Biomedical subjects

S Chakraborty

Publications and source records attributed to S Chakraborty.

At least 19 recordsLinked to original sources

PubMed2002

Evaluation of chemical constituents and free-radical scavenging activity of Swarnabhasma (gold ash), an ayurvedic drug.

From ancient times, Swarnabhasma (gold ash) has been used in several clinical manifestations including loss of memory, defective eyesight, infertility, overall body weakness and incidence of early aging. Swarnabhasma has been used by Ayurvedic physicians to treat different diseases like bronchial asthma, rheumatoid arthritis, diabetes mellitus, nervous disorders, etc. In the present investigation, Swarnabhasma was prepared after proper purification and calcination as per Ayurvedic pharmacy which consisted of Realger (As(2)S(2)), Lead oxide (Pb(3)O(4)), Pure gold (Au) and Latex of Calotropis gigantea. Qualitative analyses indicated that Swarnabhasma contained not only gold but also several microelements (Fe, Al, Cu, Zn, Co, Mg, Ca, As, Pb, etc.). Infrared spectroscopy showed that the material was free from any organic compound. The metal content in the bhasma was determined by atomic absorption spectrometry. Acute oral administration of Swarnabhasma showed no mortality in mice (up to 1 ml /20 g b.w. of Swarnabhasma suspension containing 1mg of drug). Chronic administration of Swarnabhasma also showed no toxicity as judged by SGPT, SGOT, serum creatinine and serum urea level and histological studies. In an experimental animal model, chronic Swarnabhasma-treated animals showed significantly increased superoxide dismutase and catalase activity, two enzymes that reduce free radical concentrations in the body.

Animals↗

PubMed2002

Incidence and molecular analysis of Vibrio cholerae associated with cholera outbreak subsequent to the super cyclone in Orissa, India.

An epidemiological study was carried out to find out the aetiological agent for diarrhoeal disorders in the cyclone and flood affected areas of Orissa, India. Rectal swabs collected from 107 hospitalized diarrhoea patients were bacteriologically analysed to isolate and identify the various enteropathogens. Detection of toxic genes among E. coli and V. cholerae was carried out by polymerase chain reaction (PCR) assay. Of the 107 rectal swabs analysed, 72.3% were positive for V. cholerae O1 Ogawa, 7.2% for V. cholerae O139, 1.2% for E. coli (EAggEC) and 1.2% for Shigella flexneri type 6. Using multiplex PCR assay it was found that all V. cholerae isolates were ctxA positive and El Tor biotype. Strains of V. cholerae O1 were observed to be resistant to nalidixic acid, furazolidone, streptomycin, co-trimoxazole and ampicillin. Except for nalidixic acid, the resistance pattern for O139 was identical to that of O1 strains. Representative strains of V. cholerae were further characterized by randomly amplified polymorphic DNA (RAPD) analysis and ribotyping. Both O1 and O139 V. cholerae strains exhibited the R3 pattern of ribotype and belonged to a similar pattern of RAPD compared with that of Calcutta strains. Early bacteriological and epidemiological investigations have revealed the dominance of V. cholerae O1 among the hospitalized patients in cyclone affected areas of Orissa. Drinking water scarcity and poor sanitation were thought to be responsible for these diarrhoeal outbreaks. Timely reporting and implementation of appropriate control measures could contain a vital epidemic in this area.

Cholera↗

PubMed2002

177Lu labelled polyaminophosphonates as potential agents for bone pain palliation.

Polyphosphonate ligands labelled with radioisotopes decaying by moderate energy beta emission have shown utility as palliative agents for painful bone metastasis. 177Lu (T(1/2)=6.71 d, Ebetamax=497 keV) has radionuclidic properties suitable for use in palliative therapy of bone metastasis. 177Lu was produced at a high specific activity and excellent radionuclidic purity by thermal neutron bombardment of a target prepared from natural Lu. Three polyaminomethylene phosphonate ligands, abbreviated as EDTMP, DTPMP and TTHMP, were synthesized and radiolabelled with 177Lu. Complexation parameters were optimized to achieve maximum yields (97-99.5%). All the complexes were found to retain their stability at room temperature even 14 days after preparation. Biodistribution studies of the complexes were carried out in Wistar rats. All the complexes showed significant bone uptake (6-6.5%/g in tibia at 3 h post-injection (p.i.)) with rapid clearance from blood and minimum uptake in soft tissues. These studies reveal that 177Lu complexes with the synthesized ligands have a potential use in palliative treatment of painful bone metastasis.

Animals↗

PubMed2001

In vitro dorsal root ganglia and human prostate cell line interaction: redefining perineural invasion in prostate cancer.

BACKGROUND: Little is understood regarding mechanisms of perineural invasion in prostate cancer progression. We present a novel model system and data that indicate perineural invasion is an active, specific, and reciprocal interaction between nerves and prostate cancer cells. METHODS: Mouse dorsal root ganglia (DRG) and human prostate cancer cells (Du-145, LNCaP, PC3) and stromal cells (HTS-40F) were co-cultured in Matrigel matrix. Control cultures consisted of prostate cancer and stromal cells only and DRG only. Neurite outgrowth, cell colony growth, neurite-colony contact, and retrograde extension were quantitated with dark phase microscopy and image analysis (Optimas 6.1). RESULTS: Directional outgrowth of neurites was observed projecting into DU-145 colonies within 24 hr of co-culture. Cultures with the greatest number of DU-145 cells recruited significantly more neurites and established contact earlier, indicating this process was cell-seeding density dependent. Once neurite/DU-145 cell contact was established neurite growth diminished, suggesting an active neurite recruitment by DU-145 cells. Subsequent to neurite contact, DU-145 cells migrated along neurites in a retrograde fashion into the nerve/ganglion of origin (retrograde extension) establishing perineural invasion. In addition to perineural invasion, DU-145 colony growth was elevated in DRG co-cultures relative to DU-145-only control cell cultures. Similarly, the degree of neurite outgrowth was elevated in DRG-cell co-cultures relative to DRG-only control cultures. The same observations were made with LNCaP and PC3 cells, but interactions between stromal cells and nerves were not found. CONCLUSIONS: This study shows the utility of the prostate cancer/DRG in vitro system to study specific mechanism of prostate cancer cell-nerve interaction. Moreover, these data suggest that perineural invasion mechanisms involve active and reciprocal interactions between carcinoma cells and adjacent nerve/ganglions in prostate cancer progression.

Animals↗

PubMed2001

Interaction of EVI1 with cAMP-responsive element-binding protein-binding protein (CBP) and p300/CBP-associated factor (P/CAF) results in reversible acetylation of EVI1 and in co-localization in nuclear speckles.

EVI1 is a very complex protein with two domains of zinc fingers and is inappropriately expressed in many types of human myeloid leukemias. Using reporter gene assays, several investigators showed that EVI1 is a transcription repressor, and recently it was shown that EVI1 interacts with the co-repressor carboxyl-terminal binding protein 1 (CtBP1). Earlier, we showed that the inappropriate expression of EVI1 in murine hematopoietic precursor cells leads to their abnormal differentiation and to increased proliferation. Using biochemical assays, we have identified two groups of transcription co-regulators that associate with EVI1 presumably to regulate gene expression. One group of co-regulators includes the CtBP1 and histone deacetylase. The second group includes the two co-activators cAMP-responsive element-binding protein-binding protein (CBP) and p300/CBP-associated factor (P/CAF), both of which have histone acetyltransferase (HAT) activity. All of these proteins require separate regions of EVI1 for efficient interaction, and they divergently affect the ability of EVI1 to regulate gene transcription in reporter gene assays. Confocal microscopy analysis shows that in the majority of the cells, EVI1 is nuclear and diffused, whereas in about 10% of the cells EVI1 localizes in nuclear speckles. However, in the presence of the added exogenous co-repressors histone deacetylase or CtBP1, all of the nuclei have a diffuse EVI1 staining, and the proteins do not appear to reside together in obvious nuclear structures. In contrast, when CBP or P/CAF are added, defined speckled bodies appear in the nucleus. Analysis of the staining pattern indicates that EVI1 and CBP or EVI1 and P/CAF are contained within these structures. These nuclear structures are not observed when CBP is substituted with a point mutant HAT-inactive CBP with which EVI1 also physically interacts. Finally, we show that the interaction of EVI1 with either CBP or P/CAF leads to acetylation of EVI1. These results suggest that the assembly of EVI1 in nuclear speckles requires the intact HAT activity of the co-activators.

Acetylation↗

PubMed2001

Insight to structural subsite recognition in plant thiol protease-inhibitor complexes : understanding the basis of differential inhibition and the role of water.

BACKGROUND: This work represents an extensive MD simulation / water-dynamics studies on a series of complexes of inhibitors (leupeptin, E-64, E-64-C, ZPACK) and plant cysteine proteases (actinidin, caricain, chymopapain, calotropin DI) of papain family to understand the various interactions, water binding mode, factors influencing it and the structural basis of differential inhibition. RESULTS: The tertiary structure of the enzyme-inhibitor complexes were built by visual interactive modeling and energy minimization followed by dynamic simulation of 120 ps in water environment. DASA study with and without the inhibitor revealed the potential subsite residues involved in inhibition. Though the interaction involving main chain atoms are similar, critical inspection of the complexes reveal significant differences in the side chain interactions in S2-P2 and S3-P3 pairs due to sequence differences in the equivalent positions of respective subsites leading to differential inhibition. CONCLUSION: The key finding of the study is a conserved site of a water molecule near oxyanion hole of the enzyme active site, which is found in all the modeled complexes and in most crystal structures of papain family either native or complexed. Conserved water molecules at the ligand binding sites of these homologous proteins suggest the structural importance of the water, which changes the conventional definition of chemical geometry of inhibitor binding domain, its shape and complimentarity. The water mediated recognition of inhibitor to enzyme subsites (Pn.H2O.Sn) of leupeptin acetyl oxygen to caricain, chymopapain and calotropinDI is an additional information and offer valuable insight to potent inhibitor design.

Amino Acid Chloromethyl Ketones↗

PubMed2001

Old yellow enzyme: reduction of nitrate esters, glycerin trinitrate, and propylene 1,2-dinitrate.

The reaction of the old yellow enzyme and reduced flavins with organic nitrate esters has been studied. Reduced flavins have been found to react readily with glycerin trinitrate (GTN ) (nitroglycerin) and propylene dinitrate, with rate constants at pH 7.0, 25 degrees C of 145 M(-1)s(-1) and 5.8 M(-1)s(-1), respectively. With GTN, the secondary nitrate was removed reductively 6 times faster than the primary nitrate, with liberation of nitrite. With propylene dinitrate, on the other hand, the primary nitrate residue was 3 times more reactive than the secondary residue. In the old yellow enzyme-catalyzed NADPH-dependent reduction of GTN and propylene dinitrate, ping-pong kinetics are displayed, as found for all other substrates of the enzyme. Rapid-reaction studies of mixing reduced enzyme with the nitrate esters show that a reduced enzyme--substrate complex is formed before oxidation of the reduced flavin. The rate constants for these reactions and the apparent K(d) values of the enzyme--substrate complexes have been determined and reveal that the rate-limiting step in catalysis is reduction of the enzyme by NADPH. Analysis of the products reveal that with the enzyme-catalyzed reactions, reduction of the primary nitrate in both GTN and propylene dinitrate is favored by comparison with the free-flavin reactions. This preferential positional reactivity can be rationalized by modeling of the substrates into the known crystal structure of the enzyme. In contrast to the facile reaction of free reduced flavins with GTN, reduced 5-deazaflavins have been found to react some 4--5 orders of magnitude slower. This finding implies that the chemical mechanism of the reaction is one involving radical transfers.

Catalysis↗

PubMed2001

Structure and dynamics of the alpha-lactalbumin molten globule: fluorescence studies using proteins containing a single tryptophan residue.

The fluorescence properties of three variants of alpha-lactalbumin (alpha-LA) containing a single tryptophan residue were investigated under native, molten globule, and unfolded conditions. These proteins have levels of secondary structure and stability similar to those of the wild type. The fluorescence signal in the native state is dominated by that of W104, with the signal of W60 and W118 significantly quenched by the disulfide bonds in their vicinity. In the molten globule state, the magnitude of the fluorescence signal of W60 and W118 increases, due to the loss of rigid, specific side chain packing. In contrast, the magnitude of the signal of W104 decreases in the molten globule state, perhaps due to the protonation of H107 or quenching by D102 or K108. The solvent accessibilities of individual tryptophan residues were investigated by their fluorescence emission maximum and by acrylamide quenching studies. In the native state, the order of solvent accessibility is as follows: W118 > W60 > W104. This order changes to W60 > W104 > W118 in the molten globule state. Remarkably, the solvent accessibility of W118 in the alpha-LA molten globule is lower than that in the native state. The dynamic properties of the three tryptophan residues were examined by time-resolved fluorescence anisotropy decay studies. The overall rotation of the molecule can be observed in both the native and molten globule states. In the molten globule state, there is an increase in the extent of local backbone fluctuations with respect to the native state. However, the fluctuation is not sufficient to result in complete motional averaging. The three tryptophan residues in the native and molten globule states have different degrees of motional freedom, reflecting the folding pattern and dynamic heterogeneity of these states. Taken together, these studies provide new insight into the structure and dynamics of the alpha-LA molten globule, which serves as a prototype for partially folded proteins.

Amino Acid Substitution↗

PubMed2001

Should the cytologic diagnosis of "atypical squamous cells of undetermined significance" be qualified? An assessment including comparison between conventional and liquid-based technologies.

BACKGROUND: The diagnosis of atypical squamous cells of undetermined significance (ASCUS) remains an enigma for the treating physician, because it encompasses both benign, reactive, as well as preneoplastic and possibly neoplastic conditions. To address this problem, The Bethesda System recommends qualifying the ASCUS diagnosis. This study analyzes the difference in the follow-up results between the various groups of patients with an ASCUS diagnosis as outlined by the Criteria Committee: favor premalignant (ASCUS-P), favor reactive (ASCUS-R), and unqualified (ASCUS-C). The outcome, based on follow-up biopsies and/or cytologies, for both the conventional Papanicolaou smear (CS) and liquid-based (LB) methodologies is compared. METHODS: The CS and LB biopsies and/or cytologies included 590 patients and 137 patients, respectively, who had an initial diagnosis of ASCUS. The final outcome after subsequent biopsy and cytology within a 1-year period for each methodology was tabulated. Furthermore, the addition of qualifiers for each diagnosis was tabulated for both cytology and biopsy follow-up and compared between CS and LB technologies. RESULTS: For CS, 29 patients (8.6%) were found to have squamous intraepithelial lesions (SIL) on subsequent cytologic smears, and 176 patients (63.7%) had SIL on biopsy follow-up. For LB, these numbers were 17 patients (19.1%) and 56 patients (65.1%), respectively. Regardless of the qualification used in the initial CS biopsy or cytology, over 90% of the subsequent smears resulted in a benign diagnosis. Biopsy outcomes after CS diagnoses of ASCUS-P, ASCUS-R, and ASCUS-C showed SIL in 80%, 53%, and 66% of patients, respectively. For LB diagnoses, subsequent smears detected SIL in 33% of patients with a diagnosis of ASCUS-P, 22% of patients with a diagnosis of ASCUS-R, and 12% of patients with a diagnosis of ASCUS-C. Biopsy outcomes after LB diagnoses of ASCUS-P, ASCUS-R, and ASCUS-C showed SIL in 68%, 64%, and 66% of patients, respectively. CONCLUSIONS: There appears to be no statistically valid benefit from a clinical point of view in qualifying an ASCUS interpretation by either CS or LB, except for CS evaluation of patients with a diagnosis of ASCUS-P.

Biopsy↗

PubMed2001

Genetic lesions and perturbation of chromatin architecture: a road to cell transformation.

Differential gene expression is a rigorously precise procedure that defines the developmental program of cells, tissues, organs, and of the entire organism. The correct execution of this program requires the participation of multiple and complex groups of regulators. In addition to transcription factors, which are key tools in ontogenesis by providing sequential switch of different genes, the structure of the chromatin is a dominant determinant leading to gene expression. Through the novel and insightful work of several investigators, it appears that the architecture of the chromatin spanning the genes can and does influence the efficiency of RNA transcription, and therefore of gene expression. Several new enzymatic complexes have been identified that reversibly modify the chromatin architecture by methylation, phosphorylation, and acetylation of the nucleosomal core proteins. These enzymes are crucial for the proper balance and maintenance of gene expression, and are often the target of mutations and alterations in human cancer. Here, we review briefly the current models proposing how some of these enzymes normally modify the chromatin structure and how their functional disruption leads to inappropriate gene expression and cell transformation.

Acetylation↗

PubMed2001

Cortical substrates of perceptual stability during eye movements.

We are usually unaware of retinal image motion resulting from our own movement. For instance, during slow-tracking eye movements, the world around us remains perceptually stable despite the retinal image slip induced by the eye movement. This example of perceptual invariance is achieved by subtracting an internal reference signal, reflecting the eye movement, from the retinal motion signal. If the two cancel each other, visual structures, which do not move, will also be perceived as nonmoving. If, however, the reference signal is too small or too large, a false eye-movement-induced motion of the external world will be perceived. We have exploited our ability to manipulate the size of the reference signal in an attempt to reveal the structures in visual cortex, encoding the perception of self-induced visual motion rather than the retinal motion signal. Using EEG and lately also MEG recordings in human subjects and single-unit recordings in monkeys, we have been able to show that our ability to perceive the world as stationary despite eye-movement-induced retinal image slip is based on "late" parts of the cortical hierarchy of motion processing, sparing the early stages up to cortical area MT and, among others, involving cortex at the junction between the parietal and temporal lobes close to the parieto-insular-vestibular cortex. Lesions of this network in humans render the visual system unable to compensate for the visual consequences of eye movements, giving rise to severe dizziness, whenever the eyes move smoothly.

Animals↗

PubMed2001

Substance P immunoreactivity in the enteric nervous system in Rett syndrome.

Rett syndrome is associated with profound mental retardation and motor disability in girls. It has a characteristic clinical phenotype which includes abnormalities of the autonomic nervous system. Feeding impairment and severe constipation are two symptoms of this autonomic dysfunction. Substance P, an important peptide in the autonomic nervous system, is decreased in the cerebrospinal fluid of Rett syndrome. We have demonstrated that substance P immunoreactivity is significantly decreased in Rett syndrome brain-stem and may be related to the autonomic dysfunction. In this study, we have continued the investigation of substance P in the enteric nervous system. We immunohistochemically examined the normal developing bowel in 22 controls (ages, 14 gestational weeks to 31 years) using formalin fixed tissue, with antibodies to substance P, tyrosine hydroxylase and vasoactive intestinal peptide. We compared the immunoreactivity of normal controls with 14 cases of Rett syndrome (ages, 5-41 years) and observed that the expression of substance P, tyrosine hydroxylase and vasoactive intestinal peptide immunoreactivity in the bowel in Rett syndrome was not significantly different from that of controls. This suggests that the feeding impairment and constipation in Rett syndrome relate to dysfunction of the autonomic nervous system originating outside of the bowel, in the brain-stem, as suggested by our previous study.

Adolescent↗

PubMed2001

Potential (166)Ho radiopharmaceuticals for intravascular radiation therapy (IVRT)-I: [(166)Ho] holmium labeled ethylene dicysteine.

The use of beta(-) emitting radionuclides in the control of restenosis in post angioplasty patients is currently under intense investigation at many leading cardiovascular research centers. (32)P coated metallic stents, (192)Ir wire source and balloons filled with an appropriate radionuclide solution such as of (188)Re, attached to catheter are being studied. (166)Ho has comparable radionuclidic properties to that of (188)Re, can be more easily produced and hence is an attractive alternative to (188)Re. Ethylene dicysteine complex of (166)Ho was prepared and its pharmacological behavior studied. Optimum conditions for the preparation of complex with respect to the reaction time, ligand concentration, pH of the reaction mixture as well as reaction temperature were standardized. The stability of the labeled complex at room temperature as well as at 4 degrees C was determined. Biodistribution pattern of the injected complex in Wistar rats was estimated at 10 min, 30 min and 3 h post injection. This study indicated that >90% of the injected (166)Ho-EC complex was excreted in urine within 3 h post injection, with insignificant retention in any major organ. These studies reveal that (166)Ho-EC could be a viable substitute for (188)Re compounds in radioactive liquid-filled balloon IVRT.

Animals↗

PubMed2001

Recognition and stabilization of a unique CPRI--structural motif in cucurbitaceae family trypsin inhibitor peptides: molecular dynamics based homology modeling using the X-ray structure of MCTI-II.

The high resolution crystallographic structure of MCTI-II complexed with beta trypsin (PDB entry 1MCT) was used to model the corresponding structures of the six inhibitor peptides belonging to Cucurbitaceae family (MCTI-I, LA-1, LA-2, CMTI-I, CMTI-III, CMTI-IV). Two model inhibitors, LA-1 and LA-2 were refined by molecular dynamics to estimate the average solution structure. The difference accessible surface area (DASA) study of the inhibitors with and without trypsin revealed the Arginine and other residues of the inhibitors which bind to trypsin. The hydration dynamics study of LA1 and LA2 also confirm the suitability of water molecules at the active Arg site. Moreover, the presence of a unique 3D-structural motif comprises with the four CPRI residues from the amino terminal is thought to be conserved in all the six studied inhibitors, which seems essential for the directional fixation for proper complexation of the Arg (5) residue towards the trypsin S1-binding pocket. The role of the disulphide linkage in the geometrical stabilization of CPRI (Cysteine, Proline, Arginine, Isoleucine) motif has also been envisaged from the comparative higher intra molecular Cys (3) -Cys (20) disulphide dihedral energies.

Amino Acid Motifs↗

PubMed2001

Research strategies for design and development of NSAIDs: clue to balance potency and toxicity of acetanilide compounds.

Despite the fact that many modern drug therapies are based on the concept of enzyme inhibition, inhibition of several enzymes leads to pathological disorders. Clinically used nonsteroidal anti-inflammatory drugs (NSAIDs) bind to the active site of the membrane protein, cyclooxygenase (COX) and inhibit the synthesis of prostaglandins, the mediators for causing inflammation. At the same time, inhibition of hepatic cysteine proteases by some NSAID metabolites like NAPQI is implicated in the pathogenesis of hepatotoxicity. As a part of our efforts to develop new effective NSAIDs, a comprehensive investigation starting from synthesis to the study of the final metabolism of acetanilide group of compound has been envisaged with appropriate feedback from kinetic studies to enhance our knowledge and technical competency to feed the know-how to the medicinal chemist to screen out and design new acetanilide derivatives of high potency and low toxicity. Structure-function relationship based on the interaction of acetanilide with its cognate enzyme, cyclooxygenase has been studied critically with adequate comparison with several other available crystal structures of COX-NSAID complexes. Furthermore, to make the receptor based drug design strategy a novel and comprehensive one, both the mechanism of metabolism of acetanilide and structural basis of inhibition of cysteine proteases by the reactive metabolite (NAPQI) formed by cytochrome P450 oxidation of acetanilide have been incorporated in the study. It is hoped that this synergistic approach and the results obtained from such consorted structural investigation at atomic level may guide to dictate synthetic modification with judicious balance between cyclooxygenase inhibition and hepatic cysteine protease inhibition to enhance the potential of such molecular medicine to relieve inflammation on one hand and low hepatic toxicity on the other.

Acetanilides↗

PubMed2001

Comparison of antibiogram, virulence genes, ribotypes and DNA fingerprints of Vibrio cholerae of matching serogroups isolated from hospitalised diarrhoea cases and from the environment during 1997-1998 in Calcutta, India.

This study identified 17 matching serogroups of Vibrio cholerae belonging to serogroups other than O1 and O139 isolated from human cases and from the environment during a concurrent clinical and environmental study conducted in Calcutta, a cholera endemic area. Isolates within these matching serogroups were compared by various phenotypic and genotypic traits to determine if the environment was the source of the organisms associated with the disease. Clinical strains of V. cholerae were resistant to a greater number of drugs and exhibited multi-drug resistance compared with their environmental counterparts. Except for the presence of the genes for the El Tor haemolysin and the regulatory element ToxR in most of the strains of V. cholerae examined, non-O1, non-O139 V. cholerae strains lacked most of the other known virulence traits associated with toxigenic V. cholerae O1 or O139. Restriction fragment-length polymorphism of virulence-associated genes, ribotypes and DNA fingerprints of strains of matched serogroups showed considerable diversity, although some gene polymorphisms and ribotypes of a few strains of different serogroups were similar. It is concluded that despite sharing the same serogroup, environmental and clinical isolates were genetically heterogeneous and were of different lineages.

Blotting, Southern↗

PubMed2001

Characterization of VPI pathogenicity island and CTXphi prophage in environmental strains of Vibrio cholerae.

Environmental isolates of Vibrio cholerae of eight randomly amplified polymorphic DNA (RAPD) fingerprint types from Calcutta, India, that were unusual in containing toxin-coregulated pilus or cholera toxin genes but not O1 or O139 antigens of epidemic strains were studied by PCR and sequencing to gain insights into V. cholerae evolution. We found that each isolate contained a variant form of the VPI pathogenicity island. Distinguishing features included (i) four new alleles of tcpF (which encodes secreted virulence protein; its exact function is unknown), 20 to 70% divergent (at the protein level) from each other and canonical tcpF; (ii) a new allele of toxT (virulence regulatory gene), 36% divergent (at the protein level) in its 5' half and nearly identical in its 3' half to canonical toxT; (iii) a new tcpA (pilin) gene; and (iv) four variant forms of a regulatory sequence upstream of toxT. Also found were transpositions of an IS903-related element and function-unknown genes to sites in VPI. Cholera toxin (ctx) genes were found in isolates of two RAPD types, in each case embedded in CTXphi-like prophages. Fragments that are inferred to contain only putative repressor, replication, and integration genes were present in two other RAPD types. New possible prophage repressor and replication genes were also identified. Our results show marked genetic diversity in the virulence-associated gene clusters found in some nonepidemic V. cholerae strains, suggest that some of these genes contribute to fitness in nature, and emphasize the potential importance of interstrain gene exchange in the evolution of this species.

Amino Acid Sequence↗

PubMed2001

Concomitant infection of enterotoxigenic Escherichia coli in an outbreak of cholera caused by Vibrio cholerae O1 and O139 in Ahmedabad, India.

In Ahmedabad, a major city in the state of Gujarat, India, an outbreak of acute secretory diarrhea caused by Vibrio cholerae O1 Ogawa El Tor, V. cholerae O139, and multiple serotypes of enterotoxigenic Escherichia coli (ETEC) occurred in January 2000. All of the representative V. cholerae O1 and O139 isolates examined harbored the ctxA gene (encoding the A subunit of cholera toxin) and the El Tor variant of the tcpA gene (encoding toxin-coregulated pilus). ETEC isolates of different serotypes were positive for the elt gene, encoding heat-labile enterotoxin. To further understand the molecular characteristics of the pathogens, representative isolates were examined by ribotyping and pulsed-field gel electrophoresis (PFGE). Ribotyping showed that the isolates of V. cholerae O1 Ogawa exhibited a pattern identical to that of the prevailing clone of O1 in areas where cholera is endemic in India, and all of the O139 isolates were identical to the BII clone of V. cholerae O139. PFGE of the representative O1 Ogawa isolates exhibited an identical pattern, comparable to the H pattern of the new clone of O1 reported in Calcutta, India. PFGE analysis of the V. cholerae O139 isolates showed identical patterns, but these differed from the PFGE patterns of O139 isolates reported during 1992 to 1997 in Calcutta. ETEC isolates showed genetic heterogeneity among isolates belonging to the same serotype, although the identical PFGE pattern was also observed among ETEC isolates of different serotypes. Antibiograms of the isolates were unusual, because all of the O139 isolates were resistant to nalidixic acid. Likewise, all of the E. coli isolates showed resistance to ciprofloxacin, norfloxacin, and nalidixic acid. This is a unique outbreak, and we believe that it is the first in which V. cholerae and ETEC were concomitantly involved.

Anti-Bacterial Agents↗