Identification and Characterisation of the RalA-ERp57 Interaction: Evidence for GDI Activity of ERp57

RalA is a membrane-associated small GTPase that regulates vesicle trafficking. Here we identify a specific interaction between RalA and ERp57, an oxidoreductase and signalling protein. ERp57 bound specifically to the GDP-bound form of RalA, but not the GTP-bound form, and inhibited the dissociation of GDP from RalA in vitro. These activities were inhibited by reducing agents, but no disulphide bonds were detected between RalA and ERp57. Mutation of all four of ERp57’s active site cysteine residues blocked sensitivity to reducing agents, suggesting that redox-dependent conformational changes in ERp57 affect binding to RalA. Mutations in the switch II region of the GTPase domain of RalA specifically reduced or abolished binding to ERp57, but did not block GTP-specific binding to known RalA effectors, the exocyst and RalBP1. Oxidative treatment of A431 cells with H₂O₂ inhibited cellular RalA activity, and the effect was exacerbated by expression of recombinant ERp57. The oxidative treatment significantly increased the amount of RalA localised to the cytosol. These findings suggest that ERp57 regulates RalA signalling by acting as a redox-sensitive guanine-nucleotide dissociation inhibitor (RalGDI).     

Stalk Formation and Its Inhibition in Caulobacter crescentus

Estimates of average rates of stalk formation over several generations of growth in Caulobacter crescentus showed that long-stalked Sk1 mutant and phosphate-starved wild-type cultures produce stalk material at about twice the rate of wild-type C. crescentus grown with adequate nutrients. Thus, the long stalks of Sk1 or phosphate-starved caulobacters are not merely a function of their longer doubling times. Inhibition of cell division of Sk1 418 with mitomycin C (MC) caused production of cellular filaments and resulted in inhibition of stalk formation. There was no appreciable decrease in total cell mass or in rates of ribonucleic acid and protein synthesis in the MC-treated cultures as compared with controls, but stalk formation, which is normally dependent on these processes, was severely retarded. Average stalk lengths in MC-treated Sk1 cultures were 30% of those found in control cultures. MC-produced cellular filaments were also subjected to deoxyribonucleic acid analysis and ultrastructural examination. The deoxyribonucleic acid content of MC-treated bacteria was about 50 to 60% that of untreated bacteria. Hydroxyurea also was found to produce some cellular filaments and shorter stalks, but with accompanying decreases in growth rate and yield.     

Food availability affects the strength of mutualistic host–microbiota interactions in Daphnia magna

The symbiotic gut microbial community is generally known to have a strong impact on the fitness of its host. Nevertheless, it is less clear how the impact of symbiotic interactions on the hosts'' fitness varies according to environmental circumstances such as changes in the diet. This study aims to get a better understanding of host–microbiota interactions under different levels of food availability. We conducted experiments with the invertebrate, experimental model organism Daphnia magna and compared growth, survival and reproduction of conventionalized symbiotic Daphnia with germ-free individuals given varying quantities of food. Our experiments revealed that the relative importance of the microbiota for the hosts'' fitness varied according to dietary conditions. The presence of the microbiota had strong positive effects on Daphnia when food was sufficient or abundant, but had weaker effects under food limitation. Our results indicate that the microbiota can be a potentially important factor in determining host responses to changes in dietary conditions. Characterization of the host-associated microbiota further showed that Aeromonas sp. was the most prevalent taxon in the digestive tract of Daphnia.     

IL-28A,IL-28B,and IL-29: Promising cytokines with type I interferon-like properties

IL-28A, IL-28B and IL-29 (also designated type III interferons) constitute a new subfamily within the IL-10–interferon family. They are produced by virtually any nucleated cell type, particularly dendritic cells, following viral infection or activation with bacterial components, and mediate their effects via the IL-28R1/IL-10R2 receptor complex. Although IL-28/IL-29 are closer to the IL-10-related cytokines in terms of gene structure, protein structure, and receptor usage, they display type I interferon-like anti-viral and cytostatic activities. Unlike type I interferons, the target cell populations of IL-28/IL-29 are restricted and mainly include epithelial cells and hepatocytes. These properties suggest that IL-28/IL-29 are potential therapeutic alternatives to type I interferons in terms of viral infections and tumors. This review describes the current knowledge about these cytokines.     

Invasive legume management strategies differentially impact mutualist abundance and benefit to native and invasive hosts

Determining the best management practices for plant invasions is a critical, but often elusive goal. Invasive removals frequently involve complex and poorly understood biotic interactions. For example, invasive species can leave potent legacies that influence the success of native species restoration efforts, and positive plant‐microbial feedbacks may promote continued reinvasion by an exotic species following restoration. Removal methods can vary in their effects on plant–soil feedbacks, with consequences for restoration of native species. We determined the effects of invasion by a leguminous shrub (French broom; Genista monspessulana) on the density and community composition of, and benefit conferred by, its microbial mutualists in its invading range. Densities of soil‐dwelling rhizobia were much higher in areas invaded by G. monspessulana relative to uninvaded areas, and this increased density of rhizobia fed back to increase seedling growth of both the invader and native legumes. We further compared how three techniques for removing G. monspessulana affected the densities of rhizobia relative to areas where G. monspessulana was still present. Removal by hand‐pulling reduced soil rhizobial densities, and reduced growth of one native legume, while having no effect on the growth of the invader. Overall, our results show that the consequences of restoration techniques, both above‐ and belowground, could be critical for the successful removal of an invasive legume and restoration of native species.     

New Approaches for Absolute Quantification of Stable‐Isotope‐Labeled Peptide Standards for Targeted Proteomics Based on a UV Active Tag

Targeted proteomics depends on the availability of stable isotope labeled (SIL) peptide standards, which for absolute protein quantification need to be absolutely quantified. In the present study, three new approaches for absolute quantification of SIL peptides are developed. All approaches rely on a quantification tag (Qtag) with a specific UV absorption. The Qtag is attached to the peptide during synthesis and is removed by tryptic digestion under standard proteomics workflow conditions. While one quantification method (method A) is designed to allow the fast and economic production of absolutely quantified SIL peptides, two other methods (methods B and C) are developed to enable the straightforward re‐quantification of SIL peptides after reconstitution to control and monitor known problems related to peptide solubility, precipitation, and adhesion to vials. All methods yield consistent results when compared to each other and when compared to quantification by amino acid analysis. The precise quantitation methods are used to characterize the in vivo specificity of the H3 specific histone methyltransferase EZH2.