Molecular and immunological characterization of the major outer membrane proteins of Brucella

Abstract Saccharomyces cerevisiae exponentially growing in basic or 0.7 M NaCl medium were isotopically labelled with ³⁵S-methionine, followed by protein separation and quantification by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) combined with computerised image analysis. The electrophoretic separation resolved about 650 proteins of which 13 displayed significant and at least 2-fold changes in rate of synthesis during saline growth. By sequencing of 2D-PAGE resolved proteins, one of the 8 induced spot, p42.9/5.5, was shown to correspond to the full length (containing the N-terminal extension) product of the GPD 1 gene encoding the cytoplasmic glycerol 3-phosphate dehydrogenase. The expression of the TDH 3 gene, glyceraldehyde 3-phosphate dehydrogenase, and the ENO 2 gene, enolase, decreased during growth in NaCl medium, declines hypothesised to have an impact on the flux to glycerol.     

Effects of exposure to short-term heat stress on fitness components in Drosophila melanogaster

Effects of thermal stress on survival and reproductive success in ten recently collected isofemale lines of Drosophila melanogaster were compared for flies treated as follows: always held at 25° C, placed in an incubator set at 37° C for 120 min, or exposed to 40° C in an incubator for 90 min, with or without previous exposure to 37° C. Short-term exposure to the higher temperature greatly reduced adult survival, the mating frequency of males and females, and female fecundity, which was measured as offspring produced over ten days. Male fertility, measured as the progeny produced by a female mated once, differed little among treatments. Previous exposure to a high, but non-lethal, temperature before exposure to the higher one, improved survival of males and females, and improved offspring production of females. Genetic variation was present among lines for offspring production, but genetic variation for survival was not significant, and genotype by environment interactions for fitness components of females were small. These results indicated low genetic variation in thermal resistance in the studied population, such that a threshold for temperature stress probably exists, above which local extinction is more likely than the evolution of resistance.     

Climate change in our backyards: the reshuffling of North America's winter bird communities

Much of the recent changes in North American climate have occurred during the winter months, and as result, overwintering birds represent important sentinels of anthropogenic climate change. While there is mounting evidence that bird populations are responding to a warming climate (e.g., poleward shifts) questions remain as to whether these species‐specific responses are resulting in community‐wide changes. Here, we test the hypothesis that a changing winter climate should favor the formation of winter bird communities dominated by warm‐adapted species. To do this, we quantified changes in community composition using a functional index – the Community Temperature Index (CTI) – which measures the balance between low‐ and high‐temperature dwelling species in a community. Using data from Project FeederWatch, an international citizen science program, we quantified spatiotemporal changes in winter bird communities (n = 38 bird species) across eastern North America and tested the influence of changes in winter minimum temperature over a 22‐year period. We implemented a jackknife analysis to identify those species most influential in driving changes at the community level and the population dynamics (e.g., extinction or colonization) responsible for these community changes. Since 1990, we found that the winter bird community structure has changed with communities increasingly composed of warm‐adapted species. This reshuffling of winter bird communities was strongest in southerly latitudes and driven primarily by local increases in abundance and regional patterns of colonization by southerly birds. CTI tracked patterns of changing winter temperature at different temporal scales ranging from 1 to 35 years. We conclude that a shifting winter climate has provided an opportunity for smaller, southerly distributed species to colonize new regions and promote the formation of unique winter bird assemblages throughout eastern North America.     

Inhibition of palmitate‐induced GADD34 expression augments apoptosis in mouse insulinoma cells (MIN6)

Saturated fatty acids like palmitate induce endoplasmic reticulum (ER) stress in pancreatic beta‐cells, an event linked to apoptotic loss of β‐cells in type 2 diabetes. Sustained activation of the ER stress response leads to expression of growth arrest and DNA damage‐inducible protein 34 (GADD34), a regulatory subunit of protein phosphatase 1. In the present study, we have used small interfering RNA in order to knockdown GADD34 expression in insulin‐producing MIN6 cells prior to induction of ER stress by palmitate and evaluated its consequences on RNA‐activated protein kinase‐like ER‐localized eIF2alpha kinase (PERK) signalling and apoptosis. Salubrinal, a specific inhibitor of eukaryotic initiation factor 2α (eIF2α) dephosphorylation, was used as a comparison. Salubrinal treatment augmented palmitate‐induced ER stress and increased GADD34 levels. Both GADD34 knockdown and salubrinal treatment potentiated the cytotoxic effects of palmitate as evidenced by increased DNA fragmentation and activation of caspase 3, with the fundamental difference that the former did not involve enhanced levels of GADD34. The data from this study suggest that sustained activation of PERK signalling and eIF2α phosphorylation sensitizes insulin‐producing MIN6 cells to lipoapoptosis independently of GADD34 expression levels. Copyright © 2014 John Wiley & Sons, Ltd.     

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.     

Hepatocytes release ceramide-enriched pro-inflammatory extracellular vesicles in an IRE1α-dependent manner

Nonalcoholic steatohepatitis (NASH) is a lipotoxic disease wherein activation of endoplasmic reticulum (ER) stress response and macrophage-mediated hepatic inflammation are key pathogenic features. However, the lipid mediators linking these two observations remain elusive. We postulated that ER stress-regulated release of pro-inflammatory extracellular vesicles (EVs) from lipotoxic hepatocytes may be this link. EVs were isolated from cell culture supernatants of hepatocytes treated with palmitate (PA) to induce lipotoxic ER stress, characterized by immunofluorescence, Western blotting, electron microscopy, and nanoparticle tracking analysis. Sphingolipids were measured by tandem mass spectrometry. EVs were employed in macrophage chemotaxis assays. PA induced significant EV release. Because PA activates ER stress, we used KO hepatocytes to demonstrate that PA-induced EV release was mediated by inositol requiring enzyme 1α (IRE1α)/X-box binding protein-1. PA-induced EVs were enriched in C16:0 ceramide in an IRE1α-dependent manner, and activated macrophage chemotaxis via formation of sphingosine-1-phosphate (S1P) from C16:0 ceramide. This chemotaxis was blocked by sphingosine kinase inhibitors and S1P receptor inhibitors. Lastly, elevated circulating EVs in experimental and human NASH demonstrated increased C16:0 ceramide. PA induces C16:0 ceramide-enriched EV release in an IRE1α-dependent manner. The ceramide metabolite, S1P, activates macrophage chemotaxis, a potential mechanism for the recruitment of macrophages to the liver under lipotoxic conditions.     

A twofold role for global energy gradients in marine biodiversity trends

Explanations for major biodiversity patterns have not achieved a consensus, even for the latitudinal diversity gradient (LDG), but most relate to patterns of solar energy influx into Earth systems, and its effects on temperature (as biochemical activity rates are temperature sensitive) and photosynthesis (which drives nearly all of the productivity that fuels ecosystems). Marine systems break some of the confounding correlations among temperature, latitude and biodiversity that typify the terrestrial systems that have dominated theoretical discussions and large‐scale analyses. High marine diversities occur not only in warm shallow seas where productivity may be either low or high, depending on regional features, but also in very cold deep‐sea regions, indicating that diversity is promoted by stability in temperature and in trophic resources (nutrients and food items), and more specifically by their interaction, rather than by high mean values of either variable. The common association of high diversity with stable but low to moderate annual productivity suggests that ecological specialization underlies the similarly high diversities in the shallow tropics and deep sea. Recent work on shallow‐marine bivalves is consistent with this view of decreasing specialization in less stable habitats. Lower diversities in shallow seas are associated with either high thermal seasonality (chiefly in temperate latitudes) or highly seasonal trophic supplies (at any latitude), which exclude species that are adapted to narrow ranges of those variables.