The plant alkaloid voacamine induces apoptosis-independent autophagic cell death on both sensitive and multidrug resistant human osteosarcoma cells

In our previous studies, the bisindolic alkaloid voacamine (VOA), isolated from the plant Peschiera fuchsiaefolia, proved to exert a chemosensitizing effect on cultured multidrug resistant (MDR) osteosarcoma cells exposed to doxorubicin (DOX). In particular, VOA was capable of inhibiting P-glycoprotein action in a competitive way, thus explaining the enhancement of the cytotoxic effect induced by DOX on MDR cells. Afterwards, preliminary observations suggested that such an enhancement did not involve the apoptotic process but was due instead to the induction of autophagic cell death. The results of the present investigation demonstrate that the plant alkaloid VOA is an autophagy inducer able to exert apoptosis-independent cytotoxic effect on both wild-type and MDR tumor cells. In fact, under treatment condition causing about 50 percent of cell death, no evidence of apoptosis could be revealed by microscopical observations, Annexin V-FITC labeling and analysis of PARP cleavage, whereas the same cells underwent apoptosis when treated with apoptosis inducers, such as doxorubicin and staurosporine. Conversely, VOA-induced autophagy was clearly evidentiated by electron microscopy observations, monodansylcadaverine staining, LC3 expression, and conversion. These results were confirmed by the analysis of the modulating effects of the pretreatment with autophagy inhibitors prior to VOA administration. In addition, transfection of osteosarcoma cells with siRNA against ATG genes reduced VOA cytotoxicity. In conclusion, considering the very debated dual role of autophagy in cancer cells (protective or lethal, pro- or anti- apoptotic) our findings seem to demonstrate, at least in vitro, that a natural product able to induce autophagy can be effective against drug resistant tumors, either used alone or in association with conventional chemotherapeutics.     

PI3K/mTOR signaling regulates prostatic branching morphogenesis

Prostatic branching morphogenesis is an intricate event requiring precise temporal and spatial integration of numerous hormonal and growth factor-regulated inputs, yet relatively little is known about the downstream signaling pathways that orchestrate this process. In this study, we use a novel mesenchyme-free embryonic prostate culture system, newly available mTOR inhibitors and a conditional PTEN loss-of-function model to investigate the role of the interconnected PI3K and mTOR signaling pathways in prostatic organogenesis. We demonstrate that PI3K levels and PI3K/mTOR activity are robustly induced by androgen during murine prostatic development and that PI3K/mTOR signaling is necessary for prostatic epithelial bud invasion of surrounding mesenchyme. To elucidate the cellular mechanism by which PI3K/mTOR signaling regulates prostatic branching, we show that PI3K/mTOR inhibition does not significantly alter epithelial proliferation or apoptosis, but rather decreases the efficiency and speed with which the developing prostatic epithelial cells migrate. Using mTOR kinase inhibitors to tease out the independent effects of mTOR signaling downstream of PI3K, we find that simultaneous inhibition of mTORC1 and mTORC2 activity attenuates prostatic branching and is sufficient to phenocopy combined PI3K/mTOR inhibition. Surprisingly, however, mTORC1 inhibition alone has the reverse effect, increasing the number and length of prostatic branches. Finally, simultaneous activation of PI3K and downstream mTORC1/C2 via epithelial PTEN loss-of-function also results in decreased budding reversible by mTORC1 inhibition, suggesting that the effect of mTORC1 on branching is not primarily mediated by negative feedback on PI3K/mTORC2 signaling. Taken together, our data point to an important role for PI3K/mTOR signaling in prostatic epithelial invasion and migration and implicates the balance of PI3K and downstream mTORC1/C2 activity as a critical regulator of prostatic epithelial morphogenesis.     

Cryopreservation of turkey semen by the pellet method: effects of variables such as the extender, cryoprotectant concentration, cooling time and warming temperature on sperm quality determined through principal components analysis

This study was designed to identify the best pellet cryopreservation procedure for the cryosurvival of turkey semen among 192 different treatments established by variations and permutations of seven conditions used in the freezing/thawing process. These conditions were: diluent (IGGKPh, SPh or Tselutin); dilution rate (1:3 vs. 1:4); cooling time (45 vs. 60 min); dimethylacetamide (DMA) concentration as cryoprotectant (6 vs. 8%); equilibration time in DMA (1 vs. 5 min); semen drop volume (50 vs. 80 μL) and thawing temperature (60 vs. 75 °C). Through principal components analysis (PCA), post-thaw sperm quality data (mobility, viability and membrane functional integrity) were reduced to a single output variable (Sperm Quality) indicating overall post-thaw semen quality. All treatments induced a significant reduction in semen quality after warming (P < 0.01), though one set of seven conditions, or treatment, was identified by PCA to generate the highest Sperm Quality score and a further five treatments yielded a score not significantly different (P > 0.05) from this best score. Although still not fulfilling the requirements for commercial application, our findings serve to identify the critical steps in turkey sperm cryopreservation that need to be assessed in future studies.     

Associated arthropodes in the laying hen excrement

A research about arthropode fauna in laying hen excrement was carried out in a poultry house in the State of S?o Paulo, from January 2001 to December 2002. The objective was to verify the frequency of Diptera and Coleoptera collected by Berlese funil and flutuation methods, and to show correlation between the collect methods and sazonality possible in the currents seasons. A total of 29,499 insects were collected from seventy five collects, being 16,702 Diptera (six families) and 12,797 Coleoptera (four families). Some arthropodes presented direct relation with the collect method, the seasons of the years and the rainfall precipitation, with significant increase in the number of insects collected in certain periods of the year.     

Role of c-Abl in directing metabolic versus mitogenic effects in insulin receptor signaling

c-Abl is a cytoplasmic tyrosine kinase involved in several signal transduction pathways. Here we report that c-Abl is involved also in insulin receptor signaling. Indeed, c-Abl tyrosine kinase is activated upon insulin stimulation. Inhibition of c-Abl tyrosine kinase by STI571 attenuates the effect of insulin on Akt/GSK-3beta phosphorylation and glycogen synthesis, and at the same time, it enhances the effect of insulin on ERK activation, cell proliferation, and migration. This effect of STI571 is specific to c-Abl inhibition, because it does not occur in Abl-null cells and is restored in c-Abl-reconstituted cells. Numerous evidences suggest that focal adhesion kinase (FAK) is involved in mediating this c-Abl effect. First, anti-phosphotyrosine blots indicate that c-Abl tyrosine kinase activation is concomitant with FAK dephosphorylation in response to insulin, whereas c-Abl inhibition is accompanied by FAK phosphorylation in response to insulin, a response similar to that observed with IGF-I. Second, the c-Abl effects on insulin signaling are not observed in cells devoid of FAK (FAK(-/-) cells). Taken together these results suggest that c-Abl activation by insulin, via a modification of FAK response, may play an important role in directing mitogenic versus metabolic insulin receptor signaling.     

Phospholipase A2 up-regulation during mycorrhiza formation in Tuber borchii

TbSP1 is a secreted and surface-associated phospholipase A(2) previously found to be up-regulated in C- or N-deprived free-living mycelia from the ectomycorrhizal ascomycete Tuber borchii. As nutrient limitation is considered an important environmental factor favouring the transition to symbiotic status, TbSP1 was suggested to be involved in the formation of mycorrhizas. An in vitro symbiosis system between Cistus incanus and T. borchii was set up: TbSP1 mRNA levels in free-living mycelia and in mycorrhizas sampled in different districts of the plant-fungus interaction were examined. In the same samples, TbSP1 protein expression was analysed by immunoelectron microscopy. A substantially enhanced TbSP1 mRNA expression, compared with nutrient-limited but free-living mycelia, was detected in the presence of the plant and reached maximal levels in fully developed mycorrhizas. A similar expression trend was revealed by immunolocalization experiments. We have shown that TbSP1 appears to respond to two partially overlapping yet distinct stimuli: nutrient starvation and mycorrhiza formation.     

Uncover Genetic Interactions in Caenorhabditis elegans by RNA Interference

RNA-mediated interference (RNAi) has emerged recently as one of the most powerful functional genomics tools. RNAi has been particularly effective in the nematode worm C. elegans where RNAi has been used to analyse the loss-of-function phenotypes of almost all predicted genes. In this review, we illustrate how RNAi has been used to analyse gene function in C. elegans as well as pointing to some future directions for using RNAi to examine genetic interactions in a systematic manner.     

Secretory phospholipases A2 induce neurite outgrowth in PC12 cells through lysophosphatidylcholine generation and activation of G2A receptor

We previously demonstrated that secretory phospholipase A2 (sPLA2) and lysophosphatidylcholine (LPC) exhibit neurotrophin-like neuritogenic activity in the rat pheochromocytoma cell line PC12. In this study, we further analyzed the mechanism whereby sPLA2 displays neurite-inducing activity. Exogenously added mammalian group X sPLA2 (sPLA2-X), but not group IB and IIA sPLA2s, induced neuritogenesis, which correlated with the ability of sPLA2-X to liberate LPC into the culture media. In accordance, blocking the effect of LPC by supplementation of bovine serum albumin or phospholipase B attenuated neuritogenesis by sPLA2 or LPC. Overproduction or suppression of G2A, a G-protein-coupled receptor involved in LPC signaling, resulted in the enhancement or reduction of neuritogenesis induced by sPLA2 treatment. These results indicate that the neuritogenic effect of sPLA2 is mediated by generation of LPC and subsequent activation of G2A.