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Biomedical subjects

R Mitra

Publications and source records attributed to R Mitra.

At least 37 records · Page 2Linked to original sources
PubMed1999

Morphological and cytoskeletal changes caused by non-membrane damaging cytotoxin of Vibrio cholerae on int 407 and HeLa cells.

Vibrio cholerae produces a non-membrane damaging cytotoxin (NMDCY), also known as cell rounding factor, which causes rapid rounding of cultured cells like HeLa, CHO and Vero and reportedly elicits enterotoxic activity in the rabbit ileal loop assay. Pursuing the concept that NMDCY might be an accessory factor contributing to the diarrhea caused by V. cholerae, we investigated the effect of NMDCY on Int 407 (intestinal cell line) and HeLa (non-intestinal cell line) cells using light, fluorescent and electron microscopy to gain insight into the cellular response evoked by NMDCY. Binding assays showed that NMDCY has affinity for both Int 407 and HeLa cells. Changes in the internal organelles and cytoskeletal structures of the cell lines were documented indicating changes in the secretory and metabolic function of the toxin-treated cells. Toxin-treated cells visualized under the electron microscope revealed retraction of cell body, formation of blebs on cell surface, changes in mitochondria having dilated and rarefied matrix and an extensively developed Golgi apparatus, endoplasmic reticulum and lysosomes compared to those in normal cells. Immunofluorescence study showed restructuring of microfilament network represented by actin, filamin and vinculin, as also of the microtubular component, tubulin and the intermediate filament, vimentin. Immunogold study further revealed that the toxin is internalized even within the nucleus. Moreover, a rise in the intracellular calcium level of the NMDCY-treated cells leads us to hypothesize that a cascade of events results in the final impairment of the cell machinery.

Calcium↗
PubMed1998

Characterization of non-membrane-damaging cytotoxin of non-toxigenic Vibrio cholerae O1 and its relevance to disease.

The non-membrane-damaging cytotoxin which causes dramatic cell rounding of cultured HeLa cells was purified to homogeneity from a clinical strain (WO5) of non-toxigenic Vibrio cholerae O1 Inaba belonging to the E1 Tor biotype. The purified protein has a denatured molecular weight of 35 kDa and a native molecular weight of approximately 37 kDa indicating the monomeric nature of the protein. The 15 N-terminal amino acid sequence of non-membrane-damaging cytotoxin showed complete homology to the hemagglutinin protease previously purified and characterized from V. cholerae O1. Purified non-membrane-damaging cytotoxin from V. cholerae O1 was immunologically and biochemically identical to that previously purified from V. cholerae O26. Non-membrane-damaging cytotoxin was found to be enterotoxic in rabbit ileal loop assay inducing accumulation of non-hemorrhagic fluid at 100 micrograms and elicited a concentration dependent increase in short circuit current and tissue conductance of rabbit ileal mucosa mounted on Ussing chambers. A significant serum immunoglobulin G response against non-membrane-damaging cytotoxin was elicited by patients infected with V. cholerae O139 but not with V. cholerae O1. These properties make non-membrane-damaging cytotoxin a potential virulence factor of V. cholerae which should be taken into consideration while making live, attenuated recombinant vaccine strains against cholera.

Animals↗
PubMed1998

Rapid spread of the new clone of Vibrio cholerae O1 biotype El Tor in cholera endemic areas in India.

Using molecular techniques, we investigated whether the clone of Vibrio cholerae O1 biotype El Tor which appeared in Calcutta, India, in 1994 has spread to other cholera endemic areas in the country. The ribotype of 31 of the 33 strains isolated from different parts of India during 1996 and 1997 was identical to the ribotype displayed by the new clone of V. cholerae O1 which emerged in Calcutta in 1994. Likewise, 12 of the 15 strains examined by pulsed-field gel electrophoresis (PFGE) showed identical profile to that exhibited by the new clone of O1. The restriction fragment length polymorphism (RFLP) of CTX genetic element of these strains also matched with the new clone of O1 which emerged after the outbreak of V. cholerae 0139 in Calcutta. However, two strains (AH042 and AH046) isolated from an outbreak in Ahmedabad (western India) showed different CTX RFLP but had the same ribotype and PFGE profile as the new clone, whereas one strain from Goa (G2) showed distinct ribotype and PFGE profile and the CTX RFLP was identical to the O1 strains which prevailed before the genesis of 0139 in Calcutta. The drug resistance pattern of most of the O1 strains examined in this study, except strain G2, was similar to that of the new clone of V. cholerae O1. None of the strains in this study carried plasmids. Molecular studies clearly show that the new expanded drug resistant clone of V. cholerae O1 has spread to all cholera endemic areas in India and also provide evidence for the evolution of new clones of the O1 serogroup.

Cholera↗
PubMed1998

EGF receptor signaling inhibits keratinocyte apoptosis: evidence for mediation by Bcl-XL.

Signaling through the epidermal growth factor receptor (EGFR) has been primarily implicated in the growth of epithelial cells including keratinocytes. However, the mechanism by which EGFR stimulation promotes keratinocyte cell growth is poorly understood. Here we report that human keratinocytes undergo apoptosis when incubated with the blocking EGFR monoclonal antibody 225 IgG, or PD153035, a highly specific EGFR tyrosine kinase inhibitor. Endogenous mRNA and protein levels of Bcl-XL, a member the Bcl-2 family which suppresses apoptosis, were specifically inhibited by EGFR blockade. Furthermore, stimulation of EGFR signaling through two natural ligands, transforming growth factor (TGF)-alpha and epidermal growth factor (EGF), increased the expression of Bcl-XL in quiescent keratinocytes and HaCaT cells. Finally, ectopic expression of Bcl-XL in HaCaT cells increased survival after EGFR blockade when compared to untransfected cells or HaCaT keratinocytes transfected with empty vector. These results suggest that the anti-apoptotic protein Bcl-XL plays an important role in the maintenance of keratinocyte survival in response to EGFR signaling.

Antibodies, Monoclonal↗
PubMed1998

Molecular analysis of non-O1, non-O139 Vibrio cholerae associated with an unusual upsurge in the incidence of cholera-like disease in Calcutta, India.

There was an inexplicable upsurge in the incidence of non-O1, non-O139 Vibrio cholerae among hospitalized patients admitted to the Infectious Diseases Hospital, Calcutta, India, between February and March 1996. Of the 18 strains of V. cholerae isolated during this period, 15 belonged to the non-O1, non-O139 serogroups (4 belonged to O144, 3 belonged to O11, 1 each belonged to O6, O8, O12, O19, O39, and O58, and 2 strains could not be typed), 2 belonged to the O139 serogroup, and 1 belonged to the O1 serogroup. Cell-free culture supernatants of 13 representative non-O1, non-O139 V. cholerae strains evoked a distinct cytotoxic effect on CHO and HeLa cells, and the strains examined produced the nonmembrane-damaging cytotoxin. By several PCR assays, it was determined that none of the non-O1, non-O139 strains were positive for the ctxA, zot, ace, and tcpA genes and for the genes representing the heat-labile toxin, heat-stable toxin, and verotoxin of Escherichia coli and the various variants of these genes. Studies on the clonality of non-O1, non-O139 V. cholerae strains by restriction fragment length polymorphism (RFLP) analysis of rRNA genes and of other genes (hlyA, hlyU, hlx, toxR, and attRS1) and by pulsed-field gel electrophoresis (PFGE) collectively indicate that the upsurge which occurred in February and March 1996 was caused by strains belonging to different clones. Overall, there was an excellent correlation between the results of ribotyping, RFLP analysis of various genes, and PFGE, with strains belonging to a particular serogroup showing nearly identical restriction patterns and PFGE profiles. It is clear from this study that some serogroups of V. cholerae can cause diarrhea by a mechanism quite different from that of toxigenic V. cholerae O1 and O139, and we have proposed the nomenclature of enteropathogenic V. cholerae to include these serogroups.

Bacterial Typing Techniques↗
PubMed1997

Primary adenocarcinoma of the stomach associated with peripheral neurofibromatosis: report of a case.

A 55-year old man with von Recklinghausen's disease was admitted to our hospital for investigation of weight loss, anorexia, and abdominal discomfort. Endoscopy findings revealed impairment in the extendability of the stomach by air, and barium meal studies showed a well-contracted stomach. On exploratory laparotomy, the whole of the stomach was found to be grossly shrunken with thickening of the walls suggesting a "linitis plastica" stomach. Histological findings confirmed stage III poorly differentiated diffuse infiltrative adenocarcinoma of the stomach. This report describes a rare case of "linitis plastica" stomach in a patient with peripheral neurofibromatosis (NF-1) indicating that the NF-1 disorder does not spare an individual from other common neoplastic disorders.

Adenocarcinoma↗
PubMed1997

Characteristics of two major lectins from mungbean (Vigna radiata) seeds.

Two major lectins, MBL-I and MBL-II, were purified from Vigna radiata L. seeds using ion-exchange and gel filtration chromatography techniques. MBL-I was found to be a tetramer with native M.W. of 132 kDa and subunit M.W. of 33 kDa having alpha-galactosidase activity. MBL-II consisted of two monomeric lectins with M.W. of 94 kDa and 89 kDa which were associated mainly with beta-galactosidase activity. Both MBL-I and MBL-II are D-galactose-specific lectins.

Chromatography, Gel↗
PubMed1997

CD6 dependent interactions of T cells and keratinocytes: functional evidence for a second CD6 ligand on gamma-interferon activated keratinocytes.

The CD6 glycoprotein is expressed by T lymphocytes and is hypothesized to interact with one or more ligands expressed on antigen presenting cells (APCs). We show that CD6 mediates binding of the transformed CD4+ T cell line Hut 78 to gamma-interferon activated keratinocytes (KCs). A recombinant CD6-Ig fusion protein has been reported to bind to a CD6 ligand ALCAM, but this is the first demonstration that cell-cell adhesion of human T lymphocytes can be CD6 dependent. The known CD6 ligand ALCAM (CD166) is expressed on cultured KCs but does not appear to mediate KC-Hut 78 binding, suggesting the existence of additional CD6 ligands expressed on KCs. In functional studies using autologous KCs as APCs for tetanus toxoid specific T cell clones, KCs +/- gamma-interferon are unable to stimulate autologous T cells with recall antigen. Therefore interaction of T cell CD6 with CD6 ligands on KCs does not provide sufficient co-stimulation of primed T cells to support responses to nominal antigen.

Activated-Leukocyte Cell Adhesion Molecule↗
PubMed1996

Responsiveness of human T lymphocytes to bacterial superantigens presented by cultured rheumatoid arthritis synoviocytes.

OBJECTIVE: Type B fibroblastic synoviocytes are abundant in inflamed joints of patients with rheumatoid arthritis (RA), and can secrete cytokines and other mediators of inflammation. The aim of this study was to determine whether cell lines derived from RA type B synoviocytes could also serve as accessory cells for T lymphocyte activation. METHODS: Cells from RA synoviocyte lines, with or without preculture in interferon-gamma (IFN gamma), were cultured with purified peripheral blood T cells, in the presence or absence of superantigens or other accessory cell-dependent T cell mitogens. T cell proliferation was measured by thymidine incorporation, and synoviocyte surface markers were analyzed by flow cytometry. RESULTS: RA type B synoviocyte lines were potent accessory cells for T cell responses to bacterial superantigens or lectins, and direct cell-cell contact was required. Preculture in IFN gamma augmented synoviocyte expression of major histocompatibility complex (MHC) class II molecules and of ligands for some T cell costimulatory receptors, but synoviocyte accessory cell function was evident even in the absence of IFN gamma. Blocking studies using monoclonal antibodies supported the notion of a role CD2, CD11a/CD18 and MHC class II molecules in synoviocyte-dependent T cell activation. Monoclonal antibodies against IFN gamma, interleukin-1 beta (IL-1 beta), IL-6, IL-8, and tumor necrosis factor alpha failed to block the T cell proliferative responses, but anti-IL-2 was strongly inhibitory. CONCLUSION: Cultured RA and type B synoviocytes can perform some of the functions of professional antigen-presenting cells. If such cells have similar properties in vivo, they may be important participants in activation of immune responses, in addition to their previously described synthetic and proinflammatory roles. If RA synovial tissue T cells, like normal peripheral blood T cells, can respond to superantigens presented by synoviocytes, this interaction could be important in the pathogenesis of RA.

Antigens, CD↗
PubMed1996

Temporal shifts in traits of Vibrio cholerae strains isolated from hospitalized patients in Calcutta: a 3-year (1993 to 1995) analysis.

This study presents results of a surveillance on cholera conducted with hospitalized patients admitted to the Infectious Diseases Hospital, Calcutta, India, from January 1993 to December 1995. The O139 serogroup of Vibrio cholerae dominated in 1993 but was replaced by O1 as the dominant serogroup in 1994 and 1995. The isolation rate of V. cholerae non-O1 non-O139 did not exceed 4.9% throughout the study period, while the isolation rate of the O139 serogroup in 1994 and 1995 was below 9%. No temporal clustering of any non-O1 non-O139 serogroup was observed. With the exception of 1 strain, none of the 64 strains belonging to the non-O1 non-O139 serogroup hybridized with ctx, zot, and ace gene probes, while 97.3 and 97.7% of the O139 and O1 strains, respectively, hybridized with all the three probes. Multiplex PCR studies revealed that all the O1 strains belonged to the EIT or biotype. There was a progressive increase in the cytotoxic response on CHO and HeLa cells evoked by culture supernatants of strains of V. cholerae non-O1 non-O139 isolated during 1994 and 1995 compared with the response evoked by those isolated in 1993. Dramatic shifts in patterns of resistance to antibiotics between strains of V. cholerae belonging to different serogroups and within strains of a serogroup isolated during different time periods were observed. There was a discernible increase in the incidence of multidrug-resistant strains of V. cholerae O1 isolated in 1994 and 1995 compared with that in 1993. On the basis of the results of this study, we predict the possibility of newer variants of V. cholerae emerging in the future.

Drug Resistance, Multiple↗
PubMed1995

Conformational states governing the rates of spontaneous transition mutations.

We report the results of a theoretical study, combining the results of sequence analysis and integral equation structural methods for nucleic acids in aqueous solutions, on the effects of nearest neighbors on the (T.G) mispair in solution, for 12 nearest neighbor contexts. Attempts have been made to classify the structural and energetic effects of the 5' and 3' neighbors with respect to the observed spontaneous mutation rates in vertebrates. It is found that 5' nearest neighbor is probably the most critical structural factor in facilitating or discouraging mutations. Local conformational states correlate with discrimination of bases to be excised in mispairs. Our study confirms the role of the flexibility of the DNA molecule in governing the rates of spontaneous mutations.

Base Composition↗
PubMed1993

The relationship between mutation rates for the (C-G)-->(T-A) transition and features of T-G mispair structures in different neighbor environments, determined by free energy molecular mechanics.

The results of this theoretical study combining sequence analysis and minimization with integral equation liquid structural methods indicate that the local sequence context of a T-G wobble mismatch influences the local conformation of the helix, and that conformational alterations are correlated with mutational activity. Studies on the mismatch in four different 5' and 3' neighbor contexts indicate that the nature of the 5' base to the thymine of the mispair is probably the single most critical factor in determining the structural features that facilitate or discourage mutations. When cytosine is the 5' neighbor, the helix adopts a mostly BII conformation, whereas a 5' guanine preserves the canonical BI. Structures that vary little from the BI structure on the incorporation of the mismatch have sequences that correspond to lower rates of transition, whereas those with mostly BII conformations, have sequences with high mutation rates. Subtle variations in stacking patterns around the mismatch precipitate a structural Domino-effect, with a variety of changes in conformation. The helix opens at the mismatch with increased roll angle and propeller twist, causing the thymine to migrate into the major groove and the guanine into the minor groove, exposing the heteroatomic groups to the solvent in the major and minor grooves, respectively, and allowing for some unusual hydrogen bonds. These alterations show a tentative correlation with mutation rates, implying that stacking and structure around the mismatch are important features in the discrimination by proofreading activities of canonical W-C and wobble mismatch base pairs during replication-repair. Variations in the C1'-C1' distances, high propeller twists, changes in the electrostatic complementarity leading to unusual hydrogen bonding patterns probably all correlate with detectability.

Base Composition↗
PubMed1993

Retinoic acid stimulates mouse lung development by a mechanism involving epithelial-mesenchymal interaction and regulation of epidermal growth factor receptors.

Retinoic acid (RA) stimulated proliferation of both epithelial and mesenchymal cells in cocultures isolated from developing mouse lungs. There was a corresponding increase in epithelial branching activity in organ culture of embryonic lungs exposed to similar doses of RA. Stimulation was maximal with concentrations of 1 microM and progressively decreased with either lower or higher concentrations. However, when lung cell monocultures of isolated epithelial and mesenchymal cells were exposed to RA, the mitogenic effect was observed only in the mesenchymal population. This suggests that RA may not have a direct mitogenic effect on epithelial cells but rather functions indirectly through the mesenchyme. The cellular response to RA was correlated with an increase in the expression of epidermal growth factor receptor (EGFR). Epidermal growth factor (EGF) also stimulated terminal branch formation in the developing lung. Unlike RA, EGF stimulated proliferation in both epithelial cells and mesenchymal cells in monoculture. In comparison, transforming growth factor-alpha, which also binds to the EGFR, elicited no response. We conclude that RA stimulates cell proliferation and branching activity in the developing mouse lung by a mechanism involving epithelial-mesenchymal interactions. The effect is, in part, produced by stimulation of EGFR expression, with the resulting amplification of the cellular response to EGF or other EGFR ligands. In this process the mesenchyme provides a paracrine support to the epithelium, otherwise unresponsive to RA. Further studies identified the mesenchyme as a major source of EGF in the embryonic lung, suggesting that mesenchymal EGF may represent a paracrine factor involved in the epithelial response to RA.

Animals↗
PubMed1993

Minimization of shaking-induced formation of insoluble aggregates of insulin by cyclodextrins.

Aggregation is known to complicate insulin delivery and the processing and formulation of biotechnology-derived peptide/protein drugs. Shaking-induced formation of insoluble aggregates in bovine insulin and the potential role of cyclodextrins in preventing such aggregation were investigated. Insulin, dissolved in phosphate buffer, pH 7.2, and preserved with 2 mg/ml of phenol was aggregated, in triplicate, by shaking at 450 rpm for 2.5 days on a gyratory shaker. Visible aggregation was quantitated by measuring optical density in the visible range on a spectrophotometer. Solutions were then filtered through a 0.22 mu filter and the amount of insulin remaining in filtrate was determined by HPLC. Aggregation increased at lower concentrations, with solutions turning milky at 0.5 mg/ml; HPLC assay of filtrate indicated a complete loss of insulin. Under the same conditions, except for shaking, control solutions exhibited no insulin loss, excluding absorption as a cause of the insulin loss. The use of cyclodextrins (0.5 mg/ml) to stabilize insulin was investigated. alpha-, beta-, gamma- and hydroxypropyl-beta-cyclodextrin, each at 1.5% level, were used to prevent aggregation. The efficacy of cyclodextrins in preventing aggregation (% insulin aggregated in parentheses), was: hydroxypropyl-beta- (15) approximately beta- (18) > alpha- (54). No protection was observed with gamma-cyclodextrin.

Adsorption↗