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Biomedical subjects

D Bandyopadhyay

Publications and source records attributed to D Bandyopadhyay.

At least 55 records · Page 3Linked to original sources
PubMed1998

Oxidative inactivation of gastric peroxidase by site-specific generation of hydroxyl radical and its role in stress-induced gastric ulceration.

We have shown earlier that restraint-cold stress-induced gastric ulceration in rats is caused by metal ion-dependent generation of hydroxyl radical (OH.) and oxidative inactivation of the gastric peroxidase (GPO), an important H2O2 scavenging enzyme. To study the mechanism of the oxidative damage of GPO, the purified enzyme was exposed to an OH. generating system containing Cu2+, ascorbate, and H2O2. Kinetic studies indicate that the enzyme is inactivated in a time-dependent process showing saturation with respect to Cu2+ concentration. The enzyme specifically requires Cu2+ and is not inactivated by the same concentration of Fe2+, Mn2+, or Zn2+. Sensitivity to catalase indicates the critical role of H2O2 in the inactivation. Inactivation is insensitive to superoxide dismutase, suggesting no role of superoxide. The rate of inactivation is not increased in D2O excluding the involvement of singlet oxygen in the process. However, OH. scavengers such as benzoate or mannitol cannot prevent inactivation. The results indicate a plausible generation of OH. within the enzyme molecule as the cause of inactivation. Fragmentation of peptide linkage or intramolecular crosslinking, gross change of tertiary structure, or change in intrinsic tryptophan fluorescence which occurs in "global" oxidation are not evident. Inactivation is dependent on pH and from a plot of K(obs) of inactivation against pH, the controlling role of an ionizable group of the enzyme having a pka of 7.8 could be suggested, deprotonation of which favors inactivation. Amino acid analysis shows a specific loss of two lysine residues in the inactivated enzyme. Competitive kinetic studies indicate that pyridoxal phosphate, a specific modifier of the lysine residue, prevents inactivation by competing with Cu2+ for binding at the GPO. A Cu2+ binding motif consisting at least of two lysine residues exists in GPO, which specifically binds Cu2+ and generates OH.. The radical oxidizes the lysine residues and perturbs the heme environment to cause inactivation. We suggest that oxidative damage of GPO is mediated by site-specific generation of OH. and not by the OH. generated in the bulk phase.

Animals↗
PubMed1998

Inhibition of the protein kinase C pathway promotes anti-CD95-induced apoptosis in Jurkat T cells.

The role of the basal activity of the serine/threonine protein kinase, protein kinase C (PKC) in the regulation of anti-CD95-induced apoptosis in Jurkat T cells was investigated. The PKC-specific inhibitor GF 109203X and the proposed cPKC-specific inhibitor Go 6976, in a concentration-dependent manner, increased the percentage of cells undergoing apoptosis induced by anti-CD95 mAb as demonstrated by propidium iodide (PI) staining, TUNEL assay and DNA fragmentation by gel electrophoresis. Furthermore, Go 6976 and GF 109203X abrogated phorbol myristate acetate-induced inhibition of anti-CD95-induced apoptosis. To examine the molecular mechanism by which PKC modulates anti-CD95-induced apoptosis, the effects of Go 6976 on known effector and regulatory molecules of cell death were studied. Increased recruitment of cells undergoing apoptosis was associated with enhanced anti-CD95-induced proteolytic cleavage of the most receptor-proximal cysteine protease caspase-8, subsequent cleavage and activation of the machinery protease caspase-3, and cleavage of the caspase substrates DNA-dependent protein kinase catalytic subunit, poly-(ADP-ribose) polymerase and lamin B1. CD95 and FADD protein levels in Jurkat T cells were not altered by Go 6976 treatment. In addition, Go 6976 did not alter protein levels and subcellular distribution of the anti-apoptotic molecules Bcl-2 and Bcl-xL. These data suggest indirectly that basal PKC activity acts at an early stage in the anti-CD95-induced caspase pathway to attenuate subsequent activation of downstream effector molecules and associated apoptosis in Jurkat T cells.

Antibodies, Monoclonal↗
PubMed1998

A multi-user real time inventorying system for radioactive materials: a networking approach.

A computerized system for radioisotope management and real time inventory coordinated across a large organization is reported. It handles hundreds of individual users and their separate inventory records. Use of highly efficient computer network and database technologies makes it possible to accept, maintain, and furnish all records related to receipt, usage, and disposal of the radioactive materials for the users separately and collectively. The system's central processor is an HP-9000/800 G60 RISC server and users from across the organization use their personal computers to login to this server using the TCP/IP networking protocol, which makes distributed use of the system possible. Radioisotope decay is automatically calculated by the program, so that it can make the up-to-date radioisotope inventory data of an entire institution available immediately. The system is specifically designed to allow use by large numbers of users (about 300) and accommodates high volumes of data input and retrieval without compromising simplicity and accuracy. Overall, it is an example of a true multi-user, on-line, relational database information system that makes the functioning of a radiation safety department efficient.

Computer Communication Networks↗
PubMed1998

Estrogen receptor expression and function in long-term estrogen-deprived human breast cancer cells.

Hormone-dependent breast cancer responds to primary therapies that block estrogen production or action, but tumor regrowth often occurs 12-18 months later. Additional hormonal treatments that further reduce estrogen synthesis or more effectively block its action cause additional remissions, but the mechanisms responsible for these secondary responses are not well understood. As a working hypothesis, we postulated that primary hormonal therapy induces adaptive changes, resulting in enhanced estrogen receptor (ER) expression and target gene activation and, further, that secondary treatment modalities interfere with these receptor-mediated transcriptional pathways. To test this hypothesis, we used an MCF-7 breast cancer model system involving deprivation of estradiol in culture for a prolonged period. These long-term estradiol-deprived (LTED) cells adapt by acquiring the ability to regrow in the absence of added estradiol. The experimental paradigm involved the comparison of wild-type cells with LTED cells. As endpoints, we directly assessed ER expression at the messenger RNA-, protein-, and ligand-binding levels and ER functionality by quantitating reporter gene activation and expression of endogenous estrogen target gene messenger RNA, as well as ER coactivator levels. Our data demonstrated an adaptive increase in ER expression and in basal ER functionality, as assessed by read-out of three different transfected reporters in LTED, as opposed to wild-type MCF-7 cells. Increased reporter gene read-out was dramatically inhibited by the pure antiestrogen ICI 182,780. As verification that endogenous (as well as transfected) estrogen target genes had enhanced transcription, we found that the basal levels of c-myb and c-myc message were substantially increased in LTED cells and could be inhibited by antiestrogen. Interestingly, the levels of c-myb and c-myc message in the LTED cells seemed to be increased out of proportion to the degree of ER reporter gene activation and were similar to those in wild-type cells maximally stimulated with estradiol. In addition, not all estrogen-responsive genes were activated, because transforming growth factor-alpha message level was not increased in LTED cells. Up-regulation of the steroid receptor coactivator SRC-1 did not seem to mediate the process of enhanced ER-induced transcription. Considering these observations together, we suggest that long-term estradiol deprivation causes adaptive processes that not only involve up-regulation of the ER but also influence the specificity and magnitude of activation of estrogen-responsive genes.

Breast Neoplasms↗
PubMed1997

Protein-tyrosine phosphatase 1B complexes with the insulin receptor in vivo and is tyrosine-phosphorylated in the presence of insulin.

In response to insulin, protein-tyrosine phosphatase 1B (PTPase 1B) dephosphorylates 95- and 160-180-kDa tyrosine phosphorylated (PY) proteins (Kenner, K. A., Anyanwu, E., Olefsky, J. M., and Kusari, J. (1996) J. Biol. Chem. 271, 19810-19816). To characterize these proteins, lysates from control and insulin-treated cells expressing catalytically inactive PTPase 1B (CS) were immunoadsorbed and subsequently immunoblotted using various combinations of phosphotyrosine, PTPase 1B, and insulin receptor (IR) antibodies. Anti-PTPase 1B antibodies coprecipitated a 95-kDa PY protein from insulin-stimulated cells, subsequently identified as the IR beta-subunit. Similarly, anti-IR antibodies coprecipitated the 50-kDa PY-PTPase 1B protein from insulin-treated cells. To identify PTPase 1B tyrosine (Tyr) residues that are phosphorylated in response to insulin, three candidate sites (Tyr66, Tyr152, and Tyr153) were replaced with phenylalanine. Replacing Tyr66 or Tyr152 and Tyr153 significantly reduced insulin-stimulated PTPase 1B phosphotyrosine content, as well as its association with the IR. Studies using mutant IRs demonstrated that IR autophosphorylation is necessary for the PTPase 1B-IR interaction. These results suggest that PTPase 1B complexes with the autophosphorylated insulin receptor in intact cells, either directly or within a complex involving additional proteins. The interaction requires multiple tyrosine phosphorylation sites within both the receptor and PTPase 1B.

Insulin↗
PubMed1997

Effect of cadmium on purified hepatic flavokinase: involvement of reactive -SH group(s) in the inactivation of flavokinase by cadmium.

The effect of cadmium (Cd2+) was studied in vitro on the flavokinase (ATP : riboflavin 5'-phosphotransferase, EC 2.7.1.26) activity purified from rat liver. Cadmium inhibited flavokinase activity in a concentration-dependent manner and the effect was completely reversed by increasing concentration of zinc (Zn2+), indicating a competition between Zn2+ and Cd2+ for binding with the enzyme. Further, a competition between riboflavin and Cd2+ hints at the possibility that Zn2+ and Cd2+ probably compete for the same site on the enzyme where riboflavin binds. Our studies further reveal that hepatic flavokinase contains essential, accessible and functional thiol group(s) as evidenced by a concentration-dependent inhibition by sulfhydryl reagents and protection by thiol protectors like glutathione or dithiothreitol. Furthermore, the enzyme could also be protected from the inhibitory effect of Cd2+ and Hg2+ by glutathione and dithiothreitol suggesting that Cd2+ probably interacts with reactive thiol group at or near the active site of the enzyme to cause inhibition.

Animals↗
PubMed1997

Hydroxyl radical is the major causative factor in stress-induced gastric ulceration.

The role of the metal-catalyzed production of hydroxyl radicals (OH.) on gastric ulceration caused by restraint-cold stress in rat was studied. Stress causes a 50% increase in the thiobarbituric acid reactive species (TBARS) as a measure of the lipid peroxidation, nearly 70% increase in protein oxidation as measured by its carbonyl content and about 40% decrease in the glutathione content of the fundic stomach, suggesting oxidative damage by stress. Stress also causes a time-dependent increase in the mitochondrial superoxide dismutase activity and a decrease in the peroxidase activity, both of which correlate well with the increase in the severity of ulceration as measured by the ulcer index. Specific OH. scavengers such as benzoate or dimethylsulfoxide (DMSO) and the free radical trap such as alpha-phenyl N-tert-butyl nitrone (PBN) significantly inhibit gastric ulceration suggesting the role of OH. in this oxidative damage. Desferrioxamine (DFO), a nontoxic transition metal ion chelator, protects the mucosa against stress-ulceration dose dependently. Increased level of TBARS and the inactivation of gastric peroxidase are also prevented by DFO or by antioxidants such as glutathione or vitamin E, suggesting the critical role of metal ion and OH. in the oxidative damage. A metal-catalyzed OH. generating system constituted by Cu2+, H2O2 and ascorbate (reducing equivalent of O2-) causes inactivation of the purified gastric peroxidase in vitro, which can be effectively prevented by DFO. The stress-induced activation of the superoxide dismutase is completely blocked by pretreatment with alpha-amanitin indicating an increased synthesis of the enzyme by increased transcription of its m-RNA. Quantitative measurement indicates that stress causes a fivefold increase in the generation of OH., which correlates well with the increase in ulcer index with the progress of stress. The results indicate that the stress-induced gastric ulceration is a consequence of the oxidative damage of the gastric mucosa. This is caused by the OH. generated through the metal-catalyzed Haber-Weiss reaction between O2- and H2O2, the latter being formed by the stimulation of the superoxide dismutase and inactivation of the gastric peroxidase.

Amanitins↗
PubMed1997

Effect of cadmium, mercury and copper on partially purified hepatic flavokinase of rat.

The effect of cadmium (Cd2+), mercury (Hg2+) and copper (Cu2+) was studied with partially purified flavokinase (ATP:riboflavin 5'-phosphotransferase EC 2.7.1.26) from rat liver. All the divalent heavy metal cations inhibited flavokinase activity in a concentration-dependent manner. The inhibitory effect of cadmium on the enzyme was completely reversed by increasing concentration, of Zinc (Zn2+) indicating a competition between Zn2+ and Cd2+ for binding with the enzyme. A competition between riboflavin and Cd2+ is also evident from the present investigation. These observations hint at the possibility that Zn2+ and Cd2+ probably compete for the same site on the enzyme where riboflavin binds. However, inhibition of flavokinase by Hg2+ could not be reversed by Zn2+. Our studies further reveal that hepatic flavokinase appears to contain an essential, accessible and functional thiol group(s) which is evident from a concentration dependent inhibition of activity by sulfhydryl reagents like parachloromercuribenzoate (PCMB), 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), and N-ethylmaleimide (NEM). Inhibition of flavokinase by sulfhydryl reagents were protected, except in case of NEM inhibition, when the enzyme was incubated with thiol protectors like glutathione (GSH) and dithiothreitol (DTT). Furthermore, the enzyme could also be protected from the inhibitory effect of Cd2+ and Hg2+ by GSH and DTT suggesting that Cd2+ probably interacts with a reactive thiol group at or near the active site of enzyme in bringing about its inhibitory effect.

Adenosine Triphosphate↗
PubMed1997

Insulin-induced mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) attenuates insulin-stimulated MAP kinase activity: a mechanism for the feedback inhibition of insulin signaling.

Insulin signaling involves the transient activation/inactivation of various proteins by a cycle of phosphorylation/dephosphorylation. This dynamic process is regulated by the action of protein kinases and protein phosphatases. One family of protein kinases that is important in insulin signaling is the mitogen-activated protein (MAP) kinases, whose action is reversed by specific MAP kinase phosphatases (MKPs). Insulin stimulation of Hirc B cells overexpressing the human insulin receptor resulted in increased MKP-1 mRNA levels. MKP-1 mRNA increased in a dose-dependent manner to a maximum of 3- to 4-fold over basal levels within 30 min, followed by a gradual return to basal. The mRNA induction did not require the continuous presence of insulin. The induction of MKP-1 protein synthesis followed MKP-1 mRNA induction; MKP-1 protein was maximally expressed after 120 min of insulin stimulation. MKP-1 mRNA induction by insulin required insulin receptor tyrosine kinase activity, since overexpression of an altered insulin receptor with impaired intrinsic tyrosine kinase activity prevented mRNA induction. Forskolin, (Bu)2-cAMP, 8-bromo-cAMP, and 8-(4-chlorophenylthio)-cAMP increased the MKP-1 mRNA content moderately above basal. These agents also augmented the insulin-stimulated expression of MKP-1 mRNA. However, in some cases the increase in MKP-1 mRNA expression was less than additive. Nevertheless, these results indicate that multiple signaling motifs might regulate MKP-1 expression and suggest another mechanism for the attenuation of insulin-stimulated MAP kinase activity by cAMP. Overexpression of MKP-1 in Hirc B cells inhibited both insulin-stimulated MAP kinase activity and MAP kinase-dependent gene transcription. The results of these studies led us to conclude that insulin regulates MKP-1 and strongly suggest that MKP-1 acts as a negative regulator of insulin signaling.

Animals↗
PubMed1997

Knowledge and attitude in relation to HIV/AIDS among in-service nurses of Calcutta.

Seventy-five senior nurses attending a workshop were surveyed with questionnaires and using two separate scales, their knowledge about transmission and precautionary measures, and their general attitude towards HIV/AIDS as well as willingness for patient-care were assessed. The nurses showed a satisfactory level of knowledge (mean percentage score 74.3), but misconceptions regarding disinfection and precautionary measures were present; 33% had overall negative attitudes and 24% unwilling to provide care for HIV-infected patients. Knowledge and attitude were positively correlated (r = .32). Knowledge deficits of some aspects of infection leading to fear of contagion and judgemental outlook towards HIV infection might lead to negative attitude impeding proper care. It is suggested that continuous in-service training be instituted to dispel misconceptions and to develop favourable and non-discriminatory attitude.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Evidence for a slow tertiary relaxation in the reaction of tert-butyl isocyanide with horseradish peroxidase.

The kinetics of tert-butyl isocyanide binding to the heme protein horseradish peroxidase (HRP) at 22 degrees C was examined on all time scales, from minutes to picoseconds, in aqueous borate buffer at pH 9.08. Unlike myoglobin (Mb) or hemoglobin, HRP shows two bimolecular ligand binding processes. For comparison, binding of the same ligand with Mb was measured under identical conditions. Ligand entry into the protein from the solvent in a mixing experiment is extremely slow in HRP: the bimolecular association constant is 0.04 M-1 s-1, while in Mb it is 4 x 10(3) M-1 s-1. Surprisingly, in view of that difference, picosecond and nanosecond photolyses reveal that once the ligand has reached the iron(II) site there is no difference in cage return or escape from the protein. The rate for the fastest cage return (from the contact pair) is close to 6 x 10(10) s-1 in both proteins. The rates of escape from the contact pair to form a secondary protein-caged pair are also similar: for Mb, 10 x 10(10) s-1, and for HRP, 8.5 x 10(10) s-1. The rate of rebinding from the protein-separated cage is near 4 x 10(6) s-1 in both proteins, and the rate of escape from protein to solvent is close to 3.7 x 10(6) s-1 in both. The difference between the two proteins lies in the low-millisecond time domain. After flash photolysis of HRP, there is a concentration-dependent recombination not seen in mixing experiments. This bimolecular rate constant varies slightly for different HRP preparations, being 2.6 x 10(4) or 4.0 x 10(4) M-1 s-1 in two cases, both of which are much faster than is observed in mixing experiments, namely, 0.04 M-1 s-1. In Mb, photolysis and mixing experiments consistently give the same combination rate, which is somewhat slower than the faster part of the HRP recombination. Similar measurements for the smaller ligand methyl isocyanide revealed no anomalous behavior. The interpretation proposed involves tertiary relaxation after ligand escape, which is significant in blocking the return of the large t-BuNC, but has no apparent effect on smaller ligands. Thus, HRP-t-BuNC reveals in dramatic fashion a phenomenon merely hinted at in earlier work involving the T-state binding kinetics of hemoglobin.

Horseradish Peroxidase↗
PubMed1995

Immunobiological studies on experimental visceral leishmaniasis. IV. Kinetics of evolution of disease-promoting versus host-protective cells of monocyte-macrophage lineage and their characterization.

The evolution of cells of the monocyte-macrophage lineage (MML cells) in the spleen of Leishmania donovani (LD) infected BALB/c mice was studied. Spleen cells were fractionated on a discontinuous percoll gradient and adherent cells (AC) were purified from fractionated spleen cells by adherence steps that appeared at the interfaces of 25-35%, 35-40%, 40-45% and 45-50% percoll gradients. The AC were characterized as MML cells on the basis of positive staining for non-specific esterase. Adherent cells that appeared at the interfaces of 25-35% and 40-45% were defined as A and C, respectively, and both of them showed extreme variation in a progressive infection. It was observed that A supported parasite replication whereas C remained refractory when infected with LD in vitro. Furthermore, when A cells and C cells were used as antigen-presenting cells to stimulate mixed population of IFN-gamma producing and IL-4 producing T-cells, it was observed that IL-4 and IFN-gamma were the predominating cytokine in the T-cell supernatant, respectively. Both A and C were found to be increased hand-in-hand up to 5 months of infection and from then on A decreased and C increased in their numerical strength (A-C reciprocity). The evolution of A-C reciprocity coincided with the gradual reduction in the parasitaemia in the spleen suggesting that this may contribute to the acquisition of anti-leishmania immunity.

Animals↗
PubMed1994

Quantitative brain FDG/PET studies using dynamic aortic imaging.

Positron emission tomography (PET) measurements of cerebral glucose utilization using 18F-fluorodeoxyglucose (FDG) are a useful tool in the investigation of localized brain function in normal and disease states. A major impediment to the application of FDG/PET in clinical investigation has been the need for arterial blood sampling to quantify cerebral glucose metabolism (CMRGlc). Qualitative studies, though informative in a variety of clinical settings, are of limited value for research applications and do not utilize the inherent quantitative nature of PET. We present a novel PET technique employing a whole-body PET tomograph with abdominal aortic imaging from 0 to 30 min as an alternative to arterial blood sampling to obtain the input function for cerebral metabolic rate calculations. Two or three arterial samples taken during the 10-45 min period were used to scale and extend the blood curve and the brain was imaged from 35-55 min post-injection. We performed 12 studies in which both arterial blood sampling and aortic scans were obtained. We found the correlation of global metabolic rates (GMR) when comparing the two techniques to be extremely high (R2 = 0.99). This suggests that the use of dynamic aortic imaging is less invasive and a viable alternative to arterial blood sampling in quantitative FDG/PET imaging.

Algorithms↗
PubMed1994

Prognostic association of c-erbB-2 oncogene amplification and protein overexpression in human breast cancer using archival tissues. A comparative study.

The prognostic associations of c-erbB-2 gene amplification analysed by dot-blot hybridization and of c-erbB-2 protein overexpression assessed by immunohistochemistry (IHC) were compared in 161 patients with operable breast cancer using formalin fixed paraffin embedded archival tissues. The efficiency of the dot-blot technique to detect c-erbB-2 amplification was first validated by comparing the results of dot-blot with those of Southern blot hybridization in 134 tumour samples and there was an excellent correlation. In the main series of 161 samples, where results of IHC and dot-blot were compared, 35.4% showed c-erbB-2 overexpression and 17.4% showed gene amplification. Tumours showing overexpression of c-erbB-2 protein had a significantly shorter disease-free survival (DSF) and survival(s) compared to tumours showing no overexpression. A multivariate analysis revealed that c-erbB-2 overexpression was independently correlated with poor prognosis. On the other hand, no significant association between c-erbB-2 gene amplification and DFS or S was observed. We conclude that c-erbB-2 protein overexpression assessed by IHC is a superior prognostic indicator in operable breast cancer compared to c-erbB-2 gene amplification analysed by the dot-blot technique.

Blotting, Southern↗