Wine yeast selection in the Iberian Peninsula: Saccharomyces and non-Saccharomyces as drivers of innovation in Spanish and Portuguese wine industries.

Yeast selection for the wine industry in Spain started in 1950 for the understanding of the microbial ecology, and for the selection of optimal strains to improve the performance of alcoholic fermentation and the overall wine quality. This process has been strongly developed over the last 30 years, firstly on Saccharomyces cerevisiae, and, lately, with intense activity on non-Saccharomyces. Several thousand yeast strains have been isolated, identified and tested to select those with better performance and/or specific technological properties. The present review proposes a global survey of this massive ex-situ preservation of eukaryotic microorganisms, a reservoir of biotechnological solutions for the wine sector, overviewing relevant screenings that led to the selection of strains from 12 genera and 22 species of oenological significance. In the first part, the attention goes to the selection programmes related to relevant wine-producing areas (i.e. Douro, Extremadura, Galicia, La Mancha and Uclés, Ribera del Duero, Rioja, Sherry area, and Valencia). In the second part, the focus shifted on specific non-Saccharomyces genera/species selected from different Spanish and Portuguese regions, exploited to enhance particular attributes of the wines. A fil rouge of the dissertation is the design of tailored biotechnological solutions for wines typical of given geographic areas.

Genetic regulation of the ompX porin of Salmonella Typhimurium in response to hydrogen peroxide stress.

BACKGROUND: Salmonella Typhimurium is a Gram-negative pathogen that causes a systemic disease in mice resembling typhoid fever. During its infective cycle, S. Typhimurium is phagocytized by macrophages and proliferates inside a Salmonella-containing vacuole where Salmonella is exposed and survives oxidative stress induced by H2O2 through modulation of gene expression. After exposure of Salmonella to H2O2, the expression of the porin-encoding gene ompX increases, as previously shown by microarray analysis. Expression of ompX mRNA is regulated at a post-transcriptional level by MicA and CyaR sRNAs in aerobiosis. In addition, sequence analysis predicts a site for OxyS sRNA in ompX mRNA. RESULTS: In this work we sought to evaluate the transcriptional and post-transcriptional regulation of ompX under H2O2 stress. We demonstrate that ompX expression is induced at the transcriptional level in S. Typhimurium under such conditions. Unexpectedly, an increase in ompX gene transcript and promoter activity after challenges with H2O2 does not translate into increased protein levels in the wild-type strain, suggesting that ompX mRNA is also regulated at a post-transcriptional level, at least under oxidative stress. In silico gene sequence analysis predicted that sRNAs CyaR, MicA, and OxyS could regulate ompX mRNA levels. Using rifampicin to inhibit mRNA expression, we show that the sRNAs (MicA, CyaR and OxyS) and the sRNA:mRNA chaperone Hfq positively modulate ompX mRNA levels under H2O2-induced stress in Salmonella during the exponential growth phase in Lennox broth. CONCLUSIONS: Our results demonstrate that ompX mRNA is regulated in response to H2O2 by the sRNAs CyaR, MicA and OxyS is Salmonella Typhimurium.

Genetic regulation of the ompX porin of Salmonella Typhimurium in response to hydrogen peroxide stress

BACKGROUND: Salmonella Typhimurium is a Gram negative pathogen that causes a systemic disease in mice resembling typhoid fever. During its infective cycle, S. Typhimurium is phagocytized by macrophages and proliferates inside a Salmonella containing vacuole where Salmonella is exposed and survives oxidative stress induced by H2O2 through modulation of gene expression. After exposure of Salmonella to H2O2, the expression of the porin encoding gene ompX increases, as previously shown by microarray analysis. Expression of ompX mRNA is regulated at a post transcriptional level by MicA and CyaR sRNAs in aerobiosis. In addition, sequence analysis predicts a site for OxyS sRNA in ompX mRNA. RESULTS: In this work we sought to evaluate the transcriptional and post transcriptional regulation of ompX under H2O2 stress. We demonstrate that ompX expression is induced at the transcriptional level in S . Typhimurium under such conditions. Unexpectedly, an increase in ompX gene transcript and promoter activity after challenges with H2O2 does not translate into increased protein levels in the wild type strain, suggesting that ompX mRNA is also regulated at a post transcriptional level, at least under oxidative stress. In silico gene sequence analysis predicted that sRNAs CyaR, MicA, and OxyS could regulate ompX mRNA levels. Using rifampicin to inhibit mRNA expression, we show that the sRNAs (MicA, CyaR and OxyS) and the sRNA:mRNA chaperone Hfq positively modulate ompX mRNA levels under H2O2 induced stress in Salmonella during the exponential growth phase in Lennox broth. CONCLUSIONS: Our results demonstrate that ompX mRNA is regulated in response to H2O2 by the sRNAs CyaR, MicA and OxyS is Salmonella Typhimurium.

Characterization of yeast population from unstudied natural sources in La Mancha region.

AIMS: This study aims to identify the yeast species and strains which entitled an unstudied area of Spain and evaluate the yeast species diversity richness and the genetic variety. METHODS AND RESULTS: A total of 702 yeasts were isolated from different environments in a central Spanish region (La Mancha) with diverse sources of origin (food, animals, flowers and environmental sources) during spring season. Thanks to the analysis carried out by the PCR-RFLP technique and sequencing, 35 species were identified. A neighbour-joining phylogenetic tree was created based on D1/D2 sequences. Moreover 330 strains were determined by PCR-RAPD and their profiles were analysed using the bioinformatics programme BioNumerics 7·6. The Simpson's index (D) and the genetic diversity percentage were calculated with the aim of studying the richness of the species in each environment and the genetic variety in each species. CONCLUSIONS: This study has permitted to know that the majority of the species found was Diutina rugosa while the most ubiquitous was Rhodotorula mucilaginosa which expose the dispersion capability of this species. The diversity parameters has revealed that the highest species richness was associated to environmental samples and the highest genetic variety was presented in those species with better dispersion capability or a smaller number of isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: This study permits to better understand the yeast communities in La Mancha region which gives a value the microbial potential of this region.

Acetabular Fractures in the Elderly: Midterm Outcomes of Column Stabilisation and Primary Arthroplasty.

Background. Interest in arthroplasty techniques for periarticular or intra-articular fractures in the elderly/osteoporotic patient continues to rise, including for geriatric acetabular fractures. In line with this, many acetabular fracture surgeons are now undertaking acute total hip arthroplasty in elderly/osteoporotic patients. Little is known however of the outcomes of this procedure, beyond the first year after surgery. Questions/Purposes. We determined the clinical outcomes of a series of elderly osteoporotic patients (mean age at surgery 77.4 years) treated for acetabular fractures with column fixation and simultaneous total hip arthroplasty, at a mean of 49 months after surgery. Methods. 24 patients (25 hips) were reviewed at a mean of 49 months after surgery. The surgical technique employed has previously been described. Radiographs were obtained, and clinical outcomes were assessed using Harris Hip Scores and the Merle d'Aubigné score. Results. 14 hips were available for assessment (9 deceased, 2 lost to follow-up). No patient suffered any complications beyond the perioperative period, no acetabular components were loose clinically or on latest radiographs, and the mean Harris Hip Score was 92. All but one patient scored good or excellent on the Merle d'Aubigné score. Conclusions. Column fixation and simultaneous total hip arthroplasty are a viable option for complex geriatric acetabular fractures, with encouraging midterm results. We conclude that THR is a viable long-term solution in this situation provided that the acetabular columns are stabilised prior to implantation, but more research is needed to aid in overall management decision making.

Role of COX-2-derived PGE2 on vascular stiffness and function in hypertension.

BACKGROUND AND PURPOSE: Prostanoids derived from COX-2 and EP receptors are involved in vascular remodelling in different cardiovascular pathologies. This study evaluates the contribution of COX-2 and EP1 receptors to vascular remodelling and function in hypertension. EXPERIMENTAL APPROACH: Spontaneously hypertensive rats (SHR) and angiotensin II (AngII)-infused (1.44 mg · kg(-1) · day(-1), 2 weeks) mice were treated with the COX-2 inhibitor celecoxib (25 mg · kg(-1) · day(-1) i.p) or with the EP1 receptor antagonist SC19220 (10 mg · kg(-1) · day(-1) i.p.). COX-2(-/-) mice with or without AngII infusion were also used. KEY RESULTS: Celecoxib and SC19220 treatment did not modify the altered lumen diameter and wall : lumen ratio in mesenteric resistance arteries from SHR-infused and/or AngII-infused animals. However, both treatments and COX-2 deficiency decreased the augmented vascular stiffness in vessels from hypertensive animals. This was accompanied by diminished vascular collagen deposition, normalization of altered elastin structure and decreased connective tissue growth factor and plasminogen activator inhibitor-1 gene expression. COX-2 deficiency and SC19220 treatment diminished the increased vasoconstrictor responses and endothelial dysfunction induced by AngII infusion. Hypertensive animals showed increased mPGES-1 expression and PGE2 production in vascular tissue, normalized by celecoxib. Celecoxib treatment also decreased AngII-induced macrophage infiltration and TNF-α expression. Macrophage conditioned media (MCM) increased COX-2 and collagen type I expression in vascular smooth muscle cells; the latter was reduced by celecoxib treatment. CONCLUSIONS AND IMPLICATIONS: COX-2 and EP1 receptors participate in the increased extracellular matrix deposition and vascular stiffness, the impaired vascular function and inflammation in hypertension. Targeting PGE2 receptors might have benefits in hypertension-associated vascular damage.

The ongoing relevance of acetabular fracture classification.

The most widely used classification system for acetabular fractures was developed by Judet, Judet and Letournel over 50 years ago primarily to aid surgical planning. As population demographics and injury mechanisms have altered over time, the fracture patterns also appear to be changing. We conducted a retrospective review of the imaging of 100 patients with a mean age of 54.9 years (19 to 94) and a male to female ratio of 69:31 seen between 2010 and 2013 with acetabular fractures in order to determine whether the current spectrum of injury patterns can be reliably classified using the original system. Three consultant pelvic and acetabular surgeons and one senior fellow analysed anonymous imaging. Inter-observer agreement for the classification of fractures that fitted into defined categories was substantial, (κ = 0.65, 95% confidence interval (CI) 0.51 to 0.76) with improvement to near perfect on inclusion of CT imaging (κ = 0.80, 95% CI 0.69 to 0.91). However, a high proportion of injuries (46%) were felt to be unclassifiable by more than one surgeon; there was moderate agreement on which these were (κ = 0.42 95% CI 0.31 to 0.54). Further review of the unclassifiable fractures in this cohort of 100 patients showed that they tended to occur in an older population (mean age 59.1 years; 22 to 94 vs 47.2 years; 19 to 94; p = 0.003) and within this group, there was a recurring pattern of anterior column and quadrilateral plate involvement, with or without an incomplete posterior element injury.

HuR mediates the synergistic effects of angiotensin II and IL-1ß on vascular COX-2 expression and cell migration.

BACKGROUND AND PURPOSE: Angiotensin II (AngII) and IL-1ß are involved in cardiovascular diseases through the induction of inflammatory pathways. HuR is an adenylate- and uridylate-rich element (ARE)-binding protein involved in the mRNA stabilization of many genes. This study investigated the contribution of HuR to the increased expression of COX-2 induced by AngII and IL-1ß and its consequences on VSMC migration and remodelling. EXPERIMENTAL APPROACH: Rat and human VSMCs were stimulated with AngII (0.1 µM) and/or IL-1ß (10 ng · mL(-1)). Mice were infused with AngII or subjected to carotid artery ligation. mRNA and protein levels were assayed by quantitative PCR, Western blot, immunohistochemistry and immunofluorescence. Cell migration was measured by wound healing and transwell assays. KEY RESULTS: In VSMCs, AngII potentiated COX-2 and tenascin-C expressions and cell migration induced by IL-1ß. This effect of AngII on IL-1ß-induced COX-2 expression was accompanied by increased COX-2 3' untranslated region reporter activity and mRNA stability, mediated through cytoplasmic HuR translocation and COX-2 mRNA binding. These effects were blocked by ERK1/2 and HuR inhibitors. VSMC migration was reduced by blockade of ERK1/2, HuR, COX-2, TXAS, TP and EP receptors. HuR, COX-2, mPGES-1 and TXAS expressions were increased in AngII-infused mouse aortas and in carotid-ligated arteries. AngII-induced tenascin-C expression and vascular remodelling were abolished by celecoxib and by mPGES-1 deletion. CONCLUSIONS AND IMPLICATIONS: The synergistic induction of COX-2 by AngII and IL-1ß in VSMCs involves HuR through an ERK1/2-dependent mechanism. The HuR/COX-2 axis participates in cell migration and vascular damage. HuR might be a novel target to modulate vascular remodelling.

Toll-like receptor 4 contributes to vascular remodelling and endothelial dysfunction in angiotensin II-induced hypertension.

BACKGROUND AND PURPOSE: Toll-like receptor 4 (TLR4) signalling contributes to inflammatory cardiovascular diseases, but its role in hypertension and the associated vascular damage is not known. We investigated whether TLR4 activation contributed to angiotensin II (AngII)-induced hypertension and the associated vascular structural, mechanical and functional alterations. EXPERIMENTAL APPROACH: AngII was infused (1.44 mg · kg(-1) · day(-1), s.c.) for 2 weeks in C57BL6 mice, treated with a neutralizing anti-TLR4 antibody or IgG (1 µg · day(-1); systolic BP (SBP) and aortic cytokine levels were measured. Structural, mechanical and contractile properties of aortic and mesenteric arterial segments were measured with myography and histology. RT-PCR and Western blotting were used to analyse these tissues and cultured vascular smooth muscle cells (VSMC) from hypertensive rats (SHR). KEY RESULTS: Aortic TLR4 mRNA levels were raised by AngII infusion. Anti-TLR4 antibody treatment of AngII-treated mice normalised: (i) increased SBP and TNF-α, IL-6 and CCL2 levels; (ii) vascular structural and mechanical changes; (iii) altered aortic phenylephrine- and ACh-induced responses; (iv) increased NOX-1 mRNA levels, superoxide anion production and NAD(P)H oxidase activity and effects of catalase, apocynin, ML-171 and Mito-TEMPO on vascular responses; and (v) reduced NO release and effects of L-NAME on phenylephrine-induced contraction. In VSMC, the MyD88 inhibitor ST-2825 reduced AngII-induced NAD(P)H oxidase activity. The TLR4 inhibitor CLI-095 reduced AngII-induced increased phospho-JNK1/2 and p65 NF-κB subunit nuclear protein expression. CONCLUSIONS AND IMPLICATIONS: TLR4 up-regulation by AngII contributed to the inflammation, endothelial dysfunction, vascular remodelling and stiffness associated with hypertension by mechanisms involving oxidative stress. MyD88-dependent activation and JNK/NF-κB signalling pathways participated in these alterations.

Stress-induced anhedonia is associated with an increase in Alzheimer's disease-related markers.

BACKGROUND AND PURPOSE: Stress is believed to be associated with the development of neuropsychiatric disorders, including Alzheimer's disease (AD). We have studied mechanisms implicated in vulnerability to stress and the relationship with changes in AD-related markers. EXPERIMENTAL APPROACH: Anhedonia induced by a chronic mild stress (CMS) procedure, applied for 6 weeks, was used to select rats vulnerable or resistant to stress. Sucrose intake, the Porsolt forced swimming test and cognitive deficits in the novel object recognition test (NORT) were used to characterize vulnerable and resilient rats. The antidepressant venlafaxine (20 mg·kg(-1) p.o.) or saline was administered daily during the last 2 weeks of CMS. Biochemical markers affected by stress, PKB, ERK and synaptophysin, and those associated with AD, amyloid ß-protein (Aß), ß-secretase (BACE1) and τ phosphorylation, were measured in the hippocampus. KEY RESULTS: After CMS, 40% of rats were resistant to the development of anhedonia (CMS-resistant to stress), whereas the remaining were responsive [CMS-anhedonic (CMSA)]. Only CMSA rats displayed significant increases in immobility time in the forced swimming test and cognitive deficits in the NORT, and significant decreases in synaptophysin, phosphorylated PKB and phosphorylated ERK1/2 expression in the hippocampus. Increased levels of Aß40, BACE1 and τ phosphorylation were also found only in CMSA rats. All these effects in CMSA rats were reverted by treatment with venlafaxine. CONCLUSIONS AND IMPLICATIONS: Vulnerability to stress might constitute a risk factor for the development of AD, and pharmacological treatment with venlafaxine may represent a therapeutic strategy for the treatment of stress-related disorders, including AD.

Co-inoculation of different Saccharomyces cerevisiae strains and influence on volatile composition of wines.

Wine is the result of the performance of different yeast strains throughout the fermentation in both spontaneous and inoculated processes. 22 Saccharomyces cerevisiae strains were characterized by microsatellite fingerprinting, selecting 6 of them to formulate S. cerevisiae mixed cultures. The aim of this study was to ascertain a potential benefit to use mixed cultures to improve wine quality. For this purpose yeasts behavior was studied during co-inoculated fermentations. Aromatic composition of the wines obtained was analyzed, and despite the fact that only one strain dominated at the end of the process, co-cultures released different concentrations of major volatile compounds than single strains, especially higher alcohols and acetaldehydes. Nevertheless, no significant differences were found in the type and quantity of the amino acids assimilated. This study demonstrates that the final wine composition may be modulated and enhanced by using suitable combinations of yeast strains.

Amino acid uptake by wild and commercial yeasts in single fermentations and co-fermentations.

Musts require nitrogen-containing compounds in order to ensure yeast development. This study examined the nitrogen-nutrient requirements of two commercial yeasts and three wild strains isolated from inoculated fermentations. The results showed that wild strains generally consumed lower amounts of amino acids than commercial yeasts. Most amino acids were assimilated during the exponential growth phase; only a few - including asparagine and histidine - were metabolized until the end of fermentation. The study also sought to determine whether industrial drying affected yeast nitrogen requirements.

The role of cyclooxygenase (COX)-2 derived prostanoids on vasoconstrictor responses to phenylephrine is increased by exposure to low mercury concentration.

We have previously demonstrated that chronic exposure to low-dose of mercury induced endothelial dysfunction and increased vasoconstrictor responses. The aim of this work was to investigate if mercury exposure alters contractile prostanoids production from cyclooxygenase-2 (COX-2) and its contribution to phenylephrine responses. For this, aortic segments from 3-month old Wistar rats daily treated with HgCl(2) (1(st) dose 4.6 microg/kg, subsequent dose 0.07 microg/kg/day, i.m.) or vehicle for 30 days were used. Mercury treatment did not affect systolic blood pressure but increased phenylephrine-induced vasoconstriction. The non selective COX inhibitor, indomethacin (10 micromol/l) reduced the response to phenylephrine more in aortic segments from mercury-treated than control rats. The selective COX-2 inhibitor NS 398 (1 micromol/l), the thromboxane A(2)/prostaglandin H(2) receptor (TP) antagonist SQ 29,548 (1 micromol/l), the TXA(2) synthase inhibitor furegrelate (1 micromol/l), the EP(1) receptor antagonist SC 19220 (1 micromol/l) and the AT(1) receptor antagonist losartan (10 micromol/l) reduced phenylephrine response only in vessels from mercury-treated rats. TXA(2) and PGE(2) levels were greater in the incubation medium of vessels from treated than untreated rats; NS 398 decreased these levels only in the mercury group. COX-2 protein was localized in adventitial and endothelial cells. Aortic COX-2 mRNA expression and plasma angiotensin converting enzyme activity were greater in mercury-treated rats. These results suggest that treatment with low doses of mercury increases the release of COX-2-derived vasoconstrictor prostanoids and its participation in phenylephrine responses. The increased activation of the renin-angiotensin system after mercury treatment might be associated to this increased COX-2 activity.

Altered arachidonic acid metabolism via COX-1 and COX-2 contributes to the endothelial dysfunction of penile arteries from obese Zucker rats.

BACKGROUND AND PURPOSE: The aim of the current study was to investigate the role of arachidonic acid (AA) metabolism via cyclooxygenase (COX) in the endothelial dysfunction of penile arteries from pre-diabetic, obese Zucker rats (OZR). EXPERIMENTAL APPROACH: Penile arteries from OZR and from lean Zucker rats (LZR) were mounted in microvascular myographs to assess vascular function and COX expression was determined by immunohistochemistry. KEY RESULTS: Acetylcholine (ACh) and AA elicited relaxations that were impaired in arteries from OZR. Inhibition of both COX-1 and COX-2 reduced the relaxant effects of ACh and AA in LZR but not in OZR. Inhibitors of COX-1 and of the TXA(2)/PGH(2) (TP) receptor enhanced the relaxations induced by AA in both LZR and OZR, whereas COX-2 inhibition enhanced these responses only in OZR. TP receptor blockade did not restore ACh relaxant responses in arteries from OZR. Inhibition of COX-1 increased basal tension in OZR and this contraction was blunted by TP receptor blockade. The vasoconstrictor responses to noradrenaline were augmented by indomethacin and by COX-2 inhibition in LZR but not in OZR. Immunohistochemical staining showed that both COX-1 and COX-2 are expressed in the endothelium of penile arteries from both LZR and OZR. CONCLUSIONS AND IMPLICATIONS: Vasoactive prostanoids were formed via constitutively active COX-1 and COX-2 pathways in normal rat penile arteries. Under conditions of insulin resistance, the release and/or effects of vasodilator prostanoids were impaired, contributing to the blunted endothelium-dependent vasodilatation and to the enhanced vasoconstriction.

Characterization of microbial trophic structures of two anaerobic bioreactors processing sulfate-rich waste streams.

A multi-compartment anaerobic bioreactor, designated the anaerobic migrating blanket reactor (AMBR), did not perform well in terms of chemical oxygen demand (COD) removal after an increase in sulfate load, compared to a conventional upflow anaerobic sludge blanket (UASB) reactor. The trophic structures of the bioreactors were characterized by analyzing the electron flows, formation and consumption of fermentation intermediates and terminal product (methane and hydrogen sulfide) formation. Critical performance parameters were linked to operational perturbations such as increase in sulfate load and changes in flow reversal schemes in the AMBR. Both of these manipulations affected the microbial communities, which were monitored by terminal restriction fragment length polymorphism (T-RFLP) analysis targeting the bacterial and archaeal domains. The less stable AMBR did not produce granular biomass, and in response to increased sulfate concentrations, experienced a reversal in the distribution of hydrogenotrophic methanogens that correlated with a shift in electron flow from butyrate to propionate. As this shift occurred, bacterial populations such as butyrate-producing clostridia, became predominant, thus leading to reactor imbalance. The stable UASB reactor developed and retained granules and maintained a relatively stable archaeal community. Sulfate perturbation led to the selection of a novel bacterial group (Thermotogaceae), which was most likely well adapted to the increasingly sulfidogenic conditions in the bioreactor.

Application of PCR-TTGE and PCR-RFLP for intraspecific and interspecific characterization of the genus Saccharomyces using actin gene (ACT1) primers.

In this work the actin gene was used to establish phylogenetic relationships of wider and more diffuse species of the genus Saccharomyces in food ecology by temporal temperature gradient electrophoresis (TTGE) and amplified restriction fragment length polymorphism (RFLP) analysis. Results for DNA RFLP analysis varied considerably, and some enzymes showed a high intra- and interspecific power; however, comparison of experimental results with those provided by the National Center for Biotechnology Information database disclosed a number of interesting variations. Only some experimental results matched the theoretical ones. A theoretical study of melting temperatures using available information from partial sequences of the actin gene was done. Several Saccharomyces species and strains could be distinguished using different TTGE melting points. Some degree of discrimination was achieved under different conditions, in that the Saccharomyces strains tested were separated into groups like the results obtained by PCR-RFLP.

Chronic ouabain treatment increases the contribution of nitric oxide to endothelium-dependent relaxation.

The aim of this study was to analyze the contribution of nitric oxide, prostacyclin and endothelium-dependent hyperpolarizing factor to endothelium-dependent vasodilation induced by acetylcholine in rat aorta from control and ouabain-induced hypertensive rats. Preincubation with the nitric oxide synthase inhibitor N-omega-nitro-L-arginine methyl esther (L-NAME) inhibited the vasodilator response to acetylcholine in segments from both groups but to a greater extent in segments from ouabain-treated rats. Basal and acetylcholine-induced nitric oxide release were higher in segments from ouabain-treated rats. Preincubation with the prostacyclin synthesis inhibitor tranylcypromine or with the cyclooxygenase inhibitor indomethacin inhibited the vasodilator response to acetylcholine in aortic segments from both groups. The Ca2+-dependent potassium channel blocker charybdotoxin inhibited the vasodilator response to acetylcholine only in segments from control rats. These results indicate that hypertension induced by chronic ouabain treatment is accompanied by increased endothelial nitric oxide participation and impaired endothelium-dependent hyperpolarizing factor contribution in acetylcholine-induced relaxation. These effects might explain the lack of effect of ouabain treatment on acetylcholine responses in rat aorta.

Low mercury concentrations cause oxidative stress and endothelial dysfunction in conductance and resistance arteries.

Increased cardiovascular risk after mercury exposure has been described, but the underlying mechanisms are not well explored. We analyzed the effects of chronic exposure to low mercury concentrations on endothelium-dependent responses in aorta and mesenteric resistance arteries (MRA). Wistar rats were treated with mercury chloride (1st dose 4.6 microg/kg, subsequent dose 0.07 microg.kg(-1).day(-1) im, 30 days) or vehicle. Blood levels at the end of treatment were 7.97 +/- 0.59 ng/ml. Mercury treatment: 1) did not affect systolic blood pressure; 2) increased phenylephrine-induced vasoconstriction; 3) reduced acetylcholine-induced vasodilatation; and 4) reduced in aorta and abolished in MRA the increased phenylephrine responses induced by either endothelium removal or the nitric oxide synthase (NOS) inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME, 100 microM). Superoxide dismutase (SOD, 150 U/ml) and the NADPH oxidase inhibitor apocynin (0.3 mM) decreased the phenylephrine-induced contraction in aorta more in mercury-treated rats than controls. In MRA, SOD did not affect phenylephrine responses; however, when coincubated with l-NAME, the l-NAME effect on phenylephrine response was restored in mercury-treated rats. Both apocynin and SOD restored the impaired acetylcholine-induced vasodilatation in vessels from treated rats. Endothelial NOS expression did not change in aorta but was increased in MRA from mercury-treated rats. Vascular O2(-) production, plasmatic malondialdehyde levels, and total antioxidant status increased with the mercury treatment. In conclusion, chronic exposure to low concentrations of mercury promotes endothelial dysfunction as a result of the decreased NO bioavailability induced by increases in oxidative stress. These findings offer further evidence that mercury, even at low concentrations, is an environmental risk factor for cardiovascular disease.

Role of NADPH oxidase and iNOS in vasoconstrictor responses of vessels from hypertensive and normotensive rats.

BACKGROUND AND PURPOSE: To analyse the influence of hypertension in the modulation induced by inducible NOS (iNOS)-derived NO and superoxide anion (O(2) (*-)) of vasoconstrictor responses and the sources of O(2) (*-) implicated. EXPERIMENTAL APPROACH: Vascular reactivity experiments were performed in segments of aorta from normotensive, Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR); protein and mRNA expressions were respectively measured by western blot and quantitative reverse transcription-polymerase chain reaction and O(2) (*-) production was evaluated by ethidium fluorescence. KEY RESULTS: The contractile responses to phenylephrine (1 nM-30 microM) and 5-hydroxytryptamine (0.1-100 microM) were greater in aortic segments from SHR than WKY. The selective iNOS inhibitor, 1400W (10 microM), increased the phenylephrine contraction only in WKY segments; however, iNOS protein and mRNA expressions were greater in aorta from SHR than WKY. Superoxide dismutase (SOD, 150 U ml(-1)) reduced phenylephrine and 5-hydroxytryptamine responses only in aorta from SHR; the NAD(P)H oxidase inhibitor apocynin (0.3 mM) decreased phenylephrine and 5-hydroxytryptamine responses more in vessels from SHR than WKY. Co-incubation with SOD plus 1400W potentiated the phenylephrine and 5-hydroxytryptamine responses more in segments from SHR than WKY. O(2) (*-) production was greater in aorta from SHR than WKY; apocynin abolished this difference. CONCLUSIONS AND IMPLICATIONS: Increased O(2) (*-) formation from NADP(H) oxidase in vessels from hypertensive rats contributes to the vasoconstrictor responses and counteract the increase of NO from iNOS and the consequent modulation of these responses.

Ageing affects nitric oxide synthase, cyclooxygenase and oxidative stress enzymes expression differently in mesenteric resistance arteries.

1 Our aim was to study the role of nitric oxide (NO) and arachidonic acid pathways in the alpha(1)-adrenoceptor-mediated vasoconstriction in mesenteric resistance arteries from 3--4 and 22 to 23-month-old Sprague-Dawley rats. 2 The expression of NO synthase (NOS), cyclooxygenase (COX) isoforms, soluble guanylate cyclase, superoxide dismutase and the NAD(P)H oxidase subunits p22(phox) and p 47(phox) were determined. 3 The N(G)-nitro-l-arginine methyl ester, a non-selective NOS inhibitor, shifted to the left but indomethacin and NS 398, non-selective and selective COX-2 inhibitors, shifted to the right the concentration-response curve for the vasoconstriction by phenylephrine in both age groups. 4 Ageing up-regulated endothelial NOS and p22(phox) expression but did not modify COX, soluble guanylate cyclase, superoxide dismutase and p 47(phox) expression. 5 These data suggest that the observed enhancement of eNOS protein expression could constitute a compensatory mechanism to counter-regulate a chronic loss of NO possibly through increased superoxide anion production from NAD(P)H oxidase induced by age.