The Melatonin Derivative ITH13001 Prevents Hypertension and Cardiovascular Alterations in Angiotensin II-Infused Mice.

Inflammatory mechanisms and oxidative stress seem to contribute to the pathogenesis of hypertension. ITH13001 is a melatonin-phenyl-acrylate hybrid that moderately induces the antioxidant transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) and has a potent oxidant scavenging effect compared with other derivatives of its family. Here we investigated the effect of ITH13001 on hypertension and the associated cardiovascular alterations. Angiotensin II (AngII)-infused mice were treated with ITH13001 (1 mg/kg per day, i.p.) for 2 weeks. The ITH13001 treatment prevented: 1) the development of hypertension, cardiac hypertrophy, and increased collagen and B-type natriuretic peptide (Bnp) expression in the heart; 2) the reduction of elasticity, incremental distensibility, fenestrae area, intraluminal diameter, and endothelial cell number in mesenteric resistance arteries (MRA); 3) the endothelial dysfunction in aorta and MRA; 4) the plasma and cardiovascular oxidative stress and the reduced aortic nitric oxide (NO) bioavailability; 5) the increased cardiac levels of the cytokines interleukin (IL)-1ß, IL-6, and C-C motif chemokine ligand 2 (Ccl2), the T cell marker cluster of differentiation 3 (Cd3), the inflammasome NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3), the proinflammatory enzymes inducible nitric oxide synthase (iNOS) and COX-2, the toll-like receptor 4 (TLR4) adapter protein myeloid differentiation primary response 88 (MyD88), and the nuclear factor kappa B (NF-κB) subunit p65; 6) the greater aortic expression of the cytokines tumor necrosis factor alpha (Tnf-α), Ccl2 and IL-6, Cd3, iNOS, MyD88, and NLRP3. Although ITH13001 increased nuclear Nrf2 levels and heme oxygenase 1 (HO-1) expression in vascular smooth muscle cells, both cardiac and vascular Nrf2, Ho-1, and NADPH quinone dehydrogenase 1 (Nqo1) levels remained unmodified irrespective of AngII infusion. Summarizing, ITH13001 improved hypertension-associated cardiovascular alterations independently of Nrf2 pathway activation, likely due to its direct antioxidant and anti-inflammatory properties. Therefore, ITH13001 could be a useful therapeutic strategy in patients with resistant hypertension. SIGNIFICANCE STATEMENT: Despite the existing therapeutic arsenal, only half of the patients treated for hypertension have adequately controlled blood pressure; therefore, the search for new compounds to control this pathology and the associated damage to end-target organs (cerebral, cardiac, vascular, renal) is of particular interest. The present study demonstrates that a new melatonin derivative, ITH13001, prevents hypertension development and the associated cardiovascular alterations due to its antioxidant and anti-inflammatory properties, making this compound a potential candidate for treatment of resistant hypertensive patients.

Identification and biotechnological characterisation of yeast microbiota involved in spontaneous fermented wholegrain sourdoughs.

BACKGROUND: New strategies in the cereal-based industry has brought about the elaboration of new sourdoughs with better microbial stability and safety as well as nutritional value such as those based on wholegrain flours. This has led to an increasing interest in the selection of adapted yeasts for using them as new starters. Therefore, this study aimed to isolate, identify, and characterise diverse yeast strains from wholegrain spontaneous sourdoughs. RESULTS: Three wholegrain sourdoughs (wheat, rye, and oat) were fermented and monitored for 96 h. Minimum pH values ranged from 3.1 to 3.5 while maximum yeast counts were reached at 72 h. A total of 76 yeast isolates were identified by polymerase chain reaction random amplification of polymorphic DNA (PCR-RAPD) and catalogued in six different species by sequencing the internal transcribed spacer (ITS) region. The major species were Candida glabrata, Saccharomyces cerevisiae, Kazachstania unispora, and Wickerhamomyces anomalus. The studied kinetic parameters of the growth curves (λ, G, ODmax , and µmax ) and the fermentation capacity allowed to ascertain that 12 and 5 strains, respectively, were better than baker's yeast control. The fibre assimilation ability (cellulose, xylose, and ß-glucan) was observed in the 27% of the strains and only four strains showed phytase activity. CONCLUSIONS: The yeast population in the three wholegrain sourdoughs were variable along the fermentation time. Genetic identification showed that strains and species presented a different trend for each sourdough although common species were determined (e.g., W. anomalus). Candida glabrata (4T1) and Saccharomyces cerevisiae (3A6) showed, respectively, better kinetics and impedance results than the positive control, while W. anomalus (C4) was notorious in fibre assimilation and phytase degradation. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Extracellular Tuning of Mitochondrial Respiration Leads to Aortic Aneurysm.

BACKGROUND: Marfan syndrome (MFS) is an autosomal dominant disorder of the connective tissue caused by mutations in the FBN1 (fibrillin-1) gene encoding a large glycoprotein in the extracellular matrix called fibrillin-1. The major complication of this connective disorder is the risk to develop thoracic aortic aneurysm. To date, no effective pharmacologic therapies have been identified for the management of thoracic aortic disease and the only options capable of preventing aneurysm rupture are endovascular repair or open surgery. Here, we have studied the role of mitochondrial dysfunction in the progression of thoracic aortic aneurysm and mitochondrial boosting strategies as a potential treatment to managing aortic aneurysms. METHODS: Combining transcriptomics and metabolic analysis of aortas from an MFS mouse model (Fbn1c1039g/+) and MFS patients, we have identified mitochondrial dysfunction alongside with mtDNA depletion as a new hallmark of aortic aneurysm disease in MFS. To demonstrate the importance of mitochondrial decline in the development of aneurysms, we generated a conditional mouse model with mitochondrial dysfunction specifically in vascular smooth muscle cells (VSMC) by conditional depleting Tfam (mitochondrial transcription factor A; Myh11-CreERT2Tfamflox/flox mice). We used a mouse model of MFS to test for drugs that can revert aortic disease by enhancing Tfam levels and mitochondrial respiration. RESULTS: The main canonical pathways highlighted in the transcriptomic analysis in aortas from Fbn1c1039g/+ mice were those related to metabolic function, such as mitochondrial dysfunction. Mitochondrial complexes, whose transcription depends on Tfam and mitochondrial DNA content, were reduced in aortas from young Fbn1c1039g/+ mice. In vitro experiments in Fbn1-silenced VSMCs presented increased lactate production and decreased oxygen consumption. Similar results were found in MFS patients. VSMCs seeded in matrices produced by Fbn1-deficient VSMCs undergo mitochondrial dysfunction. Conditional Tfam-deficient VSMC mice lose their contractile capacity, showed aortic aneurysms, and died prematurely. Restoring mitochondrial metabolism with the NAD precursor nicotinamide riboside rapidly reverses aortic aneurysm in Fbn1c1039g/+ mice. CONCLUSIONS: Mitochondrial function of VSMCs is controlled by the extracellular matrix and drives the development of aortic aneurysm in Marfan syndrome. Targeting vascular metabolism is a new available therapeutic strategy for managing aortic aneurysms associated with genetic disorders.

Specialized Pro-Resolving Lipid Mediators: New Therapeutic Approaches for Vascular Remodeling.

Vascular remodeling is a typical feature of vascular diseases, such as atherosclerosis, aneurysms or restenosis. Excessive inflammation is a key mechanism underlying vascular remodeling via the modulation of vascular fibrosis, phenotype and function. Recent evidence suggests that not only augmented inflammation but unresolved inflammation might also contribute to different aspects of vascular diseases. Resolution of inflammation is mediated by a family of specialized pro-resolving mediators (SPMs) that limit immune cell infiltration and initiate tissue repair mechanisms. SPMs (lipoxins, resolvins, protectins, maresins) are generated from essential polyunsaturated fatty acids. Synthases and receptors for SPMs were initially described in immune cells, but they are also present in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), where they regulate processes important for vascular physiology, such as EC activation and VSMC phenotype. Evidence from genetic models targeting SPM pathways and pharmacological supplementation with SPMs have demonstrated that these mediators may play a protective role against the development of vascular remodeling in atherosclerosis, aneurysms and restenosis. This review focuses on the latest advances in understanding the role of SPMs in vascular cells and their therapeutic effects in the vascular remodeling associated with different cardiovascular diseases.

Proteomic profiling and glycomic analysis of the yeast cell wall in strains with Aflatoxin B1 elimination ability.

The use of microorganisms for Aflatoxin B1 elimination has been studied as a new alternative tool and it is known that cell wall carried out a critical role. For that reason, cell wall and soluble intracellular fraction of eight yeasts with AFB1 detoxification capability were analysed. The quantitative and qualitative comparative label-free proteomic allowed the identification of diverse common constituent proteins, which revealed that putative cell wall proteins entailed less than 10% of the total proteome. It was possible to characterize different enzymes linked to cell wall polysaccharides biosynthesis as well as other proteins related with the cell wall organization and regulation. Additionally, the concentration of the principal polysaccharides was determined which permitted us to observe that ß-glucans concentration was higher than mannans in most of the samples. In order to better understand the biosorption role of the cell wall against the AFB1 , an antimycotic (Caspofungin) was used to damage the cell wall structure. This assay allowed the observation of an effect on the normal growth of those yeasts with damaged cell walls that were exposed to AFB1 . This effect was not observed in yeast with intact cell walls, which may reveal a protective role of this structure against mycotoxins.

Report on the 24th meeting of the ECCR 8th-9th October 2021.

The 24th meeting of the European Council for Cardiovascular Research (ECCR) was virtual and held online on October 8th and 9th, 2021. Over 130 participants including trainees, early career researchers (ECR) and established investigators from eleven European countries (Austria, Denmark, France, Germany, Italy, Netherlands, Poland, Slovenia, Spain, Turkey, U.K.), and participants also from Canada, Chile, Saudi Arabia, and the U.S.A. connected to enjoy two days of outstanding research. The meeting was opened by its president, Professor Marisol Fernandez-Alfonso from the Complutense University in Madrid and covered several topics of cardiovascular research: from vascular and metabolic aspects to novel immunological mechanisms of cardiovascular disease.

Aging-Associated miR-217 Aggravates Atherosclerosis and Promotes Cardiovascular Dysfunction.

OBJECTIVE: microRNAs are master regulators of gene expression with essential roles in virtually all biological processes. miR-217 has been associated with aging and cellular senescence, but its role in vascular disease is not understood. Approach and Results: We have used an inducible endothelium-specific knock-in mouse model to address the role of miR-217 in vascular function and atherosclerosis. miR-217 reduced NO production and promoted endothelial dysfunction, increased blood pressure, and exacerbated atherosclerosis in proatherogenic apoE-/- mice. Moreover, increased endothelial miR-217 expression led to the development of coronary artery disease and altered left ventricular heart function, inducing diastolic and systolic dysfunction. Conversely, inhibition of endogenous vascular miR-217 in apoE-/- mice improved vascular contractility and diminished atherosclerosis. Transcriptome analysis revealed that miR-217 regulates an endothelial signaling hub and downregulates a network of eNOS (endothelial NO synthase) activators, including VEGF (vascular endothelial growth factor) and apelin receptor pathways, resulting in diminished eNOS expression. Further analysis revealed that human plasma miR-217 is a biomarker of vascular aging and cardiovascular risk. CONCLUSIONS: Our results highlight the therapeutic potential of miR-217 inhibitors in aging-related cardiovascular disease.

Differential distribution and proteomic response of Saccharomyces cerevisiae and non-model yeast species to zinc.

Zinc surplus in yeast cells has been previously investigated thanks to transcriptomic studies by using traditionally Saccharomyces cerevisiae as a model. However, proteome response under zinc-replete conditions needs to be further studied in yeast. For that reason, eight yeast strains from seven different species were inoculated in zinc-depleted and zinc-replete media. The quantitative and qualitative comparative label-free proteomic analysis enabled the identification of between 2000 and 3000 proteins from each strain, and changes to the proteome ranged from 2.5% to 43.7% of identified proteins. Functional analysis (Blast2Go) has allowed the characterization of differentially abundant proteins. Common zinc-responsive proteins have been detected for the eight strains such as oxidoreductases and transferases (increased in abundance) although more of the changes detected were not shared by all the strains tested. Zinc distribution under replete conditions has been analysed in cell wall fractions, and cytoplasm plus organelles (intracellular fraction), with the latter identified to be the main zinc reservoir. Additionally, the energy dispersive spectroscopy coupled to the scanning electron microscopy technique has permitted the visualization of zinc in the whole cell. Proteomic analysis revealed that while there were some shared responses, the non-model yeast species also showed distinct proteomic profiles in zinc-replete conditions, compared to S. cerevisiae, revealing new zinc-responsive proteins in yeast.

Interleukin-17A induces vascular remodeling of small arteries and blood pressure elevation.

An important link exists between hypertension and inflammation. Hypertensive patients present elevated circulating levels of proinflammatory cytokines, including interleukin-17A (IL-17A). This cytokine participates in host defense, autoimmune and chronic inflammatory pathologies, and cardiovascular diseases, mainly through the regulation of proinflammatory factors. Emerging evidence also suggests that IL-17A could play a role in regulating blood pressure and end-organ damage. Here, our preclinical studies in a murine model of systemic IL-17A administration showed that increased levels of circulating IL-17A raised blood pressure induced inward remodeling of small mesenteric arteries (SMAs) and arterial stiffness. In IL-17A-infused mice, treatment with hydralazine and hydrochlorothiazide diminished blood pressure elevation, without modifying mechanical and structural properties of SMA, suggesting a direct vascular effect of IL-17A. The mechanisms of IL-17A seem to involve an induction of vascular smooth muscle cell (VSMC) hypertrophy and phenotype changes, in the absence of extracellular matrix (ECM) proteins accumulation. Accordingly, treatment with an IL-17A neutralizing antibody diminished SMA remodeling in a model of angiotensin II (Ang II) infusion. Moreover, in vitro studies in VSMCs reported here, provide further evidence of the direct effects of IL-17A on cell growth responses. Our experimental data suggest that IL-17A is a key mediator of vascular remodeling of the small arteries, which might contribute, at least in part, to blood pressure elevation.

Branched-chain amino acids promote endothelial dysfunction through increased reactive oxygen species generation and inflammation.

Branched-chain amino acids (BCAA: leucine, isoleucine and valine) are essential amino acids implicated in glucose metabolism and maintenance of correct brain function. Elevated BCAA levels can promote an inflammatory response in peripheral blood mononuclear cells. However, there are no studies analysing the direct effects of BCAA on endothelial cells (ECs) and its possible modulation of vascular function. In vitro and ex vivo studies were performed in human ECs and aorta from male C57BL/6J mice, respectively. In ECs, BCAA (6 mmol/L) increased eNOS expression, reactive oxygen species production by mitochondria and NADPH oxidases, peroxynitrite formation and nitrotyrosine expression. Moreover, BCAA induced pro-inflammatory responses through the transcription factor NF-κB that resulted in the release of intracellular adhesion molecule-1 and E-selectin conferring endothelial activation and adhesion capacity to inflammatory cells. Pharmacological inhibition of mTORC1 intracellular signalling pathway decreased BCAA-induced pro-oxidant and pro-inflammatory effects in ECs. In isolated murine aorta, BCAA elicited vasoconstrictor responses, particularly in pre-contracted vessels and after NO synthase blockade, and triggered endothelial dysfunction, effects that were inhibited by different antioxidants, further demonstrating the potential of BCAA to induce oxidative stress with functional impact. In summary, we demonstrate that elevated BCAA levels generate inflammation and oxidative stress in ECs, thereby facilitating inflammatory cells adhesion and endothelial dysfunction. This might contribute to the increased cardiovascular risk observed in patients with elevated BCAA blood levels.

Periarterial fat from two human vascular beds is not a source of aldosterone to promote vasoconstriction.

Mouse adipocytes have been reported to release aldosterone and reduce endothelium-dependent relaxation. It is unknown whether perivascular adipose tissue (PVAT) releases aldosterone in humans. The present experiments were designed to test the hypothesis that human PVAT releases aldosterone and induces endothelial dysfunction. Vascular reactivity was assessed in human internal mammary and renal segmental arteries obtained at surgery. The arteries were prepared with/without PVAT, and changes in isometric tension were measured in response to the vasoconstrictor thromboxane prostanoid receptor agonist U46619 and the endothelium-dependent vasodilator acetylcholine. The effects of exogenous aldosterone and of mineralocorticoid receptor (MR) antagonist eplerenone were determined. Aldosterone concentrations were measured by ELISA in conditioned media incubated with human adipose tissue with/without angiotensin II stimulation. Presence of aldosterone synthase and MR mRNA was examined in perirenal, abdominal, and mammary PVAT by PCR. U46619 -induced tension and acetylcholine-induced relaxation were unaffected by exogenous and endogenous aldosterone (addition of aldosterone and MR blocker) in mammary and renal segmental arteries, both in the presence and absence of PVAT. Aldosterone release from incubated perivascular fat was not detectable. Aldosterone synthase expression was not consistently observed in human adipose tissues in contrast to that of MR. Thus, exogenous aldosterone does not affect vascular reactivity and endothelial function in ex vivo human arterial segments, and the tested human adipose tissues have no capacity to synthesize/release aldosterone. In perspective, physiologically relevant effects of aldosterone on vascular function in humans are caused by systemic aldosterone originating from the adrenal gland.

Regulator of calcineurin 1 modulates vascular contractility and stiffness through the upregulation of COX-2-derived prostanoids.

Cyclooxygenase-2 (COX-2) derived-prostanoids participate in the altered vascular function and mechanical properties in cardiovascular diseases. We investigated whether regulator of calcineurin 1 (Rcan1) participates in vascular contractility and stiffness through the regulation of COX-2. For this, wild type (Rcan1+/+) and Rcan1-deficient (Rcan1-/-) mice untreated or treated with the COX-2 inhibitor rofecoxib were used. Vascular function and structure were analysed by myography. COX-2 and phospo-p65 expression were studied by western blotting and immunohistochemistry and TXA2 production by ELISA. We found that Rcan1 deficiency increases COX-2 and IL-6 expression and NF-κB activation in arteries and vascular smooth muscle cells (VSMC). Adenoviral-mediated re-expression of Rcan1.4 in Rcan1-/- VSMC normalized COX-2 expression. Phenylephrine-induced vasoconstrictor responses were greater in aorta from Rcan1-/- compared to Rcan1+/+ mice. This increased response were diminished by etoricoxib, furegrelate, SQ 29548, cyclosporine A and parthenolide, inhibitors of COX-2, TXA2 synthase, TP receptors, calcineurin and NF-κB, respectively. Endothelial removal and NOS inhibition increased phenylephrine responses only in Rcan1+/+ mice. TXA2 levels were greater in Rcan1-/- mice. In small mesenteric arteries, vascular function and structure were similar in both groups of mice; however, vessels from Rcan1-/- mice displayed an increase in vascular stiffness that was diminished by rofecoxib. In conclusion, our results suggest that Rcan1 might act as endogenous negative modulator of COX-2 expression and activity by inhibiting calcineurin and NF-kB pathways to maintain normal contractility and vascular stiffness in aorta and small mesenteric arteries, respectively. Our results uncover a new role for Rcan1 in vascular contractility and mechanical properties.

Oxidative Stress in Human Atherothrombosis: Sources, Markers and Therapeutic Targets.

Atherothrombosis remains one of the main causes of morbidity and mortality worldwide. The underlying pathology is a chronic pathological vascular remodeling of the arterial wall involving several pathways, including oxidative stress. Cellular and animal studies have provided compelling evidence of the direct role of oxidative stress in atherothrombosis, but such a relationship is not clearly established in humans and, to date, clinical trials on the possible beneficial effects of antioxidant therapy have provided equivocal results. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is one of the main sources of reactive oxygen species (ROS) in human atherothrombosis. Moreover, leukocyte-derived myeloperoxidase (MPO) and red blood cell-derived iron could be involved in the oxidative modification of lipids/lipoproteins (LDL/HDL) in the arterial wall. Interestingly, oxidized lipoproteins, and antioxidants, have been analyzed as potential markers of oxidative stress in the plasma of patients with atherothrombosis. In this review, we will revise sources of ROS, focusing on NADPH oxidase, but also on MPO and iron. We will also discuss the impact of these oxidative systems on LDL and HDL, as well as the value of these modified lipoproteins as circulating markers of oxidative stress in atherothrombosis. We will finish by reviewing some antioxidant systems and compounds as therapeutic strategies to prevent pathological vascular remodeling.

Activation of PPARß/δ prevents hyperglycaemia-induced impairment of Kv7 channels and cAMP-mediated relaxation in rat coronary arteries.

PPARß/δ activation protects against endothelial dysfunction in diabetic models. Elevated glucose is known to impair cAMP-induced relaxation and Kv channel function in coronary arteries (CA). Herein, we aimed to analyse the possible protective effects of the PPARß/δ agonist GW0742 on the hyperglycaemic-induced impairment of cAMP-induced relaxation and Kv channel function in rat CA. As compared with low glucose (LG), incubation under high glucose (HG) conditions attenuated the relaxation induced by the adenylate cyclase activator forskolin in CA and this was prevented by GW0742. The protective effect of GW0742 was supressed by a PPARß/δ antagonist. In myocytes isolated from CA under LG, forskolin enhanced Kv currents and induced hyperpolarization. In contrast, when CA were incubated with HG, Kv currents were diminished and the electrophysiological effects of forskolin were abolished. These deleterious effects were prevented by GW0742. The protective effects of GW0742 on forskolin-induced relaxation and Kv channel function were confirmed in CA from type-1 diabetic rats. In addition, the differences in the relaxation induced by forskolin in CA incubated under LG, HG or HG + GW0742 were abolished by the Kv7 channel inhibitor XE991. Accordingly, GW0742 prevented the down-regulation of Kv7 channels induced by HG. Finally, the preventive effect of GW0742 on oxidative stress and cAMP-induced relaxation were overcome by the pyruvate dehydrogenase kinase 4 (PDK4) inhibitor dichloroacetate (DCA). Our results reveal that the PPARß/δ agonist GW0742 prevents the impairment of the cAMP-mediated relaxation in CA under HG. This protective effect was associated with induction of PDK4, attenuation of oxidative stress and preservation of Kv7 channel function.

Genetic and phenotypic characterization of Saccharomyces spp. strains isolated in distillery plants.

In this study, the biodiversity and some interesting phenotypic properties of Saccharomyces wild yeasts isolated in distilleries, at least 100 years old, located in La Mancha (Spain), were determined. Strains were genetically characterized by RFLP-mtDNA, which confirmed a great genetic biodiversity with 73% of strains with different mtDNA profiles, highlighting the large variability found in sweet and fermented piquette substrata. The predominant species identified was S. cerevisiae, followed by S. paradoxus and S. bayanus Due to the residual sugar-alcohol extraction process using warm water, a great number of thermophilic Saccharomyces strains with a great cell vitality were found to have potential use as starters in distillery plants. Interesting technological properties such as cell vitality and growth rate at different temperatures were studied. The thermal washing process for the extraction of alcohol and reducing sugars of some raw materials contributes to the presence of Saccharomyces strains with technologically interesting properties, especially in terms of vitality and resistance to high temperatures. Due to the fact that fermentation is spontaneous, the yeast biota of these environments, Saccharomyces and non-Saccharomyces, is very varied so these ecological niches are microbial reserves of undoubted biotechnological interest.

NADPH oxidases and vascular remodeling in cardiovascular diseases.

Reactive oxygen species (ROS) are key signaling molecules that regulate vascular function and structure in physiological conditions. A misbalance between the production and detoxification of ROS increases oxidative stress that is involved in the vascular remodeling associated with cardiovascular diseases such as hypertension by affecting inflammation, hypertrophy, migration, growth/apoptosis and extracellular matrix protein turnover. The major and more specific source of ROS in the cardiovascular system is the NADPH oxidase (NOX) family of enzymes composed of seven members (NOX1-5, DUOX 1/2). Vascular cells express several NOXs being NOX-1 and NOX-4 the most abundant NOXs present in vascular smooth muscle cells. This review focuses on specific aspects of NOX-1 and NOX-4 isoforms including information on regulation, function and their role in vascular remodeling. In order to obtain a more integrated view about the role of the different NOX isoforms in different types of vascular remodeling, we discuss the available literature not only on hypertension but also in atherosclerosis, restenosis and aortic dilation.

Saccharomyces cerevisiae and metabolic activators: HXT3 gene expression and fructose/glucose discrepancy in sluggish fermentation conditions.

When exposed to mixtures of glucose and fructose, as occurs during the fermentation of grape juice into wine, Saccharomyces cerevisiae uses these sugars at different rates. Moreover, glucose and fructose are transported by the same hexose transporters (HXT), which present a greater affinity for glucose, so that late in fermentation, fructose becomes the predominant sugar. Only a few commercial fermentation activators are available to optimally solve the problems this entails. The aim of this study was to investigate the relation between HXT3 gene expression and fructose/glucose discrepancy in two different media inoculated with a commercial wine strain of S. cerevisiae in the presence of three metabolic activators. Fermentation kinetics, vitality and major metabolites were also measured. Rehydration with ergosterol improved the area under the curve and the growth rate (µ max ) in both studied media. Also, the fructose/glucose discrepancy values were improved with all activator treatments, highlighting rehydration in the presence of ascorbic acid. The yeast rehydration process was demonstrated to influence HXT3 expression under the studied conditions. Tetrahydrofolic acid treatment greatly influenced HXT3 gene expression, especially on the 12th day of the fermentation process. To a lesser extent, ergosterol and ascorbic acid also improved this parameter.

MAPK pathway activation by chronic lead-exposure increases vascular reactivity through oxidative stress/cyclooxygenase-2-dependent pathways.

Chronic exposure to low lead concentration produces hypertension; however, the underlying mechanisms remain unclear. We analyzed the role of oxidative stress, cyclooxygenase-2-dependent pathways and MAPK in the vascular alterations induced by chronic lead exposure. Aortas from lead-treated Wistar rats (1st dose: 10 µg/100g; subsequent doses: 0.125µg/100g, intramuscular, 30days) and cultured aortic vascular smooth muscle cells (VSMCs) from Sprague Dawley rats stimulated with lead (20µg/dL) were used. Lead blood levels of treated rats attained 21.7±2.38µg/dL. Lead exposure increased systolic blood pressure and aortic ring contractile response to phenylephrine, reduced acetylcholine-induced relaxation and did not affect sodium nitroprusside relaxation. Endothelium removal and L-NAME left-shifted the response to phenylephrine more in untreated than in lead-treated rats. Apocynin and indomethacin decreased more the response to phenylephrine in treated than in untreated rats. Aortic protein expression of gp91(phox), Cu/Zn-SOD, Mn-SOD and COX-2 increased after lead exposure. In cultured VSMCs lead 1) increased superoxide anion production, NADPH oxidase activity and gene and/or protein levels of NOX-1, NOX-4, Mn-SOD, EC-SOD and COX-2 and 2) activated ERK1/2 and p38 MAPK. Both antioxidants and COX-2 inhibitors normalized superoxide anion production, NADPH oxidase activity and mRNA levels of NOX-1, NOX-4 and COX-2. Blockade of the ERK1/2 and p38 signaling pathways abolished lead-induced NOX-1, NOX-4 and COX-2 expression. Results show that lead activation of the MAPK signaling pathways activates inflammatory proteins such as NADPH oxidase and COX-2, suggesting a reciprocal interplay and contribution to vascular dysfunction as an underlying mechanisms for lead-induced hypertension.

Carnitine palmitoyltransferase-1 up-regulation by PPAR-ß/δ prevents lipid-induced endothelial dysfunction.

Fatty acids cause endothelial dysfunction involving increased ROS (reactive oxygen species) and reduced NO (nitric oxide) bioavailability. We show that in MAECs (mouse aortic endothelial cells), the PPARß/δ (peroxisome- proliferator-activated receptor ß/δ) agonist GW0742 prevented the decreased A23187-stimulated NO production, phosphorylation of eNOS (endothelial nitric oxide synthase) at Ser1177 and increased intracellular ROS levels caused by exposure to palmitate in vitro. The impaired endothelium-dependent relaxation to acetylcholine in mouse aorta induced by palmitate was restored by GW0742. In vivo, GW0742 treatment prevented the reduced aortic relaxation, phosphorylation of eNOS at Ser1177, and increased ROS production and NADPH oxidase in mice fed on a high-fat diet. The PPARß/δ antagonist GSK0660 abolished all of these protective effects induced by GW0742. This agonist enhanced the expression of CPT (carnitine palmitoyltransferase)-1. The effects of GW0742 on acetylcholine- induced relaxation in aorta and on NO and ROS production in MAECs exposed to palmitate were abolished by the CPT-1 inhibitor etomoxir or by siRNA targeting CPT-1. GW0742 also inhibited the increase in DAG (diacylglycerol), PKCα/ßII (protein kinase Cα/ßII) activation, and phosphorylation of eNOS at Thr495 induced by palmitate in MAECs, which were abolished by etomoxir. In conclusion, PPARß/δ activation restored the lipid-induced endothelial dysfunction by up-regulation of CPT-1, thus reducing DAG accumulation and the subsequent PKC-mediated ROS production and eNOS inhibition.

Study of Saccharomyces cerevisiae wine strains for breeding through fermentation efficiency and tetrad analysis.

One of the issues that most concerns to both winemakers and producers of active dry yeasts is the stuck and sluggish fermentations of grape musts with high levels of sugar, reflecting the inability of inoculated yeast strain to complete the fermentation process. It is difficult to obtain a wine strain that possesses both adequate oenological and technological properties; thus, the correct approach to solving these problems is the application of breeding programs primarily focused on both properties. The first step toward this process is to characterize the phenotypic diversity between potential parental strains. In the present study, we have analyzed the fermentative behavior of 26 Saccharomyces cerevisiae wine strains in high-sugar conditions at 20 °C, using a range of tests, such as sporulation ability, spore viability, and tetrad analysis to determine the tolerance of these yeasts to several stress conditions. Most tested strains were homothallic and heterozygous for more than one character. Two auxotrophic derivatives with defects in amino acid or nucleic acid metabolism were obtained, and these strains could potentially be used for the development of hybridization techniques without using laboratory strains.