Intra-dimer cooperativity between the active site cysteines during the oxidation of peroxiredoxin 2.

Peroxiredoxin 2 (Prdx2) and other typical 2-Cys Prdxs function as homodimers in which hydrogen peroxide oxidizes each active site cysteine to a sulfenic acid which then condenses with the resolving cysteine on the alternate chain. Previous kinetic studies have considered both sites as equally reactive. Here we have studied Prdx2 using a combination of non-reducing SDS-PAGE to separate reduced monomers and dimers with one and two disulfide bonds, and stopped flow analysis of tryptophan fluorescence, to investigate whether there is cooperativity between the sites. We have observed positive cooperativity when H2O2 is added as a bolus and oxidation of the second site occurs while the first site is present as a sulfenic acid. Modelling of this reaction showed that the second site reacts 2.2 ± 0.1 times faster. In contrast, when H2O2 was generated slowly and the first active site condensed to a disulfide before the second site reacted, no cooperativity was evident. Conversion of the sulfenic acid to the disulfide showed negative cooperativity, with modelling of the exponential rise in tryptophan fluorescence yielding a rate constant of 0.75 ± 0.08 s-1 when the alternate active site was present as a sulfenic acid and 2.29 ± 0.08-fold lower when it was a disulfide. No difference in the rate of hyperoxidation at the two sites was detected. Our findings imply that oxidation of one active site affects the conformation of the second site and influences which intermediate forms of the protein are favored under different cellular conditions.

Peroxiredoxin expression and redox status in neutrophils and HL-60 cells.

Peroxiredoxins (Prxs) are thiol peroxidases with a key role in antioxidant defense and redox signaling. They could be important in neutrophils for handling the large amount of oxidants that these cells produce. We investigated the redox state of Prx1 and Prx2 in HL-60 promyelocytic cells differentiated to neutrophil-like cells (dHL-60) and in human neutrophils. HL-60 cell differentiation with dimethyl sulfoxide caused a large decrease in expression of both Prxs, and all-trans retinoic acid also decreased Prx1 expression. Prx1 was mostly reduced in dHL-60 cells. NADPH oxidase activation by phorbol myristate acetate (PMA) or ingestion of Staphylococcus aureus induced rapid oxidation to disulfide-linked dimers, and eventually hyperoxidation. The NADPH oxidase inhibitor, diphenyleneiodonium, prevented Prx1 dimerization in stimulated dHL-60 cells, and decreased the extent of oxidation under resting conditions. In contrast, Prx1 and Prx2 were present in neutrophils from human blood as disulfides, and PMA or S. aureus caused no further oxidation. They remained oxidized on incubation with diphenyleneiodonium in media. Although this suggests that Prx redox cycling could be deficient in neutrophils, thioredoxin expression and thioredoxin reductase activity were similar in neutrophils and dHL-60 cells. Additionally, neutrophil thioredoxin was initially reduced and underwent oxidation after PMA activation. Thus, although the Prxs respond to oxidant generation in dHL-60 cells, in neutrophils they appear "locked" as disulfides. On this basis we propose that neutrophil Prxs are inefficient antioxidants and contribute little to peroxide removal during the oxidative burst, and speculate that they might be involved in other cell processes.

RA Differentiation Enhances Dopaminergic Features, Changes Redox Parameters, and Increases Dopamine Transporter Dependency in 6-Hydroxydopamine-Induced Neurotoxicity in SH-SY5Y Cells.

Research on Parkinson's disease (PD) and drug development is hampered by the lack of suitable human in vitro models that simply and accurately recreate the disease conditions. To counteract this, many attempts to differentiate cell lines, such as the human SH-SY5Y neuroblastoma, into dopaminergic neurons have been undertaken since they are easier to cultivate when compared with other cellular models. Here, we characterized neuronal features discriminating undifferentiated and retinoic acid (RA)-differentiated SH-SYSY cells and described significant differences between these cell models in 6-hydroxydopamine (6-OHDA) cytotoxicity. In contrast to undifferentiated cells, RA-differentiated SH-SY5Y cells demonstrated low proliferative rate and a pronounced neuronal morphology with high expression of genes related to synapse vesicle cycle, dopamine synthesis/degradation, and of dopamine transporter (DAT). Significant differences between undifferentiated and RA-differentiated SH-SY5Y cells in the overall capacity of antioxidant defenses were found; although RA-differentiated SH-SY5Y cells presented a higher basal antioxidant capacity with high resistance against H2O2 insult, they were twofold more sensitive to 6-OHDA. DAT inhibition by 3α-bis-4-fluorophenyl-methoxytropane and dithiothreitol (a cell-permeable thiol-reducing agent) protected RA-differentiated, but not undifferentiated, SH-SY5Y cells from oxidative damage and cell death caused by 6-OHDA. Here, we demonstrate that undifferentiated and RA-differentiated SH-SY5Y cells are two unique phenotypes and also have dissimilar mechanisms in 6-OHDA cytotoxicity. Hence, our data support the use of RA-differentiated SH-SY5Y cells as an in vitro model of PD. This study may impact our understanding of the pathological mechanisms of PD and the development of new therapies and drugs for the management of the disease.

Antidepressant-like action of the bark ethanolic extract from Tabebuia avellanedae in the olfactory bulbectomized mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Tabebuia avellanedae Lorentz ex Griseb is a plant employed in tropical America folk medicine for the treatment of several diseases, including depressive disorders. AIM OF THE STUDY: To investigate the ability of Tabebuia avellanedae ethanolic extract (EET) administered chronically to cause an antidepressant-like effect in the tail suspension test (TST), a predictive test of antidepressant activity, and to reverse behavioral (hyperactivity, anhedonic-like behavior and increased immobility time in the TST) and biochemical changes induced by olfactory bulbectomy (OB), a model of depression, in mice. MATERIALS AND METHODS: Mice were submitted to OB to induce depressive-related behaviors, which were evaluated in the open-field test (hyperactivity), splash test (loss of motivational and self-care behavior indicative of an anhedonic-like behavior) and TST (increased immobility time). Phosphorylation levels of Akt, GSK-3ß, ERK1/2 and CREB, as well as BDNF immunocontent, were evaluated in the hippocampus of bulbectomized mice or sham-operated mice treated for 14 days by p.o. route with EET or vehicle. RESULTS: EET (10 and 30mg/kg) given 14 days by p.o route to mice reduced the immobility time in the TST without altering locomotor activity, an indicative of an antidepressant-like effect. EET per se increased both CREB (Ser(133)) and GSK-3ß (Ser(9)) phosphorylation (at doses of 10-30 and 30mg/kg, respectively) in sham-operated mice. OB caused hyperactivity, loss of motivational and self-care behavior, increased immobility time in the TST and an increase in CREB and ERK1 phosphorylation, as well as BDNF immunocontent. EET abolished all these OB-induced alterations except the increment of CREB phosphorylation. Akt (Ser(473)) and ERK2 phosphorylation levels were not altered in any group. CONCLUSIONS: EET ability to abolish the behavioral changes induced by OB was accompanied by modulation of ERK1 and BDNF signaling pathways, being a promising target of EET. Results indicate that this plant could constitute an attractive strategy for the management of depressive disorders, once more validating the traditional use of this plant.

Retinol and retinoic acid modulate catalase activity in Sertoli cells by distinct and gene expression-independent mechanisms.

Vitamin A (retinol) exerts a major role in several biological functions. However, it was observed that retinol induces oxidative stress on different cellular types. Catalase (EC 1.11.1.6; CAT) is a hydrogen peroxide metabolizing enzyme, and its activity and expression is widely used as an index to measure oxidative stress and perturbations in the cellular redox state. The aim of this study was to investigate the effects of retinol and its major biologically active metabolite, all-trans retinoic acid (RA), on CAT regulation. For this purpose, cultured Sertoli cells (a physiological target of vitamin A) were treated with retinol or RA. Retinol (7 microM, 14 microM) and RA (100 nM, 1 microM) enhanced intracellular reactive species production and increased CAT activity after 24 h of treatment. Retinol increased CAT immunocontent but did not alter CAT mRNA expression, while the increase in CAT activity by RA was not related to alterations in immunocontent or mRNA expression. In vitro incubation of purified CAT with retinol or RA did not alter enzyme activity.