Ethanol induces vascular relaxation via redox-sensitive and nitric oxide-dependent pathways

We investigated the role of reactive oxygen species (ROS) and nitric oxide (NO) in ethanol-induced relaxation. Vascular reactivity experiments showed that ethanol (0.03-200 mmol/L) induced relaxation in endothelium-intact and denuded rat aortic rings isolated from male Wistar rats. Pre-incubation of intact or denuded rings with L-NAME (non selective NOS inhibitor, 100 mu mol/L), 7-nitroindazole (selective nNOS inhibitor, 100 mu mol/L), ODQ (selective inhibitor of guanylyl cyclase enzyme, I mu mol/L), glibenclamide (selective blocker of ATP-sensitive K+ channels, 3 mu mol/L) and 4-aminopyridine (selective blocker of voltage-dependent K+ channels, 4-AP, 1 mmol/L) reduced ethanol-induced relaxation. Similarly, tiron (superoxide anion (O-2(-)) scavenger, 1 mmol/L) and catalase (hydrogen peroxide (H2O2) scavenger, 300 U/mL) reduced ethanol-induced relaxation to a similar extent in both endothelium-intact and denuded rings. Finally, prodifen (non-selective cytochrome P450 enzymes inhibitor, 10 mu mol/L) and 4-methylpyrazole (selective alcohol dehydrogenase inhibitor, 10 mu mol/L) reduced ethanol-induced relaxation. In cultured aortic vascular smooth muscle cells (VSMCs), ethanol stimulated generation of NO, which was significantly inhibited by L-NAME. In endothelial cells, flow cytometry studies showed that ethanol increased cytosolic Ca2+ concentration ([Ca2+]c), O-2(-) and cytosolic NO concentration ([NO]c). Tiron inhibited ethanol-induced increase in [Ca-2]c and [NO]c. The major new finding of this work is that ethanol induces relaxation via redox-sensitive and NO-cGMP-dependent pathways through direct effects on ROS production and NO signaling. These findings identify putative molecular mechanisms whereby ethanol, at pharmacological concentrations, influences vascular reactivity. (C) 2011 Elsevier Inc. All rights reserved.

Analysis of the Organic Hydroperoxide Response of Chromobacterium violaceum Reveals That OhrR Is a Cys-Based Redox Sensor Regulated by Thioredoxin

Organic hydroperoxides are oxidants generated during bacterial-host interactions. Here, we demonstrate that the peroxidase OhrA and its negative regulator OhrR comprise a major pathway for sensing and detoxifying organic hydroperoxides in the opportunistic pathogen Chromobacterium violaceum. Initially, we found that an ohrA mutant was hypersensitive to organic hydroperoxides and that it displayed a low efficiency for decomposing these molecules. Expression of ohrA and ohrR was specifically induced by organic hydroperoxides. These genes were expressed as monocistronic transcripts and also as a bicistronic ohrR-ohrA mRNA, generating the abundantly detected ohrA mRNA and the barely detected ohrR transcript. The bicistronic transcript appears to be processed. OhrR repressed both the ohrA and ohrR genes by binding directly to inverted repeat sequences within their promoters in a redox-dependent manner. Site-directed mutagenesis of each of the four OhrR cysteine residues indicated that the conserved Cys21 is critical to organic hydroperoxide sensing, whereas Cys126 is required for disulfide bond formation. Taken together, these phenotypic, genetic and biochemical data indicate that the response of C. violaceum to organic hydroperoxides is mediated by OhrA and OhrR. Finally, we demonstrated that oxidized OhrR, inactivated by intermolecular disulfide bond formation, is specifically regenerated via thiol-disulfide exchange by thioredoxin (but not other thiol reducing agents such as glutaredoxin, glutathione and lipoamide), providing a physiological reducing system for this thiol-based redox switch.

Experimental diabetes modulates collagen remodelling of joints in rats

The aim of this study was to evaluate extracellular matrix components in articular cartilage, ligaments and synovia in an experimental model of diabetes. Young Wistar rats were divided into a streptozotocin-induced (STZ; 35 mg/kg) diabetic group (DG; n=15) and a control group (CG; n=15). Weight, blood glucose and plasma anti-carboxymethyllysine were measured 70 days after STZ infusions. Knee joints, patellar ligaments, and lateral and medial collateral ligaments were isolated and stained with hematoxylineosin and Picrosirius. The total collagen content was determined by morphometry. Immunofluorescence was employed to evaluate types I, III, and V collagen in ligaments and synovial tissues and types II and XI collagen in cartilage. Results: Higher blood glucose levels and plasma anti-carboxymethyllysine were observed in DG rats when compared to those in CG rats. The final weight was significantly lower in the DG rats than in the CG rats. Histomorphometric evaluation depicted a small quantity of collagen fibers in ligaments and articular cartilage in DG rats, as well as increased collagen in synovial tissue. There was a decrease in cartilage proteoglycans in DG rats when compared with CG rats. Immunofluorescence staining revealed an increase of collagen III and V in ligaments, collagen XI in cartilage, and collagen I in synovial tissue of DG rats compared with CG rats. Conclusion: The ligaments, cartilage and synovia are highly affected following STZ-induced diabetes in rats, due the remodeling of collagen types in these tissues. This process may promote the degradation of the extracellular matrix, thus compromising joint function. Our data may help to better understand the pathogenesis of joint involvement related to diabetes.

SCG postnatal remodelling - hypertrophy and neuron number stability - in Spix's Yellow-toothed Cavies (Galea spixii)

Whilst a fall in neuron numbers seems a common pattern during postnatal development, several authors have nonetheless reported an increase in neuron number, which may be associated with any one of a number of possible processes encapsulating either neurogenesis or late maturation and incomplete differentiation. Recent publications have thus added further fuel to the notion that a postnatal neurogenesis may indeed exist in sympathetic ganglia. In the light of these uncertainties surrounding the effects exerted by postnatal development on the number of superior cervical ganglion (SCG) neurons, we have used state-of-the-art design-based stereology to investigate the quantitative structure of SCG at four distinct timepoints after birth, viz., 1-3 days, 1 month, 12 months and 36 months. The main effects exerted by ageing on the SCG structure were: (i) a 77% increase in ganglion volume; (ii) stability in the total number of the whole population of SCG nerve cells (no change - either increase or decrease) during post-natal development; (iii) a higher proportion of uninucleate neurons to binucleate neurons only in newborn animals; (iv) a 130% increase in the volume of uninucleate cell bodies; and (v) the presence of BrdU positive neurons in animals at all ages. At the time of writing our results support the idea that neurogenesis takes place in the SCG of preas, albeit it warrants confirmation by further markers. We also hypothesise that a portfolio of other mechanisms: cell repair, maturation, differentiation and death may be equally intertwined and implicated in the numerical stability of SCG neurons during postnatal development. (C) 2011 ISDN. Published by Elsevier Ltd. All rights reserved.

High sodium intake adversely affects oxidative-inflammatory response, cardiac remodelling and mortality after myocardial infarction

Objective: Enhanced sodium intake increases volume overload, oxidative stress and production of proinflammatory cytokines. In animal models, increased sodium intake favours ventricular dysfunction after myocardial infarction (MI). The aim of this study was to investigate, in human subjects presenting with ST-segment elevation MI (STEMI), the impact of sodium intake prior the coronary event. Methods: Consecutive patients (n = 372) admitted within the first 24 h of STEMI were classified by a food intake questionnaire as having a chronic daily intake of sodium higher (HS) or lower (LS) than 1.2 g in the last 90 days before MI. Plasma levels of 8-isoprostane, interleucin-2 (IL-2), tumour necrosis factor type alpha (TNF-alpha), C-reactive protein (CRP) and brain natriuretic peptide (BNP) were measured at admission and at the fifth day. Magnetic resonance imaging was performed immediately after discharge. Total mortality and recurrence of acute coronary events were investigated over 4 years of follow-up. Results: The decrease of 8-isoprostane was more prominent and the increase of IL-2, TNF-alpha and CRP less intense during the first 5 days in LS than in HS patients (p < 0.05). Sodium intake correlated with change in plasma BNP between admission and fifth day (r = 0.46; p < 0.0001). End-diastolic volumes of left atrium and left ventricle were greater in HS than in LS patients (p < 0.05). In the first 30 days after MI and up to 4 years afterwards, total mortality was higher in HS than in LS patients (p < 0.05). Conclusion: Excessive sodium intake increases oxidative stress, inflammatory response, myocardial stretching and dilatation, and short and long-term mortality after STEMI. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Cyclic Voltammetry and Computational Chemistry Studies on the Evaluation of the Redox Behavior of Parabens and other Analogues

Parabens are antimicrobial preservatives widely used in pharmaceutical, cosmetic and food industries. The alkyl chain connected to the ester group defines some important physicochemical characteristics of these compounds, including the partition coefficient and redox properties. The voltammetric and computational analyses were carried out in order to evaluate the redox behavior of these compounds and other phenolic analogues. A strong correlation between chemical substituents inductive effects of parabens with redox potentials was observed. Using cyclic voltammetry and glassy carbon working electrode, only one irreversible anodic peak was observed around 0.8 V for methylparaben (MP), ethylparaben (EP), propylparaben (PP), butylparaben (BP), benzylparaben (BzP) and p-substituted phenolic analogues. The electrodonating inductive effect of alkyl groups was demonstrated by the anodic oxidation potential shift to lower values as the carbon number increases and, therefore the parabens (and other phenolic analogues) oxidation processes to the quinonoidic forms showed great dependence on the substituent pattern.

1,4-Diamino-2-butanone, a putrescine analogue, promotes redox imbalance in Trypanosoma cruzi and mammalian cells

The putrescine analogue 1,4-diamino-2-butanone (DAB) is highly toxic to various microorganisms, including Trypanosoma cruzi. Similar to other a-aminocarbonyl metabolites. DAB exhibits pro-oxidant properties. DAB undergoes metal-catalyzed oxidation yielding H2O2, NH4+ ion, and a highly toxic alpha-oxoaldehyde. In vitro. DAB decreases mammalian cell viability associated with changes in redox balance. Here, we aim to clarify the DAB pro-oxidant effects on trypomastigotes and on intracellular T. cruzi amastigotes. DAB (0.05-5 mM) exposure in trypomastigotes, the infective stage of T. cruzi, leads to a decline in parasite viability (IC50 c.a. 0.2 mM DAB; 4 h incubation), changes in morphology, thiol redox imbalance, and increased TcSOD activity. Medium supplementation with catalase (2.5 mu M) protects trypomastigotes against DAB toxicity, while host cell invasion by trypomastigotes is hampered by DAB. Additionally, intracellular amastigotes are susceptible to DAB toxicity. Furthermore, pre-treatment with 100-500 mu M buthionine sulfoximine (BSO) of LLC-MK2 potentiates DAB cytotoxicity, whereas 5 mM N-acetyl-cysteine (NAC) protects cells from oxidative stress. Together, these data support the hypothesis that redox imbalance contributes to DAB cytotoxicity in both T. cruzi and mammalian host cells. (C) 2012 Elsevier Inc. All rights reserved.

Bixin and norbixin protect against DNA-damage and alterations of redox status induced by methylmercury exposure in vivo

Populations in the Amazon are exposed to organic mercury via consumption of contaminated foods. These ethnic groups consume a specific plant seed annatto which contains certain carotenoids. The aim of this study was to find out if these compounds (bixin, BIX and norbixin, NOR), protect against DNA-damage caused by the metal. Therefore, rats were treated orally with methylmercury (MeHg) and with the carotenoids under conditions that are relevant to humans. The animals were treated either with MeHg (30 mu g/kg/bw/day), BIX (0.110 mg/kg/bw/day), NOR (0.011.0 mg/kg/bw/day) or combinations of the metal compound and the carotenoids consecutively for 45 days. Subsequently, the glutathione levels (GSH) and the activity of catalase were determined, and DNA-damage was measured in hepatocytes and leukocytes using single cell gel electrophoresis assays. Treatment with the metal alone caused a decrease in the GSH levels (35%) and induced DNA damage, which resulted in increased DNA migration after electrophoresis in liver and blood cells, whereas no effects were seen with the carotenoids alone. When BIX or NOR were given in combination with organic mercury, the intermediate and the highest concentrations of the carotenoids (1.0 and 10.0 mg/kg/bw/day BIX and 0.1 and 1.0 mg/kg/bw/day NOR) protected against DNA-damage. Furthermore, we found with both carotenoids, a moderate increase in the GSH levels in both metal-treated and untreated animals, while the activities of catalase remained unchanged. Our results indicate that consumption of BIX and NOR may protect humans against the adverse health effects caused by exposure to organic mercury. Environ. Mol. Mutagen., 2012. (c) 2012 Wiley Periodicals, Inc.

Smoking cessation and bronchial epithelial remodelling in COPD: a cross-sectional study

Abstract Background Chronic Obstructive Pulmonary Disease (COPD) is associated with bronchial epithelial changes, including squamous cell metaplasia and goblet cell hyperplasia. These features are partially attributed to activation of the epidermal growth factor receptor (EGFR). Whereas smoking cessation reduces respiratory symptoms and lung function decline in COPD, inflammation persists. We determined epithelial proliferation and composition in bronchial biopsies from current and ex-smokers with COPD, and its relation to duration of smoking cessation. Methods 114 COPD patients were studied cross-sectionally: 99 males/15 females, age 62 ± 8 years, median 42 pack-years, no corticosteroids, current (n = 72) or ex-smokers (n = 42, median cessation duration 3.5 years), postbronchodilator FEV1 63 ± 9% predicted. Squamous cell metaplasia (%), goblet cell (PAS/Alcian Blue+) area (%), proliferating (Ki-67+) cell numbers (/mm basement membrane), and EGFR expression (%) were measured in intact epithelium of bronchial biopsies. Results Ex-smokers with COPD had significantly less epithelial squamous cell metaplasia, proliferating cell numbers, and a trend towards reduced goblet cell area than current smokers with COPD (p = 0.025, p = 0.001, p = 0.081, respectively), but no significant difference in EGFR expression. Epithelial features were not different between short-term quitters (<3.5 years) and current smokers. Long-term quitters (≥3.5 years) had less goblet cell area than both current smokers and short-term quitters (medians: 7.9% vs. 14.4%, p = 0.005; 7.9% vs. 13.5%, p = 0.008; respectively), and less proliferating cell numbers than current smokers (2.8% vs. 18.6%, p < 0.001). Conclusion Ex-smokers with COPD had less bronchial epithelial remodelling than current smokers, which was only observed after long-term smoking cessation (>3.5 years). Trial registration NCT00158847

Cyclic voltammetry and computational chemistry studies on the evaluation of the redox behavior of parabens and other analogues

Parabens are antimicrobial preservatives widely used in pharmaceutical, cosmetic and food industries. The alkyl chain connected to the ester group defines some important physicochemical characteristics of these compounds, including the partition coefficient and redox properties. The voltammetric and computational analyses were carried out in order to evaluate the redox behavior of these compounds and other phenolic analogues. A strong correlation between chemical substituents inductive effects of parabens with redox potentials was observed. Using cyclic voltammetry and glassy carbon working electrode, only one irreversible anodic peak was observed around 0.8 V for methylparaben (MP), ethylparaben (EP), propylparaben (PP), butylparaben (BP), benzylparaben (BzP) and p-substituted phenolic analogues. The electrodonating inductive effect of alkyl groups was demonstrated by the anodic oxidation potential shift to lower values as the carbon number increases and, therefore the parabens (and other phenolic analogues) oxidation processes to the quinonoidic forms showed great dependence on the substituent pattern.

The effect of beta-blockade on myocardial remodelling in Chagas' cardiomyopathy

OBJECTIVE: Chagas' disease has spread throughout Latin America because of the high rate of migration among these countries. Approximately 30% of Chagas' patients will develop cardiomyopathy, and 10% of these will develop severe cardiac damage leading to heart failure. Beta-blockade improves symptoms and survival in heart failure patients; however, its efficacy has not been well established in Chagas' disease. We evaluated the role of carvedilol in cardiac remodeling and mortality in a Chagas' cardiomyopathy animal model. METHODS: We studied Trypanosoma cruzi infection in 55 Syrian hamsters that were divided into three groups: control (15), infected (20), and infected + carvedilol (20). Animals underwent echocardiography, electrocardiography, and morphometry for collagen evaluation in ventricles stained with picrosirius red. RESULTS: The left ventricular diastolic diameter did not change between groups, although it was slightly larger in infected groups, as was left ventricular systolic diameter. Fractional shortening also did not change between groups, although it was slightly lower in infected groups. Collagen accumulation in the interstitial myocardial space was significantly higher in infected groups and was not attenuated by carvedilol. The same response was observed in the perivascular space. The survival curve showed significantly better survival in the control group compared with the infected groups; but no benefit of carvedilol was observed during the study. However, in the acute phase (up to 100 days of infection), carvedilol did reduce mortality. CONCLUSION: Carvedilol did not attenuate cardiac remodeling or mortality in this model of Chagas' cardiomyopathy. The treatment did improve survival in the acute phase of the disease.

Proline dehydrogenase regulates redox state and respiratory metabolism in Trypanosoma Cruzi

Over the past three decades, L-proline has become recognized as an important metabolite for trypanosomatids. It is involved in a number of key processes, including energy metabolism, resistance to oxidative and nutritional stress and osmoregulation. In addition, this amino acid supports critical parasite life cycle processes by acting as an energy source, thus enabling host-cell invasion by the parasite and subsequent parasite differentiation. In this paper, we demonstrate that L-proline is oxidized to Δ(1)-pyrroline-5-carboxylate (P5C) by the enzyme proline dehydrogenase (TcPRODH, E.C. 1.5.99.8) localized in Trypanosoma cruzi mitochondria. When expressed in its active form in Escherichia coli, TcPRODH exhibits a Km of 16.58±1.69 µM and a Vmax of 66±2 nmol/min mg. Furthermore, we demonstrate that TcPRODH is a FAD-dependent dimeric state protein. TcPRODH mRNA and protein expression are strongly upregulated in the intracellular epimastigote, a stage which requires an external supply of proline. In addition, when Saccharomyces cerevisiae null mutants for this gene (PUT1) were complemented with the TcPRODH gene, diminished free intracellular proline levels and an enhanced sensitivity to oxidative stress in comparison to the null mutant were observed, supporting the hypothesis that free proline accumulation constitutes a defense against oxidative imbalance. Finally, we show that proline oxidation increases cytochrome c oxidase activity in mitochondrial vesicles. Overall, these results demonstrate that TcPRODH is involved in proline-dependant cytoprotection during periods of oxidative imbalance and also shed light on the participation of proline in energy metabolism, which drives critical processes of the T. cruzi life cycle.

Impact of exercise training on redox signaling in cardiovascular diseases

Reactive oxygen and nitrogen species regulate a wide array of signaling pathways that governs cardiovascular physiology. However, oxidant stress resulting from disrupted redox signaling has an adverse impact on the pathogenesis and progression of cardiovascular diseases. In this review, we address how redox signaling and oxidant stress affect the pathophysiology of cardiovascular diseases such as ischemia-reperfusion injury, hypertension and heart failure. We also summarize the benefits of exercise training in tackling the hyperactivation of cellular oxidases and mitochondrial dysfunction seen in cardiovascular diseases

Redox regulation of the proteasome via S-glutathionylation

The proteasome is a multimeric and multicatalytic intracellular protease responsible for the degradation of proteins involved in cell cycle control, various signaling processes, antigen presentation, and control of protein synthesis. The central catalytic complex of the proteasome is called the 20S core particle. The majority of these are flanked on one or both sides by regulatory units. Most common among these units is the 19S regulatory unit. When coupled to the 19S unit, the complex is termed the asymmetric or symmetric 26S proteasome depending on whether one or both sides are coupled to the 19S unit, respectively. The 26S proteasome recognizes poly-ubiquitinylated substrates targeted for proteolysis. Targeted proteins interact with the 19S unit where they are deubiquitinylated, unfolded, and translocated to the 20S catalytic chamber for degradation. The 26S proteasome is responsible for the degradation of major proteins involved in the regulation of the cellular cycle, antigen presentation and control of protein synthesis. Alternatively, the proteasome is also active when dissociated from regulatory units. This free pool of 20S proteasome is described in yeast to mammalian cells. The free 20S proteasome degrades proteins by a process independent of poly-ubiquitinylation and ATP consumption. Oxidatively modified proteins and other substrates are degraded in this manner. The 20S proteasome comprises two central heptamers (β-rings) where the catalytic sites are located and two external heptamers (α-rings) that are responsible for proteasomal gating. Because the 20S proteasome lacks regulatory units, it is unclear what mechanisms regulate the gating of α-rings between open and closed forms. In the present review, we discuss 20S proteasomal gating modulation through a redox mechanism, namely, S-glutathionylation of cysteine residues located in the α-rings, and the consequence of this post-translational modification on 20S proteasomal function.