Syntaxin and VAMP association with lipid rafts depends on cholesterol depletion in capacitating sperm cells

Sperm cells represent a special exocytotic system since mature sperm cells contain only one large secretory vesicle, the acrosome, which fuses with the overlying plasma membrane during the fertilization process. Acrosomal exocytosis is believed to be regulated by activation of SNARE proteins. In this paper, we identified specific members of the SNARE protein family, i.e., the t-SNAREs syntaxin1 and 2, and the v-SNARE VAMP, present in boar sperm cells. Both syntaxins were predominantly found in the plasma membrane whereas v-SNAREs are mainly located in the outer acrosomal membrane of these cells. Under non-capacitating conditions both syntaxins and VAMP are scattered in well-defined punctate structures over the entire sperm head. Bicarbonate-induced in vitro activation in the presence of BSA causes a relocalization of these SNAREs to a more homogeneous distribution restricted to the apical ridge area of the sperm head, exactly matching the site of sperm zona binding and subsequent induced acrosomal exocytosis. This redistribution of syntaxin and VAMP depends on cholesterol depletion and closely resembles the previously reported redistribution of lipid raft marker proteins. Detergent-resistant membrane isolation and subsequent analysis shows that a significant proportion of syntaxin emerges in the detergent-resistant membrane (raft) fraction under such conditions, which is not the case under those conditions where cholesterol depletion is blocked. The v-SNARE VAMP displays a similar cholesterol depletion-dependent lateral and raft redistribution. Taken together, our results indicate that redistribution of syntaxin and VAMP during capacitation depends on association of these SNAREs with lipid rafts and that such a SNARE-raft association may be essential for spatial control of exocytosis and/or regulation of SNARE functioning.     

Eradication of Helicobacter pylori for the prevention of peptic ulcer rebleeding

AIM: To evaluate the effect of Helicobacter pylori eradication on ulcer bleeding recurrence in a prospective, long-term study including more than 400 patients. METHODS: Patients with peptic ulcer bleeding were prospectively included. H. pylori infection was confirmed by rapid urease test, histology or (13)C-urea breath test. Several eradication regimens were used. Ranitidine 150 mg was administered daily until eradication was confirmed by breath test 8 weeks after completing eradication therapy. Patients with therapy failure received a second or third course of therapy. Patients with eradication success did not receive maintenance anti-ulcer therapy, and were controlled yearly with a repeated breath test. RESULTS: Four hundred and twenty-two patients were followed up for at least 12 months, with a total of 906 patient-years of follow up. Mean age was 59 years, and 35% were previous nonsteroidal anti-inflammatory drug (NSAID) users. Sixty-nine percent had duodenal, 24% gastric, and 7% pyloric ulcer. Recurrence of bleeding was demonstrated in two patients at 1 year (incidence: 0.22% per patient-year of follow up), which occurred after NSAID use in both cases. CONCLUSION: Peptic ulcer rebleeding does not occur in patients with complicated ulcers after H. pylori eradication. Maintenance anti-ulcer (antisecretory) therapy is not necessary if eradication is achieved.     

Environmental evaluation of single-use and reusable cups

Goal, Scope and Background The objective of the study was to determine the environmental effects of the resuable cup used during a major event (which took place in Barcelona, Universal Forum of Cultures, 2004), compared with a single-use cup of the same composition (polypropylene) but with different physical characteristics such as mass, shape and capacity. Methods To perform the environmental evaluations and the comparison of both types of cups, the SimaPro software developed and marketed by PRé Consultants was used. The environmental evaluation of the reusable cup was compared with that of a single-use cup using the LCA methodology [6]. The functional unit used was: ‘Serving 1000 liters of draught beverages’. The objective of the study was to find the minimum number of cycles the reusable cup has to do so that its environmental impact is smaller than that of the single-use cup. Results and Conclusions Taking into account all the hypotheses put forward, the study drew the conclusion that the minimum number of uses of the reusable cup necessary for it to have a smaller environmental impact than the single-use cup is 10. The contribution of each process taking part in the entire life cycle of the cups was also studied in detail. In the case of the single-use cup, the most important contribution to all the impact categories is due to the production of polypropylene and the fabrication of the cup, except for the heavy metals category where it is due to the management of the waste coming from the cup’s use. In the case of the reusable cup being used 10 times, the contribution to the different impact categories of the waste generated by the cup’s use is negligible compared to the contribution of the fabrication and washing processes. In addition, the washing process is the one which contributes most to the ozone layer depletion, heavy metals and carcinogens categories. As the number of uses of the reusable cup increases, the contribution to all the environmental impact categories decreases. However, this reduction is not as significant for the ozone layer depletion, heavy metals and carcinogens categories. This is due to the washing process and the fact that the electrical consumption associated with it increases with the number of washings and, consequently, of uses. Recommendations and Perspectives From the environmental point of view, the reusable cup must be used at least 10 times to have less impact than the single-use cup. This is mainly due to the higher weight of the reusable cup and, therefore, the greater amount of raw material needed for its fabrication. If the LCA methodology had been introduced during the design of the reusable cup, its weight would have been lower. This modification would have resulted in a reduction of the environmental impact associated with the use of the reusable cup and, consequently, a smaller number of uses would have been necessary to attain the same level of impact as the single-use cup.     

Tor1, Sch9 and PKA downregulation in quiescence rely on Mtl1 to preserve mitochondrial integrity and cell survival

Here we show that Mtl1, member of the cell wall integrity pathway of Saccharomyces cerevisiae, plays a positive role in chronological life span (CLS). The absence of Mtl1 shortens CLS and causes impairment in the mitochondrial function. This is reflected in a descent in oxygen consumption during the postdiauxic state, an increase in the uncoupled respiration and mitochondrial membrane potential and also a descent in aconitase activity. We demonstrate that all these effects are a consequence of signalling defects suppressed by TOR1 (target of rapamycin) and SCH9 deletion and less efficiently by Protein kinase A (PKA) inactivation. Mtl1 also plays a role in the regulation of both Bcy1 stability and phosphorylation, mainly in response to glucose depletion. In postdiauxic phase and in conditions of glucose depletion, Mtl1 negatively regulates TOR1 function leading to Sch9 inactivation and Bcy1 phosphorylation converging in PKA inhibition. Slt2/Mpk1 kinase partially contributes to Bcy1 phosphorylation, although additional targets are not excluded. Mtl1 links mitochondrial dysfunction with TOR and PKA pathways in quiescence, glucose being the main signalling molecule.     

A validated LC-MS/MS assay for quantification of 24(S)-hydroxycholesterol in plasma and cerebrospinal fluid

24(S)-hydroxycholesterol [24(S)-HC] is a cholesterol metabolite that is formed almost exclusively in the brain. The concentrations of 24(S)-HC in cerebrospinal fluid (CSF) and/or plasma might be a sensitive marker of altered cholesterol metabolism in the CNS. A highly sensitive 2D-LC-MS/MS assay was developed for the quantification of 24(S)-HC in human plasma and CSF. In the development of an assay for 24(S)-HC in CSF, significant nonspecific binding of 24(S)-HC was observed and resolved with the addition of 2.5% 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) into CSF samples. The sample preparation consists of liquid-liquid extraction with methyl-tert-butyl ether and derivatization with nicotinic acid. Good linearity was observed in a range from 1 to 200 ng/ml and from 0.025 to 5 ng/ml, for plasma and CSF, respectively. Acceptable precision and accuracy were obtained for concentrations over the calibration curve ranges. Stability of 24(S)-HC was reported under a variety of storage conditions. This method has been successfully applied to support a National Institutes of Health-sponsored clinical trial of HP-β-CD in Niemann-Pick type C1 patients, in which 24(S)-HC is used as a pharmacodynamic biomarker.     

Highly efficient protein misfolding cyclic amplification

Protein misfolding cyclic amplification (PMCA) provides faithful replication of mammalian prions in vitro and has numerous applications in prion research. However, the low efficiency of conversion of PrP(C) into PrP(Sc) in PMCA limits the applicability of PMCA for many uses including structural studies of infectious prions. It also implies that only a small sub-fraction of PrP(C) may be available for conversion. Here we show that the yield, rate, and robustness of prion conversion and the sensitivity of prion detection are significantly improved by a simple modification of the PMCA format. Conducting PMCA reactions in the presence of Teflon beads (PMCAb) increased the conversion of PrP(C) into PrP(Sc) from ～10% to up to 100%. In PMCAb, a single 24-hour round consistently amplified PrP(Sc) by 600-700-fold. Furthermore, the sensitivity of prion detection in one round (24 hours) increased by 2-3 orders of magnitude. Using serial PMCAb, a 1012-fold dilution of scrapie brain material could be amplified to the level detectible by Western blotting in 3 rounds (72 hours). The improvements in amplification efficiency were observed for the commonly used hamster 263K strain and for the synthetic strain SSLOW that otherwise amplifies poorly in PMCA. The increase in the amplification efficiency did not come at the expense of prion replication specificity. The current study demonstrates that poor conversion efficiencies observed previously have not been due to the scarcity of a sub-fraction of PrP(C) susceptible to conversion nor due to limited concentrations of essential cellular cofactors required for conversion. The new PMCAb format offers immediate practical benefits and opens new avenues for developing fast ultrasensitive assays and for producing abundant quantities of PrP(Sc)in vitro.     

Throwing velocities, anthropometric characteristics, and efficacy indices of women's European water polo subchampions

Water polo is a team sport characterized by a high-intensity, intermittent activity, where technical and tactical aspect are of a great importance. For that reason, the main aim of this study was to define the anthropometrical characteristics, maximum isometric grip strength, training and competition throwing velocities, and the efficacy indices in female high-level water polo players. A second purpose was to examine the differences between the throwing velocities in training vs. European championships in the water polo female national team. Ten elite trained female water polo players participated in this study. Before the competitive phase of their season, the following measures were taken: standard anthropometry, static and dynamic training throwing velocities, and hand-grip dynamometry. In the competitive phase, efficacy indices, average and maximum throwing velocities from all the participants were also determined. Significant differences (p ≤ 0.05) were found between different training situations and different competitive throwing velocities. We concluded that elite female water polo players modify their throwing velocity depending if the throw is performed during training or competitive situation.