Synaptotagmin 8 is expressed both as a calcium-insensitive soluble and membrane protein in neurons, neuroendocrine and endocrine cells

Synaptotagmins (syt) form a large family of transmembrane proteins and some of its isoforms are known to regulate calcium-induced membrane fusion during vesicular traffic. In view of the reported implication of the isoform syt8 in exocytosis we investigated the expression, localisation and calcium-sensitivity of syt8 in secretory cells. An immunopurified antipeptide antibody was generated which is directed against a C-terminal sequence and devoid of crossreactivity towards syt1 to 12. Subcellular fractionation and immunocytochemistry revealed two forms of synaptotagmin 8 (50 and 40 kDa). Whereas the 40-kDa was present in the cytosol in brain, in PC12 and in clonal beta-cells, the 50-kDa form was localised in very typical clusters and partially colocalised with the SNARE protein Vti1a. Moreover, in primary hippocampal neurons syt8 was only found within the soma. Amplification of syt8 by RT-PCR indicated that the observed protein variants were not generated by alternative splicing of the 6th exon and are most likely linked to variations in the N-terminal region. In contrast to the established calcium sensor syt2, endogenous cytosolic syt8 and transiently expressed syt8-C2AB-eGFP did not translocate upon a raise in cytosolic calcium in living cells. Syt8 is therefore not a calcium sensor in exocytotic membrane fusion in endocrine cells.     

Effects of GPD1 overexpression in Saccharomyces cerevisiae commercial wine yeast strains lacking ALD6 genes

The utilization of Saccharomyces cerevisiae strains overproducing glycerol and with a reduced ethanol yield is a potentially valuable strategy for producing wine with decreased ethanol content. However, glycerol overproduction is accompanied by acetate accumulation. In this study, we evaluated the effects of the overexpression of GPD1, coding for glycerol-3-phosphate dehydrogenase, in three commercial wine yeast strains in which the two copies of ALD6 encoding the NADP+-dependent Mg2+-activated cytosolic acetaldehyde dehydrogenase have been deleted. Under wine fermentation conditions, the engineered industrial strains exhibit fermentation performance and growth properties similar to those of the wild type. Acetate was produced at concentrations similar to that of the wild-type strains, whereas sugar was efficiently diverted to glycerol. The ethanol yield of the GPD1 ald6 industrial strains was 15 to 20% lower than that in the controls. However, these strains accumulated acetoin at considerable levels due to inefficient reduction to 2,3-butanediol. Due to the low taste and odor thresholds of acetoin and its negative sensorial impact on wine, novel engineering strategies will be required for a proper adjustment of the metabolites at the acetaldehyde branch point.     

The Near-bottom Layer as an Ecological Boundary in Marine Ecosystems: Diversity, Taxonomic Composition and Community Definitions

The near-bottom layer of the ocean represents a boundary between two oceanic biotopes (pelagial and benthal), and as a result, the animal populations living in this habitat belong to various diverse ecological groups. There is a profusion of terms to designate the organisms which live near the sea bottom, both in relation to their behaviour and to boundary-layer hydrodynamics. Do the fauna living above the sea bottom form a true community? Should the fauna in this habitat be considered as a true community or a mixed assemblage comprised of benthic and pelagic organisms? Between 1988 and 1996, more than 500 suprabenthic hauls were taken with a modified Macer-GIROQ sledge at 15 sites in the English Channel and the Seine Estuary (5–70 m), at 13 sites on the southern edge of the Cap Ferret Canyon (Bay of Biscay, 350–1100 m), and at 8 sites on the Atlantic seamounts south of the Azores (260–2235 m). This intensive sampling permitted the collection of more than several hundred species and will serve to facilitate discussion concerning the biodiversity of the fauna collected near the sea bottom. This paper proposes that in the estuary, the near-bottom layer is colonized by a mixed assemblage of both pelagic and benthic organisms, while in the coastal and in the bathyal zones, the response to the gradual extinction of light and the decreasing benthic resuspension at near-bottom depths leads to an ecocline.     

ITGB3 shows genetic and expression interaction with SLC6A4

Autism affects more males than females and is associated with disturbances of the serotonin system. The integrin β3 (ITGB3) and serotonin transporter (SLC6A4) genes were both recently identified as male quantitative trait loci (QTLs) for serotonin levels and alleles of each have been associated with autism. Here, we use publicly available genomic resources to determine whether regulation of expression level could be the mechanism behind association between serotonin level and noncoding variation in ITGB3. We also examine whether ITGB3 might interact with SLC6A4 to contribute to autism susceptibility. Using murine and human expression data, we observe that ITGB3 and SLC6A4 expression levels are correlated (0.38<r<0.78). Moreover, genetic variation in ITGB3 is associated with expression of both ITGB3 (P=0.012) and SLC6A4 (P=0.008) in unrelated CEPH individuals. We also show preliminary evidence that genotypes at the ITGB3 and SLC6A4 loci may interact to affect autism susceptibility (P=0.033).     

Comparative study of vanA gene transfer from Enterococcus faecium to Enterococcus faecalis and to Enterococcus faecium in the intestine of mice

Vancomycin-resistant enterococci represent a large reservoir in animals because of the use of avoparcin as a growth promoter in Europe. These strains of animal origin enter the food chain and can either colonize the human gut or transfer their resistance genes to the human microbiota. In this study, we compared the transfer of vancomycin resistance from resistant animal Enterococcus faecium to sensitive human Enterococcus faecalis and E. faecium. We analysed these transfers in dibiotic mice and human faecal flora-associated mice. VanA transfer from animal E. faecium to human E. faecalis occurred in dibiotic mice. The transconjugants appeared rapidly and persisted at levels between 3.0 and 4.0 log10 colony-forming units g(-1) of faeces. In human faecal flora-associated mice, vanA gene transfer was not detected towards E. faecalis but was possible between E. faecium strains. Our experiments revealed the possibility of vanA transfer from animal E. faecium to human E. faecalis in vitro and in vivo in the intestine of dibiotic mice. However, intraspecies transfer of vanA gene seems more common than interspecies transfer among enterococci.     

Role of residual Sb(III) in meglumine antimoniate cytotoxicity and MRP1-mediated resistance

Despite the clinical use of pentavalent antimonials for more than half a century, their metabolism in mammals and mechanisms of action and toxicity remain poorly understood. It has been proposed that the more active and toxic trivalent antimony form Sb(III) plays a critical role in their antileishmanial activity and toxicity. The aim of this work was to investigate the role of residual Sb(III) both in the antileishmanial/antitumoral activities of the pentavalent meglumine antimoniate and in the MRP1 (multidrug resistance-associated protein 1)-mediated resistance to this drug. Samples of meglumine antimoniate differing in their amount of residual Sb(III) (meglumine antimoniate synthesized either from SbCl₅ or from KSb(OH)₆ as well as commercially-available meglumine antimoniate) were evaluated in vitro and in vivo on Leishmania amazonensis infections, as well as for their cytotoxicity to normal and MRP1-overexpressing GLC4 cell lines. Although in vitro the two most effective drugs contained the highest levels of Sb(III), no correlation was found in vivo between the antileishmanial activity of meglumine antimoniate and its residual Sb(III) content, suggesting that residual Sb(III) contributes only marginally to the drug antileishmanial activity. On the other hand, the GLC4 cells growth inhibition data strongly suggests a marked contribution of residual Sb(III). Additionally, the potassium salt of antimoniate (non-complexed form of Sb(V)) was found to be more cytotoxic than meglumine antimoniate. Although MRP1-overexpressing GLC4 cells showed a marked resistance to trivalent antimonials, cross-resistance to meglumine antimoniate was observed only for the products that contained relatively high levels of Sb(III) (at least 0.03% by weight), suggesting that MRP1 mediates resistance to Sb(III) but not to Sb(V). In conclusion, our data strongly suggest that residual Sb(III) in pentavalent antimonial drugs does not contribute significantly to their antileishmanial activity, but is responsible for their cytotoxic activity against mammalian cells and the MRP1-mediated resistance to these drugs.     

Denmotoxin, a three-finger toxin from the colubrid snake Boiga dendrophila (Mangrove Catsnake) with bird-specific activity

Boiga dendrophila (mangrove catsnake) is a colubrid snake that lives in Southeast Asian lowland rainforests and mangrove swamps and that preys primarily on birds. We have isolated, purified, and sequenced a novel toxin from its venom, which we named denmotoxin. It is a monomeric polypeptide of 77 amino acid residues with five disulfide bridges. In organ bath experiments, it displayed potent postsynaptic neuromuscular activity and irreversibly inhibited indirectly stimulated twitches in chick biventer cervicis nerve-muscle preparations. In contrast, it induced much smaller and readily reversible inhibition of electrically induced twitches in mouse hemidiaphragm nerve-muscle preparations. More precisely, the chick muscle alpha(1)betagammadelta-nicotinic acetylcholine receptor was 100-fold more susceptible compared with the mouse receptor. These data indicate that denmotoxin has a bird-specific postsynaptic activity. We chemically synthesized denmotoxin, crystallized it, and solved its crystal structure at 1.9 A by the molecular replacement method. The toxin structure adopts a non-conventional three-finger fold with an additional (fifth) disulfide bond in the first loop and seven additional residues at its N terminus, which is blocked by a pyroglutamic acid residue. This is the first crystal structure of a three-finger toxin from colubrid snake venom and the first fully characterized bird-specific toxin. Denmotoxin illustrates the relationship between toxin specificity and the primary prey type that constitutes the snake's diet.     

Oligomerization of EDEN-BP is required for specific mRNA deadenylation and binding

BACKGROUND INFORMATION: mRNA deadenylation [shortening of the poly(A) tail] is often triggered by specific sequence elements present within mRNA 3' untranslated regions and generally causes rapid degradation of the mRNA. In vertebrates, many of these deadenylation elements are called AREs (AU-rich elements). The EDEN (embryo deadenylation element) sequence is a Xenopus class III ARE. EDEN acts by binding a specific factor, EDEN-BP (EDEN-binding protein), which in turn stimulates deadenylation. RESULTS: We show here that EDEN-BP is able to oligomerize. A 27-amino-acid region of EDEN-BP was identified as a key domain for oligomerization. A mutant of EDEN-BP lacking this region was unable to oligomerize, and a peptide corresponding to this region competitively inhibited the oligomerization of full-length EDEN-BP. Impairing oligomerization by either of these two methods specifically abolished EDEN-dependent deadenylation. Furthermore, impairing oligomerization inhibited the binding of EDEN-BP to its target RNA, demonstrating a strong coupling between EDEN-BP oligomerization and RNA binding. CONCLUSIONS: These data, showing that the oligomerization of EDEN-BP is required for binding of the protein on its target RNA and for EDEN-dependent deadenylation in Xenopus embryos, will be important for the identification of cofactors required for the deadenylation process.     

Biphasic effect of IL-1beta on the activity of argininosuccinate synthetase in Caco-2 cells. Involvement of nitric oxide production

The expression of the argininosuccinate synthetase gene (ASS), the limiting enzyme of arginine synthesis, was previously shown to be rapidly induced by a short-term (4 h) exposure to IL-1beta in Caco-2 cells [Biochimie, 2005, 403-409]. The present report shows that, by contrast, a long-term (24 h) exposure to IL-1beta inhibited the ASS activity despite an increase in both specific mRNA level and protein amount, demonstrating a post-translational effect. Concerning the mechanism involved, we demonstrate that the inhibiting effect is linked to the production of nitric oxide (NO) induced by IL-1beta. Indeed, the inhibiting effect of IL-1beta was totally blocked in the presence of l-NMMA, an inhibitor of the inducible nitric oxide synthase, or by culturing the cells in an arginine-deprived medium. Moreover, a decrease in the ASS activity was induced by culturing the cells in the presence of SNAP, a NO donor. Conversely, blocking the action of NO by antioxidant agents, the stimulatory effect of IL-1beta on ASS activity was restored, as measured at 24 h. Finally, such an inhibiting effect of NO on ASS activity may be related, at least in part, to S-nitrosylation of the protein. The physiological relevance of the antagonistic effects of IL-1beta and NO on ASS is discussed.