The amino-terminal extensions of the longer Sendai virus C proteins modulate pY701-Stat1 and bulk Stat1 levels independently of interferon signaling

The Sendai virus (SeV) C proteins are known to interact with Stat1 to prevent interferon (IFN)-induced pY701-Stat1 formation and IFN signaling. Nevertheless, pY701-Stat1 levels paradoxically increase during SeV infection. The C proteins also induce bulk Stat1 instability in some cells, similar to rubulavirus V proteins. We have found that SeV infection increases pY701-Stat1 levels even in cells in which bulk Stat1 levels strongly decrease. Remarkably, both the decrease in bulk Stat1 levels and the increase in pY701-Stat1 levels were found to be independent of the IFN signaling system, i.e., these events occur in mutant cells in which various components of the IFN signaling system have been disabled. Consistent with this, the C-induced decrease in Stat1 levels does not require Y701 of Stat1. We present evidence that C interacts with Stat1 in two different ways, one that prevents IFN-induced pY701-Stat1 formation and IFN signaling that has already been documented, and another that induces pY701-Stat1 formation (while decreasing bulk Stat1 levels) in a manner that does not require IFN signaling. These two types of Stat1 interaction are also distinguishable by C gene mutations. In particular, the IFN signaling-independent Stat1 interactions specifically require the amino-terminal extensions of the longer C proteins. The actions of the SeV C proteins in counteracting the cellular antiviral response are clearly more extensive than previously appreciated.     

Src homology-2 domain binding assays by scintillation proximity and surface plasmon resonance

Various SH2 competitive binding assays, based on different techniques, have been described in the literature to identify and characterize SH2 ligands. The consideration that most reported methods show experimental limitations associated with assay parameters has prompted us to base our Src-SH2 inhibitor discovery program on the use of two different assays. In this study, two conceptually different biochemical methods designed to discover Src-SH2 inhibitors, respectively scintillation proximity assay (SPA) and surface plasmon resonance (SPR), have been evaluated and compared. For its high sensitivity and adaptability to automation SPA was chosen for high capacity screening (primary screen), whereas SPR was used for hits confirmation (secondary screening). However with the drastic improvement of inhibitor affinities, the limit of sensitivity was rapidly reached for the SPR assay based on the canonical pYEEI ligand. The substitution of the natural, monophosphorylated peptide ligand with a triphosphorylated peptide has allowed us to remarkably increase its sensitivity, so that molecules with nanomolar affinities could be easily differentiated in terms of IC(50) ranking. Such a new, improved SPR assay can be of great interest for the study of high affinity ligands of different SH2-based drug targets.     

Microtubule release from the centrosome in migrating cells

In migrating cells, force production relies essentially on a polarized actomyosin system, whereas the spatial regulation of actomyosin contraction and substrate contact turnover involves a complex cooperation between the microtubule (MT) and the actin filament networks (Goode, B.L., D.G. Drubin, and G. Barnes. 2000. Curr. Opin. Cell Biol., 12:63-71). Targeting and capture of MT plus ends at the cell periphery has been described, but whether or not the minus ends of these MTs are anchored at the centrosome is not known. Here, we show that release of short MTs from the centrosome is frequent in migrating cells and that their transport toward the cell periphery is blocked when dynein activity is impaired. We further show that MT release, but not MT nucleation or polymerization dynamics, is abolished by overexpression of the centrosomal MT-anchoring protein ninein. In addition, a dramatic inhibition of cell migration was observed; but, contrary to cells treated by drugs inhibiting MT dynamics, polarized membrane ruffling activity was not affected in ninein overexpressing cells. We thus propose that the balance between MT minus-end capture and release from the centrosome is critical for efficient cell migration.     

Depletion of the photosystem II core complex in mature tobacco leaves infected by the flavum strain of tobacco mosaic virus

The flavum strain of Tobacco mosaic virus (TMV) differs from the wild-type (wt) virus by causing strong yellow and green mosaic in the systemically infected developing leaves, yellowing in the fully expanded leaves, and distinct malformations of chloroplasts in both types of infected tissues. Analysis of the thylakoid proteins of flavum strain-infected tobacco leaves indicated that the chlorosis in mature leaves was accompanied by depletion of the entire photosystem II (PSII) core complexes and the 33-kDa protein of the oxygen evolving complex. The only change observed in the thylakoid proteins of the corresponding wt TMV-infected leaves was a slight reduction of the alpha and beta subunits of the ATP synthase complex. The coat proteins of different yellowing strains of TMV are known to effectively accumulate inside chloroplasts, but in this work, the viral movement protein also was detected in association with the thylakoid membranes of flavum strain-infected leaves. The mRNAs of different enzymes involved in the chlorophyll biosynthesis pathway were not reduced in the mature chlorotic leaves. These results suggest that the chlorosis was not caused by reduction of pigment biosynthesis, but rather, by reduction of specific proteins of the PSII core complexes and by consequent break-down of the pigments.     

Genetic diversity and population structure in French populations of Mycosphaerella graminicola

Mycosphaerella graminicola populations were examined in France with microsatellite markers and PCR-SSCP analysis of partial actin and β-tubulin encoding sequences. A total of 363 isolates was sampled in 2005 from 17 provinces, and genotypes from corresponding strains were characterized. Unique haplotypes comprised 84% of the population, and gene diversity was high nationwide (0.70) and locally. A moderate genetic differentiation (G(ST) = 0.18) was found and indicated that in France the M. graminicola population was more structured than in other previously studied European countries. Bayesian structure analysis identified three genetic clusters distributed among the 17 provinces. Our results highlighted the potential for the adaptation of the fungus to local conditions, leading to genetic clusters among the French population of the fungus as well as genotype flow between regional clusters.     

RIF1 Is Essential for 53BP1-Dependent Nonhomologous End Joining and Suppression of DNA Double-Strand Break Resection

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Highlights? RIF1 is essential for 53BP1-dependent CSR and fusion of dysfunctional telomeres ? BRCA1 antagonizes RIF1 in S phase to prevent error-prone repair by toxic NHEJ ? N-terminal phospho-SQ/TQ domain of 53BP1 interacts with and recruits RIF1 to DSBs ? RIF1 and 53BP1 promote NHEJ in G1 by blocking 5′ end resection of DSBs     

The Yeast Pif1 Helicase Prevents Genomic Instability Caused by G-Quadruplex-Forming CEB1 Sequences In Vivo

In budding yeast, the Pif1 DNA helicase is involved in the maintenance of both nuclear and mitochondrial genomes, but its role in these processes is still poorly understood. Here, we provide evidence for a new Pif1 function by demonstrating that its absence promotes genetic instability of alleles of the G-rich human minisatellite CEB1 inserted in the Saccharomyces cerevisiae genome, but not of other tandem repeats. Inactivation of other DNA helicases, including Sgs1, had no effect on CEB1 stability. In vitro, we show that CEB1 repeats formed stable G-quadruplex (G4) secondary structures and the Pif1 protein unwinds these structures more efficiently than regular B-DNA. Finally, synthetic CEB1 arrays in which we mutated the potential G4-forming sequences were no longer destabilized in pif1Δ cells. Hence, we conclude that CEB1 instability in pif1Δ cells depends on the potential to form G-quadruplex structures, suggesting that Pif1 could play a role in the metabolism of G4-forming sequences.     

Activity and Ranging Patterns of <Emphasis Type="Italic">Colobus angolensis ruwenzorii</Emphasis> in Nyungwe Forest,Rwanda: Possible Costs of Large Group Size

With group sizes sometimes >300 individuals, the Angolan black-and-white colobus (Colobus angolensis ruwenzorii) population in Nyungwe Forest, Rwanda is an intriguing exception to the tendency for folivores to live in smaller groups than expected relative to body size. Researchers have hypothesized that the unusually high quality of foliage at Nyungwe allows colobus there to avoid intragroup feeding competition, releasing constraints on the formation of large groups (Fimbel et al., 2001). We collected data on the activity and ranging patterns of a >300-member Nyungwe colobus group and compared our results to those from smaller groups in other black-and-white colobus (Colobus spp.) populations. Colobus at Nyungwe spent far more time feeding and moving (62%) and far less time resting (32%) than black-and-white colobus at any other site. The annual home range of the Nyungwe colobus was also many times larger (95% minimum convex polygon: 20.7 km ² ; 95% fixed kernel: 24.4 km ² ) than those for other populations. We terminated our research after the group engaged in an unprecedented migration among black-and-white colobus by moving 13 km south of their former range. Our results suggest that intragroup scramble competition may be more intense than originally believed within the large colobus groups at Nyungwe and that long periods of resource renewal may be necessary after a large colobus group passes through an area, thereby potentially helping to explain their wide ranging patterns. We discuss the socioecological convergence between the Nyungwe colobus and Chinese snub-nosed monkeys (Rhinopithecus spp.) and suggest directions for future research on the unique black-and-white colobus population at Nyungwe.

Metabolites secreted by human atherothrombotic aneurysms revealed through a metabolomic approach

Abdominal aortic aneurysm (AAA) is perma-nent and localized dilation of the abdominal aorta. Intraluminal thrombus (ILT) is involved in evolution and rupture of AAA. Complex biological processes associated with AAA include oxidative stress, proteolysis, neovascularization, aortic inflammation, cell death, and extracellular matrix breakdown. Biomarkers of growth and AAA rupture could give a more nuanced indication for surgery, unveil novel pathogenic pathways, and open possibilities for pharmacological inhibition of growth. Differential analysis of metabolites released by normal and pathological arteries in culture may help to find molecules that have a high probability of later being found in plasma and start signaling processes or be useful diagnostic/prognostic markers. We used a LC-QTOF-MS metabolomic approach to analyze metabolites released by human ILT (divided into luminal and abluminal layers), aneurysm wall (AW), and healthy wall (HW). Statistical analysis was used to compare luminal with abluminal ILT layer, ILT with AW, and AW with HW to select the metabolites exchanged between tissue and external medium. Identified compounds are related to inflammation and oxidative stress and indicate the possible role of fatty acid amides in AAA. Some metabolites (e.g., hippuric acid) had not been previously associated to aneurysm, others (fatty acid amides) have arisen, indicating a very promising line of research.     

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein’s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.