Proteome-Wide Search Reveals Unexpected RNA-Binding Proteins in Saccharomyces cerevisiae

The vast landscape of RNA-protein interactions at the heart of post-transcriptional regulation remains largely unexplored. Indeed it is likely that, even in yeast, a substantial fraction of the regulatory RNA-binding proteins (RBPs) remain to be discovered. Systematic experimental methods can play a key role in discovering these RBPs - most of the known yeast RBPs lack RNA-binding domains that might enable this activity to be predicted. We describe here a proteome-wide approach to identify RNA-protein interactions based on in vitro binding of RNA samples to yeast protein microarrays that represent over 80% of the yeast proteome. We used this procedure to screen for novel RBPs and RNA-protein interactions. A complementary mass spectrometry technique also identified proteins that associate with yeast mRNAs. Both the protein microarray and mass spectrometry methods successfully identify previously annotated RBPs, suggesting that other proteins identified in these assays might be novel RBPs. Of 35 putative novel RBPs identified by either or both of these methods, 12, including 75% of the eight most highly-ranked candidates, reproducibly associated with specific cellular RNAs. Surprisingly, most of the 12 newly discovered RBPs were enzymes. Functional characteristics of the RNA targets of some of the novel RBPs suggest coordinated post-transcriptional regulation of subunits of protein complexes and a possible link between mRNA trafficking and vesicle transport. Our results suggest that many more RBPs still remain to be identified and provide a set of candidates for further investigation

Kinin B1 Receptor Enhances the Oxidative Stress in a Rat Model of Insulin Resistance: Outcome in Hypertension, Allodynia and Metabolic Complications

BACKGROUND: Kinin B(1) receptor (B(1)R) is induced by the oxidative stress in models of diabetes mellitus. This study aims at determining whether B(1)R activation could perpetuate the oxidative stress which leads to diabetic complications. METHODS AND FINDINGS: Young Sprague-Dawley rats were fed with 10% D-Glucose or tap water (controls) for 8-12 weeks. A selective B(1)R antagonist (SSR240612) was administered acutely (3-30 mg/kg) or daily for a period of 7 days (10 mg/kg) and the impact was measured on systolic blood pressure, allodynia, protein and/or mRNA B(1)R expression, aortic superoxide anion (O(2)(*-)) production and expression of superoxide dismutase (MnSOD) and catalase. SSR240612 reduced dose-dependently (3-30 mg/kg) high blood pressure in 12-week glucose-fed rats, but had no effect in controls. Eight-week glucose-fed rats exhibited insulin resistance (HOMA index), hypertension, tactile and cold allodynia and significant increases of plasma levels of glucose and insulin. This was associated with higher aortic levels of O(2)(*-), NADPH oxidase activity, MnSOD and catalase expression. All these abnormalities including B(1)R overexpression (spinal cord, aorta, liver and gastrocnemius muscle) were normalized by the prolonged treatment with SSR240612. The production of O(2)(*-) in the aorta of glucose-fed rats was also measured in the presence and absence of inhibitors (10-100 microM) of NADPH oxidase (apocynin), xanthine oxidase (allopurinol) or nitric oxide synthase (L-NAME) with and without Sar[D-Phe(8)]des-Arg(9)-BK (20 microM; B(1)R agonist). Data show that the greater aortic O(2)(*-) production induced by the B(1)R agonist was blocked only by apocynin. CONCLUSIONS: Activation of kinin B(1)R increased O(2)(*-) through the activation of NADPH oxidase in the vasculature. Prolonged blockade of B(1)R restored cardiovascular, sensory and metabolic abnormalities by reducing oxidative stress and B(1)R gene expression in this model

Anti-oxidant enzyme activities and expression and oxidative damage in patients with non-immediate reactions to drugs

Adverse drug reactions with an immunological basis (ADRIB) may involve activation of other concomitant, non-specific mechanisms, amplifying the specific response and contributing to the severity and duration. One concomitant mechanism could be the generation of reactive oxygen species (ROS) and/or their detoxification by anti-oxidants, including anti-oxidant enzymes. We analysed the activity of the anti-oxidant enzymes Cu/Zn-superoxide dismutase (SOD), catalase (CAT) and cellular glutathione peroxidase (GPX), as well as certain markers of oxidative damage (thiobarbituric acid reactive substances (TBARS) and carbonyl content) in peripheral blood mononuclear cells from patients with non-immediate ADRIB using spectrophotometric methods and the anti-oxidant enzymes expression by quantitative real-time reverse transcription–polymerase chain reaction. SOD activity and expression were increased in all types of non-immediate reactions (urticaria, maculopapular exanthema and toxic epidermal necrolysis). Regarding oxidative damage, TBARS were increased in urticaria and maculopapular exanthema, and carbonyl groups in all types of reactions. Our observations indicate that oxidative damage occurs in non-immediate reactions. Carbonyl stress and the inadequacy of the anti-oxidant defences are probable causes