Modification of the detrimental effect of TNF‐α on human endothelial progenitor cells by fasudil and Y27632

Exposure of human endothelial progenitor cells (EPCs) to tumor necrosis factor‐α (TNF‐α) reduced their number and biological activity. Yet, signal transduction events linked to TNF‐α action are still poorly understood. To address this issue, we examined the possible effect of fasudil and Y27632, two inhibitors of Rho kinase pathway, which is involved in endothelial dysfunction, atherosclerosis, and in‐ flammation. Results demonstrated that incubation with fasudil starting from 50 μM but not Y27632 determined a dose‐dependent improvement of EPC number during exposure to TNF‐α (P < 0.05 vs. TNF‐α alone). Analysis of the signal transduction pathway activated by TNF‐α revealed that the increased expression of p‐p38 was not significantly altered by fasudil. Instead, fasudil blocked the TNF‐α induced phosphorylation of Erk1/2 (P < 0.05 vs. TNF‐α) as well as the inhibitor of Erk1/2‐specific phosphorylated form, i.e., PD98059 (P < 0.05 vs. TNF‐α). These results were confirmed by analysis of these kinases by confocal microscopy. Finally, 2D‐DIGE and MALDI‐TOF/TOF analysis of EPCs treated with fasudil revealed increased expression levels of an actin‐related protein and an adenylyl cyclase associated protein and decreased expression levels of proteins related to radical scavenger and nucleotide metabolism. These findings suggest that fasudil positively affects EPC number and that other major signals might take part to this complex pathway. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:351–360, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20345     

Evaluation of intra-specific diversities in Oenococcus oeni through analysis of genomic and expressed DNA

In winemaking Oenococcus (O.) oeni is the most frequent species of lactic acid bacteria (LAB) associated with malolactic fermentation (MLF). Several studies have demonstrated that O. oeni is a quite homogeneous species and strains are difficult to differentiate especially when isolates from the same region are analyzed. In this study, the molecular biodiversity of O. oeni isolated from wines of the same region (Aglianico produced in Basilicata Region, Southern Italy) was evaluated with the aim of designing a molecular approach for discrimination and characterization of the isolates at the strain level. Three molecular techniques were applied: random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), restriction endonucleases analysis-pulsed field gel electrophoresis (REA-PFGE) and differential display PCR (DD-PCR). The results obtained by RAPD-PCR confirmed the difficulty in differentiating isolates. By means of REA-PFGE a higher polymorphism, often related to the origin (winery) of strains, was revealed. However, on analyzing strains isolated from the same winery, only in some cases was more than one REA-PFGE pattern obtained. By analyzing dendrograms constructed on the basis of DD-PCR profiles differentiation of strains isolated from the same winery, in some cases, could be accomplished. The reliability of the DD-PCR in the differentiation of closely related strains suggests that this method could represent an alternative and/or additional tool to other molecular methods, such as REA-PFGE, for fine characterization of oenococcal strains.     

Pivotal Role of the RB Family Proteins in in Vitro Thyroid Cell Transformation

Rat thyroid differentiated cells (PC Cl 3) are an excellent model system with which to study the interaction between differentiation and cell transformation. We previously demonstrated that PC Cl 3 cells expressing the adenovirus E1A gene no longer depend on thyrotropin for growth and do not express thyroid differentiation markers. Here we show that an E1A mutant unable to bind the RB protein failed to transform the PC Cl 3 cells. Conversely, mutations in the E1A p300 interacting region did not affect its transforming ability. The pivotal role of RB family proteins in the thyroid cell transformation is supported by the thyrotropin independence induced by the E7 gene of human papilloma virus type 16, but not by a mutated form in the RB-binding region.     

Kallidin applied to the human nasal mucosa produces algesic response not blocked by capsaicin desensitization.

Various kinins (dissolved in 50 microliters) were applied to the nasal mucosa of healthy human volunteers to test the algesic and proinflammatory effects of these peptides in an intact human tissue. [des-Arg9]-bradykinin (0.5 mumol) was found to be inactive, while bradykinin (0.05-0.5 mumol) and especially kallidin (0.005-0.5 mumol) induced: (a) a mild painful sensation described as burning and pricking (latency 30 s, duration 3-5 min), (b) perception of pulsatility and obstruction in the nasal cavity (onset 1 min, duration 6-8 min). Substance P (0.5 mumol) and neurokinin A (0.5 mumol) produced slight obstruction and weak pulsatile sensation but not pain. Capsaicin (0.05 nmol) produced pain and secretion of fluid, but not pulsatile sensation. The effects of kallidin were not affected by repeated (to induce desensitization) applications of capsaicin (0.5 mumol). Likewise, ipratropium bromide (80 mg in 100 microliters) did not affect responses to kallidin. In an intact human tissue, kallidin produces various effects, including an algesic response, that are apparently independent from activation of B1 receptors and from desensitization of capsaicin-sensitive primary afferents.     

Antibacterial activity of rufloxacin in theStaphylococcus aureus rat granuloma pouch model

The protective effects of rufloxacin againstStaphylococcus aureus-induced infections were compared with those of ciprofloxacin in the granuloma pouch model in the rat. Two strains with different in vitro sensitivity to the drugs were studied. Rufloxacin concentrations persisted longer than ciprofloxacin in the exudate in the pouch cavity and were about eight times higher. Equal doses of rufloxacin and ciprofloxacin had similar antibacterial activities. However, rufloxacin inhibitedStaphylococcus aureus bacterial growth significantly longer than did ciprofloxacin.     

New indole amide derivatives as potent CRTH2 receptor antagonists

A new series of indole amide acting as hCRTH2 receptor ligands had been explored and are described herein. Several amide derivatives displaying low nanomolar activity in hCRTH2 binding and whole blood assays were identified. They were found to behave as a full antagonists, exhibiting good selectivity over related prostaglandin receptors. Also, prototypical compounds in this novel series which displayed acceptable CYP profiles and were orally bioavailable in rats were identified.     

Sperm storage and use in polyandrous females of the globally invasive fruitfly, Ceratitis capitata

The medfly, Ceratitis capitata, is an invasive species in which polyandry, associated with sperm precedence, is a common behaviour in the wild. In this species, characterized by internal fertilization, we disclose how the sperm from two males are stored in the female storage organs and how they are used in terms of paternity outcome. The experiments were designed to furnish comparable and unbiased estimates of sperm numbers and progeny in twice-mated females. Results are incorporated in a model through which it is possible to relate the amount of stored sperm with the progeny of twice-mated females. The results show that polyandrous medfly females conserve equal amounts of sperm from the two males to fertilize their eggs. However, we observed a clear advantage of the second male's sperm in siring progeny, which interestingly decreases in favor of the first male as ovipositions progress. The results enable us to exclude differential sperm mortality and suggest that it is the mechanics governing the storage organs which causes the initial, but decreasing second male sperm precedence during the female reproductive life. These outcomes allow us to correlate sperm use in polyandrous females with the mating strategies and invasiveness of this fly.     

Vitamin D sensitizes breast cancer cells to the action of H2O2: mitochondria as a convergence point in the death pathway

Calcitriol, the hormonal form of vitamin D3, sensitizes breast cancer cells to reactive oxygen species (ROS)-dependent cytotoxicity induced by various anticancer modalities. This effect could be due to increased generation of ROS and/ or to increased sensitivity of the target cells to ROS. This work examined the effect of calcitriol on the damage inflicted on breast cancer cells by the direct action of ROS represented by H2O2. Treatment of MCF-7 cells with H2O2 resulted in activation of caspase 7 as well as induction of caspase-independent cell death. Both were enhanced by 48-72 h of pretreatment with calcitriol. This effect was not due to modulation of H2O2 degradation or to a specific effect on *OH-mediated cytotoxicity. The H2O2-induced drop in mitochondrial membrane potential and release of cytochrome c were enhanced by calcitriol. These findings indicate that calcitriol sensitizes breast cancer cells to ROS-induced death by affecting event(s) common to both caspase-dependent and -independent modes of cell death upstream to mitochondrial damage.