Structural and functional studies of RegB, a new member of a family of sequence-specific ribonucleases involved in mRNA inactivation on the ribosome

The RegB endoribonuclease participates in the bacteriophage T4 life cycle by favoring early messenger RNA breakdown. RegB specifically cleaves GGAG sequences found in intergenic regions, mainly in translation initiation sites. Its activity is very low but can be enhanced up to 100-fold by the ribosomal 30 S subunit or by ribosomal protein S1. RegB has no significant sequence homology to any known protein. Here we used NMR to solve the structure of RegB and map its interactions with two RNA substrates. We also generated a collection of mutants affected in RegB function. Our results show that, despite the absence of any sequence homology, RegB has structural similarities with two Escherichia coli ribonucleases involved in mRNA inactivation on translating ribosomes: YoeB and RelE. Although these ribonucleases have different catalytic sites, we propose that RegB is a new member of the RelE/YoeB structural and functional family of ribonucleases specialized in mRNA inactivation within the ribosome.     

Control and regulation of KplE1 prophage site-specific recombination: a new recombination module analyzed

KplE1 is one of the 10 prophage regions of Escherichia coli K12, located at 2464 kb on the chromosome. KplE1 is defective for lysis, but it is fully competent for excisive recombination. In this study, we have mapped the binding sites of the recombination proteins, namely IntS, TorI, and IHF on attL and attR, and the organization of these sites suggests that the intasome is architecturally different from the lambda canonical form. We also measured the relative contribution of these proteins to both excisive and integrative recombination by using a quantitative in vitro assay. These experiments show a requirement of the TorI excisionase for excisive recombination and of the IntS integrase for both integration and excision. Moreover, we observed a strong influence of the supercoiled state of the substrates. The KplE1 recombination module, composed of the integrase and excisionase genes together with the attL and attR DNA regions, is highly similar to that of several phages infecting various E. coli strains as well as Shigella flexneri and Shigella sonnei. The in vitro recombination data reveal that HK620 and KplE1 att sequences are exchangeable. This study thus defines a new site-specific recombination module, and implications for the mechanism and regulation of recombination are discussed.     

HER2 status in ovarian carcinomas: a multicenter GINECO study of 320 patients

Background

Despite a typically good response to first-line combination chemotherapy, the prognosis for patients with advanced ovarian cancer remains poor because of acquired chemoresistance. The use of targeted therapies such as trastuzumab may potentially improve outcomes for patients with ovarian cancer. HER2 overexpression/amplification has been reported in ovarian cancer, but the exact percentage of HER2-positive tumors varies widely in the literature. In this study, HER2 gene status was evaluated in a large, multicentric series of 320 patients with advanced ovarian cancer, including 243 patients enrolled in a multicenter prospective clinical trial of paclitaxel/carboplatin-based chemotherapy.

Methodology/Principal Findings

The HER2 status of primary tumors and metastases was evaluated by both immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) analysis of paraffin-embedded tissue on conventional slides. The prognostic impact of HER2 expression was analyzed. HER2 gene was overexpressed and amplified in 6.6% of analyzed tumors. Despite frequent intratumoral heterogeneity, no statistically significant difference was detected between primary tumors and corresponding metastases.

Conclusions/Significance

Our results show that the decision algorithm usually used in breast cancer (IHC as a screening test, with equivocal results confirmed by FISH) is appropriate in ovarian cancer. In contrast to previous series, HER2-positive status did not influence outcome in the present study, possibly due to the fact that patients in our study received paclitaxel/carboplatin-based chemotherapy. This raises the question of whether HER2 status and paclitaxel sensitively are linked.     

The perpetuation and epidemic recurrence of communicable diseases in human populations

Recurrence of communicable diseases is a looming threat for human populations. Factors explaining the recurrences are partially known, involving demographics, biology, and complex relationships with the environment, but no comprehensive theory exists today. Here, we review some recent results obtained in modelling studies with a view to understanding better the mechanisms of perpetuation. Factors intrinsic to the interaction of pathogen and host have regained interest in this respect, especially with multiple pathogen and multiple population interactions. Extrinsic factors, including pure demography and environmental forcing are also strong predictors. With increasingly detailed data available, large-scale integrated models will help sorting out the multiple influences on recurrence.     

Is autophagy the key mechanism by which the sphingolipid rheostat controls the cell fate decision?

Sphingolipids are major constituents of biological membrane and some of them behave as second messengers involved in the cell fate decision. Ceramide and sphingosine 1-phosphate (S1P) constitute a rheostat system in which ceramide promotes cell death and S1P increases cell survival. We have shown that both sphingolipids are able to trigger autophagy with opposing outcomes on cell survival. Here we discuss and speculate on the diverging functions of the autophagic pathways induced by ceramide and S1P, respectively.     

Potential relationship between glutathione metabolism and flocculation in the yeast Kluyveromyces lactis

Reduced glutathione (GSH) is involved in biochemical and physiological processes in cells. Flocculation is an important mechanism in microorganisms. The present study concerned the potential relationship between GSH metabolism and flocculation. Two yeast strains, a flocculent (Kluyveromyces lactis 5c) and a nonflocculent (Kluyveromyces lactis 5a) strain, were used. The level of intracellular GSH measured during the growth period was significantly higher in the nonflocculent than in the flocculent strain; in contrast, the flocculent strain exhibited brighter staining of vacuoles than the nonflocculent strain when observed using epifluorescence microscopy. Compounds acting either on flocculation (EDTA, galactose) or on GSH metabolism (buthionine sulfoximine, and N-acetylcysteine) were tested on the flocculent strain during the growth period. Both EDTA and galactose fully inhibited flocculation and induced GSH overproduction of 58% and 153%, respectively. Buthionine sulfoximine decreased GSH level by 76% but had no effect on flocculation; N-acetylcysteine increased the GSH level and flocculation by 106% and 41%, respectively. Combination of EDTA and N-acetylcysteine produced similar effects than with each of them. Combination of galactose and N-acetylcysteine increased the GSH level but decreased flocculation. These results demonstrated that GSH homeostasis is linked to the flocculation mechanism. A hypothesis related to stress is given.     

Impact of urban fragmentation on the genetic structure of the eastern red-backed salamander

Urban development is a major cause of habitat loss and fragmentation. Few studies, however, have dealt with fragmentation in an urban landscape. In this paper, we examine the genetic structure of isolated populations of the eastern red-backed salamander (Plethodon cinereus) in a metropolitan area. We sampled four populations located on a mountain in the heart of Montréal (Québec, Canada), which presents a mosaic of forested patches isolated by roads, graveyards and buildings. We assessed the genetic structure of these populations using microsatellite loci and compared it to the genetic structure of four populations located in a continuous habitat in southern Québec. Our results indicate that allelic richness and heterozygosity are lower in the urban populations. Exact differentiation tests and pairwise F _ST also show that the populations found in the fragmented habitat are genetically differentiated, whereas populations located in the continuous habitat are genetically homogeneous. These results raise conservation concerns for these populations as well as for rare or threatened species inhabiting urban landscapes.     

Seleno-independent glutathione peroxidases. More than simple antioxidant scavengers

Glutathione peroxidases (GPXs, EC 1.11.1.9) were first discovered in mammals as key enzymes involved in scavenging of activated oxygen species (AOS). Their efficient antioxidant activity depends on the presence of the rare amino-acid residue selenocysteine (SeCys) at the catalytic site. Nonselenium GPX-like proteins (NS-GPXs) with a Cys residue instead of SeCys have also been found in most organisms. As SeCys is important for GPX activity, the function of the NS-GPX can be questioned. Here, we highlight the evolutionary link between NS-GPX and seleno-GPX, particularly the evolution of the SeCys incorporation system. We then discuss what is known about the enzymatic activity and physiological functions of NS-GPX. Biochemical studies have shown that NS-GPXs are not true GPXs; notably they reduce AOS using reducing substrates other than glutathione, such as thioredoxin. We provide evidence that, in addition to their inefficient scavenging action, NS-GPXs act as AOS sensors in various signal-transduction pathways.     

The 'pair of sugar tongs' site on the non-catalytic domain C of barley alpha-amylase participates in substrate binding and activity

Some starch-degrading enzymes accommodate carbohydrates at sites situated at a certain distance from the active site. In the crystal structure of barley alpha-amylase 1, oligosaccharide is thus bound to the 'sugar tongs' site. This site on the non-catalytic domain C in the C-terminal part of the molecule contains a key residue, Tyr380, which has numerous contacts with the oligosaccharide. The mutant enzymes Y380A and Y380M failed to bind to beta-cyclodextrin-Sepharose, a starch-mimic resin used for alpha-amylase affinity purification. The K(d) for beta-cyclodextrin binding to Y380A and Y380M was 1.4 mm compared to 0.20-0.25 mm for the wild-type, S378P and S378T enzymes. The substitution in the S378P enzyme mimics Pro376 in the barley alpha-amylase 2 isozyme, which in spite of its conserved Tyr378 did not bind oligosaccharide at the 'sugar tongs' in the structure. Crystal structures of both wild-type and S378P enzymes, but not the Y380A enzyme, showed binding of the pseudotetrasaccharide acarbose at the 'sugar tongs' site. The 'sugar tongs' site also contributed importantly to the adsorption to starch granules, as Kd = 0.47 mg.mL(-1) for the wild-type enzyme increased to 5.9 mg.mL(-1) for Y380A, which moreover catalyzed the release of soluble oligosaccharides from starch granules with only 10% of the wild-type activity. beta-cyclodextrin both inhibited binding to and suppressed activity on starch granules for wild-type and S378P enzymes, but did not affect these properties of Y380A, reflecting the functional role of Tyr380. In addition, the Y380A enzyme hydrolyzed amylose with reduced multiple attack, emphasizing that the 'sugar tongs' participates in multivalent binding of polysaccharide substrates.