The rapid activation of N-Ras by α-thrombin in fibroblasts is mediated by the specific G-protein Gαi2–Gβ1–Gγ5 and occurs in lipid rafts

α-thrombin is a potent mitogen for fibroblasts and initiates a rapid signal transduction pathway leading to the activation of Ras and the stimulation of cell cycle progression. While the signaling events downstream of Ras have been studied in significant detail and appear well conserved across many species and cell types, the precise molecular events beginning with thrombin receptor activation and leading to the activation of Ras are not as well understood. In this study, we examined the immediate events in the rapid response to α-thrombin, in a single cell type, and found that an unexpected degree of specificity exists in the pathway linking α-thrombin to Ras activation. Specifically, although IIC9 cells express all three Ras isoforms, only N-Ras is rapidly activated by α-thrombin. Further, although several Gα subunits associate with PAR1 and are released following stimulation, only Gα_i2 couples to the rapid activation of Ras. Similarly, although IIC9 cells express many Gβ and Gγ subunits, only a subset associates with Gα_i2, and of those, only a single Gβγ dimer, Gβ₁γ₅, participates in the rapid activation of N-Ras. We then hypothesized that co-localization into membrane microdomains called lipid rafts, or caveolae, is at least partially responsible for this degree of specificity. Accordingly, we found that all components localize to lipid rafts and that disruption of caveolae abolishes the rapid activation of N-Ras by α-thrombin. We thus report the molecular elucidation of an extremely specific and rapid signal transduction pathway linking α-thrombin stimulation to the activation of Ras.     

Molecular modeling of flexible arm‐mediated interactions between bacterial chemoreceptors and their modification enzyme

Sensory adaptation in bacterial chemotaxis is mediated by methylation and demethylation of specific glutamyl residues in the cytoplasmic domain of chemoreceptors. Methylation is catalyzed by methyltransferase CheR. In E. coli and related organisms, methylation sufficiently rapid to be physiologically effective requires a carboxyl terminal pentapeptide sequence on the receptor being modified or, via adaptational assistance, on a neighboring homodimer in a receptor cluster. Pentapeptide‐enhanced methylation is thought to be mediated by a ～30 residue, potentially disordered sequence that serves as a flexible arm connecting the receptor body and pentapeptide‐bound methyltransferase, thus allowing diffusionally restricted enzyme to reach methyl‐accepting sites. However, it was not known how many or which sites on the same or neighboring receptors were accessible to the tethered enzyme. We investigated using molecular modeling and found that, in a hexagonal array of trimers of receptor dimers, CheR tethered to a dimer of chemoreceptor Tar by its native 30‐residue flexible‐arm sequence could reach all methyl‐accepting sites on the dimer to which it was tethered plus 48 methyl‐accepting sites distributed among nine neighboring dimers, equivalent to the total sites carried by six receptors. This modeling‐determined methylation neighborhood of one enzyme‐binding dimer and six neighbors corresponds precisely with the experimentally identified neighborhood of seven. Thus, the experimentally observed adaptational assistance can occur by docking of pentapeptide‐bound, diffusionally restricted enzyme to methyl‐accepting sites on neighboring receptors. Our analysis introduces the notion that physiologically relevant adaptational assistance could occur even if only a subset of sites on a particular receptor are within reach.     

Characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> from deep ground water in a cholera endemic area in Central India

A total of 8 out of 11 deep ground water samples collected from different villages in Central India were found contaminated with Vibrio cholerae non O1, non O139. In a multiplex PCR, isolates were found positive for ompW gene but negative for ctxAB and rfbO1 genes. However, isolates from two places were positive for tcp and zot genes, indicating their intestinal colonization and toxigenic potential. Antibiotic susceptibility studies revealed that all isolates were multidrug resistant. Although, none of the isolates was found PCR positive for the mobile genetic elements, class 1 integrons and SXT constins. The results of this study corroborated that deep ground water can also be an important reservoir of V. cholerae in plane endemic areas, suggesting a continuous monitoring of water samples for timely prevention of the disease.     

Comparative in-vitro biodegradation studies of epoxy and its silicone blend by selected microbial consortia

A total of six bacterial isolates were developed into two consortia and tested for utilization of epoxy silicone blends (ESBs; % w/w: 3.0) and epoxy as the sole carbon source. In-vitro biodegradation studies in minimal broth revealed that higher biomass and more sustained growth of consortia were obtained in the presence of epoxy and/or ESBs when these were incubated under aerobic conditions for 15 days. Treated samples were analyzed by Fourier transform infrared spectroscopy (FTIR) and simultaneous thermogravimetric–differential thermogravimetry–differential thermal analysis (TG–DTG–DTA), which indicated the breakage and formation of bonds in the polymer backbone. Moreover, a weight loss of 34.17 and 36.9% was found in epoxy and ESBs, respectively after 15 days of treatment with consortium-1. Further, in-vitro growth statistics study revealed more CFU count at mid-logarithmic phase in the presence of epoxy/ESBs unlikely to the absence of the polymers. However, the generation time was not affected. In the present study, consortium-1, comprising of Microbacterium sp., Pseudomonas putida and Bacterium Te 68R showed better biodegradation in comparison to consortium-2, wherein, P. putida and Pseudomonas aeruginosa were present. Overall, these results suggest that epoxy/ESBs polymers could be degraded by a biologically mediated process if a suitable consortium is used.     

Nevirapine Resistance and Breast-Milk HIV Transmission: Effects of Single and Extended-Dose Nevirapine Prophylaxis in Subtype C HIV-Infected Infants

Background

Daily nevirapine (NVP) prophylaxis to HIV-exposed infants significantly reduces breast-milk HIV transmission. We assessed NVP-resistance in Indian infants enrolled in the “six-week extended-dose nevirapine” (SWEN) trial who received single-dose NVP (SD-NVP) or SWEN for prevention of breast-milk HIV transmission but who also acquired subtype C HIV infection during the first year of life.

Methods/Findings

Standard population sequencing and cloning for viral subpopulations present at ≥5% frequency were used to determine HIV genotypes from 94% of the 79 infected Indian infants studied. Timing of infection was defined based on when an infant''s blood sample first tested positive for HIV DNA. SWEN-exposed infants diagnosed with HIV by six weeks of age had a significantly higher prevalence of NVP-resistance than those who received SD-NVP, by both standard population sequencing (92% of 12 vs. 38% of 29; p = 0.002) and low frequency clonal analysis (92% of 12 vs. 59% of 29; p = 0.06). Likelihood of infection with NVP-resistant HIV through breast-milk among infants infected after age six weeks was substantial, but prevalence of NVP-resistance did not differ among SWEN or SD-NVP exposed infants by standard population sequencing (15% of 13 vs. 15% of 20; p = 1.00) and clonal analysis (31% of 13 vs. 40% of 20; p = 0.72). Types of NVP-resistance mutations and patterns of persistence at one year of age were similar between the two groups. NVP-resistance mutations did differ by timing of HIV infection; the Y181C variant was predominant among infants diagnosed in the first six weeks of life, compared to Y188C/H during late breast-milk transmission.

Conclusions/Significance

Use of SWEN to prevent breast-milk HIV transmission carries a high likelihood of resistance if infection occurs in the first six weeks of life. Moreover, there was a continued risk of transmission of NVP-resistant HIV through breastfeeding during the first year of life, but did not differ between SD-NVP and SWEN groups. As with SD-NVP, the value of preventing HIV infection in a large number of infants should be considered alongside the high risk of resistance associated with extended NVP prophylaxis.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00061321","term_id":"NCT00061321"}}NCT00061321     

Immunological Biosensor for Detection of Vibrio cholerae O1in Environmental Water Samples

Environmental surveillance for the presence of Vibrio cholerae O1 is of utmost importance for the effective public health protection of cholera. In the present study, an amperometric immunosensor was developed for detection of Vibrio cholerae in environmental samples by using disposable screen-printed electrodes (SPEs). For this purpose, the experiments done include fabrication of SPEs by using carbon ink, electrochemical characterization of electrodes, optimization of dilutions of antibodies and immobilization of antibody. V. cholerae O1 bacteria were spiked in various environmental water samples in known number. The seeded samples were filtered through a 0.22 μm membrane, and the filters enriched in alkaline peptone water for 6 h and then used directly for detection of V. cholerae using the immunosensor. The immunosensor could detect as few as 8 c.f.u./ml in hand-pump water (ground water) and seawater, and 80 c.f.u./ml in sewer water and tap water. The total time taken in this detection assay was 55 min. Thus, the proposed method is simple and can be used for environmental monitoring of V.␣cholerae.     

Arylanthranilodinitriles: a new biaryl class of antileishmanial agents

A series of anthranilodinitrile-based biaryls were synthesized and evaluated in vitro against extracellular promastigotes and intracellular amastigotes of Leishmania donovani. Among various screened compounds, a biaryl with trifluoromethyl group 5f showed 83% inhibition against promastigotes and 70% inhibition against amastigotes of L. donovani at 8 and 20microg/mL concentrations, respectively.     

Role of zinc in mitigating the toxic effects of chlorpyrifos on hematological alterations and electron microscopic observations in rat blood

The present study determined the protective potential of zinc in attenuating the toxicity induced by chlorpyrifos in rat blood. Male Sparque Dawley (SD) rats received either oral chlorpyrifos (13.5 mg/kg body weight) treatment every alternate day, zinc alone (227 mg/l in drinking water) or combined chlorpyrifos plus zinc treatment for a total duration of 8 weeks. The effects of different treatments were studied on various parameters in rat blood including haemoglobin (Hb) levels, total leukocyte count (TLC), differential leukocyte count (DLC), zinc protoporphyrins (ZPP), serum trace elemental concentrations and Scanning Electron Microscopic (SEM) observation of the blood cells. Chlorpyrifos treatment to normal control animals resulted in a significant decrease in TLC and ZPP concentration after 4 and 8 weeks. Chlorpyrifos treated animals also showed significant neutrophilia and lymphopenia after 8 weeks of toxicity. In addition, a significant decrease in serum zinc and iron concentrations were observed following chlorpyrifos intoxication, however, these animals responded with increased serum copper levels following the toxic treatment with this organophosphate. SEM studies of the red blood cells from chlorpyrifos treated animals indicated marked alterations in the topographical morphology of the various cell types, with the prominent feature being common aniscocytosis of the erythrocytes. Oral zinc treatment to the chlorpyrifos treated animals significantly improved the total leukocyte, neutrophil and lymphocyte counts, as well as the otherwise reduced concentrations of ZPP and the levels of various serum trace elements. Protective effects of zinc were also evident in the electron microscopic observations where most blood cell types depicted reverted to a close to the normal appearance. Based upon these data, the present study is first of its kind and suggests that zinc treatment considerably attenuates chlorpyrifos induced toxicity induced in restoring the altered hematological indices and morphological changes.