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Abstract: The inactivation of the homotetrameric cytosolic alcohol dehydrogenase I from Kluyveromyces lactis (KlADH I) by naturally occurring disulfides, oxidized glutathione, cystine and cystamine, was studied. The inactivation was fully reversed by dithiothreitol. The nicotinamide coenzyme, but not the substrate ethanol, protected KlADH I from inactivation. Gel filtration experiments and SDS-PAGE analysis, also, revealed that enzyme inactivation coincides with inter-subunits disulfide bond formation which are noticeably enhanced after prolonged oxidation with GSSG. Moreover, oxidized KlADH I, as its reduced state, retained the tetrameric stucture and appears mainly as a dimer under non-reducing SDS-PAGE. Conversely, KlADH I Cys278Ile mutant is unaffected by disulfides treatment. Therefore, in vitro, KlADH I wild-type could exist in two reversible forms: reduced (active) and oxidized (inactive), in which the Cys278 residues of each tetramer are linked by disulfide bonds. The redox state of KlADH I could represent the path for modulating its activity and then a regulatory step of glycolysis under hypoxic conditions. It might be hypothesized that KlADH I could represent an important target in redox signaling of Kluyveromyces lactis cell by inhibiting, under oxidative stress, the glycolytic pathway in favor of the pentose-phosphate shunt to restore its reducing potential. [Copyright &y& Elsevier]

Abstract: In order to discover molecular biomarkers in radiation response we investigated the effects of X-radiation on radioresistant K562 cells by using a comparative proteomic analysis. In treated cells 29 up-regulated and 10 down-regulated proteins were detected by image analysis and identified by mass spectrometry. Elongation factor 1 alpha 1 and stress-70 protein showed a 6.2 and 5.4 fold increase respectively in treated cells. Additional proteins such us pi and omega classes glutathione transferases, ATP synthase D chain, were also found to be up-regulated, suggesting that the enzyme belonging to the cellular detoxification system against oxidative stress and energetic metabolism may have a key role in the cellular response to radiation injury. This data set may provide a useful tool to design a combined chemo- and radiotherapic strategy against leukemia disease. [Copyright &y& Elsevier]

Abstract: We used proteomic approach to analyze the protein profile of human follicular fluid (HFF) obtained from 25 normo-ovulatory women undergoing assisted reproduction techniques due to a male infertility factor. In all HFF samples analyzed we found 695 common spots distributed in the 3 to 10 pH range and in the 10–200 kDa range. Only 625 of these spots were also present in the plasma. We used MALDI-TOF-MS analysis to unequivocally assign 183 HFF/plasma matched spots and 27 HFF/plasma unmatched spots. A large number of acute-phase proteins, including transferrin, ceruloplasmin, afamin, hemopexin, haptoglobin and plasma amyloid protein, were identified in HFF in relatively high concentration supporting the hypothesis that mammalian ovulation can be compared to an inflammatory event. We also identified several important antioxidant enzymes; i.e., catalase, superoxide dismutase, glutathione transferase, paraoxonase, heat shock protein 27 and protein disulfide isomerase. This indicates that during maturation the human follicle is well protected against toxic injury due to oxidative stress. [Copyright &y& Elsevier]