Genomic instability is associated with natural life span variation in Saccharomyces cerevisiae

Increasing genomic instability is associated with aging in eukaryotes, but the connection between genomic instability and natural variation in life span is unknown. We have quantified chronological life span and loss-of-heterozygosity (LOH) in 11 natural isolates of Saccharomyces cerevisiae. We show that genomic instability increases and mitotic asymmetry breaks down during chronological aging. The age-dependent increase of genomic instability generally lags behind the drop of viability and this delay accounts for approximately 50% of the observed natural variation of replicative life span in these yeast isolates. We conclude that the abilities of yeast strains to tolerate genomic instability co-vary with their replicative life spans. To the best of our knowledge, this is the first quantitative evidence that demonstrates a link between genomic instability and natural variation in life span.     

Cathepsin L inhibitor I blocks mitotic chromosomes decondensation during cleavage cell cycles of sea urchin embryos

We have previously reported that sperm histones (SpH) degradation after fertilization is catalyzed by a cystein-protease (SpH-protease). Its inhibition blocks the degradation of SpH in vivo and also aborts sea urchin development at the initial embryonic cell cycles. It remains unknown if this effect is a consequence of the persistence of SpH on zygotic chromatin, or if this protease is involved per-se in the progression of the embryonic cell cycles. To discriminate among these two options we have inhibited this protease at a time when male chromatin remodeling was completed and the embryos were engaged in the second cell cycle of the cleavage divisions. The role of this enzyme in cell cycle was initially analyzed by immuno-inhibiting its SpH degrading activity in one of the two blastomeres after the initial cleavage division, while the other blastomere was used as a control. We found that in the blastomere injected with the anti-SpH-protease antibodies the cytokinesis was arrested, the chromatin failed to decondense after mitosis and BrdU incorporation into DNA was blocked. Since the N-terminal sequence and the SpH protease was homologous to the cathepsin L (Cat L) family of proteases, we subsequently investigated if the deleterious effect of the inhibition of this protease is related to its Cat L activity. In this context we analyzed the effect of Cat L inhibitor I (Z-Phe-Phe-CH(2)F) on embryonic development. We found that the addition of 100 uM of this inhibitor to the embryos harvested at the time of the initial cleavage division (80 min p.i.) mimics perfectly the effects of the immuno-inhibition of this enzyme obtained by microinjecting the anti-SpH-protease antibodies. Taken together these results indicate that the activity of this protease is required for embryonic cell cycle progression. Interestingly, we observed that when this protease was inhibited the chromatin decondensation after mitosis was abolished indicating that the inhibition of this enzyme affects chromosomes decondensation after mitosis.     

Selectivity in the mechanism of action of antimicrobial mastoparan peptide Polybia-MP1

Many potent antimicrobial peptides also present hemolytic activity, an undesired collateral effect for the therapeutic application. Unlike other mastoparan peptides, Polybia-MP1 (IDWKKLLDAAKQIL), obtained from the venom of the social wasp Polybia paulista, is highly selective of bacterial cells. The study of its mechanism of action demonstrated that it permeates vesicles at a greater rate of leakage on the anionic over the zwitterionic, impaired by the presence of cholesterol or cardiolipin; its lytic activity is characterized by a threshold peptide to lipid molar ratio that depends on the phospholipid composition of the vesicles. At these particular threshold concentrations, the apparent average pore number is distinctive between anionic and zwitterionic vesicles, suggesting that pores are similarly formed depending on the ionic character of the bilayer. To prospect the molecular reasons for the strengthened selectivity in Polybia-MP1 and its absence in Mastoparan-X, MD simulations were carried out. Both peptides presented amphipathic alpha-helical structures, as previously observed in Circular Dichroism spectra, with important differences in the extension and stability of the helix; their backbone solvation analysis also indicate a different profile, suggesting that the selectivity of Polybia-MP1 is a consequence of the distribution of the charged and polar residues along the peptide helix, and on how the solvent molecules orient themselves according to these electrostatic interactions. We suggest that the lack of hemolytic activity of Polybia-MP1 is due to the presence and position of Asp residues that enable the equilibrium of electrostatic interactions and favor the preference for the more hydrophilic environment.     

Antineoplastic 31-norcycloartanones from Solanum cernuum vell

Triterpenoids with 31-norcycloartanone structure were isolated for the first time from the Solanum genus. Cycloeucalenone and 24-oxo-31-norcycloartanone were the main constituents of the dichloromethane extract of Solanum cernuum Vell. leaves [7% (w/w) and 1.47% (w/w)]. Both triterpenoids were tested against human tumour cell lines, and 24-oxo-31-norcycloartanone was significantly active and selective against the lung tumour cell line NCI-H460 with total growth inhibition at 1.10 microg/mL, growth inhibition 50 at 0.19 microg/mL and lethal concentration 50 at 8.43 microg/mL, while cycloeucalenone showed poor activity. A homologous series of alkanes (C25-C34), beta-sitosterol, and the xanthophyll lutein were also identified. The antiulcer activity was assayed for the dichloromethane extract. In the gastric ulcer model induced by 95% ethanol, administration of 500, 1000 and 2000 mg/kg/po dichloromethane extract gave ulcer lesion indices of, respectively, 38.2, 61.0 and 81.9%, while carbenoxolone inhibited 88.9% at 200 mg/kg. In the gastric ulcer model induced by indomethacin the dichloromethane extract showed a small percentage of lesion inhibition. The ethanol extract was also analyzed and was mainly composed of glycoalkaloids, peptides and disaccharides.     

Effects of 2(RS)-n-propylthiazolidine-4(R)-carboxylic acid on 4-hydroxy-2-nonenal-induced apoptotic T cell death

4-Hydroxy-2-nonenal (HNE), the aldehydic product of lipid peroxidation, is associated with multiple immune dysfunctions, such as HIV and hepatitis C virus infection. HNE-induced immunosuppression could be due to a decrease in CD4+ T lymphocyte activation or proliferation. Glutathione (GSH) is the most abundant endogenous antioxidant in cells, and an adduct between HNE and GSH has been suggested to be a marker of oxidative stress. Our earlier studies showed that HNE induced cytotoxicity and Akt inactivation, which led to the enhancement of FasL expression and concomitantly decreased cellular FLICE-like inhibitory protein (c-FLIP(S)) levels. In this study, we found that HNE caused intracellular GSH depletion in Jurkat T cells, and we further investigated the role of 2(RS)-n-propylthiazolidine-4(R)-carboxylic acid (PTCA), a GSH prodrug, in attenuating HNE-induced cytotoxicity in CD4+ T lymphocytes. The results show that PTCA protected against HNE-induced apoptosis and depletion of intracellular GSH. PTCA also suppressed FasL expression through increasing levels of Akt kinase as well as antiapoptotic c-FLIP(S) and decreasing the activation of type 2 protein serine/threonine phosphatase. Taken together, these data demonstrate a novel correlation between GSH levels and Akt activation in T lymphocyte survival, which involves FasL down-regulation and c-FLIP(S) expression through increasing intracellular GSH levels. This suggests that PTCA could potentially be used in the treatment of oxidative stress-induced immunosuppressive diseases.     

Secretome of HepG2 cells infected with dengue virus: implications for pathogenesis

Viral hemorrhagic fever is a clinical syndrome that poses serious global health threat. Among the causative agents, dengue virus (DV) has the highest incidence rate and its infection is the major cause of viral hemorrhagic fever in the world. Although the pathophysiological mechanisms of DV-induced diseases are not yet understood, it is well accepted that liver is a site of viral replication. In this study, we used proteomics to analyze infection of a hepatic cell lineage, HepG2, with DV, focusing on the secreted proteins. 1D-electrophoresis and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) were used, allowing the identification of a total of 107 proteins, among which 35 were found only in control secretome and 24 only in infected cells secretome. To validate these data, we performed 2D-eletrophoresis followed by MALDI-TOF/TOF, resulting in the identification of 20 proteins, 8 of them confirming LC-MS/MS results. We discuss the results obtained taking into account the proteins previously described in the secretome of HepG2 cells, proteins present in human plasma and proteins of interest for dengue pathogenesis. Altogether the data presented here provide clues for the progress in the understanding of the role of liver secretion in the progression of the disease.     

Identification, cloning and functional characterization of a novel dermonecrotic toxin (phospholipase D) from brown spider (Loxosceles intermedia) venom

Brown spider bites are associated with lesions including dermonecrosis, gravitational spreading and a massive inflammatory response, along with systemic problems that may include hematological disturbances and renal failure. The mechanisms by which the venom exerts its noxious effects are currently under investigation. It is known that the venom contains a major toxin (dermonecrotic toxin, biochemically a phospholipase D) that can experimentally induce dermonecrosis, inflammatory response, animal mortality and platelet aggregation. Herein, we describe cloning, heterologous expression, purification and functionality of a novel isoform of the 33 kDa dermonecrotic toxin. Circular dichroism analysis evidenced correct folding for the toxin. The recombinant toxin was recognized by whole venom serum antibodies and by a specific antibody to a previously described dermonecrotic toxin. The identified toxin was found to display phospholipase activity and dermonecrotic properties. Additionally, the toxin caused a massive inflammatory response in rabbit skin dermis, evoked platelet aggregation, increased vascular permeability, caused edema and death in mice. These characteristics in combination with functional studies for other dermonecrotic toxins illustrate that a family of dermonecrotic toxins exists, and includes a novel member with high activity that may be useful for future structural and functional studies.     

Men Gain Additional Psychological Benefits by Adding Exercise to a Weight‐Loss Program

Objective: Adding exercise to a comprehensive weight‐loss program might not only attenuate any psychological distress associated with weight‐loss attempts but also may provide psychological benefits. This study examined whether a diet‐plus‐exercise weight‐loss program improved psychological outcomes more than a diet‐only weight‐loss program or an assessment‐only control group. Research Methods and Procedures: This study was part of a larger 1‐year randomized weight‐loss trial examining the effects of diet and exercise on cardiovascular disease risk factors in 264 overweight adults. Psychological measures specific to weight control (e.g., cognitive restraint, disinhibition, hunger, and body dissatisfaction) as well as traditional measures of psychological distress (e.g., symptoms of depression, anxiety, and stress) were obtained at baseline and 1 year. Results: Men and women in either weight‐loss program reported greater restraint, less disinhibition, and less hunger at 1 year than those in no program. Men in the diet‐plus‐exercise program experienced additional increases in restraint and decreases in hunger than did men in the diet‐only program. Women in the diet‐plus‐exercise program did not experience additional psychological benefits specific to weight control than those in the diet‐only program, despite increases in aerobic capacity. Discussion: The pattern seen for overweight men in the diet‐plus‐exercise program at 1 year—greater restraint, less disinhibition, and less hunger—is similar to the pattern seen in successful weight maintainers. These results underscore the need for innovative strategies that will enhance and sustain the pattern of psychological benefits specific to weight control associated with successful weight loss, especially for overweight women.     

Actin-Based Motility of Intracellular Microbial Pathogens

A diverse group of intracellular microorganisms, including Listeria monocytogenes, Shigella spp., Rickettsia spp., and vaccinia virus, utilize actin-based motility to move within and spread between mammalian host cells. These organisms have in common a pathogenic life cycle that involves a stage within the cytoplasm of mammalian host cells. Within the cytoplasm of host cells, these organisms activate components of the cellular actin assembly machinery to induce the formation of actin tails on the microbial surface. The assembly of these actin tails provides force that propels the organisms through the cell cytoplasm to the cell periphery or into adjacent cells. Each of these organisms utilizes preexisting mammalian pathways of actin rearrangement to induce its own actin-based motility. Particularly remarkable is that while all of these microbes use the same or overlapping pathways, each intercepts the pathway at a different step. In addition, the microbial molecules involved are each distinctly different from the others. Taken together, these observations suggest that each of these microbes separately and convergently evolved a mechanism to utilize the cellular actin assembly machinery. The current understanding of the molecular mechanisms of microbial actin-based motility is the subject of this review.