrose solution, containing 2 mM Ca2+ (0.046 min[-1]). After 30 minutes, the [Ca2+]i of fiber cells exposed to Ringer's solution, containing 2 mM Ni2+ (574.7+/-29 nM; n = 7) or Zn2+ (402.6+/-77 nM; n = 7) was significantly lower (P < 0.001) compared with that in fiber cells exposed to Ringer's solution alone (1995+/-461 nM, n = 10). In Ringer's solution, leupeptin delayed globulization without significantly affecting the increase in [Ca2+]i. The [Ca2+]i of fiber cells isolated from outer and inner cortex and suspended in Hepes-sucrose was comparable; however, after 15 minutes of exposure to Ringer's solution, [Ca2+]i in fibers from the outer cortex was approximately three times higher than [Ca2+]i in those from the inner cortex. CONCLUSIONS: Exposure to high (millimolar) concentrations of calcium in the external medium leads to an increase in [Ca2+]i of isolated individual fiber cells, which precedes disintegrative globulization. The protective effects of Na+-free and K+-free solutions on globulization appear to be due to a lower rate of increase of [Ca2+]i. Part of the calcium influx may be mediated by L-type calcium channels and by Na-Ca exchange, operating in reverse. Proteolytic inhibitors do not affect the increase in [Ca2+]i but delay globulization by inhibiting calcium-mediated proteolysis. The isolated fiber cells and the disintegrated globules maintain a 100- to 300-fold gradient of calcium across their plasma membranes.

Aniline Compounds↗
PubMed1996

The Global Oscillation Network Group (GONG) Project

Helioseismology requires nearly continuous observations of the oscillations of the solar surface for long periods of time in order to obtain precise measurements of the sun's normal modes of oscillation. The GONG project acquires velocity images from a network of six identical instruments distributed around the world. The GONG network began full operation in October 1995. It has achieved a duty cycle of 89 percent and reduced the magnitude of spectral artifacts by a factor of 280 in power, compared with single-site observations. The instrumental noise is less than the observed solar background.

Journal Article↗
PubMed1996

Technetium-99m dextran: a promising new protein-losing enteropathy imaging agent.

The purpose of this study was to evaluate technetium-99m dextran (99mTc-Dx; molecular weight 81000) as a prospective protein-losing enteropathy (PLE) imaging agent. Twenty-two patients with diseases commonly associated with PLE and 12 healthy control subjects underwent intravenous 99mTc-Dx scintigraphy. All of the 22 test patients showed significant radiotracer accumulation in the intestines within 3-4 h post injection. The focal, regional or generalised nature of the enteropathy and involvement of the large or small intestine could be identified in most cases. Four of the 12 apparently healthy subjects also showed minimal accumulation in the abdominal area occurring late in the study period. This could have been physiological, related to food habits or due to unsuspected intestinal worms. We attribute the high sensitivity of 99mTc-Dx to its relatively fast blood (background) clearance. The radiotracer may have several other advantages over 99mTc-labelled human serum albumin in imaging PLE.

Adult↗
PubMed1996

Scintiscan demonstration of localized bowel loop inflammation. Comparison of Tc-99m dextran, Tc-99m citrate, and Tc-99m human immunoglobulin G.

A 60-year-old man with localized small bowel inflammation underwent abdominal scintigraphy with Tc-99m human polyclonal immunoglobulin G and two experimental inflammation-seeking agents, Tc-99m dextran and Tc-99m citrate. The abnormal loop was visualized in all the three studies. Additionally, the Tc-99m dextran study revealed exudation and luminal transit of the tracer suggestive of regional protein-losing enteropathy. The authors conclude that Tc-99 dextran and Tc-99 citrate are clinically suitable inflammation-seeking agents that need further evaluation, especially for locating abdominal inflammations.

Citrates↗