Wnt4 action in gonadal development and sex determination

Wnt4 is a growth factor involved in multiple developmental processes such as the formation of the kidney, adrenal, mammary gland, pituitary and the female reproductive system. During mammalian embryogenesis, Wnt4 is expressed in the gonads of both sexes before sex determination events take place and is subsequently down-regulated in the male gonad. Inactivation of the Wnt4 gene in mice has revealed that it is involved at several steps of female reproductive development. Wnt4 is implicated in Müllerian duct regression, the formation of sex-specific vasculature, the inhibition of steroidogenesis and in sex-specific cell migration events. A mouse model of sex-reversal has partially unravelled the molecular pathways in which Wnt4 operates during the development of the female reproductive system. However, the specific molecular mechanism of action of Wnt4 during gonadal development remains unknown. This and downstream signaling pathways involved in Wnt4 action during female gonad development are reviewed and models of Wnt4 action are proposed for Müllerian duct formation, sex-specific vasculature development, and sex determination events. Further identification of critical downstream effectors of the Wnt4 signaling pathway in mouse models and in patients with sex-reversal conditions could help in understanding sex-reversal pathologies in humans.     

TNFα-initiated oxidative/nitrative stress mediates cardiomyocyte apoptosis in traumatic animals

Whole body non-penetrating trauma causes myocardial infarction in humans and mechanical trauma (MT) results in cardiac dysfunction in animals. Our recent study demonstrated that incubation of cardiomyocytes with plasma isolated from MT animals causes significant cardiomyocyte apoptosis that can be blocked by neutralization of TNFα. The present study attempted to obtain direct in vivo evidence to support that overproduction of TNFα plays a causative role in trauma-induced cardiomyocyte apoptosis. Non-lethal MT caused significant TNFα overproduction (2.4-fold at 1.5 h after MT) and increased cardiomyocyte apoptosis (starting 3 h and peaking 12 h after MT). Pharmacological inhibition of TNFα with etanercept or TNFα gene deletion reduced post-trauma myocyte apoptosis (P < 0.01). Expression of iNOS and NADPH oxidase, overproduction of NO and , and excessive protein nitration in the MT heart were all significantly reduced in etanercept-treated or TNFα^−/− mice, suggesting that oxidative/nitrative stress may contribute to TNFα-initiated myocyte apoptosis in MT hearts. Additional experiments demonstrated that inhibiting iNOS (1400W) or NADPH oxidase (apocynin), or scavenging peroxynitrite (FP15) significantly reduced myocyte apoptosis in MT animals (P < 0.01). Collectively, these data demonstrated that non-lethal mechanical trauma caused significant TNFα production that in turn stimulated myocardial apoptosis via oxidative/nitrative stress.     

ARIA2: automated NOE assignment and data integration in NMR structure calculation

Modern structural genomics projects demand for integrated methods for the interpretation and storage of nuclear magnetic resonance (NMR) data. Here we present version 2.1 of our program ARIA (Ambiguous Restraints for Iterative Assignment) for automated assignment of nuclear Overhauser enhancement (NOE) data and NMR structure calculation. We report on recent developments, most notably a graphical user interface, and the incorporation of the object-oriented data model of the Collaborative Computing Project for NMR (CCPN). The CCPN data model defines a storage model for NMR data, which greatly facilitates the transfer of data between different NMR software packages. Availability: A distribution with the source code of ARIA 2.1 is freely available at http://www.pasteur.fr/recherche/unites/Binfs/aria2.     

Liver response of the Antarctic fish (<Emphasis Type="Italic">Notothenia coriiceps</Emphasis> Richardson, 1844) to hepatotrophic factors

The Notothenia coriiceps liver is commonly infected with parasites, reducing the hepatic mass and inducing the regeneration. In order to better understand the effect of nutrient influx on hepatic regeneration at 0°C, a usual mammal hepatotrophic factor (HF) solution was injected into ten fish (HF group), while ten fish were injected with saline solution (control), twice a day, for 15 days. The liver and carcass weight were measured, and samples were obtained for histological studies. The HF group presented a higher liver/carcass weight (62.5%) than control group. This increase in liver mass was due mainly to hepatocytes hypertrophy, including nuclear size increase and cytoplasmic inclusions of glycogen. Hyperplasia is also observed, although to a lesser extent. The hepatic reaction to HF in Antarctic fish was here demonstrated for the first time, helping to understand the liver response to seasonal nutrient.     

Chromosomal rearrangements in wheat: their types and distribution.

Four hundred and sixty polyploid wheat accessions and 39 triticale forms from 37 countries of Europe, Asia, and USA were scored by C-banding for the presence of translocations. Chromosomal rearrangements were detected in 70 of 208 accessions of tetraploid wheat, 69 of 252 accessions of hexaploid wheat, and 3 of 39 triticale forms. Altogether, 58 types of major chromosomal rearrangements were identified in the studied material; they are discussed relative to 11 additional translocation types described by other authors. Six chromosome modifications of unknown origin were also observed. Among all chromosomal aberrations identified in wheat, single translocations were the most frequent type (39), followed by multiple rearrangements (9 types), pericentric inversions (9 types), and paracentric inversions (3 types). According to C-banding analyses, the breakpoints were located at or near the centromere in 60 rearranged chromosomes, while in 52 cases they were in interstitial chromosome regions. In the latter case, translocation breakpoints were often located at the border of C-bands and the euchromatin region or between two adjacent C-bands; some of these regions seem to be translocation "hotspots". Our results and data published by other authors indicate that the B-genome chromosomes are involved in translocations most frequently, followed by the A- and D-genome chromosomes; individual chromosomes also differ in the frequencies of translocations. Most translocations were detected in 1 or 2 accessions, and only 11 variants showed relatively high frequencies or were detected in wheat varieties of different origins or from different species. High frequencies of some translocations with a very restricted distribution could be due to a "bottleneck effect". Other types seem to occur independently and their broad distribution can result from selective advantages of rearranged genotypes in diverse environmental conditions. We found significant geographic variation in the spectra and frequencies of translocation in wheat: the highest proportions of rearranged genotypes were found in Central Asia, the Middle East, Northern Africa, and France. A low proportion of aberrant genotypes was characteristic of tetraploid wheat from Transcaucasia and hexaploid wheat from Middle Asia and Eastern Europe.     

Pre-spliceosomal binding of U1 small nuclear ribonucleoprotein (RNP) and heterogenous nuclear RNP E1 is associated with suppression of a growth hormone receptor pseudoexon

Pseudoexons occur frequently in the human genome. This paper characterizes a pseudoexon in the GH receptor gene. Inappropriate activation of this pseudoexon causes Laron syndrome. Using in vitro splicing assays, pseudoexon silencing was shown to require a combination of a weak 5' pseudosplice-site and splicing silencing elements within the pseudoexon. Immunoprecipitation experiments showed that specific binding of heterogenous nuclear ribonucleoprotein E1 (hnRNP E1) and U1 small nuclear ribonucleoprotein (snRNP) in the pre-spliceosomal complex was associated with silencing of pseudoexon splicing. The possible role of hnRNP E1 was further supported by RNA interference experiments in cultured cells. Immunoprecipitation experiments with three other pseudoexons suggested that pre-spliceosomal binding of U1 snRNP is a potential general mechanism of suppression of pseudoexons.     

Inactivation of a subpopulation of human neutrophils by exposure to ultrahigh-molecular-weight polyethylene wear debris

Polymorphonuclear neutrophils, a first line of defence against invading microbial pathogens, may be attracted by inflammatory mediators triggered by ultrahigh-molecular-weight polyethylene (UHMWPE) wear particles released from orthopaedic prostheses. Phagocytosis of UHMWPE particles by neutrophils may indirectly compromise their phagocytic-bactericidal mechanisms, thus enhancing host susceptibility to microbial infections. In an in vitro assay, pre-exposure of purified human neutrophils to UHMWPE micrometre- and submicrometre-sized wear particles interfered with subsequent Staphylococcos aureus uptake in a heterogeneous way, as assessed by a dual label fluorescence microscopic assay that discriminated intracellular rhodamine-labelled UHMWPE particles from fluorescein isothiocyanate-labelled S. aureus. Indeed, a higher percentage (44%) of neutrophils having engulfed UHMWPE particles lost the ability to phagocytize S. aureus, compared with UHMWPE-free neutrophils (<3%). Pre-exposure of neutrophils to UHMWPE wear particles did not impair but rather stimulated their oxidative burst response in a chemoluminescence assay. The presence of UHMWPE wear particles did not lead to significant overall consumption of complement-mediated opsonic factors nor decreased surface membrane display of neutrophil complement receptors. In conclusion, engulfment of UHMWPE wear particles led to inactivation of S. aureus uptake in nearly half of the neutrophil population, which may potentially impair host clearance mechanisms against pyogenic infections.     

The effect of n-3 PUFA on eNOS activity and expression in Ea hy 926 cells

The aim of this study was to evaluate the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on NO synthase (eNOS) activation in Ea hy 926 endothelial cells. EPA or DHA (0-80 microM), added to the culture medium during 24h, were dose-dependently incorporated into the cells. In control medium, eNOS activity (evaluated by the citrulline assay) and eNOS phosphorylation on Ser 1177 were correlated. They were increased by 10 microM histamine and prevented by 20 microM lysophosphatidylcholine (LPC). By contrast, EPA or DHA increased basal phosphorylation without affecting eNOS activity in non-stimulated cells, but dose-dependently decreased this activity in histamine-stimulated cells without modifying the phosphorylation level. Furthermore, EPA and DHA did not prevent the deleterious effects of LPC on histamine stimulation. In conclusion, incorporation of EPA and DHA could be deleterious for endothelial cells by deregulating the activation of eNOS and preventing NO liberation.     

A FRET analysis to unravel the role of cholesterol in Rac1 and PI 3-kinase activation in the InlB/Met signalling pathway

The signalling pathway for the hepatocyte growth factor receptor, Met/HGF-R, is hijacked by the bacterial surface protein InlB to induce Listeria monocytogenes entry into non-phagocytic cells. We previously showed that Listeria invades host cells by interacting with specialized microdomains of the host plasma membrane called lipid rafts. In this study, we analysed in living cells signalling events that are crucial for Listeria entry using a fluorescence resonance energy transfer-based microscopic method. Phosphoinositide (PI) 3-kinase activity and Rac1 signalling induced by Listeria interacting with epithelial cells were monitored as well as signalling induced by soluble InlB and the Met natural ligand HGF. We found that InlB and HGF induced similar kinetics of PI 3-kinase and Rac1 activation. PI 3-kinase activation was upstream and independent of Rac1 activation. Cholesterol-depletion experiments were performed to address the role of lipid rafts in Met signalling. The amount of 3'-phosphoinositides produced by PI 3-kinase was not affected by cholesterol depletion, while their membrane dynamic was cholesterol-dependent. Rac1 activation, downstream from PI 3-kinase, was cholesterol-dependent suggesting that the spatial distribution of 3'-phosphoinositides within membrane microdomains is critical for Rac1 activation and consequently for F-actin assembly at bacterial entry site.