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Biomedical subjects

R Mitra

Publications and source records attributed to R Mitra.

At least 19 recordsLinked to original sources
PubMed2006

Brd4 is required for e2-mediated transcriptional activation but not genome partitioning of all papillomaviruses.

Bromodomain protein 4 (Brd4) has been identified as the cellular binding target through which the E2 protein of bovine papillomavirus type 1 links the viral genome to mitotic chromosomes. This tethering ensures retention and efficient partitioning of genomes to daughter cells following cell division. E2 is also a regulator of viral gene expression and a replication factor, in association with the viral E1 protein. In this study, we show that E2 proteins from a wide range of papillomaviruses interact with Brd4, albeit with variations in efficiency. Moreover, disruption of the E2-Brd4 interaction abrogates the transactivation function of E2, indicating that Brd4 is required for E2-mediated transactivation of all papillomaviruses. However, the interaction of E2 and Brd4 is not required for genome partitioning of all papillomaviruses since a number of papillomavirus E2 proteins associate with mitotic chromosomes independently of Brd4 binding. Furthermore, mutations in E2 that disrupt the interaction with Brd4 do not affect the ability of these E2s to associate with chromosomes. Thus, while all papillomaviruses attach their genomes to cellular chromosomes to facilitate genome segregation, they target different cellular binding partners. In summary, the E2 proteins from many papillomaviruses, including the clinically important alpha genus human papillomaviruses, interact with Brd4 to mediate transcriptional activation function but not all depend on this interaction to efficiently associate with mitotic chromosomes.

Animals↗
PubMed2005

Dose perturbations due to contrast medium and air in mammosite treatment: an experimental and Monte Carlo study.

In the management of early breast cancer, a partial breast irradiation technique called MammoSite (Proxima Therapeutic Inc., Alpharetta, GA) has been advocated in recent years. In MammoSite, a balloon implanted at the surgical cavity during tumor excision is filled with a radio-opaque solution, and radiation is delivered via a high dose rate brachytherapy source situated at the center of the balloon. Frequently air may be introduced during placement of the balloon and/or injection of the contrast solution into the balloon. The purpose of this work is to quantify as well as to understand dose perturbations due to the presence of a high-Z contrast medium and/or an air bubble with measurements and Monte Carlo calculations. In addition, the measured dose distribution is compared with that obtained from a commercial treatment planning system (Nucletron PLATO system). For a balloon diameter of 42 mm, the dose variation as a function of distance from the balloon surface is measured for various concentrations of a radio-opaque solution (in the range 5%-25% by volume) with a small volume parallel plate ion chamber and a micro-diode detector placed perpendicular to the balloon axis. Monte Carlo simulations are performed to provide a basic understanding of the interaction mechanism and the magnitude of dose perturbation at the interface near balloon surface. Our results show that the radio-opaque concentration produces dose perturbation up to 6%. The dose perturbation occurs mostly within the distances <1 mm from the balloon surface. The Plato system that does not include heterogeneity correction may be sufficient for dose planning at distances > or = 10 mm from the balloon surface for the iodine concentrations used in the MammoSite procedures. The dose enhancement effect near the balloon surface (<1 mm) due to the higher iodine concentration is not correctly predicted by the Plato system. The dose near the balloon surface may be increased by 0.5% per cm3 of air. Monte Carlo simulation suggests that the interface effect (enhanced dose near surface) is primarily due to Compton electrons of short range (<0.5 mm). For more accurate dosimetry in MammoSite delivery, the dose perturbation due to the presence of a radio-opaque contrast medium and air bubbles should be considered in a brachytherapy planning system.

Artifacts↗
PubMed2004

The in vivo synthesis and accumulation of lectin in developing seeds of black gram (Vigna mungo L. Hepper).

Black gram (Vigna mungo L. Hepper) seed contains two D-galactose-specific lectin species, BGL-I and BGL-II, identified on the basis of elution from ion exchange column and immunochemical cross-reactivity. BGL-I consisted of two monomeric lectins, BGL-I-1 and BGL-1-2, of relative molecular weights 94 and 89 kDa, respectively. BGL-II is another monomeric lectin with a molecular weight of 83 kDa. The in vivo synthesis studies using pulse-chase experiment showed that BGL-II lectin was synthesized as early as 14 days after flowering (DAF). The 94-kDa BGL-I-1 lectin was synthesized around 17 DAF. There was no cotranslational or posttranslational modification of the lectin proteins. The amount of lectin in developing seeds was determined by radial immunodiffusion assay technique. The maximum amount of lectin per seed was found at 28 DAF.

Food Analysis↗
PubMed2003

Potential for rhizofiltration of uranium using hairy root cultures of Brassica juncea and Chenopodium amaranticolor.

Hairy root cultures of Brassica juncea and Chenopodium amaranticolor were developed by genetic transformation using Agrobacterium rhizogenes. The stable, transformed root systems demonstrated a high growth rate of 1.5-3.0 g/g dry weight/day in Murashige and Skoog medium. In the present study, hairy root system was used for removal of uranium from the solution of concentration up to 5,000 microM. The results indicated that the hairy roots could remove uranium from the aqueous solution within a short period of incubation. B. juncea could take up 20-23% of uranium from the solution containing up to 5,000 microM, when calculated on g/g dry weight basis. C. amaranticolor showed a slow and steady trend in taking up uranium, with 13% uptake from the solution of 5,000 microM concentration. Root growth was not affected up to 500 microM of uranium nitrate over a period of 10 days.

Biodegradation, Environmental↗
PubMed2002

Purification and characterization of a lectin from wild sunflower (Helianthus tuberosus L.) tubers.

A lectin (HTTL) was isolated from Helianthus tuberosus L. (wild sunflower) tubers using ion-exchange chromatography, gel filtration, and affinity chromatography. The lectin agglutinated both untreated and trypsin-treated rabbit erythrocytes and did not agglutinate human blood cells of groups A, B, and O. The gel filtration showed the native molecular mass of 72 kDa and subunit molecular masses of 17 and 18.5 kDa on 12% SDS-PAGE. The lectin activity was inhibited by D-mannose. The tetrameric protein revealed a unique characteristic by forming a broad zone of protein in native PAGE at pH 8.3, which dissociated into seven subunits of varying e/m ratios on acid gel at pH 4.3. These seven bands revealed two polypeptide species of molecular masses 17 and 18.5 kDa on 12% SDS-PAGE, as in the case of the native protein. The result indicated that of the seven subunits, three were homotetramers of 17 kDa, one was a homotetramer of 18.5 kDa, and three were heterotetramers of 17 and 18.5 kDa. The lectin was thermostable with broad pH optima (pH 4-8) and had no requirement for divalent metal cations for its activity. The amino acid composition showed that the lectin contained higher amounts of glycine, alanine, and lysine, but no methionine. The sugar content was estimated to be 5.3% mannose equivalent. The HTTL was mitogenic to mouse spleen (total) cells at 25 microg/ml concentration. The lectin showed characteristics different from those of the earlier reported H. tuberosus tuber lectins and hence opens up a new avenue to investigate the structure-function relationship of lectin in Helianthus species.

Agglutination Tests↗
PubMed2002

Can human fetal cortical brain tissue transplant (up to 20 weeks) sustain its metabolic and oxygen requirements in a heterotopic site outside the brain? A study of 12 volunteers with Parkinson's disease.

BACKGROUND: Neural and stem cell transplantation is emerging as a potential treatment for neurodegenerative diseases from Parkinson's to Huntington's disease. Stereotactic placement of dopaminergic neurons in the caudate-putamen (striatum), is being attempted in centers of excellence and has proved to be beneficial. Basic research using cell transplantation indicates that structural development mechanisms seen in immature brains, i.e., fetal brains, can also function in the adult brain. The adult brain consumes 15% of the resting cardiac output for its metabolic needs. While most human tissues can sustain an anaerobic assault for a few minutes up to 30 minutes, a sudden total lack of oxygen supply to the brain cells in an adult will render the person unconscious within five to ten seconds. Our team has been working on the problem of human fetal tissue response to antigenic assault for the last two decades. In the present series, 12 patients with prolonged histories of Parkinsonism, who were not responding to anti-Parkinsonian drugs, and could not afford costly stereotactic surgery or deep brain stimulation and other modalities of recent Parkinson's disease treatment, were enrolled in the study. MATERIALS AND METHOD: After obtaining proper informed consents from the patients or their guardians and from the multidisciplinary ethical committee, the patients, varying in age from 45 to 75 years and suffering for many years with Parkinsonism, were enrolled in the heterotopic brain tissue transplant programme. We followed standard antiseptic, aseptic and premedication protocols, after selecting a proposed site of transplantation of the brain in the axillary fold of the skin, under local infiltration anaesthesia. In an adjacent OR, a fetus was collected from a consenting patient undergoing hysterotomy and ligation (before 20 weeks), under general anaesthesia. Within a minute of hysterotomy, the fetal brain tissue was dissected, and under the guidance of the operative microscope, 1 g of fetal cortical brain tissue was dissected and weighed in an electronic machine. The tissue was collected from around 1 cm of the frontal opercula of the developing human fetal brain and grafted in the already dissected and prepared subcutaneous site in the axilla and the skin was closed. Hematological parameters (Hgb; total count, Tc; differential count, Dc; erythocyte sedimentation rate, ESR) were estimated sequentially up to one month. A small portion of the transplanted tissue was retrieved after one to two months, and a serial histological study was done along with a clinical assessment of the disease condition as per the specifications of the Unified Parkinson's Disease Rating Scale. The results were matched with the pre-transplant ratings of the individual cases. Presenting dyskinesia was also rated (0-4), on the basis of objective criteria assessment like walking, putting on a coat, lifting a cup to drink, etc. RESULTS AND ANALYSIS: Initially 30 patients suffering from advanced Parkinson's disease (PD) were approached after getting the necessary clearance from the institutional multidisciplinary ethical committee; however, we have been able to arrange transplantation in only 12 cases so far. These patients were evaluated at the pre- and one month post-transplant period by the Unified Parkinson's Disease Rating Scale (0-108) and the minimum score was 40 in the motor portion of the unified scale at the pre-transplant state. Evaluation of the patients after one month revealed mild improvement of the pre-transplant scoring (up to 33.3%) in 41.6% of the cases, and moderate improvement (up to 66.6%) in another 41.6% of the cases. While 16.8% of the cases did not show any improvement from the basal score, i.e., the pre-transplant score, there was a definite sense of well being and rise in weight (2-4 pounds) noted in each case and there was also a reduction of the L-Dopa dosage in 75% of the cases. There was also a 58.3% improvement in the bradykinesia scoring from the pre-transplant level. What is intriguing is the survival, growth and proliferation of the grafted fetal brain tissue in the HLA- and sex-randomized adult axilla without any immunosuppressive support. Not a single histological study of the fetal brain tissues after removal from the axilla showed any signs of graft vs. host or inflammatory reaction (Figures 1-9) but there were features of growth of the transplanted cortical brain tissue along with its different components like neurogenesis, gliogenesis, early neovascularisation and angiogenesis, etc. There was also no systemic leucocytosis or lymphocytosis. DISCUSSION AND CONCLUSION: Histological evidence at the transplanted tissue site suggests that fetal cortical brain tissue can sustain life in sex-randomized, HLA-randomized adult hosts, without the support of immuno-suppressive drugs and the tacit support of the blood-CSF and blood-brain barrier and other specific requirements of adult brain cells in the skull. Whether the clinical improvement in PD is transient or long lasting is presently under investigation along with basic questions like, is it due to transplanted fetal dopaminergic or non-dopaminergic neurons or is it the growth factors and the cytokine mediated hitherto unknown reactions causing the clinical improvement.

Aged↗
PubMed2001

To move or not to move: measurements of prostate motion by urethrography using MRI.

PURPOSE: Urethrography is commonly used to aid in definition of the prostate apex during CT simulation for prostate cancer. If the position of the prostate were altered by the urethrogram itself, then systematic error could be introduced into the patient's treatment. Sagittal MRI scans were acquired immediately before and after a localization urethrogram to determine the extent of displacement. METHODS AND MATERIALS: Thirteen patients underwent sagittal T2-weighted fast spin echo MRI scans. Patients were scanned supine in an alpha cradle cast in the treatment position. The prostate was contoured by 3 different observers to determine the apex location on the central sagittal MRI section and the center of mass relative to an immobile bony landmark. Statistical multivariate analysis was performed to establish if there was a net displacement of the prostate (systematic error), and to determine the margin required to cover the random prostate position within a 95% confidence interval. RESULTS: There was no significant systematic motion of either the prostate nor its apex in either the anterior-posterior or superior-inferior directions. The average motion of the prostate center of mass was 0.04 +/- 0.40 cm (1 SD) and 0.01 +/- 0.33 cm in the anterior-posterior and superior-inferior direction, respectively. The corresponding figures for location of the apex were 0.05 +/- 0.30 cm and 0.01 +/- 0.33 cm, respectively. The statistical analysis revealed that a margin of 2 mm is sufficient to cover any random motion of the prostate that could occur as a result of the urethrogram 95% of the time. CONCLUSION: Urethrography during CT simulation for prostate cancer does not cause significant prostate displacement or systematic error in planning and delivering external-beam radiation.

Adenocarcinoma↗
PubMed2001

Activated macrophages migrate to the subcutaneous tumor site via the peritoneum: a novel route of cell trafficking.

Spontaneous regression of AK-5 tumor in syngeneic hosts reported earlier involves the interplay of Th1-type cytokines and cell-mediated immunity. Upon subcutaneous transplantation of AK-5 cells, there was accumulation of immune cells in the peritoneum, of which macrophages were the predominant type and were found to be in a hyperactive state. They released macrophage-derived tumoricidal mediators like NO, O2(-), and ONOO(-) which exhibited potent cytotoxic activity against AK-5 cells in vitro. Interestingly, there was a dramatic disappearance of these hyperactive cells from the peritoneal cavity which correlated well with the onset of tumor regression at the subcutaneous site. Direct labeling of these cells in the peritoneum by the tracking dye PKH26 showed their migration to the tumor site. Similarly, frozen tumor sections when scanned under confocal microscope clearly exhibited fluorescent macrophages embedded into the tumor. Immunohistochemical sections of these intratumoral macrophages showed nitrotyrosine residues, indicating their contribution in the free-radical-mediated AK-5 cell death, thereby leading to successful tumor remission. These observations suggest a directional migration of the hyperactivated peritoneal population to the tumor site. We have also confirmed the influx of macrophages and other immune cells into the peritoneum after sc transplantation of Meth A tumor cells in Balb/c mice. Our studies suggest a role for the peritoneal compartment in imparting appropriate stimulus to the immune cells prior to their participation in the antitumor immune response. These studies suggest a novel route of macrophage trafficking via the peritoneum.

Animals↗
PubMed2001

Enhanced pulmonary delivery of insulin by lung lavage fluid and phospholipids.

Pulmonary delivery appears to be the most promising non-parenteral route of insulin administration. In this work, we investigated the enhancement of insulin absorption in the presence of phospholipids and lung lavage fluid in vivo and in vitro. In-vitro experiments of insulin uptake by type II cells showed a significantly enhanced absorption in presence of lavage fluid, compared to various buffer preparations. The same trend was obtained with in-vivo studies of tracheal instillation of insulin. The incorporation of phospholipids as absorption enhancers in 1,2-dipalmitoyl phosphatidylcholine (DPPC) dispersion was compared to blank liposomes. A significantly higher blood glucose decrease was observed with a DPPC-insulin physical mixture compared to liposome, suggesting a possible effect of the phospholipid chain physical state on the insulin in-vivo absorption.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2001

Do race and gender influence the use of invasive procedures?

OBJECTIVE: To assess the influence of race and gender influence on the use of invasive procedures in patients with acute myocardial infarction (AMI) in community hospitals. DESIGN: Prospective, observational. SETTING: Five mid-Michigan community hospitals. PATIENTS: All patients (838) identified with AMI between January 1994 and April 1995 in 1 of these hospitals. MEASUREMENTS AND MAIN RESULTS: After adjusting for age, hospital of admission, insurance type, severity of AMI, and comorbidity, using white men as the reference group, the rate of being offered cardiac catheterization (CC) was 0.88 (95% confidence interval [95% CI], 0.60 to 1.29) for white women; 0.79 (95% CI, 0.41 to 1.50) for black men; and 1.14 (95% CI, 0.53 to 2.45)for black women. Among patients who underwent CC, after also adjusting for coronary artery anatomy, the rate of being offered angioplasty, using white men as the reference group, was 1.22 (95% CI, 0.75 to 1.98) for white women; 0.61 (5% CI, 0.29 to 1.28, P =.192) for black men; and 0.40 (95% CI, 0.14 to 1.13) for black women The adjusted rate of being offered bypass surgery was 0.47 (95% CI, 0.24 to 0.89) for white women; 0.36 (95% CI, 0.12 to 1.06) for black men; and 0.37 (95% CI, 0.11 to 1.28)for black women. CONCLUSIONS: Our study shows that white women are less likely than white men to be offered bypass surgery after AMI. Although black men and women with AMI are less likely than white men to be offered percutaneous transluminal coronary angioplasty or coronary artery bypass grafting in both unadjusted and adjusted analyses, these findings did not reach statistical significance. Our study is limited in power due to the small number of blacks in the sample.

Black or African American↗
PubMed2001

Purification and characterization of a hemagglutinin isolated from the leaves of Chenopodium (Chenopodium amaranticolor).

A hemagglutinin was isolated and purified from the leaves of Chenopodium (Chenopodium amaranticolor) using ion-exchange chromatography and affinity chromatography on fetuin-agarose matrix. It agglutinated rabbit erythrocytes. The hemagglutinin had a native molecular mass of 58 kDa, as estimated by gel filtration and showed a single band of molecular mass of 33 kDa on SDS-PAGE. It showed hemagglutination activity over the pH range 3-12 and was found to be stable up to 70 degrees C. On isoelectrofocussing, the pI of this hemagglutinin was estimated to be 5.25. However, it was found to contain seven charge variants when isoelectrofocussing was performed in presence of 6M urea.

Chenopodium↗
PubMed2001

Characterization and biological activities of Chenopodium leaf hemagglutinin (CLH).

A hemagglutinin (CLH) having native molecular mass of 58 kDa and subunit molecular mass of 33 kDa had been purified from the leaves of Chenopodium amaranticolor. The protein agglutinated rabbit erythrocytes and no agglutination was observed with any of the groups A, B or O of human blood. The amino acid composition revealed that CLH was rich in aspartic acid, glutamic acid, glycine and phenylalanine and also significant amount of methionine. The N-terminal amino acid sequence analysis showed that CLH had no homology with any of the plant hemagglutinins studied so far. It was inactive towards human peripheral blood cells but mitogenic for mouse spleen B-lymphocytes. CLH inhibited protein synthesis in rat thymocytes at high concentration. CLH did not inhibit TMV infection of leaves indicating absence of antiviral properties.

Amino Acids↗
PubMed2000

Lipid emulsions as vehicles for enhanced nasal delivery of insulin.

The objective of this work is to explore lipid emulsion based formulations of insulin as an enhancer of nasal absorption. Insulin was incorporated into the aqueous phases of water-in-oil (w/o) and oil-in-water (o/w) emulsions. The formulations were perfused through the nasal cavity of rats in situ. Enhancement of insulin absorption was observed when insulin was incorporated into the continuous aqueous phase of an o/w emulsion. The presence of a small fraction of oil droplets along with insulin in the aqueous phase appeared to favor insulin absorption. When the oil phase constitutes the external phase, as in w/o emulsion, no insulin absorption was noted. Inhibition of insulin absorption might arise from a rate limiting barrier effect of the membrane completely covered by a stagnant oil layer. The in situ model was validated by in vivo experiments, which also revealed an increase in insulin absorption with o/w emulsions. However at lower insulin doses there was no statistically significant enhancing effect. In situ perfusion experiments across rat nasal pathway appear to be an appropriate model to study the enhancement effect of nasal formulations.

Administration, Intranasal↗
PubMed2000

Mechanism of inhibition of HIV-1 integrase by G-tetrad-forming oligonucleotides in Vitro.

The G-tetrad-forming oligonucleotides and have been identified as potent inhibitors of human immunodeficiency virus type 1 integrase (HIV-1 IN) activity (Rando, R. F., Ojwang, J., Elbaggari, A., Reyes, G. R., Tinder, R., McGrath, M. S., and Hogan, M. E. (1995) J. Biol. Chem. 270, 1754-1760; Mazumder, A., Neamati, N., Ojwang, J. O., Sunder, S., Rando, R. F., and Pommier, Y. (1996) Biochemistry 35, 13762-13771; Jing, N., and Hogan, M. E. (1998) J. Biol. Chem. 273, 34992-34999). To understand the inhibition of HIV-1 IN activity by the G-quartet inhibitors, we have designed the oligonucleotides and, composed of three and four G-quartets with stem lengths of 19 and 24 A, respectively. The fact that increasing the G-quartet stem length from 15 to 24 A kept inhibition of HIV-1 IN activity unchanged suggests that the binding interaction occurs between a GTGT loop domain of the G-quartet inhibitors and a catalytic site of HIV-1 IN, referred to as a face-to-face interaction. Docking the NMR structure of (Jing and Hogan (1998)) into the x-ray structure of the core domain of HIV-1 IN, HIV-1 IN-(51-209) (Maignan, S., Guilloteau, J.-P. , Qing, Z.-L., Clement-Mella, C., and Mikol, V. (1998) J. Mol. Biol. 282, 359-368), was performed using the GRAMM program. The statistical distributions of hydrogen bonding between HIV-1 IN and were obtained from the analyses of 1000 random docking structures. The docking results show a high probability of interaction between the GTGT loop residues of the G-quartet inhibitors and the catalytic site of HIV-1 IN, in agreement with the experimental observation.

Base Composition↗
PubMed2000

Clonal dissemination of Vibrio parahaemolyticus displaying similar DNA fingerprint but belonging to two different serovars (O3:K6 and O4:K68) in Thailand and India.

Active surveillance of Vibrio parahaemolyticus infection among hospitalized patients in Calcutta, India, showed the appearance of the O4:K68 serovar for the first time in March 1998 alongside the continued predominant incidence of the O3:K6 serovar. Strains belonging to both these serovars have been reported to possess pandemic potential. The genomes of O3:K6 and O4:K68 strains and for comparison, non-O3:K6 and non-O4:K68 strains isolated from two different countries, India and Thailand, were examined by different molecular techniques to determine their relatedness. The O3:K6 and O4:K68 strains from Calcutta and Bangkok carried the tdh gene but not the trh gene. Characterization of representative strains of these two serovars by ribotyping and by arbitrarily primed-polymerase chain reaction (AP-PCR) showed that the isolates had identical ribotype and DNA fingerprint. Pulsed-field gel electrophoresis (PFGE) performed with the same set of strains yielded nearly similar restriction fragment length polymorphism (RFLP) patterns for the O3:K6 and O4:K68 isolates from Calcutta and Thailand. Phylogenetic analysis of the NotI RFLP showed that the O3:K6 and O4:K68 strains formed a cluster with 78-91% similarity thus indicating close genetic relationship between the two different serovars isolated during the same time-frame but from widely separated geographical regions. The non-O3:K6 and non-O4:K68, in contrast, showed different ribotype, AP-PCR and PFGE patterns.

DNA Fingerprinting↗
PubMed2000

Virulence genes in environmental strains of Vibrio cholerae.

The virulence of a pathogen is dependent on a discrete set of genetic determinants and their well-regulated expression. The ctxAB and tcpA genes are known to play a cardinal role in maintaining virulence in Vibrio cholerae, and these genes are believed to be exclusively associated with clinical strains of O1 and O139 serogroups. In this study, we examined the presence of five virulence genes, including ctxAB and tcpA, as well as toxR and toxT, which are involved in the regulation of virulence, in environmental strains of V. cholerae cultured from three different freshwater lakes and ponds in the eastern part of Calcutta, India. PCR analysis revealed the presence of these virulence genes or their homologues among diverse serotypes and ribotypes of environmental V. cholerae strains. Sequencing of a part of the tcpA gene carried by an environmental strain showed 97.7% homology to the tcpA gene of the classical biotype of V. cholerae O1. Strains carrying the tcpA gene expressed the toxin-coregulated pilus (TCP), demonstrated by both autoagglutination analysis and electron microscopy of the TCP pili. Strains carrying ctxAB genes also produced cholera toxin, determined by monosialoganglioside enzyme-linked immunosorbent assay and by passage in the ileal loops of rabbits. Thus, this study demonstrates the presence and expression of critical virulence genes or their homologues in diverse environmental strains of V. cholerae, which appear to constitute an environmental reservoir of virulence genes, thereby providing new insights into the ecology of V. cholerae.

Amino Acid Sequence↗
PubMed2000

Cell vacuolation, a manifestation of the El tor hemolysin of Vibrio cholerae.

Culture supernatants of nontoxigenic nonepidemic clinical strains of Vibrio cholerae belonging to diverse serogroups were found to induce vacuolation of nonconfluent HeLa cells. The vacuoles became prominent 18 h after introduction of culture supernatant, and vacuolated cells survived for 48 h and then died. Only a fraction of the vacuolated cells took up neutral red dye, implying that there were differences in the vacuolar microenvironment. Further tests showed that the factor responsible for vacuolation was heat labile and proteinaceous. Vacuolating activity was completely neutralized by antibody to hemolysin of V. cholerae but not by antibody to vacuolating cytotoxin of Helicobacter pylori. Partial purification of the vacuolating factor led to elution of fractions, which showed both hemolytic and vacuolating activity. PCR amplification and cloning of the hemolysin structural gene (hlyA) into Escherichia coli DH5alpha led to isolation of clones producing cell vacuolating factor in a cell-associated form. Further, a null insertion mutation in the hlyA gene of a high-vacuolating-factor-producing strain led to complete abolition of both cell vacuolating and hemolytic activities. These analyses establish vacuolation as a potentially important but previously unrecognized property of V. cholerae El Tor hemolysin.

Animals↗