The deSUMOylase SENP7 promotes chromatin relaxation for homologous recombination DNA repair

SUMO conjugation is known to occur in response to double‐stranded DNA breaks in mammalian cells, but whether SUMO deconjugation has a role remains unclear. Here, we show that the SUMO/Sentrin/Smt3‐specific peptidase, SENP7, interacts with the chromatin repressive KRAB‐associated protein 1 (KAP1) through heterochromatin protein 1 alpha (HP1α). SENP7 promotes the removal of SUMO2/3 from KAP1 and regulates the interaction of the chromatin remodeler CHD3 with chromatin. Consequently, in the presence of CHD3, SENP7 is required for chromatin relaxation in response to DNA damage, for homologous recombination repair and for cellular resistance to DNA‐damaging agents. Thus, deSUMOylation by SENP7 is required to promote a permissive chromatin environment for DNA repair.     

Proteins of the kidney microvillar membrane. Asymmetric labelling of the membrane by lactoperoxidase-catalysed radioiodination and by photolysis of 3,5-di[125I]iodo-4-azidobenzenesulphonate.

Two methods were used to label pig kidney microvillar membrane proteins from the luminal and cytoplasmic surfaces of closed membrane vesicles. The first method was lactoperoxidase-catalysed radioiodination. The enzyme reagents, lactoperoxidase and glucose oxidase, were positioned inside the vesicles before sealing or externally after sealing, iodination being initiated by the subsequent addition of glucose and 125I-. After resolution of the labelled proteins by electrophoresis in the presence of dodecyl sulphate, asymmetric labelling patterns on radioautographs were observed. However, the major disadvantage of this method is the high degree of intramembrane labelling of the fatty acid chains of membrane lipids, a reaction that undermines any conclusions about the location of the label in that region of the protein supposedly exposed at the surface of the membrane. The second method overcame this disadvantage. A new hydrophilic photoreagent, 3,5-di[125I]iodo-4-azidobenzesulphonate, was synthesized via the intermediate, diazotized 3,5-di[125I]iodosulphanilic acid. It was transported by a Na+-dependent system into microvillar vesicles, thus permitting labelling from either side of the membrane when the vesicles were photolysed. The labelling of membrane lipids was less than with the first method and was essentially confined to the polar headgroups. The activity of several microvillar peptidases survived the labelling reaction and they could be identified in the immunoprecipitates after resolution of the detergent-solubilized membrane proteins by crossed-immunoelectrophoresis. Treatment with papain converted the detergent-solubilized form of susceptible enzymes into the proteinase-solubilized form, which lacked the intramembrane domain and any portion exposed at the cytoplasmic surface. Radioautography established that aminopeptidases M and A, dipeptidyl peptidase IV and neutral endopeptidase were transmembrane proteins. This novel approach to the investigation of membrane topology may be applicable to other complex membranes.     

Anticodon sequence mutants of Escherichia coli initiator tRNA: effects of overproduction of aminoacyl-tRNA synthetases,methionyl-tRNA formyltransferase,and initiation factor 2 on activity in initiation

Anticodon sequence mutants of Escherichia coli initiator tRNA initiate protein synthesis with codons other than AUG and amino acids other than methionine. Because the anticodon sequence is, in many cases, important for recognition of tRNAs by aminoacyl-tRNA synthetases, the mutant tRNAs are aminoacylated in vivo with different amino acids. The activity of a mutant tRNA in initiation in vivo depends on (i) the level of expression of the tRNA, (ii) the extent of aminoacylation of the tRNA, (iii) the extent of formylation of the aminoacyl-tRNA to formylaminoacyl-tRNA (fAA-tRNA), and (iv) the affinity of the fAA-tRNA for the initiation factor IF2 and the ribosome. Previously, using E. coli overproducing aminoacyl-tRNA synthetases, methionyl-tRNA formyltransferase, or IF2, we identified the steps limiting the activity in initiation of mutant tRNAs aminoacylated with glutamine and valine. Here, we have identified the steps limiting the activity of mutant tRNAs aminoacylated with isoleucine and phenylalanine. The combined results of experiments involving a variety of initiation codons (AUG, UAG, CAG, GUC, AUC, and UUC) provide support to the hypothesis that the ribosome.fAA-tRNA complex can act as an intermediate in initiation of protein synthesis. Comparison of binding affinities of various fAA-tRNAs (fMet-, fGln-, fVal-, fIle-, and fPhe-tRNAs) to IF2 using surface plasmon resonance supports the idea that IF2 can act as a carrier of fAA-tRNA to the ribosome. Other results suggest that the C1xA72 base pair mismatch, unique to eubacterial and organellar initiator tRNAs, may also be important for the binding of fAA-tRNA to IF2.     

Prolonged Ex vivo expansion and differentiation of naïve murine CD43− B splenocytes

Ex vivo expansion of naive primary B cells is still a challenge, yet would open new possibilities for in vitro studies of the immune response or the production of monoclonal antibodies. In our hands, unstimulated murine B cells did not expand in significant numbers, while culture viability decreased rapidly within a few days. Activation mimicking in vivo stimulation through either T cell‐independent or T‐cell dependent signaling, led to several division cycles, albeit accompanied by irreversible differentiation. By co‐culturing B cells under moderate hypothermia (30°C) on live feeder fibroblasts expressing recombinant CD40 ligand (CD154) and by repeatedly transferring cultured B cells to new feeder cell cultures, we could extend the growth of primary mouse B cells compared to cultures maintained at 37°C. B cells under these conditions showed an activated phenotype as shown by the presence of AID and IRF4, two factors required for IgH class switch recombination in antigen‐activated B cells. In contrast to cells cultured at 37°C, B cells under hyperthermia did surprisingly not differentiate into Blimp‐1 expressing plasmablasts. Thus, the repeated batch process under hyperthermic conditions represents a first step towards the development of a continuous cultivation system for the expansion of primary B cells. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:978–989, 2016     

Myxococcus xanthus Displays Frz-Dependent Chemokinetic Behavior during Vegetative Swarming

Myxococcus xanthus has been shown to utilize both directed (tactic) and undirected (kinetic) movements during different stages of its complex life cycle. We have used time-lapse video microscopic analysis to separate tactic and kinetic behaviors associated specifically with vegetatively swarming cells. Isolated individual cells separated by a thin agar barrier from mature swarms showed significant increases in gliding velocity compared to that of similar cells some distance from the swarm. This orthokinetic behavior was independent of the frequency of reversals of gliding direction (klinokinesis) but did require both the Frz signal transduction system and S-motility. We propose that M. xanthus uses Frz-dependent, auto-orthokinetic behavior to facilitate the dispersal of cells under conditions where both cell density and nutrient levels are high.     

Reward mechanisms in obesity: new insights and future directions

Food is consumed in order to maintain energy balance at homeostatic levels. In addition, palatable food is also consumed for its hedonic properties independent of energy status. Such reward-related consumption can result in caloric intake exceeding requirements and is considered a major culprit in the rapidly increasing rates of obesity in developed countries. Compared with homeostatic mechanisms of feeding, much less is known about how hedonic systems in brain influence food intake. Intriguingly, excessive consumption of palatable food can trigger neuroadaptive responses in brain reward circuitries similar to drugs of abuse. Furthermore, similar genetic vulnerabilities in brain reward systems can increase predisposition to drug addiction and obesity. Here, recent advances in our understanding of the brain circuitries that regulate hedonic aspects of feeding behavior will be reviewed. Also, emerging evidence suggesting that obesity and drug addiction may share common hedonic mechanisms will also be considered.     

A trait-based approach across the native and invaded range to understand plant invasiveness and community impact

Alien plant species invasiveness and impact on diversity (i.e. species richness and composition) can be driven by the altered competitive interactions experienced by the invader in its invaded range compared to its native range. Trait-based competition effects on invasiveness can be mediated through size-asymmetric competition, i.e. a trait suit of the invader that drives competitive dominance, and through ‘niche differences', i.e. trait differentiation and thus minimized competition between invader and the invaded community. In terms of invasion impact, size-asymmetric competition is expected to result in competitive exclusion of co-occurring subordinate species, whereas ‘niche differences' might result in competitive exclusion of the most functionally similar co-occurring species. Although observational work does not allow the full disentanglement of both trait-based effects, it does allow to verify the occurrence of expected theoretical trait patters. In this study, we explored the trait-based competition effects on invasiveness and diversity impact for Rosa rugosa in both its invaded range in Belgium and its native range in Japan, based on seven functional traits across 100 vegetation plots. Following the predictions for enhanced invasiveness, we found much lower functional overlap between R. rugosa and the co-occurring species in the invaded range compared to the native range. This likely also explains the absence of diversity impact in its native range. Despite the absence of changes in species richness in the invaded range, the invader did strongly impact species composition of invaded communities. This impact occurred through strong shade tolerance responses, suggesting size-asymmetric competition effects and cover loss of co-occurring dominant species, next to exclusion of co-occurring species most functionally similar to the invader; suggesting niche difference effects. In conclusion, this case-study illustrates how exploring functional trait patterns across a species native and invaded range can help in understanding how trait-based competition processes can affect invasiveness and community impact.     

Evaluating the Evidence for Hormesis: A Statistical Perspective

It is possible to account for hormesis under current regulatory guidelines by invoking criteria for departure from default risk assessment procedures. However, past experience suggests that it will be difficult to amass enough evidence for hormesis in an individual case to permit departure from default procedures. Accordingly, hormesis is likely to be important in agency risk assessments only if guidelines are modified to incorporate hormesis as a default assumption. This could be appropriate if hormesis is determined to be a universal or near-universal phenomenon. Although there is ample evidence that hormesis occurs in many specific situations, the overall prevalence of hormesis is very difficult to evaluate based on currently available data. The lack of a valid statistical test for hormesis is a major limitation when evaluating evidence for hormesis. The attempts at estimating the prevalence of hormesis reviewed herein did not adequately control for false positives and/or may have had inadequate power to detect hormesis. Some suggestions are made for constructing a database and analyzing the data therein that would provide more readily interpretable information on the prevalence of hormesis.