Loss of hydrophobic motif and activation loop phosphorylation is a consequence and not the mechanism of S6 kinase 1 inhibition by rapamycin.

S6K1 regulation associates a central role with dynamics of sequential phosphorylations at the hydrophobic motif (T412) and activation loop (T252) of the enzyme, such that the hydrophobic motif phosphorylation supposedly brought about by mTOR- kinase, primes the enzyme for PDK1 dependent phosphorylation at the activation loop for its full activation. Accordingly loss of hydrophobic motif phosphorylation attributed to TOR- kinase inhibition, with resultant loss of activation loop phosphorylation is the hypothesis put forward to explain the mechanism of rapamycin inhibition. Our recent observation that rapamycin continues to inhibit S6K1 in the absence of either phosphorylation, together with the evidence that phosphorylation at activation loop may occur prior to that of hydrophobic motif raises serious questions about the proposed mechanism of rapamycin inhibition. Here, we show that rapamycin fails to effect preferential loss of either phosphorylation and the two instead exhibit equal sensitivity to rapamycin both in time and quantum. We further show that of activation loop and hydrophobic motif phosphorylations turnover in an interdependent manner so as to exhibit all or none pattern of loss to rapamycin. Using insect cell expression system, we further substantiate their interdependent turnover and provide evidence that the two phosphorylations are brought about in a coordinate and not sequential manner. These data together with the observation that both kinases that cause hydrophobic motif and activation loop phosphorylations in insect or mammalian cells are completely insensitive to inhibition by rapamycin, suggest that their loss is a consequence and not the mechanism of rapamycin inhibition in accordance with the model proposed herein.

Prevalence of myopia in students of srinagar city of kashmir, India.

BACKGROUND: Myopia is a common ocular disorder. Prevalence data with regard to myopia is scarce in India and almost nonexistent in Kashmir. OBJECTIVE: To determine the prevalence of myopia in Srinagar City and to evaluate risk factors associated with the disease. METHODS: 38 schools in the Srinagar were selected randomly and students were examined by our optometrist team. Children with refractive error of -0.25 D to -5.9 D were considered myopic, while those with -6 D and above were considered high myopic. STATISTICAL ANALYSIS USED: χ2 Tests were used as appropriate to test whether potential risk factors were significantly associated with myopia. Odds ratios (OR) and 95% confidence intervals (95% CI) were calculated for risk factors that were independently associated with myopia in this population. RESULTS: A total of 4,360 students of mean age 12.11 (95% confidence interval [CI] = 11.99 - 12.22: range, 7-18) participated in the study. Myopia was found in 4.74% students. Increasing age was associated with the increased risk of having myopia. Girl students were more likely to have myopia than boys (OR = 1.52). The prevalence of myopia among girls was more than that of boys. Students from low socioeconomic conditions were having higher prevalence of myopia than their counterparts from higher socioeconomic counterparts. CONCLUSION: Reduced vision because of myopia is an important health problem in students in Srinagar City. Most of these students do not have the necessary correction spectacles. Effective strategies are needed to eliminate the cause of a significant visual problem.

Effect of nifedipine administration on the functional capacity of neutrophils.

Administration of nifedipine to mice over a period of six months caused a significant (p < 0.05) decrease in neutrophilic functions viz superoxide generation, coupled to NADPH oxidase activity as well as NADPH production by HMP shunt. Properties like chemotaxis and phagocytosis showed a similar decrease. From this study, it is seen that nifedipine causes neutrophil functional abrogation which is therefore an apparent concern for the prolonged usage of the drug. However, relevance of the mouse model to clinical situation needs further investigation.

Effect of exogenous copper on lipid peroxidation in rat hepatocytes. Possible involvement of protein kinase C.

We have investigated the direct effect of copper on malondialdehyde formation in rat isolated hepatocytes. Copper was found to decrease the cell viability with concomitant production of malondialdehyde in a time related manner. In addition the protein kinase C activator, PMA, was found to have a synergistic effect with copper on rat hepatocytes. These results indicate that protein kinase C may be important in mediating hepatotoxicity after exposure to copper.

Decrease of myocardial infarct size with desferrioxamine: possible role of oxygen free radicals in its ameliorative effect.

The ability of an iron chelator, desferrioxamine, to inhibit the infarct size in in vivo rat heart was assessed. Anaesthetised rats were subjected to coronary artery ligation (CAL) for 72 hr and infarct size was measured macroscopically using TTC staining. Systolic blood pressure and ECG were monitored. Desferrioxamine (10 mg/kg and 20 mg/kg i.v.) administered half an hour after CAL markedly reduced the infarct size. However, drug treatment did not alter the systolic blood pressure of animals. In addition, desferrioxamine in vitro and in vivo demonstrated an inhibition of rat PMN-evoked and luminol-enhanced chemiluminescence. The capacity of desferrioxamine to impair the generation or to scavenge directly oxygen free radicals may be responsible for its beneficial effect on myocardial infarct size in rats.

Intracellular cAMP determines the extent of degradation and not the synthesis of collagen by rat hepatocytes.

Intracellular collagen degradation in normal rat hepatocytes was exponentially stimulated by db-cAMP (10-100 microM). The effect was manifested as a decrease (p less than 0.01) in net collagen production. The extent of degradation directly co-related with the intracellular cAMP levels, only up to a threshold concentration (16.2 +/- 1.3 p moles/10(6) cells) elicited by 100 microM of db-cAMP. Higher concentrations induced no further increment. Forskolin adenylate cyclase activator (10-50 microM), produced similar effects demonstrating cAMP dependence of the phenomenon. Both db-cAMP as well as Forskolin stimulated collagen degradation (p less than 0.05) in hepatocytes from rats administered CCL4. However, the extent of stimulation was significantly (p less than 0.01) less compared to that observed in normal hepatocytes. Our data demonstrates that elevated cAMP levels regulate net collagen content by signalling intracellular collagen degradation and not synthesis.

Collagen-stimulated superoxide production: evidence for coupled mobilization of calcium.

Superoxide production by human neutrophils was stimulated by rat liver collagen. The stimulation was exponentially related to the collagen concentration, with maximal effect at 150 micrograms/ml. The collagen-induced effect was significantly enhanced by the presence of Ca2+ in the medium. Verapamil--a calcium channel blocker--caused a dose-dependent inhibition of superoxide production by collagen-stimulated neutrophils. Collagen-induced stimulation was associated with a transient rise in cytosolic free Ca2+ independent of the presence of Ca2+ in the medium. Depletion of intracellular calcium caused a significant decrease in superoxide activity; however, replenishment of Ca2+ in the medium significantly overcame the inhibition. These changes were associated with a direct binding of [14C]collagen with the neutrophils. Our data suggest that collagen-neutrophil interaction couples superoxide production with the process of Ca2+ mobilization and that this interaction may play a physiologic role in neutrophil stimulation.

Nifedipine administration impairs natural resistance of mice to Salmonella typhimurium.

Administration of Ca2+ channel blockers in cardiac disorders and the central role of Ca2+ in modulating neutrophil functions, prompted us to investigate whether administration of nifedipine to mice would alter their natural resistance to infectious agents like Salmonella typhimurium. Neutrophil chemiluminescence (CL) in response to S. typhimurium was significantly (p less than 0.01) decreased in mice fed with nifedipine (0.015 mg/kg body weight) over a period of six months. Intracellular killing of S. typhimurium by isolated neutrophils also decreased significantly (p less than 0.01) and exponentially with nifedipine administration, representing a 42% fall at six months. In addition the drug administration lowered the survival rate of animals following challenge by a lethal dose of S. typhimurium (LD50 = 1 x 10(4) bacteria/animal). Our data suggest that long term administration of nifedipine lowers the natural resistance of mice to S. typhimurium owing to impaired neutrophil functions.

Nifedipine impairs neutrophil respiratory burst by a mechanism other than calcium channel blockade.

Superoxide production by mice neutrophils was inhibited by nifedipine exposure in a dose dependent manner. The inhibition of Ca2+ uptake elicited by nifedipine did not appear to account for the observed effect as the extracellular Ca2+ enrichment and depletion did not produce a significant reversal of the inhibition. Cytosolic free Ca2+ as measured by Quin 2AM fluorescence did not show any significant change, indicating that the effect was independent of the inhibition of Ca2+ influx. In addition nifedipine caused a significant inhibition (p less than 0.01) in NADPH oxidase activity. Our data indicates that nifedipine inhibits superoxide production independent of inhibiting Ca2+ inflow and supports the hypothesis that Ca2+ antagonists affect cellular functions by non Ca2+ mediated process as well.