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Biomedical subjects

A Bhatnagar

Publications and source records attributed to A Bhatnagar.

At least 37 records · Page 2Linked to original sources
PubMed2000

The dynamin-dependent, arrestin-independent internalization of 5-hydroxytryptamine 2A (5-HT2A) serotonin receptors reveals differential sorting of arrestins and 5-HT2A receptors during endocytosis.

5-Hydroxytryptamine 2A (5-HT2A) receptors, a major site of action of clozapine and other atypical antipsychotic medications, are, paradoxically, internalized in vitro and in vivo by antagonists and agonists. The mechanisms responsible for this paradoxical regulation of 5-HT2A receptors are unknown. In this study, the arrestin and dynamin dependences of agonist- and antagonist-mediated internalization were investigated in live cells using green fluorescent protein (GFP)-tagged 5-HT2A receptors (SR2-GFP). Preliminary experiments indicated that GFP tagging of 5-HT2A receptors had no effect on either the binding affinities of several ligands or agonist efficacy. Likewise, both the native receptor and SR2-GFP were internalized via endosomes in vitro. Experiments with a dynamin dominant-negative mutant (dynamin K44A) demonstrated that both agonist- and antagonist-induced internalization were dynamin-dependent. By contrast, both the agonist- and antagonist-induced internalization of SR2-GFP were insensitive to three different arrestin (Arr) dominant-negative mutants (Arr-2 V53D, Arr-2-(319-418), and Arr-3-(284-409)). Interestingly, 5-HT2A receptor activation by agonists, but not antagonists, induced greater Arr-3 than Arr-2 translocation to the plasma membrane. Importantly, the agonist-induced internalization of 5-HT2A receptors was accompanied by differential sorting of Arr-2, Arr-3, and 5-HT2A receptors into distinct plasma membrane and intracellular compartments. The agonist-induced redistribution of Arr-2 and Arr-3 into intracellular vesicles and plasma membrane compartments distinct from those involved in 5-HT2A receptor internalization implies novel roles for Arr-2 and Arr-3 independent of 5-HT2A receptor internalization and desensitization.

Animals↗
PubMed2000

Selective recognition of glutathiolated aldehydes by aldose reductase.

In this study, the selectivity and specificity of aldose reductase (AR) for glutathionyl aldehydes was examined. Relative to free aldehydes, AR was a more efficient catalyst for the reduction of glutathiolated aldehydes. Reduction of glutathionyl propanal [gammaGlu-Cys(propanal)-Gly] was more efficient than that of Gly-Cys(propanal)-Gly and gamma-aminobutyric acid-Cys(propanal)-Gly suggesting a possible interaction between alpha-carboxyl of the conjugate and AR. Two active site residues, Trp20 or Ser302, were identified by molecular modeling as potential sites of this interaction. Mutations containing tryptophan-to-phenylalanine (W20F) and serine-to-alanine (S302A) substitutions did not significantly affect reduction of free aldehydes but decreased the catalytic efficiency of AR for glutathiolated aldehydes. Combined mutations indicate that both Trp20 and Ser302 are required for efficient catalysis of the conjugates. The decrease in efficiency due to W20F mutation with glutathionyl propanal was not observed with gamma-aminobutyric-Cys(propanal)-Gly or Gly-Cys-(propanal)-Gly, indicating that Trp20 is involved in binding the alpha-carboxyl of the conjugate. The effect of the S302A mutation was less severe when gammaGlu-Cys(propanal)-Glu rather than glutathionyl propanal was used as the substrate, consistent with an interaction between Ser302 and Gly-3 of the conjugate. These observations suggest that glutathiolation facilitates aldehyde reduction by AR and enhances the range of aldehydes available to the enzyme. Because the N-terminal carboxylate is unique to glutathione, binding of the conjugate with the alpha-carboxyl facing the bottom of the alpha/beta-barrel may assist in the exclusion of unrelated peptides and proteins.

Aldehyde Reductase↗
PubMed2000

Pentoxiphylline reduces nitric oxide production among patients with HIV infection.

Tumour necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) levels are elevated among patients with human immunodeficiency virus (HIV) infection. TNF-alpha is known to lower NO production. In this study we used a TNF-alpha inhibitor, pentoxiphylline, to treat patients with HIV infection who were free of opportunistic infections and see if NO production was altered with this drug. NO production was determined by spectrophotometric analysis using nitrite and citrulline as surrogate markers and TNF-alpha levels were determined by ELISA before and after 4 weeks of the treatment. Nineteen patients (ten males, mean age 36.6+/-5.2 years) and 16 age and sex matched healthy controls were studied. Mean CD4 counts of patients were 206.5 mm(3). Nitrite level among patients at recruitment was 99.7+/-26.5 nmol/ml (range 50-167 nmol/ml) and was significantly higher than 46.4+/-16.2 nmol/ml; the value of healthy controls (P<0.05). Patient levels declined significantly to 44. 2+/-19.7 nmol/ml (range 10-106.6 nmol/ml) following 4 weeks of therapy (P<0.01). Citrulline level at recruitment was 810.8+/-425.8 nmol/ml (range 366.6-1888.7 nmol/ml), which was significantly higher than 488.6+/-224.5 nmol/ml, the level of controls (P<0.01). There was a statistically significant decrease in these levels among patients to 533.6+/-299.5 nmol/ml (range 250-163.4 nmol/ml) after 4 weeks of therapy (P<0.01). TNF-alpha levels showed a significant decline in the OD values from 0.34+/-0.22 at the start of therapy to 0.24+/-0.18 (P<0.05). We conclude that the use of pentoxiphylline is associated with decrease in TNF-alpha levels and NO production.

Adult↗
PubMed2000

Metabolism of lipid peroxidation product, 4-hydroxynonenal (HNE) in rat erythrocytes: role of aldose reductase.

Lipid peroxidation represents a significant source of erythrocyte dysfunction and aging. Because the toxicity of lipid peroxidation appears to be in part due to aldehydic end products, we examined, in rat erythrocytes, the metabolism of 4-hydroxy-trans-2-nonenal (HNE), one of the most abundant and toxic lipid-derived aldehydes. Packed erythrocytes, 0.1 ml, completely metabolized 20 nmoles of HNE in 20 min. The glutathione conjugate of HNE and 4-hydroxynonanoic acid (HNA) represented 70 and 25% of the total metabolism, respectively. Approximately 70% of the metabolites were extruded to the medium. Upon electrospray ionization mass spectrometry, the glutathione conjugate resolved into two distinct species corresponding to glutathionyl HNE (GS-HNE) and glutathionyl 1,4-dihydroxynonene (GS-DHN). The concentration of GS-DHN formed was twice that of GS-HNE. Inhibition of aldose reductase by sorbinil and tolrestat led to a selective decrease in the formation of GS-DHN, although the extent of HNE glutathiolation was unaffected. Inhibitors of aldehyde or alcohol dehydrogenase, i.e., cyanamide and 4-methyl pyrazole, had no effect on the formation of HNA and GS-DHN, indicating that these enzymes are not significant participants in the erythrocyte HNE metabolism. Thus, oxidation to HNA, conjugation with glutathione, and further reduction of the conjugate by aldose reductase appear to be the major pathways of HNE metabolism in erythrocytes. These pathways may be critical determinants of erythrocyte toxicity due to lipid peroxidation-derived aldehydes.

Aldehyde Reductase↗
PubMed2000

Cyclooxygenase-2 mediates the cardioprotective effects of the late phase of ischemic preconditioning in conscious rabbits.

We examined the role of cyclooxygenase-2 (COX-2) in the late phase of ischemic preconditioning (PC). A total of 176 conscious rabbits were used. Ischemic PC (six cycles of 4-min coronary occlusions/4-min reperfusions) resulted in a rapid increase in myocardial COX-2 mRNA levels (+231 +/- 64% at 1 h; RNase protection assay) followed 24 h later by an increase in COX-2 protein expression (+216 +/- 79%; Western blotting) and in the myocardial content of prostaglandin (PG)E(2) and 6-keto-PGF(1alpha) (+250 +/- 85% and +259 +/- 107%, respectively; enzyme immunoassay). Administration of two unrelated COX-2 selective inhibitors (NS-398 and celecoxib) 24 h after ischemic PC abolished the ischemic PC-induced increase in tissue levels of PGE(2) and 6-keto-PGF(1alpha). The same doses of NS-398 and celecoxib, given 24 h after ischemic PC, completely blocked the cardioprotective effects of late PC against both myocardial stunning and myocardial infarction, indicating that COX-2 activity is necessary for this phenomenon to occur. Neither NS-398 nor celecoxib lowered PGE(2) or 6-keto-PGF(1alpha) levels in the nonischemic region of preconditioned rabbits, indicating that constitutive COX-1 activity was unaffected. Taken together, these results demonstrate that, in conscious rabbits, up-regulation of COX-2 plays an essential role in the cardioprotection afforded by the late phase of ischemic PC. Therefore, this study identifies COX-2 as a cardioprotective protein. The analysis of arachidonic acid metabolites strongly points to PGE(2) and/or PGI(2) as the likely effectors of COX-2-dependent protection. The recognition that COX-2 mediates the antistunning and antiinfarct effects of late PC impels a reassessment of current views regarding this enzyme, which is generally regarded as detrimental.

6-Ketoprostaglandin F1 alpha↗
PubMed2000

Kinetic and structural characterization of the glutathione-binding site of aldose reductase.

Aldose reductase (AR), a member of the aldo-keto reductase superfamily, has been implicated in the etiology of secondary diabetic complications. However, the physiological functions of AR under euglycemic conditions remain unclear. We have recently demonstrated that, in intact heart, AR catalyzes the reduction of the glutathione conjugate of the lipid peroxidation product 4-hydroxy-trans-2-nonenal (Srivastava, S., Chandra, A., Wang, L., Seifert, W. E., Jr., DaGue, B. B., Ansari, N. H., Srivastava, S. K., and Bhatnagar, A. (1998) J. Biol. Chem. 273, 10893-10900), consistent with a possible role of AR in the metabolism of glutathione conjugates of aldehydes. Herein, we present several lines of evidence suggesting that the active site of AR forms a specific glutathione-binding domain. The catalytic efficiency of AR in the reduction of the glutathione conjugates of acrolein, trans-2-hexenal, trans-2-nonenal, and trans,trans-2,4-decadienal was 4-1000-fold higher than for the corresponding free alkanal. Alterations in the structure of glutathione diminished the catalytic efficiency in the reduction of the acrolein adduct, consistent with the presence of specific interactions between the amino acid residues of glutathione and the AR active site. In addition, non-aldehydic conjugates of glutathione or glutathione analogs displayed active-site inhibition. Molecular dynamics calculations suggest that the conjugate adopts a specific low energy configuration at the active site, indicating selective binding. These observations support an important role of AR in the metabolism of glutathione conjugates of endogenous and xenobiotic aldehydes and demonstrate, for the first time, efficient binding of glutathione conjugates to an aldo-keto reductase.

Aldehyde Reductase↗
PubMed2000

Nuclear imaging methods for non-invasive drug monitoring.

Functional imaging techniques provide complimentary information to that provided by structural studies such as MRI and CT. Functional imaging is based upon known parameters such as physiology, metabolism, biochemistry, pharmacology, and any other biological process. As such, this methodology plays a major role in understanding the basic mechanisms of a multitude of disorders, accurate diagnosis of certain diseases, and developing effective treatment for serious illnesses such as cancer and central nervous system maladies. Although this type of imaging can be performed with various modalities, nuclear procedures have played the leading role in this discipline. Advances made in labeling various radionuclides to biologically important compounds, and development of sophisticated instruments have substantially contributed to the growth of the field of functional imaging. The introduction of positron emission topography (PET), which is based on imaging of compounds labeled with elements such as carbon, nitrogen, and fluorine, has added a major dimension to the evolution of the discipline. This review deals with a brief introduction to the methodologies utilized with radiolabeled tracers and then deals with specific applications of this technology. These applications include assessment of blood flow and metabolism, receptor imaging, elucidating the pathophysiologic process, evaluating role of labeled therapeutic agents, and the potential of these techniques in the development of novel biologic therapies. Functional imaging with radiolabeled tracers will play an increasingly important role in modern medicine, and its impact will be substantial in the management of patients with various disorders.

Drug Monitoring↗
PubMed2000

Impact of attenuation correction on the accuracy of FDG-PET in patients with abdominal tumors: a free-response ROC analysis.

The aim of this study was to evaluate image quality and lesion detectability with and without attenuation correction in patients with abdominal tumors, using a free-response receiver operating characteristic (FROC) methodology. Thirty-four patients with various abdominal tumors were evaluated (11 men, 23 women, median age 48 years). Whole-body emission scans were performed 68 min (35-102 min) after intravenous injection of 4.3 MBq/kg fluorine-18 fluorodeoxyglucose (FDG). Images were reconstructed using the OS-EM algorithm and corrected for attenuation either using postinjection singles transmission (n=27) or by calculation and body outline (n=7). Total scan duration did not exceed 70 min. Studies were read independently by four observers unaware of any clinical data. The uncorrected (UC) images were systematically read before the attenuation-corrected (AC) images. All studies were given an image quality score ranging from 1 (unreadable) to 5 (excellent). Each focus of increased activity was then localized and given a probability of malignancy using a five-point scale. The average image quality score was similar for both UC and AC images. At the time of the positron emission tomography (PET) scans, 127 lesions (63 liver metastases, 9 retroperitoneal lesions, 50 peritoneal or bowel lesions, and 5 pancreatic carcinomas) were revealed by pathological or correlative studies. The areas under the FROC curves were consistently greater for AC images (range 0.8663-0.8867) than for UC images (range 0.7774 -0.8613). Overall, the difference between the AC images and the UC images was significant (P=0.019). In particular, correction for attenuation increased the sensitivity regardless of the location of the lesions. In conclusion, correction for attenuation significantly improves the diagnostic accuracy of FDG-PET for abdominal staging of neoplasms, without impairing the image quality.

Abdominal Neoplasms↗
PubMed2000

Involvement of aldose reductase in vascular smooth muscle cell growth and lesion formation after arterial injury.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) is an important feature of atherosclerosis, restenosis, and hypertension. Although multiple mediators of VSMC growth have been identified, few effective pharmacological tools have been developed to limit such growth. Recent evidence indicating an important role for oxidative stress in cell growth led us to investigate the potential role of aldose reductase (AR) in the proliferation of VSMCs. Because AR catalyzes the reduction of mitogenic aldehydes derived from lipid peroxidation, we hypothesized that it might be a potential regulator of redox changes that accompany VSMC growth. Herein we report several lines of evidence suggesting that AR facilitates/mediates VSMC growth. Stimulation of human aortic SMCs in culture with mitogenic concentrations of serum, thrombin, basic fibroblast growth factor, and the lipid peroxidation product 4-hydroxy-trans-2-nonenal (HNE) led to a 2- to 4-fold increase in the steady-state levels of AR mRNA, a 4- to 7-fold increase in AR protein, and a 2- to 3-fold increase in its catalytic activity. Inhibition of the enzyme by sorbinil or tolrestat diminished mitogen-induced DNA synthesis and cell proliferation. In parallel experiments, the extent of reduction of the glutathione conjugate of HNE to glutathionyl-1,4-dihydroxynonene in HNE-exposed VSMCs was decreased by serum starvation or sorbinil. Immunohistochemical staining of cross sections from balloon-injured rat carotid arteries showed increased expression of AR protein associated with the neointima. The media of injured or uninjured arteries demonstrated no significant staining. Compared with untreated animals, rats fed sorbinil (40 mg. kg(-1). d(-1)) displayed a 51% and a 58% reduction in the ratio of neointima to the media at 10 and 21 days, respectively, after balloon injury. Taken together, these findings suggest that AR is upregulated during growth and that this upregulation facilitates growth by enhancing the metabolism of secondary products of reactive oxygen species.

Aldehyde Reductase↗
PubMed1999

Radionuclide imaging of cardiac pathology: a mechanistic perspective.

Cardiac imaging is performed for a variety of indications using different radiotracers and ligands. The most common is the use of thallium-201 and Tc-labeled tracers for myocardial perfusion imaging. Its uses are manifold, and range from the diagnosis of coronary artery disease to the risk stratification of patients and prognostication. Ischemia is being imaged with nitroimidazole compounds, myocardial necrosis with antimyosin and myocardial infiltration with gallium-labeled leukocytes and somatostatin receptor analogs. The status of myocardial innervation has been evaluated with radiolabeled MIBG. Metabolism in the heart in the normal and ischemic state can be studied with PET and SPECT using fatty acid and glucose analogs. The expression of accessory molecules on the surface of cardiac myocytes in patients with cardiac allograft rejection is now possible. Clearly, much has been accomplished for imaging the heart and pathology related to it. The past has been satisfying, the present interesting and the future promises to be even more exciting!

Journal Article↗
PubMed1999

Structural and kinetic determinants of aldehyde reduction by aldose reductase.

Aldose reductase (AR) is a member of the aldo-keto reductase superfamily. Due to its ability to catalyze the formation of sorbitol from glucose during hyperglycemic and hypertonic stress, the aldose-reducing property of AR has been accepted as its main physiological and pathological function. Nonetheless, AR is a poor catalyst for glucose reduction and displays active-site properties unexpected of a carbohydrate-binding protein. We, therefore, examined the catalytic properties of AR with a series of naturally occurring aldehydes, compatible in their hydrophobicity to the large apolar active site of the enzyme. Our results show that recombinant human AR is an efficient catalyst for the reduction of medium- to long-chain unbranched saturated and unsaturated aldehydes. The enzyme displayed selective preference for saturated aldehydes, such as hexanal, and unsaturated aldehydes, such as trans-2-octenal and nonenal as well as their 4-hydroxy derivatives. Short-chain aldehydes such as propanal and acrolein were reduced less efficiently. Branched derivatives of acrolein or its glutathione conjugate (GS-propanal) were, however, reduced with high efficiency. In the absence of NADPH, the alpha, beta unsaturated aldehydes caused covalent modification of the enzyme. On the basis of electrospray mass spectrometric analysis of the wild-type and site-directed mutants of AR (in which the solvent exposed cysteines were individually replaced with serine), the site of modification was identified to be the active-site residue, Cys 298. The unsaturated aldehydes, however, did not modify the enzyme bound to NADPH and did not inactivate the enzyme during catalysis. Modeling studies indicate that the large hydrophobic active site of AR can accommodate a large number of aldehydes without changes in the structure of the binding site or movement of side chains. High hydrophobicity due to long alkyl chains or apolar substituents appears to stabilize the interaction of the aldehyde substrates with the enzyme. Apparently, such hydrophobic interactions provide substrate selectivity and catalytic efficiency of the order achievable by hydrogen bonding. Since several of the aldehydes reduced by AR are either environmental and pharmacological pollutants or products of lipid peroxidation, the present studies provide the basis of future investigations on the role of AR in regulating aldehyde metabolism particularly under pathological states associated with oxidative stress and/or aldehyde toxicity.

Aldehyde Reductase↗
PubMed1999

Can the standardized uptake value characterize primary brain tumors on FDG-PET?

The aim of this study was to evaluate the usefulness of measuring the standardized uptake value (SUV) in primary brain tumors on fluorine-18 fluorodeoxyglucose (FDG) positron emission tomography (PET) scans. Two groups of patients were studied. Whole-brain glucose cerebral metabolic rates (wCMRs) and SUVs were obtained in 20 normal subjects. Twenty-seven patients with histology-proven malignant primary CNS tumors (high-grade gliomas n=22, primitive neuroectodermal tumors n=3, ependymomas n=2) were also studied. The degree of FDG uptake was assessed by visual inspection and thereafter regions of interest were placed over the lesion, the contralateral cortex and white matter and the whole brain. Average (avg) and maximum (max) pixel values were determined in each site. Based on these measurements, SUV, tumor to cortex (T/C) and tumor to white matter (T/WM) activity ratios were calculated. There was no correlation between wCMRs (4.55+/-0.36 mg min(-1) 100 g(-1)) and wSUVs (5.41+/-0.43) in the normal subjects (r=0.18, P=0.45). In the second group, 17 lesions were described as definitely and seven as probably malignant. However, SUVs in these tumors and in the contralateral cortex were not significantly different. Although the SUVs were generally higher in the tumor than in the contralateral white matter, there was a significant overlap between the values. The range of the SUVs was wide: 2.54-11.8 for the tumors, 2.98-9.96 for the cortex and 1.87-6.76 for the white matter. SUVs in the normal cortex were negatively correlated with blood glucose level at the time of the injection. SUVs in the whole brain and in the cortex were lower in patients previously treated by irradiation, even months after completion of the treatment. No correlation was detectable between any of the SUVs and the age of the patients, tumor type, time post injection, use of dexamethasone, patient weight, dose injected and visual score. With cutoff levels of 1.5 for T max/WM and 0.6 for T max/C, the sensitivity of the activity ratios was 74% and 96% respectively. In conclusion, SUVs do not correlate with CMRs across subjects and appear to be of limited value in characterizing brain tumors. Visual assessment and measurement of the activity ratios currently remain the most reliable methods of analysis.

Adult↗
PubMed1999

Superantigen-induced T cell responses in acute rheumatic fever and chronic rheumatic heart disease patients.

CD4+ and CD8+ T cells from healthy donors, acute rheumatic fever (ARF) and chronic rheumatic heart disease (CRHD) patients responded variably to a superantigen from Streptococcus pyogenes--Streptococcal pyrogenic erythrogenic toxin A (SPE-A). In vitro culture of CD4+ T cells from ARF patients (CD4-ARF) with SPE-A exhibited a Th1 type of response as they produced high levels of IL-2, while CD4+ T cells from CRHD patients (CD4-RHD) secreted IL-4 and IL-10 in large amounts, i.e. Th2 type of cytokine profile. The skewing of human CD4+ T cells (in response to SPE-A stimulation) to Th1 or Th2 type reflects the role of the two subsets in a disorder with differing intensities at the two extremes of the spectrum. Moreover, the anergy induction experiments revealed that CD8-ARF and CD8-RHD undergo anergy (to different extents), whereas CD4+ T cells do not, in response to re-stimulation by SPE-A. These results initially demonstrate that both CD4+ and CD8+ T cells respond differentially to SPE-A, and hence it is an important observation with respect to the pathogenesis of ARF/CRHD. Anergy in CD8+ T cells in the presence of SPE-A in vitro goes a step further to show the clinical relevance of these cells and their possible role in suppression of the disease.

Acute Disease↗