Nonesterified cholesterol content of lysosomes modulates susceptibility to oxidant-induced permeabilization

Reactive oxygen species (ROS) can induce lysosomal membrane permeabilization (LMP). Photoirradiation of murine hepatoma 1c1c7 cultures preloaded with the photosensitizer NPe6 generates singlet oxygen within acidic organelles and causes LMP and the activation of procaspases. Treatment with the cationic amphiphilic drugs (CADs) U18666A, imipramine, and clozapine stimulated the accumulation of filipin-stainable nonesterified cholesterol/sterols in late endosomes/lysosomes, but not in mitochondria. Concentration-response studies demonstrated an inverse relationship between lysosomal nonesterified cholesterol/sterol contents and susceptibility to NPe6 photoirradiation-induced intracellular membrane oxidation, LMP, and activation of procaspase-9 and -3. Similarly, the kinetics of restoration of NPe6 photoirradiation-induced LMP paralleled the losses of lysosomal cholesterol that occurred upon replating U18666A-treated cultures in CAD-free medium. Consistent with the oxidation of lysosomal cholesterol, filipin staining in U18666A-treated cultures progressively decreased with increasing photoirradiating light dose. U18666A also suppressed the induction of LMP and procaspase activation by exogenously added hydrogen peroxide. However, neither U18666A nor imipramine suppressed the induction of apoptosis by agents that did not directly induce LMP. These studies indicate that lysosomal nonesterified cholesterol/sterol content modulates susceptibility to ROS-induced LMP and possibly does so by being an alternative target for oxidants and lowering the probability of damage to other lysosomal membrane lipids and/or proteins.     

Report from the second cytomegalovirus and immunosenescence workshop

The Second International Workshop on CMV & Immunosenescence was held in Cambridge, UK, 2-4th December, 2010. The presentations covered four separate sessions: cytomegalovirus and T cell phenotypes; T cell memory frequency, inflation and immunosenescence; cytomegalovirus in aging, mortality and disease states; and the immunobiology of cytomegalovirus-specific T cells and effects of the virus on vaccination. This commentary summarizes the major findings of these presentations and references subsequently published work from the presenter laboratory where appropriate and draws together major themes that were subsequently discussed along with new areas of interest that were highlighted by this discussion.     

Selenium effects on arsenic cytotoxicity and protein phosphorylation in human kidney cells using chip-based nanoLC-MS/MS

Although arsenic toxicity is well known, little is known of how it exerts its effects at the proteome level. Protein phosphorylation is an important post-translational modification in the regulation of cell signaling. Despite the importance of protein phosphorylation, the identification and characterization of phosphorylated proteins, as influenced by interaction between arsenic and selenium species have not been fully studied. The aim of this study is to identify phosphorylation in arsenic toxified cells, with and without selenium present. Here, we identify the phosphorylated proteins related to post translational modifications (PTMs) after inorganic arsenic (iAs) and selenomethionine (SeMet) were inoculated together with HEK293 human kidney cells. In this study, using TiO(2)-based nanoLC-phosphochip? coupled to ESI-MS we observed phosphorylated peptide enrichment and significant reduction in sample complexity. The identification of phosphorylated proteins in highly complex digests of cell lysate were markedly different with As toxification only, or when in the presence of SeMet. Several phosphorylation sites and proteins are identified using Spectrum Mill and Mascot protein data base search engines. Cytotoxicity studies showed that SeMet significantly reduces the cytotoxic effect of iAs in HEK293 cells, while inorganic selenium did not.     

Protein profiling underscores immunological functions of uterine cervical mucus plug in human pregnancy

The cervical mucus plug (CMP) differs from the cervical secretions of non-pregnant women, and is the ultimate sealant of the uterine cavity during pregnancy. Although several studies have analyzed biochemical properties of large glycoproteins in the CMP, comprehensive information about its protein composition is yet unavailable. We hypothesized that protein profiling of the CMP could provide key clues to its physiological functions in pregnancy. For this purpose, five CMPs obtained from women in labor at term were analyzed by LC-MS/MS. Out of 291 total proteins identified, 137 were detected in two or more samples, which included S100A8, S100A9, and complement proteins (C3, C4a, C4b, C6, and C8g). Several proteins, which have not been described in the cervical mucus of non-pregnant women or in cervicovaginal fluids, such as CD81 antigen and pregnancy zone protein, were also identified. Gene ontology analysis of identified proteins showed significant enrichment of 28 biological processes such as 'activation of plasma proteins involved in acute inflammatory response' and 'positive regulation of cholesterol esterification'. We report the proteome of CMPs from pregnant women at term for the first time, and the overall findings strongly suggest an important role for the CMP in the maintenance of pregnancy and parturition.     

Indigenous arbuscular mycorrhizal fungal assemblages protect grassland host plants from pathogens

Plant roots can establish associations with neutral, beneficial and pathogenic groups of soil organisms. Although it has been recognized from the study of individual isolates that these associations are individually important for plant growth, little is known about interactions of whole assemblages of beneficial and pathogenic microorganisms associating with plants.We investigated the influence of an interaction between local arbuscular mycorrhizal (AM) fungal and pathogenic/saprobic microbial assemblages on the growth of two different plant species from semi-arid grasslands in NE Germany (Mallnow near Berlin). In a greenhouse experiment each plant species was grown for six months in either sterile soil or in sterile soil with one of three different treatments: 1) an AM fungal spore fraction isolated from field soil from Mallnow; 2) a soil pathogen/saprobe fraction consisting of a microbial community prepared with field soil from Mallnow and; 3) the combined AM fungal and pathogen/saprobe fractions. While both plant species grew significantly larger in the presence of AM fungi, they responded negatively to the pathogen/saprobe treatment. For both plant species, we found evidence of pathogen protection effects provided by the AM fungal assemblages. These results indicate that interactions between assemblages of beneficial and pathogenic microorganisms can influence the growth of host plants, but that the magnitude of these effects is plant species-specific.     

Seasonal variation of Ralstonia solanacearum biovar 2 populations in a Spanish river: recovery of stressed cells at low temperatures

The presence of Ralstonia solanacearum biovar 2 in the watercourses of European countries is increasing, but little is known about its ecology in aquatic habitats. The detection of this pathogen in 2000 in one Spanish river led us to study its population density at different locations on the river over a period of 3 years. During 2000 and 2001, the pathogen was recovered at low densities (10 to 80 CFU/ml) by direct plating on modified SMSA agar from water samples at 14 degrees C or higher, but its isolation was usually unsuccessful at temperatures below 9 degrees C. To monitor the pathogen's abundance in winter, we used two liquid selective media for enrichment (at 29 and 35 degrees C) and compared them by using spiked river water samples: modified Wilbrink broth (MWB) was more efficient than modified SMSA broth for double-antibody-sandwich indirect enzyme-linked immunosorbent assay (DASI-ELISA) detection of R. solanacearum. Enrichment in MWB at both temperatures allowed us to recover R. solanacearum cells that were nonculturable on solid media up to 25 days after their entry into the viable but nonculturable state. When we applied this technique to water samples during the cold months of 2001 and 2002, we obtained the best detection results by the most-probable-number method after enrichment at 35 degrees C with MWB. The enrichment protocol was combined with DASI-ELISA and validated by Co-PCR to detect both naturally and artificially starved and cold-stressed cells in water, which were still infective. Overall, the data from this study demonstrate the effects of temperature variation on the population and culturability of R. solanacearum cells on solid media and their survival at low temperatures.     

Regulation of membrane trafficking by a novel Cdc42-related protein in Caenorhabditis elegans epithelial cells

Rho GTPases are mainly known for their implication in cytoskeleton remodeling. They have also been recently shown to regulate various aspects of membrane trafficking. Here, we report the identification and the characterization of a novel Caenorhabditis elegans Cdc42-related protein, CRP-1, that shows atypical enzymatic characteristics in vitro. Expression in mouse fibroblasts revealed that, in contrast with CDC-42, CRP-1 was unable to reorganize the actin cytoskeleton and mainly localized to trans-Golgi network and recycling endosomes. This subcellular localization, as well as its expression profile restricted to a subset of epithelial-like cells in C. elegans, suggested a potential function for this protein in polarized membrane trafficking. Consistent with this hypothesis, alteration of CRP-1 expression affected the apical trafficking of CHE-14 in vulval and rectal epithelial cells and sphingolipids (C(6)-NBD-ceramide) uptake and/or trafficking in intestinal cells. However, it did not affect basolateral trafficking of myotactin in the pharynx and the targeting of IFB-2 and AJM-1, two cytosolic apical markers of intestine epithelial cells. Hence, our data demonstrate a function for CRP-1 in the regulation of membrane trafficking in a subset of cells with epithelial characteristics.     

Oral albuterol dosing during the latter stages of a resistance exercise program

Subjects performed isoload variable resistance exercise (REX) 3 days per week. After 10 weeks, they received a double-blind albuterol (n = 11) or placebo (n = 11) capsule assignment with no crossover and continued training. During the first week of capsule administration, dosages were increased from 4 mg to 16 mg daily and then maintained for 14 days. At weeks 0, 10, and 13, we measured upper arm and thigh cross-sectional area, knee and elbow extensor and flexor (KE, KF, EE, EF) strength at 3 angular velocities, and lean body mass. Data after 10 weeks showed insignificant between-group differences. From weeks 10-13, as subjects continued REX training, albuterol evoked higher (p < 0.05) KE-KF strength gains at multiple velocities versus placebo dosing. A higher lean body mass trend also occurred with albuterol from weeks 10-13. Results suggest that albuterol augments REX to provide greater strength gains from hypertrophic factors than an REX-placebo assignment.