Vitamin C and E supplementation does not affect heat shock proteins or endogenous antioxidants in trained skeletal muscles during 12 weeks of strength training.

BACKGROUND: Supplementation with large doses of antioxidants, such as vitamin C and E, has been shown to blunt some adaptations to endurance training. The effects of antioxidant supplementation on adaptations to strength training is sparsely studied. Herein we investigated the effects of vitamin C and E supplementation on acute stress responses to exercise and adaptation to traditional heavy load strength training. METHODS: In a double blind placebo-controlled design, twenty-eight, young, trained males and females were randomly assigned to receive either vitamin C and E (C: 1000 mg, E: 235 mg, per day) or placebo supplements, and underwent strength training for 10 weeks. After five weeks, a subgroup conducted a strength training session to investigate acute stress responses. Muscle samples were obtained to investigate changes in stress responses and in proteins and mRNA related to the heat shock proteins (HSPs) or antioxidant enzymes. RESULTS: The acute responses to the exercise session revealed activation of the NFκB pathway indicated by degradation of IκBα in both groups. Vitamin C and E supplementation had, however, no effects on the acute stress responses. Furthermore, ten weeks of strength training did not change muscle αB-crystallin, HSP27, HSP70, GPx1 or mnSOD levels, with no influence of supplementation. CONCLUSIONS: Our results showed that although vitamin C and E supplementation has been shown to interfere with training adaptations, it did not affect acute stress responses or long-term training adaptations in the HSPs or antioxidant enzymes in this study.

The transcriptional co-activator SND1 is a novel regulator of alternative splicing in prostate cancer cells.

Splicing abnormalities have profound impact in human cancer. Several splicing factors, including SAM68, have pro-oncogenic functions, and their increased expression often correlates with human cancer development and progression. Herein, we have identified using mass spectrometry proteins that interact with endogenous SAM68 in prostate cancer (PCa) cells. Among other interesting proteins, we have characterized the interaction of SAM68 with SND1, a transcriptional co-activator that binds spliceosome components, thus coupling transcription and splicing. We found that both SAM68 and SND1 are upregulated in PCa cells with respect to benign prostate cells. Upregulation of SND1 exerts a synergic effect with SAM68 on exon v5 inclusion in the CD44 mRNA. The effect of SND1 on CD44 splicing required SAM68, as it was compromised after knockdown of this protein or mutation of the SAM68-binding sites in the CD44 pre-mRNA. More generally, we found that SND1 promotes the inclusion of CD44 variable exons by recruiting SAM68 and spliceosomal components on CD44 pre-mRNA. Inclusion of the variable exons in CD44 correlates with increased proliferation, motility and invasiveness of cancer cells. Strikingly, we found that knockdown of SND1, or SAM68, reduced proliferation and migration of PCa cells. Thus, our findings strongly suggest that SND1 is a novel regulator of alternative splicing that promotes PCa cell growth and survival.

Alternative splicing modulates autoinhibition and SH3 accessibility in the Src kinase Fyn.

Src family kinases are central regulators of a large number of signaling pathways. To adapt to the idiosyncrasies of different cell types, these kinases may need a fine-tuning of their intrinsic molecular control mechanisms. Here, we describe on a molecular level how the Fyn kinase uses alternative splicing to adapt to different cellular environments. Using structural analysis, site-directed mutagenesis, and functional analysis, we show how the inclusion of either exon 7A or 7B affects the autoinhibition of Fyn and how this changes the SH3-dependent interaction and tyrosine phosphorylation of Sam68, with functional consequences for the Sam68-regulated survival of epithelial cells. Our results illustrate a novel mechanism of evolution that may contribute to the complexity of Src kinase regulation.

PATZ1 gene has a critical role in the spermatogenesis and testicular tumours.

PATZ1 is a recently discovered zinc finger protein that, due to the presence of the POZ domain, acts as a transcriptional repressor affecting the basal activity of different promoters. To gain insights into its biological role, we generated mice lacking the PATZ1 gene. Male PATZ1(-/-) mice were unfertile, suggesting a crucial role of this gene in spermatogenesis. Consistently, most of adult testes from these mice showed only few spermatocytes, associated with increased apoptosis, and complete absence of spermatids and spermatozoa, with the subsequent loss of tubular structure. The analysis of PATZ1 expression, by northern blot, western blot and immunohistochemistry, revealed its presence in Sertoli cells and, among the germ cells, exclusively in the spermatogonia. Since PATZ1 has been indicated as a potential tumour suppressor gene, we also looked at its expression in tumours deriving from testicular germ cells (TGCTs). Although expression of PATZ1 protein was increased in these tumours, it was delocalized in the cytoplasm, suggesting an impaired function. These results indicate that PATZ1 plays a crucial role in normal male gametogenesis and that its up-regulation and mis-localization could be associated to the development of TGCTs.

The RNA-binding protein Sam68 contributes to proliferation and survival of human prostate cancer cells.

The tyrosine kinase Src is frequently activated in advanced human prostate carcinomas and its activation correlates with tyrosine phosphorylation of the RNA-binding protein Sam68. Herein, we have investigated the expression and function of Sam68 in human prostate cancer cells. Analysis of specimens obtained from 20 patients revealed that Sam68 is upregulated at the protein level in 35% of the samples. Real-time polymerase chain reaction confirmed the results at the mRNA level in most patients. Downregulation of Sam68 by RNAi in LNCaP prostate cancer cells delayed cell cycle progression and reduced the proliferation rate. Moreover, depletion of Sam68 sensitized cells to apoptosis induced by DNA-damaging agents. Similarly, stable cell lines expressing a truncated GFP-Sam68(GSG) protein displayed reduced growth rates and higher sensitivity to cisplatin-induced apoptosis. Microarray analyses revealed that a subset of genes involved in proliferation and apoptosis were altered when Sam68 was knocked down in LNCaP cells. Our results indicate that Sam68 expression supports prostate cancer cells proliferation and survival to cytotoxic agents.

Mutational analysis of the iron binding site of Saccharomyces cerevisiae ferroxidase Fet3. An in vivo study.

The role of residues predicted to be involved in the binding of iron by the yeast ferroxidase Fet3 has been studied by site-directed mutagenesis. The effect of Fet3 mutations E185A, E185Q, Y354F, D409V and H489D has been investigated in vivo by kinetic analyses of high affinity iron uptake. Our results indicate that Glu-185 is critical for the binding of iron, since substitution of this residue with Ala or Gln strongly affects both growth and the kinetic parameters of high affinity iron uptake, greatly increasing K(m). Mutations Y354F and D409V result in less severe alteration of high affinity iron uptake, while mutant H489D is unable to grow under conditions of iron limitation.

Cloning of Pichia pastoris Fet3: insights into the high affinity iron uptake system.

High-affinity iron uptake by yeast cells appears to require the presence of a complex formed on the plasma membrane by the multicopper oxidase Fet3 and the permease Ftr1 which work together to allow iron to enter safely inside the cell. The Pichia pastoris ferroxidase Fet3 has been cloned and it has been found to display high sequence similarity to other yeast multicopper oxidases, including all the predicted ligands for the catalytic copper atoms and for the iron substrate. P. pastoris appears to possess a high-affinity iron uptake system similar to that of S. cerevisiae, as far as regulation of expression is concerned. However, the P. pastoris high-affinity iron uptake system presents a K(m) value for iron almost ten times higher than that of S. cerevisiae, possibly to control iron fluxes over a wider range of concentrations of this metal, in order to avoid toxic iron overloading.