Slit antagonizes netrin-1 attractive effects during the migration of inferior olivary neurons

Inferior olivary neurons (ION) migrate circumferentially around the caudal rhombencephalon starting from the alar plate to locate ventrally close to the floor-plate, ipsilaterally to their site of proliferation. The floor-plate constitutes a source of diffusible factors. Among them, netrin-1 is implied in the survival and attraction of migrating ION in vivo and in vitro. We have looked for a possible involvement of slit-1/2 during ION migration. We report that: (1) slit-1 and slit-2 are coexpressed in the floor-plate of the rhombencephalon throughout ION development; (2) robo-2, a slit receptor, is expressed in migrating ION, in particular when they reach the vicinity of the floor-plate; (3) using in vitro assays in collagen matrix, netrin-1 exerts an attractive effect on ION leading processes and nuclei; (4) slit has a weak repulsive effect on ION axon outgrowth and no effect on migration by itself, but (5) when combined with netrin-1, it antagonizes part of or all of the effects of netrin-1 in a dose-dependent manner, inhibiting the attraction of axons and the migration of cell nuclei. Our results indicate that slit silences the attractive effects of netrin-1 and could participate in the correct ventral positioning of ION, stopping the migration when cell bodies reach the floor-plate.     

Proteomic analysis of CA1 and CA3 regions of rat hippocampus and differential susceptibility to intermittent hypoxia

The CA1 and CA3 regions of the hippocampus markedly differ in their susceptibility to hypoxia in general, and more particularly to the intermittent hypoxia that characterizes sleep apnea. Proteomic approaches were used to identify proteins differentially expressed in the CA1 and CA3 regions of the rat hippocampus and to assess changes in protein expression following a 6-h exposure to intermittent hypoxia (IH). Ninety-nine proteins were identified, and 15 were differentially expressed in the CA1 and the CA3 regions. Following IH, 32 proteins in the CA1 region and only 7 proteins in the more resistant CA3 area were up-regulated. Hypoxia-regulated proteins in the CA1 region included structural proteins, proteins related to apoptosis, primarily chaperone proteins, and proteins involved in cellular metabolic pathways. We conclude that IH-mediated CA1 injury results from complex interactions between pathways involving increased metabolism, induction of stress-induced proteins and apoptosis, and, ultimately, disruption of structural proteins and cell integrity. These findings provide initial insights into mechanisms underlying differences in susceptibility to hypoxia in neural tissue, and may allow for future delineation of interventional strategies aiming to enhance neuronal adaptation to IH.     

Y-chromosome analysis in Egypt suggests a genetic regional continuity in Northeastern Africa

The geographic location of Egypt, at the interface between North Africa, the Middle East, and southern Europe, prompted us to investigate the genetic diversity of this population and its relationship with neighboring populations. To assess the extent to which the modern Egyptian population reflects this intermediate geographic position, ten Unique Event Polymorphisms (UEPs), mapping to the nonrecombining portion of the Y chromosome, have been typed in 164 Y chromosomes from three North African populations. The analysis of these binary markers, which define 11 Y-chromosome lineages, were used to determine the haplogroup frequencies in Egyptians, Moroccan Arabs, and Moroccan Berbers and thereby define the Y-chromosome background in these regions. Pairwise comparisons with a set of 15 different populations from neighboring European, North African, and Middle Eastern populations and geographic analysis showed the absence of any significant genetic barrier in the eastern part of the Mediterranean area, suggesting that genetic variation and gene flow in this area follow the "isolation-by-distance" model. These results are in sharp contrast with the observation of a strong north-south genetic barrier in the western Mediterranean basin, defined by the Gibraltar Strait. Thus, the Y-chromosome gene pool in the modern Egyptian population reflects a mixture of European, Middle Eastern, and African characteristics, highlighting the importance of ancient and recent migration waves, followed by gene flow, in the region.     

Epstein-Barr virus mRNA export factor EB2 is essential for production of infectious virus

The splicing machinery which positions a protein export complex near the exon-exon junction mediates nuclear export of mRNAs generated from intron-containing genes. Many Epstein-Barr virus (EBV) early and late genes are intronless, and an alternative pathway, independent of splicing, must export the corresponding mRNAs. Since the EBV EB2 protein induces the cytoplasmic accumulation of intronless mRNA, it is tempting to speculate that EB2 is a viral adapter involved in the export of intronless viral mRNA. If this is true, then the EB2 protein is essential for the production of EBV infectious virions. To test this hypothesis, we generated an EBV mutant in which the BMLF1 gene, encoding the EB2 protein, has been deleted (EBV(BMLF1-KO)). Our studies show that EB2 is necessary for the production of infectious EBV and that its function cannot be transcomplemented by a cellular factor. In the EBV(BMLF1-KO) 293 cells, oriLyt-dependent DNA replication was greatly enhanced by EB2. Accordingly, EB2 induced the cytoplasmic accumulation of a subset of EBV early mRNAs coding for essential proteins implicated in EBV DNA replication during the productive cycle. Two herpesvirus homologs of the EB2 protein, the herpes simplex virus type 1 protein ICP27 and, the human cytomegalovirus protein UL69, only partly rescued the phenotype of the EBV(BMLF1-KO) mutant, indicating that some EB2 functions in virus production cannot be transcomplemented by ICP27 and UL69.     

A collagen-like surface glycoprotein is a structural component of the Bacillus anthracis exosporium

Bacillus anthracis, the aetiological agent of anthrax, is a Gram-positive spore-forming bacterium. The exosporium is the outermost integument surrounding the mature spore. Here, we describe the purification and the characterization of an immunodominant protein of the spore surface. This protein was abundant, glycosylated and part of the exosporium. The amino-terminal sequence was determined and the corresponding gene was identified. It encodes a protein of 382 amino acid residues, the central part of which contains a region of GXX motifs presenting similarity to mammalian collagen proteins. Thus, this collagen-like surface protein was named BclA (for Bacillus collagen-like protein of anthracis). BclA was absent from vegetative cells; it was detected only in spores and sporulating cells. A potential promoter, dependent on the sigma factor sigma(K), which is required for a variety of events late in sporulation, was found upstream from the bclA gene. A bclA deletion mutant was constructed and analysed. Electron microscopy studies showed that BclA is a structural component of the filaments covering the outer layer of the exosporium.     

Genetic diversity and molecular detection of three toxic dinoflagellate genera (Alexandrium,Dinophysis, and Karenia) from French coasts

The objectives of this study were 1) to study the genetic diversity of the Alexandrium, Dinophysis and Karenia genera along the French coasts in order to design probes targeting specific DNA regions, and 2) to apply PCR-based detection to detect these three toxic dinoflagellate genera in natural samples. Genetic diversity of these toxic taxa was first studied from either cultures or cells isolated from Lugol-fixed field samples. By this way, partial sequences of the large ribosomal subunit (LSU rDNA) including the variable domains D1 and D2 of A. minutum, Alexandrium species inside the tamarensis complex, the D. acuminata complex and K. mikimotoi were obtained. Next, specific primers were designed for a selection of toxic algae and used during semi-nested PCR detection. This method was tested over a 3-month period on water samples from the Bay of Concarneau (Brittany, France) and on sediment from the Antifer harbor (The English Channel, France). Specificity and sensitivity of this molecular detection were evaluated using the occurrence of target taxa reported by the IFREMER (Institut Fran?ais de Recherche pour l'Exploitation de la Mer) monitoring network based on conventional microscopic examination. This work presents the first results obtained on the biogeographical distribution of genotypes of these three toxic genera along the French coasts.     

Tumour class prediction and discovery by microarray-based DNA methylation analysis

Aberrant DNA methylation of CpG sites is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation occurs in a tumour type-specific manner. However, large-scale analysis of candidate genes has so far been hampered by the lack of high throughput assays for methylation detection. We have developed the first microarray-based technique which allows genome-wide assessment of selected CpG dinucleotides as well as quantification of methylation at each site. Several hundred CpG sites were screened in 76 samples from four different human tumour types and corresponding healthy controls. Discriminative CpG dinucleotides were identified for different tissue type distinctions and used to predict the tumour class of as yet unknown samples with high accuracy using machine learning techniques. Some CpG dinucleotides correlate with progression to malignancy, whereas others are methylated in a tissue-specific manner independent of malignancy. Our results demonstrate that genome-wide analysis of methylation patterns combined with supervised and unsupervised machine learning techniques constitute a powerful novel tool to classify human cancers.     

Proteomic analysis reveals alterations in the renal kallikrein pathway during hypoxia-induced hypertension

Obstructive sleep apnea syndrome (OSAS), a disorder characterized by episodic hypoxia (EH) during sleep, is associated with systemic hypertension. We used proteomic analysis to examine differences in rat kidney protein expression during EH, and their potential relationship to EH-induced hypertension. Young male Sprague-Dawley rats were exposed to either EH or sustained hypoxia (SH) for 14 (EH14/SH14) and 30 (EH30/SH30) days. Mean arterial blood pressure was significantly increased only in EH30 (p < 0.0002). Kidney proteins were resolved by two-dimensional-PAGE and were identified by MALDI-MS. Renal expression of kallistatin, a potent vasodilator, was down-regulated in all animals. Expression of alpha-1-antitrypsin, an inhibitor of kallikrein activation, was up-regulated in EH but down-regulated in SH. Western blotting showed significant elevation of B(2)-bradykinin receptor expression in all normotensive animals but remained unchanged in hypertensive animals. Proteins relevant to vascular hypertrophy, such as smooth muscle myosin and protein-disulfide isomerase were up-regulated in EH30 but were down-regulated in SH30. These data indicate that EH induces changes in renal protein expression consistent with impairment of vasodilation mediated by the kallikrein-kallistatin pathway and vascular hypertrophy. In contrast, SH-induced changes suggest the kallikrein- and bradykinin-mediated compensatory mechanisms for prevention of hypertension and vascular remodeling. To test the hypothesis suggested by the proteomic data, we measured the effect of EH on blood pressure in transgenic hKLK1 rats that overexpress human kallikrein. Transgenic hKLK1 animals were protected from EH-induced hypertension. We conclude that EH-induced hypertension may result, at least in part, from altered regulation of the renal kallikrein system.     

Arsenite oxidase,an ancient bioenergetic enzyme

Operons coding for the enzyme arsenite oxidase have been detected in the genomes from Archaea and Bacteria by Blast searches using the amino acid sequences of the respective enzyme characterized in two different beta-proteobacteria as templates. Sequence analyses show that in all these species, arsenite oxidase is transported over the cytoplasmic membrane via the tat system and most probably remains membrane attached by an N-terminal transmembrane helix of the Rieske subunit. The biochemical and biophysical data obtained for arsenite oxidase in the green filamentous bacterium Chloroflexus aurantiacus allow a structural model of the enzyme's membrane association to be proposed. Phylogenies for the two constituent subunits (i.e., the molybdopterin-containing and the Rieske subunit) of the heterodimeric enzyme and their respective homologs in DMSO-reductase, formate dehydrogenase, nitrate reductase, and the Rieske/cytb complexes were calculated from multiple sequence alignments. The obtained phylogenetic trees indicate an early origin of arsenite oxidase before the divergence of Archaea and Bacteria. Evolutionary implications of these phylogenies are discussed.