Nucleotide exchange and cGMP phosphodiesterase activation by pertussis toxin inactivated transducin.

Transducin, the signal coupling protein of retinal rod photoreceptor cells, is one of a family of G proteins that can be inactivated by pertussis toxin. We have investigated the nature of this inactivation in order to determine (1) whether it requires the toxin-catalyzed transfer of ADP-ribose from NAD+ to cysteine-347 of the alpha subunit and (2) whether it involves locking the alpha subunit in the inactive conformation characteristic of its GDP-bound state, or is limited to disruption of binding to photoexcited rhodopsin (R*). Our results indicate that all observed effects of pertussis toxin treatment, including a shift in the electrophoretic mobility of transducin's alpha subunit and functional inactivation, require NAD+ and that the appearance of the shift parallels incorporation of ADP-ribose. We have also found that, apart from interactions with photoexcited rhodopsin, the functional properties of ADP-ribosylated transducin are essentially the same as those of unmodified transducin. Normal spontaneous nucleotide exchange kinetics and the ability to activate cGMP phosphodiesterase are preserved following quantitative ADP-ribosylation, as are the abilities to hydrolyze GTP, to bind to a dye affinity column, and to display enhanced fluorescence upon addition of Al3+ and F-. Thus, ADP-ribosylation merely blocks catalysis of transducin nucleotide exchange by R* and does not lock transducin in an inactive state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apoptosis induced by modulation in selenium status involves p38 MAPK and ROS: implications in spermatogenesis

Selenium has been linked to cell survival and apoptosis. Apoptosis plays an important role in spermatogenesis. Evidence suggests that reactive oxygen species induce apoptotic pathways. Although the mechanism by which oxidants mediate apoptosis is not well defined, the mitogen-activated protein kinase (MAPK) and caspase pathways have been implicated in apoptosis. Thus, this study was designed, keeping in view the critical balance between cell proliferation and apoptosis for normal spermatogenesis, and the requirement of selenium for the maintenance of male fertility. The intracellular selenium status was modulated by feeding selenium-deficient and -excess diet for 8 weeks. Involvement of p38 MAPK and ROS was monitored. Apoptotic factors like caspases and Bcl-2 were also analyzed. It was observed that the selenium levels were altered along with an increase in ROS generation and lipid peroxidation. mRNA expression of p38, caspases 3, and 8 increased, whereas that for Bcl-2 decreased. Western immunoblot analysis and immunohistochemical localization studies for p38 showed a similar increase. Integrity of DNA was altered in the form of apoptotic cells. Thus, the results presented in this study suggest that sodium selenite causes apoptosis and the toxicity of selenite is mediated by increase in ROS. Morevoer, ROS generation is associated with increased expression of p38, caspases 3 and 8, and decreased Bcl-2 expression. Our data indicate that p38 participates in testicular apoptosis and that selenium is required for maintenance of the critical balance between cell death and proliferation.     

Screening of potential probiotic lactic acid bacteria and production of amylase and its partial purification

Probiotics are the healthy living bacteria when administered in adequate amounts confers health benefits in the host. The main objective of present study was to screen the bacteria for potential probiotic characters and enzyme production. The probiotic characters like tolerance to low pH, bile salts, antibiotic sensitivity, hydrophobicity and auto-aggregation properties were evaluated. Among all isolates Lactobacillus fermentum and Lactobacillus sp G3_4_1TO2 showed maximum potential probiotic characters and produced amylase enzyme by observing the halo zone around the colonies with the diameter 0.9?mm and 1.23?mm. Lactobacillus sp G3_4_1TO2 produced maximum amylase when compared with Lb. fermentum. The protein yield was 55.4% with the specific activity of 88.9 U/mg and obtained 40.8% purification fold. The molecular weight of amylase enzyme determined by SDS PAGE was 95,000?Da. From the present study it was considered that Lactobacillus sp G3_4_1TO2 was a potential probiotic bacteria producing maximum amylase enzyme.