Exposure of myosin subfragment 1 (S1) to 3-morpholinosydnonimine (SIN-1) produced a time-dependent inhibition of the F-actin-stimulated S1 Mg 2+ -ATPase activity, reaching 50% inhibition with 46.7 ( 8.3 µM SIN-1 for 8.7 µM S1, that is, at a SIN-1/S1 molar ratio of approximately 5.5. The inhibition was due to the peroxynitrite produced by SIN-1 decomposition because (1) decomposed SIN-1 was found to have no effect on S1 ATPase activity, (2) addition of SIN-1 in the presence of superoxide dismutase and catalase fully prevented inhibition by SIN-1, and (3) micromolar pulses of chemically synthesized peroxynitrite produced inhibition of F-actin-stimulated S1 Mg 2+ -ATPase activity. In parallel, SIN-1 produced the inhibition of the nonphysiological Ca 2+ -dependent and K + /EDTA-dependent S1 ATPase activity of S1 and, therefore, suggested that the inhibition of F-actin-stimulated S1 Mg 2+ -ATPase activity is produced by the oxidation of highly reactive cysteines of S1 (Cys 707 and Cys 697 ), located close to the catalytic center. This point was further confirmed by the titration of S1 cysteines with 5,5′-dithiobis(2-nitrobenzoic acid) and by the parallel decrease of Cys 707 labeling by 5-(iodoacetamido)fluorescein, and it was reinforced by the fact that other common protein modifications produced by peroxynitrite, for example, protein carbonyl and nitrotyrosine formation, were barely detected at the concentrations of SIN-1 that produced more than 50% inhibition of the F-actin-stimulated S1 Mg 2+ -ATPase activity. Differential scanning calorimetry of S1 (untreated and treated with different SIN-1 concentrations) pointed out that SIN-1, at concentrations that generate micromolar peroxynitrite fluxes, impaired the ability of ADP‚V 1 to induce the intermediate catalytic transition state and also produced the partial unfolding of S1 that leads to an enhanced susceptibility of S1 to trypsin digestion, which can be fully protected by 2 mM GSH.

The aim of this study was to investigate whether the effects of aging on oxidative stress markers and expression of major oxidant and antioxidant enzymes associate with impairment of renal function and increases in blood pressure. To explore this, we determined age-associated changes in lipid peroxidation (urinary malondialdehyde), plasma and urinary hydrogen peroxide (H2O2) levels, as well as renal H2O2 production, and the expression of oxidant and antioxidant enzymes in young (13 weeks) and old (52 weeks) male Wistar Kyoto (WKY) rats. Urinary lipid peroxidation levels and H2O2 production by the renal cortex and medulla of old rats were higher than their young counterparts. This was accompanied by overexpression of NADPH oxidase components Nox4 and p22phox in the renal cortex of old rats. Similarly, expression of superoxide dismutase (SOD) isoforms 2 and 3 and catalase were increased in the renal cortex from old rats. Renal function parameters (creatinine clearance and fractional excretion of sodium), diastolic blood pressure and heart rate were not affected by aging, although slight increases in systolic blood pressure were observed during this 52-week period. It is concluded that overexpression of renal Nox4 and p22phox and the increases in renal H2O2 levels in aged WKY does not associate with renal functional impairment or marked increases in blood pressure. It is hypothesized that lack of oxidative stress-associated effects in aged WKY rats may result from increases in antioxidant defenses that counteract the damaging effects of H2O2.

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Gene therapy has long been heralded as the new hope to evolve from symptomatic care of genetic pathologies to a full cure. Recent successes in using gene therapy for treating several ocular and haematopoietic pathologies have shown the great potential of this approach that, in the early days, relied on the use of viral vectors, which were considered by many to be undesirable for human treatment. Therefore, there is considerable interest and effort in developing non-viral vectors, with efficiency close to that of viral vectors. The aim of this study was to develop suitable non-viral carriers for gene therapy to treat pathologies affecting the retina. In this study poly(2-(N,N-dimethylamino)ethyl methacrylate), PDMAEMA was synthesized by reversible addition-fragmentation chain transfer (RAFT) and the in vitro cytocompatibility and transfection efficiency of a range of polymer:DNA ratios evaluated using a retinal cell line; in vivo biocompatibility was evaluated by ocular injection in C57BL/6 mice. The results showed that through RAFT, it is possible to produce a defined-size polymer that is compatible with cell viability in vitro and capable of efficiently directing gene expression in a polymer-DNA ratio-dependent manner. When injected into the eyes of mice, these vectors induced a transient, mild inflammation, characteristic of the implantation of medical devices. These results form the basis of future studies where RAFT-synthesized PDMAEMA will be used to deliver gene expression systems to the retina of mouse models of retinal pathologies. Copyright © 2014 John Wiley & Sons, Ltd.

Background and purpose: This study evaluated the signalling coupled to the a 1 -adrenoceptor-induced stimulation of the Cl À / HCO 3 À exchanger in hypertension. Experimental approach: The Na þ -independent HCO 3 À transport system activity was assayed as the initial rate of pH i recovery after an alkaline load (CO 2 /HCO 3 removal) in immortalized renal proximal tubular epithelial cells from spontaneously hypertensive rat (SHR) and their normotensive control (Wistar Kyoto rat; WKY). Key results: Noradrenaline increased Cl À /HCO 3 À exchanger activity with EC 50 values of 0.6 and 5.3 mM in SHR and WKY cells, respectively. These effects were abolished by prazosin, but not by yohimbine. Phenylephrine increased Cl À /HCO 3 À exchanger activity in SHR and WKY cells (EC 50 of 2.6 and 4.9 mM, respectively). Phenylephrine-mediated increase in Cl À /HCO 3 À exchanger activity in WKY and SHR cells was inhibited by protein kinase C (PKC), MAPK/ERK kinase (MEK) and p38 mitogen-activated protein kinase (p38 MAPK) inhibitors. The expression of a 1A -and a 1B -adrenoceptors was identical in WKY and SHR cells. SHR cells generated more H 2 O 2 than WKY cells. In SHR cells, the NADPH oxidase inhibitor apocynin reduced their increased ability to generate H 2 O 2 and abolished their hypersensitivity to phenylephrine, but failed to affect basal Cl À /HCO 3 À exchanger activity. H 2 O 2 -dependent stimulation of Cl À /HCO 3 À exchange activity was significantly higher in SHR than in WKY cells. Conclusions and implications: Differences between WKY and SHR cells on their sensitivity to a 1 -adrenoceptor stimulation did not correlate with the abundance of a 1A -and a 1B -adrenoceptors and may be related to the increased generation of H 2 O 2 , which may amplify the response downstream of a 1 -adrenoceptor activation.

Abstract-The present study tested the hypothesis that angiotensin II (Ang II)-induced oxidative stress and Ang II-stimulated Cl Ϫ /HCO 3 Ϫ exchanger are increased and related to the differential membrane Ang II type 1 (AT 1 ) receptor and reduced nicotinamide-adenine dinucleotide phosphate oxidase expression in immortalized renal proximal tubular epithelial (PTE) cells from the spontaneously hypertensive rat (SHR) relative to its normotensive control (Wistar Kyoto rat [WKY]

It has been suggested that alterations in Na + ,K + -ATPase mediate the development of several aging-related pathologies, such as hypertension and diabetes. Thus, we evaluated Na + ,K + -ATPase function and H 2 O 2 production in the renal cortex and medulla of Wistar Kyoto (WKY) rats at 13, 52 and 91 weeks of age. Creatinine clearance, proteinuria, urinary excretion of Na + and K + and fractional excretion of Na + were also determined.The results show that at 91 weeks old WKY rats had increased creatinine clearance and did not have proteinuria. Despite aging having had no effect on urinary Na + excretion, urinary K + excretion was increased and fractional Na + excretion was decreased with age. In renal proximal tubules and isolated renal cortical cells, 91 weeks old rats had decreased Na + ,K + -ATPase activity when compared to 13 and 52 weeks old rats. In renal medulla, 91 weeks old rats had increased Na + ,K + -ATPase activity, paralleled by an increase in protein expression of α 1 -subunit of Na + ,K + -ATPase. In addition, renal H 2 O 2 production increased with age and at 91 weeks of age renal medulla H 2 O 2 production was significantly higher than renal cortex production.The present work demonstrates that although at 91 weeks of age WKY rats were able to maintain Na + homeostasis, aging was accompanied by alterations in renal Na + ,K + -ATPase function. The observed increase in oxidative stress may account, in part, for the observed changes. Possibly, altered Na + ,K + -ATPase renal function may precede the development of age-related pathologies and loss of renal function.