The trafficking of prosaposin (SGP-1) and GM2AP to the lysosomes of TM4 Sertoli cells is mediated by sortilin and monomeric adaptor proteins

Prosaposin (SGP-1) and GM2 activator protein (GM2AP) are soluble sphingolipid activator proteins (SAPs) that are targeted to the lysosomal compartment of Sertoli cells to aid hydrolases in the breakdown of glycosphingolipids. To reach the lysosome, most soluble proteins must interact with the mannose 6-phosphate receptor (MPR). To be sorted from the Golgi, the MPR must bind to the Golgi associated, gamma-adaptin homologous, ARF binding proteins (GGAs), a group of monomeric adaptor proteins responsible for the recruitment of clathrin. It is well established, however, that the lysosomes of I-cell disease (ICD) patients have near normal levels of several lysosomal proteins, including prosaposin and GM2AP. ICD results from a mutation in the phosphotransferase that adds mannose 6-phosphate to hydrolases. Thus, prosaposin and GM2AP can traffic to lysosomes in a MPR independent manner. Previous work has demonstrated that an interaction with sphingomyelin in the Golgi membrane is necessary for the targeting of prosaposin by an unknown receptor. Using a TM4 Sertoli cell line, we tested the hypothesis that prosaposin and GM2AP are targeted to the lysosomal compartment via the sortilin receptor, which has been recently shown to have a GGA binding motif. Interestingly, dominant-negative GGAs, unable to bind clathrin to shuttle from the Golgi, prevented the trafficking of prosaposin and GM2AP to lysosomes. A dominant negative construct of sortilin lacking the GGA binding domain retained prosaposin and GM2AP in the Golgi. In conclusion, our results showed that the trafficking of prosaposin and GM2AP to the lysosome is dependent on sortilin.     

Effects of azadirachtin/Neemazal on different stages and adult life table parameters of Trichogramma cacoeciae (Hymenoptera: Trichogrammatidae)

The effects of azadirachtin/Neemazal on adults, emergence, and life table parameters of Trichogramma cacoeciae Marchal were studied. The adults were exposed to fresh residues of the insecticide applied on glass plates. Based on the dose-response study, the LC50 value was 1330 ppm or 13.3 microg (AI)/ml. The effect of Neemazal on three developmental stages of the parasitoid was tested by dipping parasitized Sitotroga cerealella (Olivier) and Cydia pomonella (L.) eggs at the field-recommended concentration 3, 6, and 9 d after parasitization corresponding to larval, prepupal, and pupal stages. The emergence of adult parasitoids was adversely affected in both hosts, but the adverse effect was more in S. cerealella eggs compared with C. pomonella. The adult emergence was reduced by 73.3 and 33.76% in Sitotroga and Cydia eggs compared with controls, respectively. Longevity and progeny production of the emergent adults did not differ significantly from control. Neemazal affected stable population parameters (r(m), T, and DT) significantly. The intrinsic rate of increase for the control and Neemazal-exposed populations was 0.340 and 0.335 female offspring per female per day, respectively. Because some of postemergence life table parameters of adults were significantly reduced by the insecticide treatment, emergence rate of the parasitoid from treated eggs is not an adequate measure of ecological selectivity, and field studies are needed to determine the actual impact of neem on T. cacoeciae.     

Mathematical algorithm for discovering states of expression from direct genetic comparison by microarrays

Highly specific direct genome-scale expression discovery from two biological samples facilitates functional discovery of molecular systems. Here, expression data from cDNA arrays are ranked and curve-fitted. The algorithm uses filters based on the derivatives (slopes) of the curve fits. The rules are set to (i) filter the largest number of artifactual ratios from same-to-same datasets and (ii) maximize discovery from direct comparisons of different samples. The unsupervised discovery is optimized without lowering specificity. The false discovery rates are significantly lower than other methods. The discovered states of genetic expression facilitate functional discovery and are validated by real-time RT–PCR. Better quality improves sensitivity.     

Criticality and synchrony of fluctuations in rhythmical brain activity: pretransitional effects in epileptic patients

In extending our previous results demonstrating critical fluctuations in electropathophysiological brain activity prior to onset of partial epileptic seizures, we used once again Hakens approach to self-organized complex systems, which resorts to identifying an order parameter. A renormalized density in phase space and a renormalized differential density were defined and substituted for the Lerner density, which we used previously. Fluctuations in electropathophysiological activity, consisting of periods of high activity recorded from both depth and scalp electrodes in six presurgical patients, were characterized on different time scales. Extension in space of the fluctuations was characterized by observing their synchronous occurrence in different areas of the brain. By simultaneously establishing the fluctuations criticality, we were observing synchronous instabilities. Criticality followed from characterizing the time course of the synchronously arising fluctuations, when approaching seizure onset (i) by their slowing, for the longer-lasting ones (up to about 2 min), and (ii) by their increase in rate of production, for those of shorter duration (up to 20–30 s). Critical behavior may be displayed in favorable cases over more than 1 h prior to seizure onset. Other effects are also described that in some cases may interfere with the simple time course of fluctuations and must be given further consideration, particularly with a view to application to seizure anticipation. Because periods of high activity are involved, any marker of increased electropathophysiological activity in the brain may, in principle, play the role of an order parameter or of an approximate order parameter, e.g., a signals root-mean-square amplitude, or its excess energy content. Such markers may be put to use for the fluctuations they display or merely for their average time evolution prior to seizure onset. We compared the time evolution, prior to onset, of the excess production rate of synchronies and of the signals excess energy content, both averaged over signal sections of a chosen duration. In view of potential noninvasive applications, those scalp electrodes showing effects similar to the observations we made at the epileptogenic focus were identified, and the time course of their order parameters prior to seizure onset was analyzed.     

Neuropeptide Y induced increase of cytosolic and nuclear Ca2+ in heart and vascular smooth muscle cells

It was reported that neuropeptide Y (NPY) affects cardiac and vascular smooth muscle (VSM) function probably by increasing intracellular Ca2+. In this study, using fura-2 microfluorometry and fluo-3 confocal microscopy techniques for intracellular Ca2+ measurement, we attempted to verify whether the action of NPY receptor's stimulation in heart and VSM cells modulates intracellular Ca2+ and whether this effect is mediated via the Y1 receptor type. Using spontaneously contracting single ventricular heart cells of 10-day-old embryonic chicks and the fluo-3 confocal microscopy Ca2+ measurement technique to localize cytosolic ([Ca]c) and nuclear ([Ca]n) free Ca2+ level and distribution, 10-10 M of human (h) NPY significantly (P < 0.05) increased the frequency of cytosolic and nuclear Ca2+ transients during spontaneous contraction. Increasing the concentration of hNPY (10(-9) M) did not further increase the frequency of Ca2+ transients. The L-type Ca2+ channel blocker, nifedipine (10(-5) M), significantly (P < 0.001) blocked the spontaneous rise of intracellular Ca2+ in the absence and presence of hNPY (10(-10) and 10(-9) M). However, the selective Y1 receptor antagonist, BIBP3226 (10(-6) M), significantly decreased the hNPY-induced (10(-10) and 10(-9) M) increase in the frequency of Ca2+ transients back to near the control level (P < 0.05). In resting nonworking heart and human aortic VSM cells, hNPY induced a dose-dependent sustained increase of basal resting intracellular Ca2+ with an EC50 near 10(-9) M. This sustained increase was cytosolic and nuclear and was completely blocked by the Ca2+ chelator EGTA, and was significantly decreased by the Y1 receptor antagonist BIBP3226 in both heart (P < 0.05) and VSM (P < 0.01) cells. These results strongly suggest that NPY stimulates the resting basal steady-state Ca2+ influx through the sarcolemma and induces sustained increases of cytosolic and nuclear calcium, in good part, via the activation of the sarcolemma membrane Y1 receptor type in both resting heart and VSM cells. In addition, NPY also increased the frequency of Ca2+ transients during spontaneous contraction of heart cells mainly via the activation of the Y1 receptor type, which may explain in part the active cardiovascular action of this peptide.     

A convenient synthesis of 2'-deoxy-2-fluoroadenosine; a potential prodrug for suicide gene therapy

A convenient synthesis of 2'-deoxy-2-fluoro-adenosine (1) is described. Deaminative fluorination of 2-aminoadenosine (2) followed by silylation of the 3', 5'-hydroxyl groups gave the corresponding 2-fluoroadenosine derivative 4 in good yield. Thiocarbonylation of 4 to thiocarbonylimidazolyl derivative 5a followed by treatment with an excess of tris(trimethylsilyl)silane (TTMSS) and tert-butyl peroxide in toluene at 80 degrees C was found to affect an efficient deoxygenation to the corresponding 2'-deoxy derivative 6. Desilylation of 6 by Et4NF in CH3CN afforded 1 in high yield.     

Improvement in raw sago starch degrading enzyme production from Acremonium sp. endophytic fungus using carbon and nitrogen sources

Attempts were made to improve the growth of endophytic fungus Acremonium sp. and its raw sago starch degrading enzyme (RSSDE) production using different nitrogen and carbon sources at varying pH values and temperatures. It was observed that growth and enzyme activity levels were highest with peptone and sodium nitrate as the nitrogen sources and raw sago starch as the carbon source of which the optimum concentrations were 0.5 g/l, 3 g/l, and 20 g/l, respectively. Cell growth and RSSDE production reached their optimum at pH 5.0 and incubation temperature of 30 degrees C. Under these conditions, the enzyme production was significantly increased by 19- to 22-folds compared to the activity obtained in the original basal medium.