Caspase-8 binding to cardiolipin in giant unilamellar vesicles provides a functional docking platform for bid

Caspase-8 is involved in death receptor-mediated apoptosis in type II cells, the proapoptotic programme of which is triggered by truncated Bid. Indeed, caspase-8 and Bid are the known intermediates of this signalling pathway. Cardiolipin has been shown to provide an anchor and an essential activating platform for caspase-8 at the mitochondrial membrane surface. Destabilisation of this platform alters receptor-mediated apoptosis in diseases such as Barth Syndrome, which is characterised by the presence of immature cardiolipin which does not allow caspase-8 binding. We used a simplified in vitro system that mimics contact sites and/or cardiolipin-enriched microdomains at the outer mitochondrial surface in which the platform consisting of caspase-8, Bid and cardiolipin was reconstituted in giant unilamellar vesicles. We analysed these vesicles by flow cytometry and confirm previous results that demonstrate the requirement for intact mature cardiolipin for caspase-8 activation and Bid binding and cleavage. We also used confocal microscopy to visualise the rupture of the vesicles and their revesiculation at smaller sizes due to alteration of the curvature following caspase-8 and Bid binding. Biophysical approaches, including Laurdan fluorescence and rupture/tension measurements, were used to determine the ability of these three components (cardiolipin, caspase-8 and Bid) to fulfil the minimal requirements for the formation and function of the platform at the mitochondrial membrane. Our results shed light on the active functional role of cardiolipin, bridging the gap between death receptors and mitochondria

Prevention of falls and fractures in old people by administration of calcium and vitamin d. randomized clinical trial

<p>Abstract</p> <p>Background</p> <p>There are many studies that associate vitamin D serum levels in older persons with muscle strength, physical performance and risk of fractures and falls. However, current evidence is insufficient to make a general recommendation for administrating calcium and vitamin D to older persons. The objective of this study is to determine the effectiveness of calcium and vitamin D supplementation in improving musculoskeletal function and decreasing the number of falls in person aged over 65 years.</p> <p>Methods/Design</p> <p>Phase III, randomized, double blind, placebo-controlled trial to evaluate the efficacy of already marketed drugs in a new indication. It will be performed at Primary Care doctor visits at several Healthcare Centers in different Spanish Health Areas. A total of 704 non-institutionalized subjects aged 65 years or older will be studied (sample size calculated for a statistical power of 80%, alpha error 0.05, annual incidence of falls 30% and expected reduction of 30% to 20% and expected loss to follow up of 20%). The test drug containing 800 IU of vitamin D and 1000 mg of calcium will be administered daily. The control group will receive a placebo. The subjects will be followed up over two years. The primary variable will be the incidence of spontaneous falls. The secondary variables will include: consequences of the falls (fractures, need for hospitalization), change in calcidiol plasma levels and other analytical determinations (transaminases, PTH, calcium/phosphorous, albumin, creatinine, etc.), change in bone mass by densitometry, change in muscle strength in the dominant hand and change in musculoskeletal strength, risk factors for falls, treatment compliance, adverse effects and socio-demographic data.</p> <p>Discussion</p> <p>The following principles have been considered in the development of this Project: the product data are sufficient to ensure that the risks assumed by the study participants are acceptable, the study objectives will probably provide further knowledge on the problem studied and the available information justifies the performance of the study and its possible risk for the participants.</p> <p>If calcium and vitamin D supplementation is effective in the prevention of falls and fractures in the elderly population, a recommendation may be issued with the aim of preventing some of the consequences of falls that affect quality of life and the ensuing personal, health and social costs.</p> <p>Trial Registration</p> <p>ClinicalTrials.gov: <a href="http://www.clinicaltrials.gov/ct2/show/NCT01452243">NCT01452243</a></p> <p>Clinical trial authorized by the Spanish Medicines Agency: EudraCT number 2006-001643-63.</p

Evolutionary and pulsational properties of white dwarf stars

Abridged. White dwarf stars are the final evolutionary stage of the vast majority of stars, including our Sun. The study of white dwarfs has potential applications to different fields of astrophysics. In particular, they can be used as independent reliable cosmic clocks, and can also provide valuable information about the fundamental parameters of a wide variety of stellar populations, like our Galaxy and open and globular clusters. In addition, the high densities and temperatures characterizing white dwarfs allow to use these stars as cosmic laboratories for studying physical processes under extreme conditions that cannot be achieved in terrestrial laboratories. They can be used to constrain fundamental properties of elementary particles such as axions and neutrinos, and to study problems related to the variation of fundamental constants. In this work, we review the essentials of the physics of white dwarf stars. Special emphasis is placed on the physical processes that lead to the formation of white dwarfs as well as on the different energy sources and processes responsible for chemical abundance changes that occur along their evolution. Moreover, in the course of their lives, white dwarfs cross different pulsational instability strips. The existence of these instability strips provides astronomers with an unique opportunity to peer into their internal structure that would otherwise remain hidden from observers. We will show that this allows to measure with unprecedented precision the stellar masses and to infer their envelope thicknesses, to probe the core chemical stratification, and to detect rotation rates and magnetic fields. Consequently, in this work, we also review the pulsational properties of white dwarfs and the most recent applications of white dwarf asteroseismology.Comment: 85 pages, 28 figures. To be published in The Astronomy and Astrophysics Revie

Genetic variability of hepatitis C virus before and after combined therapy of interferon plus ribavirin

We present an analysis of the selective forces acting on two hepatitis C virus genome regions previously postulated to be involved in the viral response to combined antiviral therapy. One includes the three hypervariable regions in the envelope E2 glycoprotein, and the other encompasses the PKR binding domain and the V3 domain in the NS5A region. We used a cohort of 22 non-responder patients to combined therapy (interferon alpha-2a plus ribavirin) for which samples were obtained before initiation of therapy and after 6 or/and 12 months of treatment. A range of 25-100 clones per patient, genome region and time sample were sequenced. These were used to detect general patterns of adaptation, to identify particular adaptation mechanisms and to analyze the patterns of evolutionary change in both genome regions. These analyses failed to detect a common adaptive mechanism for the lack of response to antiviral treatment in these patients. On the contrary, a wide range of situations were observed, from patients showing no positively selected sites to others with many, and with completely different topologies in the reconstructed phylogenetic trees. Altogether, these results suggest that viral strategies to evade selection pressure from the immune system and antiviral therapies do not result from a single mechanism and they are likely based on a range of different alternatives, in which several different changes, or their combination, along the HCV genome confer viruses the ability to overcome strong selective [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]

Relationships between heavy metal concentrations in three different body fluids and male reproductive parameters: a pilot study

<p>Abstract</p> <p>Background</p> <p>Animal studies have shown the reproductive toxicity of a number of heavy metals. Very few human observational studies have analyzed the relationship between male reproductive function and heavy metal concentrations in diverse biological fluids.</p> <p>Methods</p> <p>The current study assessed the associations between seminal and hormonal parameters and the concentration of the 3 most frequent heavy metal toxicants (lead, cadmium and mercury) in three different body fluids. Sixty one men attending infertility clinics that participated in a case-control study to explore the role of environmental toxins and lifestyles on male infertility were analyzed. Concentration of lead, cadmium and mercury were measured in blood and seminal plasma and whole blood using anodic stripping voltammetry and atomic absorption spectrophotometry. Serum samples were analyzed for follicle-stimulating hormone, luteinizing hormone and testosterone. Semen analyses were performed according to World Health Organization criteria. Mann-Whitney test and Spearman's rank correlations were used for unadjusted analyses. Multiple linear regression models were performed controlling for age, body mass index and number of cigarettes per day.</p> <p>Results</p> <p>There were no significant differences between cases and controls in the concentrations of heavy metals in any of the three body fluids. In multivariate analyses using all subjects no significant associations were found between serum hormone levels and metal concentrations. However there was a significant positive association between the percentage of immotile sperms and seminal plasma levels of lead and cadmium.</p> <p>Conclusions</p> <p>Our results suggest that the presence of lead and cadmium in the reproductive tract of men may be related to a moderate alteration of their seminal parameters.</p

Extremely Long-Lived Stigmas Allow Extended Cross-Pollination Opportunities in a High Andean Plant

High-elevation ecosystems are traditionally viewed as environments in which predominantly autogamous breeding systems should be selected because of the limited pollinator availability. Chaetanthera renifolia (Asteraceae) is an endemic monocarpic triennial herb restricted to a narrow altitudinal range within the high Andes of central Chile (3300–3500 m a.s.l.), just below the vegetation limit. This species displays one of the larger capitulum within the genus. Under the reproductive assurance hypothesis, and considering its short longevity (monocarpic triennial), an autogamous breeding system and low levels of pollen limitation would be predicted for C. renifolia. In contrast, considering its large floral size, a xenogamous breeding system, and significant levels of pollen limitation could be expected. In addition, the increased pollination probability hypothesis predicts prolonged stigma longevity for high alpine plants. We tested these alternative predictions by performing experimental crossings in the field to establish the breeding system and to measure the magnitude of pollen limitation in two populations of C. renifolia. In addition, we measured the stigma longevity in unpollinated and open pollinated capitula, and pollinator visitation rates in the field. We found low levels of self-compatibility and significant levels of pollen limitation in C. renifolia. Pollinator visitation rates were moderate (0.047–0.079 visits per capitulum per 30 min). Although pollinator visitation rate significantly differed between populations, they were not translated into differences in achene output. Finally, C. renifolia stigma longevity of unpollinated plants was extremely long and significantly higher than that of open pollinated plants (26.3±2.8 days vs. 10.1±2.2, respectively), which gives support to the increased pollination probability hypothesis for high-elevation flowering plants. Our results add to a growing number of studies that show that xenogamous breeding systems and mechanisms to increase pollination opportunities can be selected in high-elevation ecosystems

Inhibition of PbGP43 expression may suggest that gp43 is a virulence factor in Paracoccidioides brasiliensis

ABSTARCT: Glycoprotein gp43 is an immunodominant diagnostic antigen for paracoccidioidomycosis caused by Paracoccidioides brasiliensis. It is abundantly secreted in isolates such as Pb339. It is structurally related to beta-1,3-exoglucanases, however inactive. Its function in fungal biology is unknown, but it elicits humoral, innate and protective cellular immune responses; it binds to extracellular matrix-associated proteins. In this study we applied an antisense RNA (aRNA) technology and Agrobacterium tumefaciens-mediated transformation to generate mitotically stable PbGP43 mutants (PbGP43 aRNA) derived from wild type Pb339 to study its role in P. brasiliensis biology and during infection. Control PbEV was transformed with empty vector. Growth curve, cell vitality and morphology of PbGP43 aRNA mutants were indistinguishable from those of controls. PbGP43 expression was reduced 80-85% in mutants 1 and 2, as determined by real time PCR, correlating with a massive decrease in gp43 expression. This was shown by immunoblotting of culture supernatants revealed with anti-gp43 mouse monoclonal and rabbit polyclonal antibodies, and also by affinity-ligand assays of extracellular molecules with laminin and fibronectin. In vitro, there was significantly increased TNF-α production and reduced yeast recovery when PbGP43 aRNA1 was exposed to IFN-γ-stimulated macrophages, suggesting reduced binding/uptake and/or increased killing. In vivo, fungal burden in lungs of BALB/c mice infected with silenced mutant was negligible and associated with decreased lung ΙΛ-10 and IL-6. Therefore, our results correlated low gp43 expression with lower pathogenicity in mice, but that will be definitely proven when PbGP43 knockouts become available.

Two cold inducible genes encoding lipid transfer protein LTP4 from barley show differential responses to bacterial pathogens

The barley genesHvLtp4.2 andHvLtp4.3 both encode the lipid transfer protein LTP4 and are less than 1 kb apart in tail-to-tail orientation. They differ in their non-coding regions from each other and from the gene corresponding to a previously reportedLtp4 cDNA (nowLtp4.1). Southern blot analysis indicated the existence of three or moreLtp4 genes per haploid genome and showed considerable polymorphism among barley cultivars. We have investigated the transient expression of genesHvLtp4.2 andHvLtp4.3 following transformation by particle bombardment, using promoter fusions to the-glucuronidase reporter sequence. In leaves, activities of the two promoters were of the same order as those of the sucrose synthase (Ss1) and cauliflower mosaic virus 35S promoters used as controls. Their expression patterns were similar, except thatLtp4.2 was more active thanLtp4.3 in endosperm, andLtp4.3 was active in roots, whileLtp4.2 was not. The promoters of both genes were induced by low temperature, both in winter and spring barley cultivars. Northern blot analysis, using theLtp4-specific probe, indicated thatXanthomonas campestris pv.translucens induced an increase over basal levels ofLtp4 mRNA, whilePseudomonas syringae pv.japonica caused a decrease. TheLtp4.3-Gus promoter fusion also responded in opposite ways to these two compatible bacterial pathogens, whereas theLtp4.2-Gus construction did not respond to infectio

The stellar and sub-stellar IMF of simple and composite populations

The current knowledge on the stellar IMF is documented. It appears to become top-heavy when the star-formation rate density surpasses about 0.1Msun/(yr pc^3) on a pc scale and it may become increasingly bottom-heavy with increasing metallicity and in increasingly massive early-type galaxies. It declines quite steeply below about 0.07Msun with brown dwarfs (BDs) and very low mass stars having their own IMF. The most massive star of mass mmax formed in an embedded cluster with stellar mass Mecl correlates strongly with Mecl being a result of gravitation-driven but resource-limited growth and fragmentation induced starvation. There is no convincing evidence whatsoever that massive stars do form in isolation. Various methods of discretising a stellar population are introduced: optimal sampling leads to a mass distribution that perfectly represents the exact form of the desired IMF and the mmax-to-Mecl relation, while random sampling results in statistical variations of the shape of the IMF. The observed mmax-to-Mecl correlation and the small spread of IMF power-law indices together suggest that optimally sampling the IMF may be the more realistic description of star formation than random sampling from a universal IMF with a constant upper mass limit. Composite populations on galaxy scales, which are formed from many pc scale star formation events, need to be described by the integrated galactic IMF. This IGIMF varies systematically from top-light to top-heavy in dependence of galaxy type and star formation rate, with dramatic implications for theories of galaxy formation and evolution.Comment: 167 pages, 37 figures, 3 tables, published in Stellar Systems and Galactic Structure, Vol.5, Springer. This revised version is consistent with the published version and includes additional references and minor additions to the text as well as a recomputed Table 1. ISBN 978-90-481-8817-