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Biomedical subjects

A N Ghosh

Publications and source records attributed to A N Ghosh.

At least 37 records · Page 2Linked to original sources

Sequential changes in gut mucosa of rabbits infected with Vibrio cholerae O139 Bengal: an ultrastructural study.

Adhesion and subsequent colonisation are important events in the infection by Vibrio cholerae O139 Bengal. To determine in details the pathological changes in the gut mucosa, an epidemic strain of O139 Bengal was inoculated in a rabbit ileal loop model. Electron microscopic studies were done at different time intervals after inoculation of the strain to see the histological changes at the ultrastructural level. From 10 hours onwards, cellular invasive processes with presence of bacteria in the lamina propria and other associated inflammatory changes were revealed.

Animals↗

Integration of the DNA of a novel filamentous bacteriophage VSK from Vibrio cholerae 0139 into the host chromosomal DNA.

An unusual filamentous bacteriophage, VSK, containing single-stranded, circular DNA as its genome was isolated from Vibrio cholerae 0139 strains P07 and B04. Unlike other single-stranded DNA phages, VSK can integrate its genome into the chromosome of the host and enter into a lysogenic state. The double-stranded replicative form (RF) of the single-stranded phage DNA was isolated. A restriction map of the VSK RF DNA was constructed using HaeII, AvaII, ClaI and XbaI. By Southern blot analysis of the chromosomal DNA of the lysogen using labeled phage DNA as probe, the attachment site (attP) on the viral genome was also identified.

Bacteriophages↗

Purification of a cell-associated hemagglutinin from Shigella dysenteriae type 1.

A cell-associated hemagglutinin (HA) was isolated and purified from a clinical isolate of Shigella dysenteriae type 1 by affinity chromatography on a fetuin-agarose column. The purified hemagglutinin produced a single-stained protein band of around 66 kDa in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). In an immunodiffusion test, HA-antisera produced a single precipitin band against the purified HA without exhibiting any reactivity towards lipopolysaccharide (LPS) of S. dysenteriae type 1 strain. Inhibition of the hemagglutination by the glycoproteins fetuin, asialofetuin and a sugar derivative N-acetyl-neuraminic acid but not by simple sugars, suggested the specific requirement of complex carbohydrate for binding. Electron micrographs of the purified HA revealed a morphology typical of globular protein.

Cells↗

Phage specific for Vibrio cholerae O139 Bengal.

From the stool of a Vibrio cholerae O139 Bengal-infected patient, a phage that specifically lysed capsulated V. cholerae O139 strains only was isolated. The phage is useful for the confirmatory diagnosis of V. cholerae O139 infection and for the differentiation of variants that lack the capsule.

Bacteriophages↗

Electron microscopic studies on Vibrio cholerae O139.

We conducted studies to investigate the surface architecture of V. cholerae O139 using electron microscopy and compared it with O1 and other serogroups of V. cholerae. The bacterium is comma-shaped and has a single polar flagellum and morphologically resembles the classical and E1Tor biotypes of V. cholerae O1. High power electron microscopy showed a few pili, 5 to 7 nm in diameter, and 2 to 3 in number per bacterium. The presence of a capsule on electron microscopy of ultrathin sections of V. cholerae O139 treated with polycationic ferritin clearly distinguished the O139 serogroup from the O1 serogroup which are not encapsulated. Immunoelectron microscopy further revealed that an anti-O139 monoclonal antibody of the IgG2a isotype bound specifically only to an O139 strain but not to any other serogroup of V. cholerae indicating that O139 has unique epitopes not found in other serogroups of V. cholerae.

Bacterial Capsules↗

Studies on adhesion, haemagglutination and other biological properties of Vibrio cholerae O139.

The adhesive capabilities of eight Vibrio cholerae O139 epidemic strains to isolated rabbit intestinal epithelial cells (RIEC) were observed to be high similar to those observed with a Vibrio cholerae O1 strain isolated from patients. Toxin production by the strains, measured by accumulation of fluid in rabbit ileal loop model, was high and the toxin was lethal as the animal expired within 6 h. Culture filtrates of the strains exhibited the presence of vascular permeability factor which produce induration and necrosis in the adult rabbit and guinea pig skin. All the strains showed high to moderate haemagglutinin titres against chicken erythrocytes and produced El Tor-like haemolysin. SDS-PAGE of the outer membrane preparation of the strains showed the presence of major protein component at 38 kDa region. The lethality of the toxin, high adhesive activity, shifting of the major outer membrane protein band and production of thermolabile haemolysin on Wagatsuma agar were the major variations of these epidemic strains from V. cholerae O1 and V. cholerae non-O1 strains isolated previously.

Animals↗

Colonization ability & intestinal pathology of rabbits orally fed with Vibrio cholerae O139 Bengal.

The colonization ability of a representative epidemic strain of V. cholerae O139 Bengal was studied in the oral rabbit colonization model and the nature of colonization in the ileal and jejunal tissues was examined ultrastructurally. Results of the colonization study and ileal loop assay indicated that the strain proliferates and colonizes the small intestine of the rabbit mucosal surface. Further, the electronmicroscopic study revealed the disruptive effect of the strain on the apical membrane of the epithelial cells. The results of this study suggested that apart from colonization, invasion of the bacteria was important in the pathogenesis of V. cholerae O139 mediated infections.

Animals↗

Purification, pore-forming ability, and antigenic relatedness of the major outer membrane protein of Shigella dysenteriae type 1.

The major outer membrane protein (MOMP), the most abundant outer membrane protein, was purified to homogeneity from Shigella dysenteriae type 1. The purification method involved selective extraction of MOMP with sodium dodecyl sulfate in the presence of 0.4 M sodium chloride followed by size exclusion chromatography with Sephacryl S-200 HR. MOMP was found to form hydrophilic diffusion pores by incorporation into artificial liposome vesicles composed of egg yolk phosphatidylcholine and dicetylphosphate, indicating that MOMP of S. dysenteriae type 1 exhibited significant porin activity. However, the liposomes containing heat-denatured MOMP were barely active. The molecular weight of MOMP found by size exclusion chromatography was 130,000, and in sodium dodecyl sulfate-10% polyacrylamide gel it moved as an oligomer of 78,000 molecular weight. Upon boiling, fully dissociated monomers of 38,000 molecular weight were seen for S. dysenteriae type 1. However, among the four Shigella spp., the monomeric MOMP generated upon boiling ranged from 38,000 to 35,000 in molecular weight. Antibody raised in BALB/c mice immunized with MOMP of S. dysenteriae type 1 reacted strongly with purified MOMP of S. dysenteriae type 1 in an enzyme-linked immunosorbent assay (ELISA). The antibody reacted with whole-cell preparations of S. dysenteriae type 1 in an ELISA, suggesting that MOMP possessed surface components. Moreover, MOMP could be visualized on the bacterial surface by immunoelectron microscopy with anti-MOMP antibody. S. dysenteriae type 1 MOMP-specific immunoglobulin eluted from MOMP bound to a nitrocellulose membrane was found to cross-react with MOMP preparations of S. flexneri, S. boydii, and S. sonnei, indicating that MOMPs were antigenically related among Shigella species. The strong immunogenicity, surface exposure, and antigenic relatedness make MOMP of Shigella species an immunologically significant macromolecule for study.

Animals↗

Ultrastructural changes in HeLa cells associated with enteroadherent Escherichia coli isolated from infants with diarrhoea in Calcutta.

In the present study, we compared two enteroadherent Escherichia coli (EAEC) not belonging to the classical enteropathogenic O serotypes with respect to their attachment to and invasion of HeLa cells. Depending on the pattern of adherence to HeLa cells, one of the strains showed localized adherence (LA), and the other showed diffuse adherence (DA). Electronmicroscopic study showed that LA-EAEC produced the intimate attaching and effacing lesions and intracellular penetration in cultured HeLa cells. In contrast, DA-EAEC exhibited fimbrially-mediated adhesion to HeLa cells. Both LA and DA possessed morphologically distinct fimbriae. LA-EAEC expressed rod-like fimbriae, whereas fibrillar fimbriae were observed in DA strain. Ultrastructural study showed that the mechanisms of invasion by both the strains were different. LA-EAEC strains invaded the cells after pedestal formation and were enclosed in membrane-bound vacuoles. In contrast, DA-EAEC showed no membrane dissolution and pedestal formation during internalization of HeLa cells. A carefully controlled study will be necessary to establish the pathogenic role of DA-EAEC in diarrhoea.

Bacterial Adhesion↗

Biophysical characterization of Vibrio El Tor typing phage e5.

Vibrio cholerae typing phage e5, which can lyse only the El Tor strains of V. cholerae, was characterized. The phage had a polyhedral head 51 nm in diameter and a short tail 13 nm in length. It contained 13 structural polypeptides, with the molecular mass of the major component being 50 kDa. Phage chromosome comprised a 38.5-kb linear double-stranded DNA molecule with unique termini, as determined by restriction fragment analysis and electron microscopy, and had a G+C content of 35.5%. A physical map was constructed with the restriction endonucleases HaeII and HpaII. Adsorption of the phage to its host followed a biphasic kinetics and its intracellular growth was characterized by a latent period of 15 min and a burst size of 100 particles per infected cell. The phage was found to be moderately thermotolerant.

Bacteriophages↗

Vibriophage D10 contains non-permuted DNA with cohesive ends.

Phage D10, a Vibrio cholerae O-1 El Tor group X phage, is one of the five newly isolated phages used in the phage typing scheme developed for V. cholerae O-1 biotype El Tor and belongs to the Myoviridae family. From electron microscopic studies it is shown that phage D10 has a DNA genome of 32 +/- 0.2 kb. This is the first report where it has been shown by the construction of a partial denaturation map that this vibriophage genome is nonpermuted and has cohesive ends. The location of the ends of the DNA in the phage head has also been inferred.

DNA, Viral↗

New phage typing scheme for Vibrio cholerae O1 biotype El Tor strains.

The conventional phage typing scheme proposed by S. Basu and S. Mukerjee (Experientia 24:299-300, 1968) has been used routinely for identification of the strains at the Vibrio Phage Reference Laboratory since 1968. However, because of limitations of this scheme, a new phage typing scheme using five newly isolated phages was incorporated into the conventional scheme. A different definition of routine test dilution (almost confluent lysis) was found to be more useful than the one previously used (confluent lysis). The 1,000 strains tested could be clustered into 27 types with the five new phages. With the new scheme of 10 phages (5 new phages and 5 phages of Basu and Mukerjee), the 1,000 strains could be grouped into 146 types. The new phages were different from each other and also from those of Basu and Mukerjee, as revealed by lytic pattern, electron microscopy, restriction endonuclease digestion, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and antiphage antiserum studies. With the new typing scheme, 99.6% of the strains were typeable. Phage type 115 was the most common and includes 119 (11.9%) of the 1,000 strains tested. Next most common were phage types 142 (9.4%), 143 (7.0%), 104 and 116 (both 5.4%), 3 (5.3%), 5 (4.1%), 4 (3.9%), 24 (2.1%), and 100 (1.7%). The larger number of types would be useful for further classification of the strains for epidemiological purposes. This newly developed scheme is highly applicable to, and could be widely adopted for, phage typing of Vibrio cholerae O1 biotype El Tor strains.

Antibodies, Viral↗

N-acetyl-D-glucosamine-specific lectin purified from Vibrio cholerae 01.

An N-acetyl-D-glucosamine-specific cell associated hemagglutinin (HA) was isolated and purified from a strain of Vibrio cholerae 01 by chitin affinity chromatography followed by separation on Bio Gel P-150. A single stained protein band of 47 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was observed with the purified HA. HA-antisera produced a single precipitin band against the purified HA in an immunodiffusion test without exhibiting any reactivity towards purified lipopolysaccharide (LPS). Purified HA, used as solid-phase antigen in an enzyme-linked immunosorbent assay (ELISA), reacted strongly with HA-antisera but cross-reacted negligibly with antisera raised against purified LPS. Hemagglutinating activity of the purified HA was highly sensitive to N-acetyl-D-glucosamine. The immunogold-labelling method using HA-antisera confirmed the location of the HA on the surface of the bacterial cells. The HA-antisera reacted with a protein component of the homologous outer membrane preparation. A significant inhibition was observed in the adhesive capability of the V. cholerae 01 strain to isolated rabbit intestinal epithelial cells (RIEC) in vitro when the later were pre-treated with the purified HA.

Acetylglucosamine↗

Chlorpromazine induced aggregation of normal human hemoglobin. Electron microscopic evidence.

Normal human hemoglobin exceeding a certain minimum concentration (called critical aggregation concentration) undergoes aggregation in presence of the psychotherapeutic drug chlorpromazine (CPZ). The critical aggregation concentration decreases with the increase of CPZ concentration. Electron micrographs of CPZ-treated hemoglobin clearly indicate that the aggregates of hemoglobin are in filamentous form of average width 75 +/- 8 A. A possible mechanism for such aggregation has been discussed.

Chlorpromazine↗

A 4-kilobase congo red binding plasmid DNA fragment of Shigella dysenteriae 1 suppresses the growth and cell differentiation in Escherichia coli.

A 4-kilobase congo red binding plasmid DNA fragment of pCAT 120 of Shigella dysenteriae 1 was transferred to an Escherichia coli K12 strain by transformation. Transformants were unable to grow in any liquid broth medium. Electron microscopic studies revealed that the transformants grown on tryptic soy agar were associated in clusters after cell division. Normal cell separation among the transformants in comparison with recipient E. coli K12 was only observed when the growth medium was supplemented with sterile culture filtrate of the recipient strain. An unknown factor(s) required for cell separation located on the chromosome was suppressed by a 4-kb congo red binding plasmid DNA (pCAT 120) fragment of S. dysenteriae 1.

Cell Division↗

Electrophoretic mobility and immunoblot analysis of the outer membrane proteins of Aeromonas hydrophila, A. sobria and A. caviae.

The outer membrane profiles of three species of the genus Aeromonas were examined by means of SDS-PAGE and immunoblotting to identify species-specific polypeptides and antigens which could presumably be applied to differentiate Aeromonas spp. at the species or subspecies level. Profiles on an 11% discontinuous SDS-PAGE showed common band sharing at the 52 kD position. Species-specific bands for the three strains could also be detected. Immunoblots using heterologous LPS-adsorbed polyclonal antisera revealed demarcated common and uncommon antigens within the three species. Outer membrane preparations were immunoblotted against whole cell polyclonal antisera. The previously documented host pathogenicity of A. sobria correlated well with the immunoblots which showed antigenicity, especially due to the LPS, when compared with the other two species.

Aeromonas↗

Purification and characterization of a novel hemagglutinin from Vibrio cholerae.

A lectin with strong hemagglutinating activity toward erythrocytes of several animal species was isolated from an 18-h culture supernatant of a diarrheagenic strain, V2, of non-O1 Vibrio cholerae. The hemagglutinin (HA) was purified free of lipopolysaccharide by salt fractionation followed by gel filtration, hydrophobic interaction chromatography, and, finally, gel filtration in the presence of urea and deoxycholate. The purification procedure resulted in an HA preparation with 80-fold enhancement of specific activity. The HA consisted of noncovalently bound subunits of Mr 62,000 and behaved essentially as a single component with pI 6.0. Nonpolar and acidic amino acids contributed 46 and 24%, respectively, to the total amino acid residues. Electron micrographs of the HA showed it to consist of large, nonstoichiometric aggregates' of disklike molecules of 10-nm diameter. Inhibition of the HA by the glycoproteins fetuin, asialofetuin, and mucin, but not by ovalbumin and simple sugars, suggested the specific requirement of complex carbohydrates for binding. Rabbit antisera to the purified HA inhibited the hemagglutinating activities of the crude cell-free HA preparations, but not cell-associated HA activities of the parent (V2) or of other O1 and non-O1 V. cholerae strains. This suggested that the released and cell-associated HA activities were mediated by antigenically distinct components. Immunoblotting experiments showed that the antisera recognized a polypeptide component of Mr 62,000 in the cell envelope preparations of the parent and several other V. cholerae O1 and non-O1 strains. These data suggested that the HA was a nonfimbrial lectin of somatic origin with no protease activity and was apparently distinct from V. cholerae HAs described so far.

Amino Acids↗