Phytochelatin signal tranduction and cadmium chelation in plants

As plantas utilizam diversas estratégias de sinalização para reconhecer e responder aos stresses ambientais. A maioria das vias de transdução de sinais partilham um sinal genérico, normalmente a modulação dos níveis intracelulares de Ca2+. Esta por sua vez pode iniciar uma cascata de fosforilação proteica que finalmente afecta as proteínas directamente envolvidas na protecção celular ou culmina em factores de transcrição que vão determinar a resposta fisiológica ao stresse. A percepção destes sinais e a compreensão de como estes podem activar as respostas adaptativas são factores-chave para a tolerância das plantas a stresses abióticos. Um dos principais stresses abóticos que restrigem o crescimento das plantas é a presença de metais pesados. A produção de fitoquelatinas e a subsequente quelação dos metais é o mecanismo mais conhecido de tolerância ao stresse metálico em plantas. Fitoquelatinas (PCs) são péptidos com grupos tiol que são sintetizados através da transpeptidação da glutationa (GSH), pela acção da enzima fitoquelatina sintase (PCS). No entanto, até ao momento, as vias de sinalização que levam à síntese de fitoquelatinas e à percepção do stresse metálico são pouco compreendidas. Dentro deste contexto, o presente trabalho foi elaborado com o intuito de elucidar a via de sinalização através da qual o cádmio é detectado pelas células vegetais e induz a síntese de PCs. Quase todos, os estudos de stresses abióticos em plantas apontam para o facto de a sua sinalização se basear nos mesmos tipos de sinais moleculares, nomeadamente a sinalização por cálcio, a fosforilação proteica e a indução de espécies reactivas de oxigénio (ROS). Trabalhos recentes sugerem que a sinalização de PCs poderá envolver todos estes parâmetros. Assim, uma primeira abordagem foi efectuada para compreender a síntese de PCs na espécie Arabidopsis thaliana, através da monitorizaçção da actividade de enzimas relacionadas, a γ-EC sintetase, GSH sintetase e a PC sintase (PCS), assim como o tempo necessário para o elongamento das PCs e a sua acumulação. Seguidamente, ao longo deste processo foi analisada a expressão de sinais específicos, associados com sinais de cálcio, fosforilação proteica e sinalização por ROS. A importância destes factores na síntese de PCs foi também avaliada através do uso de moduladores farmacológicos de cálcio e fosfatases proteicas e também pela indução de stresse oxidativo. Os resultados demonstraram novos dados sobre o papel do cálcio e da fosforilação proteica na produção de PCs e na síntese de GSH, revelando que a actvidade da PCS é regulada por fosforilação e que a sinalização de cálcio pode mediar a síntese de GSH. O envolvimento da sinalização de ROS na síntese de GSH, atráves de crosstalk com a sinalização de cálcio também foi proposta. Assim, os resultados aqui apresentados descrevem uma possível via de sinalização de cádmio nas plantas e da indução de fitoquelatinas. Este trabalho poderá ser portanto muito útil na implementação de novas metodologias de agricultura sustentável e práticas de fitorremediação em solos contaminados com metais pesados.

Sustainable viticulture in Bairrada appellation: vineyard and harvest year effects on grapes oenological potential

The sustainable viticulture of a region passes, among other aspects, for maximizing the varieties potential minimizing subsequent interventions during winemaking, which should contribute to the production of quality wines maintaining their typicity and rationalizing costs. The detailed knowledge of each Appellation specificities, namely vineyard parcel (soil type and topographical peculiarities) and harvest climatic conditions is crucial for sustainability in this sector. Thus, in line with this current trend, the starting point for the development of this PhD thesis was to evaluate the oenological potential of different varieties cultivated throughout Bairrada Appellation (Portugal). During maturation several changes in grape varieties occur, namely berries become sweeter, less acidic, and they develop flavour, aroma and colour properties. The development of these characteristics is essential to define grapes oenological potential, i.e. to estimate the possibility of their usage to produce specific wines. A three years sampling plan was designed to evaluate the effect of harvest year and parcel characteristics on V. vinifera cv. Arinto, Bical, Sauvignon Blanc, Baga, Castelão, Touriga Nacional, and Sousão grapes composition. For each variety, 3 parcels with different characteristics were selected. Several physicochemical parameters were evaluated, during maturation: berry weight, pH, titratable acidity, sugar and phenolic contents, antiradical activity, and volatile composition (free fraction). Special attention was devoted to grapes at technologic maturity, since, besides these parameters, glycosidically-linked fraction was also considered. By using the results obtained at technologic maturity, a comprehensive approach was applied to identify the significance of harvest and parcel characteristics effects on each variety composition. Considering all the parameters under study, it may be highlighted some significant differences. According to the obtained results determined during maturation, it was possible to conclude that Arinto, Bical and Sauvignon Blanc grapes from parcels with clay-sandy and clay-calcareous soils have higher phenolic content and antiradical activity. Otherwise, Sauvignon Blanc presented similar volatile composition for grapes cultivated in the 3 parcels, while Arinto and Bical exhibited higher volatile content in grapes from claysandy and clay-calcareous soils. For Baga, Castelão and Touriga Nacional red varieties, grapes with higher phenolic content, antiradical activity, and volatile content were obtained from clayey and clay-calcareous soils. Furthermore, for Touriga Nacional, parcels altitude seems also to modulate grapes composition. Beyond parcel effect, harvest year conditions also influence grapes composition: 2011 harvest was related with lower phenolic and volatile contents, as well as lower antiradical activity.For grapes collected at technologic maturity, analysis of variance-simultaneous component analysis (ASCA) was applied combining all the parameters under study, in order to assess the influence of harvest and parcel characteristics on each variety oenological potential. The results obtained using this comprehensive approach is closely related with those observed during maturation and revealed that harvest was the main factor that influenced grapes composition (53% to 68% of the total data set variance) followed by parcel characteristics, explaining ca. 15-19% of the total data set variance. The oenological potential of each variety may be different from one parcel to another, i.e., clay-sandy and clay-calcareous related-environments seem to favour Arinto and Bical white grapes composition, but for the red varieties, grapes composition was favoured by clayey and clay-calcareous soils. Besides, also higher altitude seems to favour Touriga Nacional grapes composition. Sauvignon Blanc seems to be a variety well adapted to the different parcel characteristics. In order to go forward in the valuation of these varieties, the aroma properties of 6 monovarietal wines were studied based on an aroma network-approach, linking molecular data related to volatile composition and aroma data about the key odor active molecules. This approach allowed to identify different wine aroma properties and to infer about the consumer’s sensory perception. It was found that aroma properties differ from one wine variety to another: while Arinto and Sauvignon Blanc wine exhibited higher tree fruity, sweety and flowery aromas, related essentially with ester compounds and C13 norisoprenoids, the opposite was obtained for Bical wine, corroborating the aroma sensory perceptions of the trained panel. Sauvignon Blanc also exhibited higher toasted aromas (related with thiols, mainly with 2-methyl-3-furanthiol). Touriga Nacional red wine exhibited higher tree, tropical, and berry fruits notes (sensory described as sweet fruits), toasted and flowery aromas, while these are similar for the other red wines under study. Besides Portuguese Bairrada wines, this aroma network approach is a tool that can be used to explain the aroma properties of wines worldwide. The grape and wine data generated under the present PhD thesis, in the context of Bairrada Appellation, shows the unique character of each variety, and may be used by growers and wine producers as a support for decisionmaking based on objective criteria, increasing the sustainability in this sector. For instance, it is possible to take advantage of the natural resources and produce products with different characteristics obtained from the same variety, minimizing costs during the winemaking process.

Evaluation of the role of glutathione in the lead-induced toxicity in saccharomyces cerevisiae

The effect of intracellular reduced glutathione (GSH) in the lead stress response of Saccharomyces cerevisiae was investigated. Yeast cells exposed to Pb, for 3 h, lost the cell proliferation capacity (viability) and decreased intracellular GSH level. The Pb-induced loss of cell viability was compared among yeast cells deficient in GSH1 (∆gsh1) or GSH2 (∆gsh2) genes and wild-type (WT) cells. When exposed to Pb, ∆gsh1 and ∆gsh2 cells did not display an increased loss of viability, compared with WT cells. However, the depletion of cellular thiols, including GSH, by treatment of WT cells with iodoacetamide (an alkylating agent, which binds covalently to thiol group), increased the loss of viability in Pb-treated cells. In contrast, GSH enrichment, due to the incubation of WT cells with amino acids mixture constituting GSH (l-glutamic acid, l-cysteine and glycine), reduced the Pb-induced loss of proliferation capacity. The obtained results suggest that intracellular GSH is involved in the defence against the Pb-induced toxicity; however, at physiological concentration, GSH seems not to be sufficient to prevent the Pb-induced loss of cell viability.

Développement d'une méthode de marquage protéique par fluorescence

Le marquage protéique par fluorescence est une méthode de choix permettant d’étudier l’évolution des protéines depuis leur synthèse cellulaire jusqu’à leur dégradation, en plus de rendre possible leur localisation ainsi que la visualisation des interactions entre protéines. De cet intérêt certain ont découlé différentes techniques de marquage, dont celle présentement développée dans le groupe Keillor. Le principe de celle-ci repose sur la réaction entre deux maléimides portés par un fluorogène et une séquence peptidique cible, laquelle contient deux résidus cystéines séparés par une distance appropriée. Suite à cette double addition de thiols du peptide sur les maléimides du fluorogène, la fluorescence latente de ce dernier est régénérée, menant au marquage covalent de la protéine d’intérêt. Afin d’optimiser la spécificité et la sensibilité de cette méthode de marquage, la synthèse de nouveaux fluorogènes et l’étude de l’efficacité de quench de la fluorescence par les maléimides est présentement en cours dans les laboratoires du groupe Keillor.

Caractérisation et étude de la régulation d’une isoforme cytosolique de peroxyrédoxine chez les solanacées

Les peroxyrédoxines (PRXs) forment une famille de peroxydases communes à tous les organismes vivants et ubiquitaires dans la cellule. Leur particularité provient d’un ou deux résidus cystéines accomplissant un cycle d’oxydo-réduction à l’aide d’un donneur d’électron. Ces protéines thiols sensibles au potentiel redox sont impliquées dans le mécanisme de détoxification du H2O2, une molécule oxydante induite lors de situations de stress. Les PRXs pourraient être induites par le stress et régulées par phosphorylation. En effet, des expérimentations in vitro ont démontré que la nucléoside diphosphate kinase 1 (NDPK1) a la capacité de phosphoryler une PRX cytosolique de pomme de terre. Ce mémoire décrit les travaux expérimentaux effectués pour caractériser la fonction de la PRX. Pour cela, le clonage d’une isoforme a été effectué, suivi d’une caractérisation biochimique et d’une étude d’expression de la protéine. Les données de séquençage révèlent qu’il s’agit d’une PRX de type II phylogénétiquement liée aux PRXs cytosoliques. L’ADNc codant pour cette peroxyrédoxine (PRX1) a été cloné chez Solanum chacoense. Une protéine recombinante portant une étiquette (6xHis) en N-terminale a été produite. Des essais enzymatiques ont confirmé la fonction antioxydante de la protéine recombinante et un anticorps polyclonal a été généré chez le lapin puis utilisé en conjonction avec un anticorps anti-NDPK1 pour déterminer les patrons d’expression généraux de ces protéines chez Solanum lycopersicum et Solanum tuberosum lors de situations de stress. Les données démontrent que les deux protéines sont généralement co-exprimées mais pas co-régulées et que la PRX1 est induite en certaines situations de stress.

La bioaccumulation d’une nanoparticule d’argent (nAg) par l’algue verte Chlamydomonas reinhardtii : distinguer la contribution de la particule de celle de l’ion Ag+

L’explosion de la nanotechnologie a permis l’intégration d’une multitude de nanoparticules dans des produits de consommation. Les nanoparticules d’argent (nAg) sont les plus utilisées à ces fins, selon les derniers recensements disponibles. La plupart des études toxicologiques, à ce jour, ont fait état de l’implication très évidente de l’ion Ag+ dans la toxicité aigüe des nAg; cependant, quelques études ont mis en évidence des effets toxicologiques dus aux nAg. Il y a un certain consensus à propos d’un risque de contamination des eaux douces via leur rejet par les effluents des réseaux d’aqueducs. Puisque les concentrations en Ag+ sont généralement très faibles dans les eaux douces (de l’ordre du pg L-1), de par la formation de complexes non-labiles avec des thiols (organiques et inorganiques) et des sulfures, la toxicité inhérente aux nAg pourrait ne pas être négligeable- comparativement aux tests en laboratoires. Cette étude s’intéressait donc aux mécanismes de bioaccumulation d’argent par l’algue verte C. reinhardtii suite à l’exposition à des nAg de 5 nm (enrobage d’acide polyacrylique). La bioaccumulation d’argent pour l’exposition à Ag+ servait de point de comparaison; également, les abondances de l’ARNm de l’isocitrate lyase 1 (ICL1) et de l’ARNm de Copper Transporter 2 (CTR2) étaient mesurées comme témoins biologiques de la bioaccumulation de Ag+. Les expériences ont été menées en présence d’un tampon organique (NaHEPES, 2 x 10-2 M; Ca2+, 5x 10-5 M) à pH de 7,00. Pour des expositions à temps fixe de 2 heures, la bioaccumulation d’argent pour nAg était supérieure à ce qui était prédit par sa concentration initiale en Ag+; cependant, il n’y avait pas de différence d’abondance des ARNm de ICL1 et de CTR2 entre nAg et Ag+. D’un autre côté, pour une exposition à temps variables, la bioaccumulation d’argent pour nAg était supérieure à ce qui était prédit par sa concentration initiale en Ag+ et une augmentation de l’abondance de l’ARNm de ICL1 était notée pour nAg. Cependant, il n’y avait aucune différence significative au niveau de l’abondance de l’ARNm de CTR2 entre nAg et une solution équivalente en Ag+. L’ajout d’un fort ligand organique (L-Cystéine; log K= 11,5) à une solution de nAg en diminuait radicalement la bioaccumulation d’argent par rapport à nAg-sans ajout de ligand. Par contre, l’abondance des ARNm de ICL1 et de CTR2 étaient stimulées significativement par rapport à une solution contrôle non-exposée à nAg, ni à Ag+. Les résultats suggéraient fortement que les nAg généraient des ions Ag+ au contact de C. reinhardtii.

Inhibition réversible et photomarquage de la transglutaminase tissulaire

La transglutaminase tissulaire est une enzyme dépendante du calcium qui catalyse la formation de liens isopeptidiques, entre les chaînes latérales de résidus glutamine et lysine, permettant, par le fait même, la réticulation des protéines dans les systèmes biologiques. Elle joue un rôle, entre autres, dans l’endocytose, la régulation du développement des cellules, et même dans l’apoptose. Néanmoins, une dérégulation de l’activité biologique de cette enzyme peut entrainer différentes pathologies, comme la formation de cataractes, de plaques amyloïdes dans la maladie d’Alzheimer, ou encore peut mener au développement de la maladie céliaque. C’est pourquoi une meilleure connaissance du mécanisme d’action de cette enzyme et la possibilité de réguler son action à l’aide de substrats ou d’inhibiteurs sont nécessaires. Dans cette optique, une méthode d’expression et de purification de la transglutaminase humaine a été développée, permettant de travailler directement avec la cible pharmacologique désirée. De plus, une étude du mode d’inhibition et de liaison d’une classe d’inhibiteurs réversibles précédemment découverte dans le groupe, soit la famille des trans-cinnamoyles, a permis d’identifier que la puissance de ces molécules est influencée par la présence du calcium et qu’une inhibition dépendante du temps est observée, en lien avec un potentiel équilibre conformationnel lent de la transglutaminase. D’un autre côté, la susceptibilité à une attaque nucléophile par des thiols de cette classe de molécule rend leur potentiel pharmacologique grandement diminué, et c’est pourquoi une nouvelle famille de molécules a été identifiée, basée sur un squelette ynone, avec une valeur d’IC50 très prometteuse de 2,6 μM, en faisant un des meilleurs inhibiteurs réversibles de la transglutaminase développés à ce jour. Finalement, une stratégie de photomarquage jumelée à une analyse de spectrométrie de masse en tandem a été développée pour la découverte du site de liaison du substrat dérivé de la lysine, dans le but de mieux comprendre le mécanisme complexe de cette enzyme.

Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis

Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N-hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes. Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response.

Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis

Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N-hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes, Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response.

Flavor-protein binding: disulfide interchange reactions between ovalbumin and volatile disulfides

The irreversible binding of selected sulfur-containing flavor compounds to proteins was investigated in aqueous solutions containing ovalbumin and a mixture of disulfides (diethyl, dipropyl, dibutyl, diallyl, and 2-furfuryl methyl) using solid-phase micro-extraction (SPME). In systems which had not been heated, the recovery of disulfides from the headspace above the protein at the native pH (6.7) was similar to that from an aqueous blank. However, significant losses were observed when the pH of the solution was increased to 8.0. When the protein was denatured by heating, much greater losses were observed and some free thiols were produced. In similar heat-denatured systems at pH 2.0, no losses of disulfides were observed. Disulfides containing allyl or furfuryl groups were more reactive than saturated alkyl disulfides. Interchange reactions between protein sulfhydryl groups and the disulfides are believed to be responsible for the loss of the disulfides.

Inactivation of the antibacterial and cytotoxic properties of silver ions by biologically relevant compounds

There has been a recent surge in the use of silver as an antimicrobial agent in a wide range of domestic and clinical products, intended to prevent or treat bacterial infections and reduce bacterial colonization of surfaces. It has been reported that the antibacterial and cytotoxic properties of silver are affected by the assay conditions, particularly the type of growth media used in vitro. The toxicity of Ag+ to bacterial cells is comparable to that of human cells. We demonstrate that biologically relevant compounds such as glutathione, cysteine and human blood components significantly reduce the toxicity of silver ions to clinically relevant pathogenic bacteria and primary human dermal fibroblasts (skin cells). Bacteria are able to grow normally in the presence of silver nitrate at >20-fold the minimum inhibitory concentration (MIC) if Ag+ and thiols are added in a 1:1 ratio because the reaction of Ag+ with extracellular thiols prevents silver ions from interacting with cells. Extracellular thiols and human serum also significantly reduce the antimicrobial activity of silver wound dressings Aquacel-Ag (Convatec) and Acticoat (Smith & Nephew) to Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli in vitro. These results have important implications for the deployment of silver as an antimicrobial agent in environments exposed to biological tissue or secretions. Significant amounts of money and effort have been directed at the development of silver-coated medical devices (e.g. dressings, catheters, implants). We believe our findings are essential for the effective design and testing of antimicrobial silver coatings.

Effect of high-hydrostatic pressure and pH treatments on the emulsification properties of gum arabic

This study investigated the emulsification properties of the native gums and those treated at high pressure (800 MPa) both at their “natural” pH (4.49 and 4.58, respectively) and under “acidic and basic” pH (2.8 and 8.0) conditions. The emulsification behaviour of KLTA gum was found to be superior to that of the GCA gum. High pressure and pH treatment changed the emulsification properties of both gums. The acidic amino acids in gum arabic were shown to play an important role in their emulsification behaviour, and mechanisms of emulsification for the two gums were suggested to be different. The highly “branched” nature of the carbohydrate in GCA gum was also thought to be responsible for the “spreading” of droplet size distributions observed. Coomassie brilliant blue binding was used to indicate conformational changes in protein structure and Ellman’s assay was used to estimate any changes in levels of free thiols present.

Key bioactive reaction products of the NO/H2S interaction are S/N-hybrid species, polysulfides, and nitroxyl

Experimental evidence suggests that nitric oxide (NO) and hydrogen sulfide (H2S) signaling pathways are intimately intertwined, with mutual attenuation or potentiation of biological responses in the cardiovascular system and elsewhere. The chemical basis of this interaction is elusive. Moreover, polysulfides recently emerged as potential mediators of H2S/sulfide signaling, but their biosynthesis and relationship to NO remain enigmatic. We sought to characterize the nature, chemical biology, and bioactivity of key reaction products formed in the NO/sulfide system. At physiological pH, we find that NO and sulfide form a network of cascading chemical reactions that generate radical intermediates as well as anionic and uncharged solutes, with accumulation of three major products: nitrosopersulfide (SSNO−), polysulfides, and dinitrososulfite N-nitrosohydroxylamine-N-sulfonate (SULFI/NO), each with a distinct chemical biology and in vitro and in vivo bioactivity. SSNO− is resistant to thiols and cyanolysis, efficiently donates both sulfane sulfur and NO, and potently lowers blood pressure. Polysulfides are both intermediates and products of SSNO− synthesis/decomposition, and they also decrease blood pressure and enhance arterial compliance. SULFI/NO is a weak combined NO/nitroxyl donor that releases mainly N2O on decomposition; although it affects blood pressure only mildly, it markedly increases cardiac contractility, and formation of its precursor sulfite likely contributes to NO scavenging. Our results unveil an unexpectedly rich network of coupled chemical reactions between NO and H2S/sulfide, suggesting that the bioactivity of either transmitter is governed by concomitant formation of polysulfides and anionic S/N-hybrid species. This conceptual framework would seem to offer ample opportunities for the modulation of fundamental biological processes governed by redox switching and sulfur trafficking.

Nitrosopersulfide (SSNO-) targets soluble guanylyl cyclase and induces vasodilation in vivo

Background Recent experimental evidence suggests that nitric oxide (NO) and hydrogen sulfide signaling pathways are intimately intertwined particularly in the vasculature, with mutual attenuation or potentiation of biological responses under control of the soluble guanylyl cyclase (sGC) / phopshodiesterase (PDE) pathway. There is now compelling evidence that part of the NO/sulfide cross talk has a chemical foundation via the formation of S/N-hybrid molecules including thionitrous acid (HSNO) and nitrosopersulfde (SSNO-). The aim of this study was to characterize the bioactive products of the interaction between sulfide and NO metabolites targeting sGC that may potentially regulate vasodilation. Results We found that the chemical interaction of sulfide with NO or nitrosothiols leads to formation of S/N-hybrid metabolites including SSNO- via intermediate formation of HSNO. Contrary to a recent report in the literature but consistent with the transient nature of HSNO, its formation was not detectable by high-resolution mass spectrometry under physiologically relevant conditions. SSNO- is also formed in non-aqueous media by the reaction of nitrite with oxidized sulfur species including colloidal sulfur and polysulfides. SSNO- is stable in the presence of high concentrations of thiols, release NO, and activates sGC in RFL-6 cells in an NO-dependent fashion. Moreover, SSNO- is a potent vasodilator in aortic rings in vitro and lowers blood pressure in rats in vivo. The presence of high concentrations of SOD or thiols does not affect SSNO- mediated sGC activation, while it potentiates and inhibits the effects of the nitroxyl (HNO) donor Angeli's salt, suggesting that HNO release from SSNO- is not involved in sGC activation. Conclusion The reaction between NO and sulfide leads to fomation of S/N-hybrid molecules including SSNO-, releasing NO, activating sGC and inducing vasodilation. SSNO- is considerably more stable than HSNO at pH 7.4 and thus a more likely biological mediator that can account for the chemical cross-talk between NO and sulfide.

Dependence of the product on the P-P ligand in reactions of [RuCl(3)(NO)(P-P)] complexes (P-P = aromatic diphosphines) with 2-mercaptopyridine

This study presents the syntheses and characterization of 2-mercaptopyridine (pyS(-)) complexes containing ruthenium(II) with the following general formula [Ru(pyS)(2)(P-P)], P-P = (c-dppen) = cis-1,2-bis(diphenylphosphino)ethylene) (1); (dppe)=1,2-bis(diphenylphosphino)ethane (2); (dppp)=1,3-bis(diphenylphosphino)propane (3) and (dppb) = 1,4-bis(diphenylphosphino)butane (4). The complexes were synthesized from the mer- or fac-[RuCl(3)(NO)(P-P)] precursors in the presence of triethylamine in methanol solution with dependence of the product on the P-P ligand. The reaction of pyS- with a ruthenium complex containing a bulky aromatic diphosphine dppb disclosed a major product with a dangling coordinated dppbO-P, the [Ru(pyS)(2)(NO)(eta(1)-dppbO-P)]PF(6) (5). In addition, this work also presents and discusses the spectroscopic and electrochemical behavior of 1-5. and report the X-ray structures for I and S. (C) 2009 Elsevier Ltd. All rights reserved.