Robo4 signaling in endothelial cells implies attraction guidance mechanisms

Roundabouts (robo) are cell-surface receptors that mediate repulsive signaling mechanisms at the central nervous system midline. However, robos may also mediate attraction mechanisms in the context of vascular development. Here, we have performed structure-function analysis of roundabout4 (Robo4), the predominant robo expressed in embryonic zebrafish vasculature and found by gain of function approaches in vitro that Robo4 activates Cdc42 and Rac1 Rho GTPases in endothelial cells. Indeed, complementary robo4 gene knockdown approaches in zebrafish embryos show lower amounts of active Cdc42 and Rac1 and angioblasts isolated from these knockdown embryos search actively for directionality and guidance cues. Furthermore, Robo4-expressing endothelial cells show morphology and phenotype, characteristic of Rho GTPase activation. Taken together, this study suggests that Robo4 mediates attraction-signaling mechanisms through Rho GTPases in vertebrate vascular guidance.     

Inhibition by suramin of protein synthesis in vitro. Ribosomes as the target of the drug

Suramin, a drug widely used both as a therapeutic agent and in research, inhibits translation in eukaryotic cell-free systems from rabbit reticulocyte lysate (IC(50)=142-241 microM). Suramin affects both initiation (block of 43S pre-initiation complex formation) and elongation (impairment of poly(U) translation). The drug induces an increase in the pools of ribosomal subunits and the formation of high molecular weight ribosomal complexes, thus causing the disappearance of polysomes. Ribosomes isolated from suramin-treated translating mixtures are inactivated. [(3)H]Suramin binds to ribosomes and to isolated 60S and 40S ribosomal subunits (116, 106 and 3 binding sites, respectively) showing higher affinity for the small subunit (K(d)=2 microM).     

Vetiver oil production correlates with early root growth

To gain insight into essential oil metabolism we analyzed the oil produced in Vetiver (Vetiveria zizanioides (L.) Nash) roots during early growth. Planting of Vetiver in the Campania region of southern Italy was performed in the spring using Vetiver culms with short roots and approximately 20 cm leaves. During the first two years of growth Vetiver essential oil was hydrodistilled from root samples collected at intervals of two months starting from transplantation. The production of Vetiver oil was constant during the first six months. Over the next two months there was a twofold increase in production of essential oil, which anticipated a progressive decrease during the cold months (from January to April 2003). The analysis of the Vetiver oil showed the presence of 49 constituents, mainly the tricyclic sesquiterpenes khusimol and zizanoic acid, and the bicyclic sesquiterpenes (E)-isovalencenol, junenol, juniper camphor, nootkatone and α-vetivone. These results suggest that Vetiver essential oil production is closely related to the metabolism of plant roots, which is affected by changes in environmental temperatures.     

Analysis of the shift of the transmission pattern for hepatitis C in a community in Central Italy

Anti-HCV and HCV-RNA prevalence among adults from an isolated Central Italian community were 16.3% and 9.0%; 3.0%, 2.3% among 25-to-49-year-olds, 27.6%, 14.7% among older subjects. 1b genotype prevailed (88.5%). 1a, la/1b, 4 were also isolated. Seropositivity was associated with age > 50 years, public dental health care, glass syringe use, surgical interventions. The transmission pattern seems to be shifting from that typical of Southern Italy (high prevalence, particularly in the elderly, 1b genotype predominant, various routes of transmission involved, cohort effect), to that typical of Europe (lower prevalence, particularly among younger adults, other genotypes involved, association with intravenous drug use and immigrants).     

Design, synthesis and SAR of a novel series of benzimidazoles as potent NPY Y5 antagonists

A novel class of benzimidazole NPY Y5 receptor antagonists was prepared exploiting a privileged spirocarbamate moiety. The structure-activity relationship of this series and efforts to achieve a profile suitable for further development and an appropriate pharmacokinetic profile in rat are described. Optimisation led to the identification of the brain penetrant, orally bioavailable Y5 antagonist 9b which significantly inhibited the food intake induced by a Y5 selective agonist with a minimal effective dose of 30mg/kg po.     

Pro-Inflammatory Profile of Preeclamptic Placental Mesenchymal Stromal Cells: New Insights into the Etiopathogenesis of Preeclampsia

The objective of the present study was to evaluate whether placental mesenchymal stromal cells (PDMSCs) derived from normal and preeclamptic (PE) chorionic villous tissue presented differences in their cytokines expression profiles. Moreover, we investigated the effects of conditioned media from normal and PE-PDMSCs on the expression of pro-inflammatory Macrophage migration Inhibitory Factor (MIF), Vascular Endothelial Growth Factor (VEGF), soluble FMS-like tyrosine kinase-1 (sFlt-1) and free β-human Chorionic Gonadotropin (βhCG) by normal term villous explants. This information will help to understand whether anomalies in PE-PDMSCs could cause or contribute to the anomalies typical of preeclampsia.

Methods

Chorionic villous PDMSCs were isolated from severe preeclamptic (n = 12) and physiological control term (n = 12) placentae. Control and PE-PDMSCs’s cytokines expression profiles were determined by Cytokine Array. Control and PE-PDMSCs were plated for 72 h and conditioned media (CM) was collected. Physiological villous explants (n = 48) were treated with control or PE-PDMSCs CM for 72 h and processed for mRNA and protein isolation. MIF, VEGF and sFlt-1 mRNA and protein expression were analyzed by Real Time PCR and Western Blot respectively. Free βhCG was assessed by immunofluorescent.

Results

Cytokine array showed increased release of pro-inflammatory cytokines by PE relative to control PDMSCs. Physiological explants treated with PE-PDMSCs CM showed significantly increased MIF and sFlt-1 expression relative to untreated and control PDMSCs CM explants. Interestingly, both control and PE-PDMSCs media induced VEGF mRNA increase while only normal PDMSCs media promoted VEGF protein accumulation. PE-PDMSCs CM explants released significantly increased amounts of free βhCG relative to normal PDMSCs CM ones.

Conclusions

Herein, we reported elevated production of pro-inflammatory cytokines by PE-PDMSCs. Importantly, PE PDMSCs induced a PE-like phenotype in physiological villous explants. Our data clearly depict chorionic mesenchymal stromal cells as central players in placental physiopathology, thus opening to new intriguing perspectives for the treatment of human placental-related disorders as preeclampsia.     

Identification of a pepducin acting as S1P3 receptor antagonist

Sphingosine‐1‐phosphate (S1P) is a bioactive lipid with key functions in the immune, inflammatory, and cardiovascular systems. S1P exerts its action through the interaction with a family of five known G protein‐coupled receptors, named S1P_1–5. Among them, S1P₃ has been implicated in the pathological processes of a number of diseases, including sepsis and cancer. KRX‐725 (compound 1) is a pepducin that mimics the effects of S1P by triggering specifically S1P₃. Here, aiming to identify novel S1P₃ antagonists, we carried out an alanine scanning analysis to address the contribution of the side chains of each amino acid residue to the peptide function. Then, deleted peptides from both the C‐ and N‐terminus were prepared in order to determine the minimal sequence for activity and to identify the structural requirements for agonistic and, possibly, antagonistic behaviors. The pharmacological results of the Ala‐scan derived compounds (2–10) suggested a high tolerance of the pepducin 1 to amino acid substitutions. Importantly, the deleted peptide 16 has the ability to inhibit, in a dose‐dependent manner, both pepducin 1‐induced vasorelaxation and fibroblast proliferation. Finally, a computational analysis was performed on the prepared compounds, showing that the supposed antagonists 16 and 17 appeared to be aligned with each other but not with the others. These results suggested a correlation between specific conformations and activities. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.