IgE receptor type I-dependent regulation of a Rab3D-associated kinase: a possible link in the calcium-dependent assembly of SNARE complexes

Following activation through high affinity IgE receptors (FcepsilonRI), mast cells release, within a few minutes, their granule content of inflammatory and allergic mediators. FcepsilonRI-induced degranulation is a SNARE (soluble N-ethylmaleimide attachment protein receptors)-dependent fusion process. It is regulated by Rab3D, a subfamily member of Rab GTPases. Evidence exists showing that Rab3 action is calcium-regulated although the molecular mechanisms remain unclear. To obtain an understanding of Rab3D function we have searched for Rab3D-associated effectors that respond to allergic triggering through FcepsilonRI. Using the RBL-2H3 mast cell line we detected a Ser/Thr kinase activity, termed here Rak3D (from Rab3D-associated kinase), because it was specifically co-immunoprecipitated with anti-Rab3D antibody. Rak3D activity, as measured by its auto- or transphosphorylation, was maximal in resting cells and decreased upon stimulation. The down-regulation of the observed activity was blocked with EGTA, but not with other degranulation inhibitors, suggesting that its activity functions downstream of calcium influx. We found that Rak3D phosphorylates the NH(2)-terminal regulatory domain of the t-SNARE syntaxin 4, but not syntaxin 2 or 3. The phosphorylation of syntaxin 4 decreased its binding to its partner SNAP23. Thus, we propose a novel phosphorylation-dependent mechanism by which Rab3D controls SNARE assembly in a calcium-dependent manner.     

Protein Tyrosine Phosphatase-PEST and β8 Integrin Regulate Spatiotemporal Patterns of RhoGDI1 Activation in Migrating Cells

Directional cell motility is essential for normal development and physiology, although how motile cells spatiotemporally activate signaling events remains largely unknown. Here, we have characterized an adhesion and signaling unit comprised of protein tyrosine phosphatase (PTP)-PEST and the extracellular matrix (ECM) adhesion receptor β8 integrin that plays essential roles in directional cell motility. β8 integrin and PTP-PEST form protein complexes at the leading edge of migrating cells and balance patterns of Rac1 and Cdc42 signaling by controlling the subcellular localization and phosphorylation status of Rho GDP dissociation inhibitor 1 (RhoGDI1). Translocation of Src-phosphorylated RhoGDI1 to the cell''s leading edge promotes local activation of Rac1 and Cdc42, whereas dephosphorylation of RhoGDI1 by integrin-bound PTP-PEST promotes RhoGDI1 release from the membrane and sequestration of inactive Rac1/Cdc42 in the cytoplasm. Collectively, these data reveal a finely tuned regulatory mechanism for controlling signaling events at the leading edge of directionally migrating cells.     

Xenopus Shugoshin 2 regulates the spindle assembly pathway mediated by the chromosomal passenger complex

Shugoshins (Sgo) are conserved proteins that act as protectors of centromeric cohesion and as sensors of tension for the machinery that eliminates improper kinetochore-microtubule attachments. Most vertebrates contain two Sgo proteins, but their specific functions are not always clear. Xenopus laevis Sgo1, XSgo1, protects centromeric cohesin from the prophase dissociation pathway. Here, we report the identification of XSgo2 and show that it does not regulate cohesion. Instead, we find that it participates in bipolar spindle assembly. Both Sgo proteins interact physically with the Chromosomal Passenger Complex (CPC) containing Aurora B, a key regulator of mitosis, but the functional consequences of such interaction are distinct. XSgo1 is required for proper localization of the CPC while XSgo2 positively contributes to its activation and the subsequent phosphorylation of at least one key substrate for bipolar spindle assembly, the microtubule depolymerizing kinesin MCAK (Mitotic Centromere-Associated Kinesin). Thus, the two Xenopus Sgo proteins have non-overlapping functions in chromosome segregation. Our results further suggest that this functional specificity could rely on the association of XSgo1 and XSgo2 with different regulatory subunits of the PP2A complex.     

MDC1 maintains genomic stability by participating in the amplification of ATM-dependent DNA damage signals

MDC1 functions in checkpoint activation and DNA repair following DNA damage. To address the physiological role of MDC1, we disrupted the MDC1 gene in mice. MDC1-/- mice recapitulated many phenotypes of H2AX-/- mice, including growth retardation, male infertility, immune defects, chromosome instability, DNA repair defects, and radiation sensitivity. At the molecular level, H2AX, MDC1, and ATM form a positive feedback loop, with MDC1 directly mediating the interaction between H2AX and ATM. MDC1 binds phosphorylated H2AX through its BRCT domain and ATM through its FHA domain. Through these interactions, MDC1 accumulates activated ATM flanking the sites of DNA damage, facilitating further ATM-dependent phosphorylation of H2AX and the amplification of DNA damage signals. In the absence of MDC1, many downstream ATM signaling events are defective. These results suggest that MDC1, as a signal amplifier of the ATM pathway, is vital in controlling proper DNA damage response and maintaining genomic stability.     

Novel series of substituted biphenylmethyl urea derivatives as MCH-R1 antagonists for the treatment of obesity

We have designed and synthesized two novel series of MCH-R1 antagonists based on a substituted biphenylmethyl urea core. SAR was explored, suggesting that optimal binding with the receptor was achieved when the biphenylmethyl group and the linker were substituted on the same nitrogen of the urea moiety. Compound 1-(3'-cyano-4-biphenylmethyl)-3-(2-hydroxy-1,1-dimethylethyl)-1-{2-[1-(4-methylbenzyl)-4-piperidinyl]ethyl}urea 2t showed the best antagonist binding activity to the MCH-R1 with a 43 nM K(i).     

Plants Probiotics as a Tool to Produce Highly Functional Fruits: The Case of Phyllobacterium and Vitamin C in Strawberries

The increasing interest in the preservation of the environment and the health of consumers is changing production methods and food consumption habits. Functional foods are increasingly demanded by consumers because they contain bioactive compounds involved in health protection. In this sense biofertilization using plant probiotics is a reliable alternative to the use of chemical fertilizers, but there are few studies about the effects of plant probiotics on the yield of functional fruits and, especially, on the content of bioactive compounds. In the present work we reported that a strain of genus Phyllobacterium able to produce biofilms and to colonize strawberry roots is able to increase the yield and quality of strawberry plants. In addition, the fruits from plants inoculated with this strain have significantly higher content in vitamin C, one of the most interesting bioactive compounds in strawberries. Therefore the use of selected plant probiotics benefits the environment and human health without agronomical losses, allowing the production of highly functional foods.     

The Helicobacter pylori VacA cytotoxin activates RBL-2H3 cells by inducing cytosolic calcium oscillations

Helicobacter pylori causes an acute inflammatory response followed by chronic infection of the human gastric mucosa. Identification of the bacterial molecules endowed with a pro-inflammatory activity is essential to a molecular understanding of the pathogenesis of H. pylori associated diseases. The vacuolating cytotoxin A (VacA) induces mast cells to release pro-inflammatory cytokines. Here, we show that VacA activates the mast cell line RBL-2H3 by rapidly inducing an oscillation of the level of cytosolic calcium with exocytosis of secretory granules. Cytosolic calcium derives mainly from intracellular stores. VacA also stimulates a calcium-dependent production of pro-inflammatory cytokines, including tumour necrosis factor alpha (TNF-alpha). These observations indicate that VacA may act as a pro-inflammatory factor of H. pylori at very early stages of the innate immune response.     

Karyotypic differentiation via 2n reduction and a finding of a case of triploidy in anurans of the genus <Emphasis Type="Italic">Engystomops</Emphasis> (Anura,Leiuperidae)

The genus Engystomops is divided into two groups, namely the Duovox clade and the Edentulus clade. The species of Edentulus clade have karyotypes with 2n = 22, while E. pustulatus and E. puyango, which belong to Duovox clade, have 2n = 20. To investigate if 2n = 20 is a synapomorphy of Duovox clade, we cytogenetically analyzed all the species of this group, except for E. puyango, in the present study. All of them had 2n = 20, differing from the species of Edentulus clade. Since the species already karyotyped of the genus Physalaemus, which is considered to be the sister group of Engystomops, also have 2n = 22, we conclude that the 2n reduction is a synapomorphy of Duovox clade. Despite the karyotypes of all the species of Duovox clade were very similar, they varied in the NOR pattern. In E. coloradorum, an additional NOR was found in one homologue of the chromosome pair 10 exclusively in all females, indicating that this could possibly be a sexual pair of the ZZ/ZW system. Also in this species, it was found the first case of natural polyploidy of the genus Engystomops.     

Black flower coloration in wild Lisianthius nigrescens: its chemistry and ecological consequences

The major pigments responsible for the flower color within the black flowered Gentianaceae, Lisianthius nigrescens, were characterized by HPLC and chemical analyses HPLC analysis showed one major and one minor anthocyanin and 3 major and 3 minor flavone glycosides. The anthocyanins [delphinidin-3-O-rhamnol(1-6)galactoside and its 5-O-glucoside] comprised an extraordinary 24% of the dry weight of wild collected L. nigrescens corallas, and were accompanied in a 1:1 ratio by a range of apigenin and luteolin 8-C-glucosides and their 7-O-methyl ethers. The high levels of anthocyanins and flavones (and their co-pigmentation) is thought to account for the almost complete absorption of both UV and visible wavebands observed by reflectance photography.