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Biomedical subjects

A K Chakrabarti

Publications and source records attributed to A K Chakrabarti.

At least 19 recordsLinked to original sources
PubMed2000

Novel endogenous inhibitor of sulfur mustard-stimulated protease in cultured human epidermal keratinocytes: possible application in vesicant intervention.

Protease stimulation at the dermal-epidermal junction may be responsible for the skin blistering (vesication) action of sulfur mustard (HD). We have purified a protease to homogeneity from cultured normal human epidermal keratinocytes (NHEK) exposed to 300 microM HD. In this report, we describe the results of our studies on purification and characterization of an endogenous inhibitor of HD-stimulated protease in NHEK. Purification to homogeneity was accomplished by chromatographic separation of the dialyzed Triton X-100-solubilized inhibitor using ion-exchange DEAE-cellulose. Analysis of the purified inhibitor by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed one polypeptide with an apparent molecular mass of 116 kDa. Activity of the inhibitor was screened by incubating different column elute fractions with protease purified from the same cells. Preliminary results showed that the purified inhibitor effectively inhibited the protease isolated from NHEK, whereas other naturally occurring inhibitors, e.g. soybean trypsin-chymotrypsin inhibitors, elafin and aprotinin, were ineffective. Although complete characterization and regulation of this inhibitor remain to be resolved, this purification may be a major step towards developing a specific protective measure against HD-induced toxicity.

Blister↗
PubMed2000

Development and evaluation of a phage typing scheme for Vibrio cholerae O139.

The scenario of cholera that existed previously changed in 1992 and 1993 with the emergence of toxigenic Vibrio cholerae O139 in India. The genesis of the new serogroup formed the impetus to search for O139 phages in and around the country. A total of five newly isolated phages lytic to V. cholerae O139 strains were used for the development of this phage typing scheme. These phages differed from each other and also differed from the existing O1 phages in their lytic patterns, morphologies, restriction endonuclease digestion profiles, and immunological criteria. With this scheme, 500 V. cholerae O139 strains were evaluated for their phage types, and almost all strains were found to be typeable. The strains clustered into 10 different phage types, of which type 1 (38.2%) was the dominant type, followed by type 2 (22.4%) and type 3 (18%). Additionally, a comparative study of phage types in 1993 and 1994 versus those from 1996 to 1998 for O139 strains showed a higher percentage of phage type 1 (40.5%), followed by type 3 (18.8%) during the period between 1993 and 1994, whereas phage type 2 (32. 1%) was the next major type during the period from 1996 to 1998. This scheme comprising five newly isolated phages would be another useful tool in the study of the epidemiology of cholera caused by V. cholerae O139.

Bacteriophage Typing↗
PubMed1999

Distribution of phage type of Vibrio cholerae O1 biotype ElTor in Indian scenario (1991-98).

During the period 1991-98, distribution of biotype, serotype and phage type of V. cholerae O1 strains isolated from different parts of the country and referred to the National Institute of Cholera and Enteric Diseases, Calcutta were studied. Of the 8101 strains received, 5613 (69.2%) were subjected to phage typing. All these strains belonged to the biotype ElTor and Ogawa was the predominant serotype (96.08%). The strains were clustered into only two types--types 2 and 4 and around 10 per cent strains remained untypable. However, using the new scheme, all these strains were found to be typable and 8 major types were recognized of which type number 27 was the predominant type (66.12%). The distribution of a common type throughout the country suggests that a particular clone of V. cholerae O1 is probably circulating all over India. A constant monitoring through phage typing is necessary to observe the emergence of any new clone of V. cholerae O1 in India.

Bacterial Typing Techniques↗
PubMed1998

Purification and characterization of protease activated by sulfur mustard in normal human epidermal keratinocytes.

A membrane-bound protease induced by sulfur mustard in cultured normal human epidermal keratinocytes (NHEK) was purified and partially characterized. Maximum enzyme stimulation occurred at 16 hr after normal human epidermal keratinocytes were exposed to 300 microM sulfur mustard. Purification to homogeneity of the protease was accomplished by Triton X-100 solubilization, ultracentrifugation, and dialysis, followed by ion-exchange chromatography through DEAE-cellulose and finally hydrophobic column chromatography through phenyl Sepharose. Analysis of the purified enzyme by SDS-PAGE revealed a single polypeptide at the 80 kDa region. Further investigation of biochemical properties showed that a synthetic serine-specific Chromozym TRY peptide and the physiological protein laminin were good substrates for this enzyme. Moreover, this enzyme was inhibited mostly by the serine-protease inhibitors leupeptin and di-isopropyl fluorophosphate and not by the cysteine protease inhibitor E-64 or the metalloprotease inhibitor 1,10-phenanthroline (Component H, CH), indicating the serine protease nature of this enzyme. This enzyme had a pH optimum in the range of 7.0 to 8.0. Amino acid sequencing of the purified enzyme revealed that this enzyme belongs to the endopeptidase family (serine protease), and is homologous with a mammalian-type bacterial serine endopeptidase that can preferentially cleave K-X, including K-P. These results suggest that serine-protease stimulation may be one of the mechanisms of mustard-induced skin blister formation, and that some specific serine-protease inhibitors may be useful for the treatment of this sulfur mustard toxicity.

Amino Acid Sequence↗
PubMed1997

Immunolocalization of cytoplasmic and myelin mcalpain in transfected Schwann cells: II. Effect of withdrawal of growth factors.

We have examined the reversal of the regulatory effect of growth factors on calpain/calpastatin activity in transfected Schwann cells (tSc) after their subsequent withdrawal. Removal of nerve growth factor (NGF) or cyclic adenosine monophosphate (cAMP) from tSc resulted in a smaller loss of mu calpain (37%) and mcalpain (36.5 %) activity compared to treated cells from which the growth factors were not withdrawn. The mu calpain activity increased approximately 12% following withdrawal of acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) at 24 hr, while the increased mcalpain activity was more than 30-40% compared with that of cells that were continuously treated. The activity of both isoforms returned to their normal levels (untreated) at 48-72 hr following withdrawal of various growth factors, including NGF, cAMP, aFGF, bFGF, platelet-derived growth factor aa (PDGFaa), and PDGFbb. The inhibitory activity of calpastatin was greater than control following withdrawal of NGF, cAMP, PDGFaa, or PDGFbb at 24 hr and this inhibitory activity was less with treatment by aFGF and bFGF. The control activity was restored at 48 hr following withdrawal of these factors. The intensity of the cytoplasmic calpain immunoreactivity was significantly decreased in the nuclear and non-nuclear regions of the cytoplasm, respectively, following withdrawal of cAMP at 144 hr. Removal of bFGF from the medium resulted in an increase of cytoplasmic calpain immunoreactivity in the nuclear regions and cytoplasm, while there was dramatic loss of myelin calpain immunoreactivity from both the nuclear region and cytoplasm. The changes in calpain activity and immunoreactivity in tSc following withdrawal of growth factors suggest that release of calpain from membrane to cytosol may be regulated by these factors.

Animals↗
PubMed1997

Immunolocalization of cytoplasmic and myelin mcalpain in transfected Schwann cells: I. Effect of treatment with growth factors.

We have examined the effect of growth factors on the activity and localization of calpain in transfected Schwann cells (tSc). Axolemma-enriched fraction, cAMP, or NGF showed concentration-dependent inhibition of both mu calpain and mcalpain activity. In contrast, both acidic FGF and basic FGF stimulated mu calpain (37%) and mcalpain (58%) of tSC while PDGF-aa and PDGF-bb inhibited both calpain activities. The inhibitor (calpastatin) activity was approximately 90% following treatment with NGF, cAMP, PDGF-aa, and PDGF-bb compared to control while this activity was 40% with FGF-treated samples. Immunofluorescence studies indicated localization of cytoplasmic calpain in the nuclear region following growth factor treatment in the cytoplasm. Growth factor treatment caused a decrease in the intensity of calpain immunoreactivity. Treatment with cAMP or FGF resulted in strong immunoreactivity of mcalpain in the nuclear region and cytoplasm compared to untreated. The growth factors did not cause translocation of calpain to the outer surface of the cell membrane. The increased immunoreactivity seen with myelin calpain antibody was greater than cytosolic antibody. The changes seen in calpain activity and immunoreactivity following treatment with growth factors suggest that these factors may regulate calpain-calpastatin expression and translocation to the membrane for interaction with lipids for enzyme activation.

Animals↗
PubMed1997

Isolation of Vibrio cholerae 0139 phages to develop a phage typing scheme.

Five V. cholerae 0139 phages isolated from different parts of India have been used for phage typing study. A strain isolated from Nagpur city (NPR-4) was used as the host for phage propagation. All but one of the 260 strains of V. cholerae 0139 were found to be typeable and could be clustered into 8 distinct phage types as revealed by lytic patterns. Phage type 1 was the predominant type (61.15%) followed by type 2 (18.46%). The strains isolated from Madras exhibited 7 out of 8 phage types. These newly isolated phages could be adopted for phage typing of V. cholerae 0139 strains as an epidemiological tool.

Bacteriophage Typing↗
PubMed1997

Comparison between the multiplex PCR, sensitivity to biotype specific phages & polymyxin B for biotyping of Vibrio cholerae O1.

A total of 196 Vibrio cholerae O1 strains isolated between 1970 and 1996 were biotyped by multiplex PCR, susceptibility to polymyxin B and sensitivity to biotype specific phages. We modified the multiplex PCR by increasing the primer concentration of tcpA to improve the results. Comparison of the results of modified multiplex PCR and sensitivity to biotype specific phages and to polymyxin B showed that multiplex PCR was as efficient as phage typing for biotyping of V. cholerae O1. All the strains of V. cholerae O1 could be accurately distinguished based on polymyxin B sensitivity. Thus our results show that susceptibility of strains of V. cholerae O1 to polymyxin B is the easiest method to biotype V. cholerae O1 and is feasible in most laboratories when compared with multiplex PCR and sensitivity to biotype specific phages.

Anti-Bacterial Agents↗
PubMed1996

Regulation of brain m calpain Ca2+ sensitivity by mixtures of membrane lipids: activation at intracellular Ca2+ level.

Combinations of certain phospholipids and gangliosides increase the specific activity of m calpain and can activate m calpain at 1 to 10 microM Ca2+ concentration. However, this level of calcium is still greater than the normal intracellular calcium level. We have used combinations of lipids to demonstrate the m calpain activity at the physiological Ca2+ level. GD1a (100 microM) and cerebroside (Cerb; 750 microM; 1:7.5) mixture was the most effective. At 0.5 microM to 1.0 microM Ca2+ concentrations, 15-20% of the maximal activity was detected for the purified myelin and cytosolic m calpains. Other combinations were GD1a (100 microM), GM1 (100 microM), Cerb (750 microM), sulfatide (Sulf; 750 microM), and phosphatidylinositol (PI; 300 microM) at a ratio of 1:1: 7.5:7.5:3, respectively. These lipid mixtures stimulated calpain activity at three- to tenfold less calcium concentration than control. The other mixtures, including GD1a:Sulf (1:9) > GD1a:PI (1:4) > PI:Sulf (1:5) > Cerb:Sulf (1:5) and PI:Cerb (1:2.5), also stimulated calpain activity at 1.0 microM Ca2+ concentration. Triton X-100, oxidized glutathione (GSSG), and calpain activator did not affect the Ca2+ requirement. Liposomes containing GD1a, Cerb, and m calpain also showed recognizable calpain activity at a significantly reduced Ca2+ concentration (0.4 microM), confirming the glycolipid-mediated enzyme modulation. These studies indicate that specific lipid mixtures can stimulate m calpain activity at an intracellular level of Ca2+.

Animals↗
PubMed1996

Biological activity & interaction of Vibrio cholerae bacteriophages in rabbit ileal loop.

A set of ten V. cholerae EITor phages is in routine use for phage typing of V. cholerae O1 biotype EITor strains. These phages were used in rabbit ileal loop experiment to investigate whether these phages have any prophylactic value as regards their lytic capability on V. cholerae strains. The phages were found to have no prophylactic use as they were unable to lyse the standard bacterial strain V. cholerae MAK 757.

Animals↗
PubMed1995

Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain.

Blood↗
PubMed1993

Calcium-activated neutral proteinase (calpain) in rat brain during development: compartmentation and role in myelination.

The activity of both forms (microM and mM Ca(2+)-sensitive) of calcium-activated neutral proteinase (calpain) was determined in developing rat brain. Triton X-100 did not affect mcalpain activity at the earlier ages (1-5 days postpartum) whereas mcalpain activity significantly increased at 16 days and older. The mcalpain activity in brain was negligible at earlier ages (1-7 days) and the peak activity occurred between 16 and 30 days after birth. The peak activity of mcalpain in myelin was found between 16 and 30 days of age and myelin from rats older than 30 days contained 40-50% of the brain mcalpain activity. In contrast, 70-80% of the brain mcalpain activity was in cytosol at younger ages (1-10 days) and decreased to 30% with increasing age (90 days). On the other hand, mu calpain was found mainly (65-75%) associated with a membrane fraction (microsomes) before 10 days and the majority of the activity was found in cytosol (68%) between 16 and 30 days. Immunoblot studies revealed mcalpain in both myelin and cytosol from developing rat brain. These results indicate that mcalpain is present in myelin and suggest that it may be involved in the formation of myelin sheath.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1993

Malignant orbital tumours: observation in north Bengal.

Sixty-one cases of malignant orbital tumours were analysed retrospectively in relation to the incidence, age, sex, race, anatomical site of origin of the tumours and their histological types in North Bengal. Retinoblastoma is the commonest type (65%) followed by adenocarcinoma (10%), squamous cell and basal cell carcinoma (8.8% each). Other tumours were rarely encountered.

Adenocarcinoma↗
PubMed1992

Calcium-activated neutral proteinase (calpain) activity in C6 cell line: compartmentation of mu and m calpain.

Calcium-activated neutral proteinase (calpain) activity was determined, including in cytosol and membrane fractions, in rat glioma C6 cell line. The mu and m forms of calpain were separated by DEAE and phenylsepharose column chromatography and with removal of the endogenous inhibitor calpastatin. C6 cells contained more mcalpain than the mu isoform. More than 70% of mcalpain activity was membrane-associated and 20% was cytosolic. Isolated plasma membrane also contained 69% of the mcalpain activity. In contrast, approximately 80% of mucalpain activity was cytosolic and 16% was membranous. Half-maximal activity for mu and mcalpain was obtained at 1 microM and 0.2 mM CaCl2, respectively. Trypsin dissociation of cells reduced activity. Triton X-100 stimulated mcalpain activity of the whole homogenate and the membrane pellet but not of the cytosol. Activity of the myelin marker enzyme adenosine 2'3'-cyclic nucleotide 3'-phosphohydrolase (CNPase), was also found in C6 cells. The identification of calpain and CNPase in C6 cells is in keeping with an interpretation that C6 differentiation resembles, at least in part, that of the myelin-forming oligodendroglial cells.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1992

Effects of detergents on Ca(2+)-activated neural proteinase activity (calpain) in neural and non-neural tissue: a comparative study.

Calcium activated neutral proteinase (mcalpain) activity was determined in brain and other tissue of rat. More than 60% of the brain mcalpain activity was present in the particulate fraction while only 30% was in cytosol. In contrast, particulate fractions of liver, kidney, muscle, and heart contained about 8-12% of tissue mcalpain activity while 88% was present in cytosol. Removal of the endogenous inhibitor calpastatin increased the tissue mcalpain activity severalfold. Triton X-100 and deoxycholate (DOC) stimulated the neural calpain activity by ten-fold while activity in non-neural tissue was unaffected. Incubation with other detergents, e.g. Triton N-57 and thioglucopyranoside, stimulated brain calpain activity five-fold while Brij-35 did not have any effect. Sodiumdodecylsulphate (SDS), on the other hand, inhibited the enzyme activity. Brain contained the lowest calpain activity compared to non-neural tissue. The calpain activity in muscle, kidney and heart was three-fold greater than liver. Immunoblot identification of the enzyme revealed that calpain was predominantly in the particulate fraction and less in cytosol of brain while it was present mainly in cytosol and less in the pellet fractions of non-neural tissue.

Animals↗