Nitric oxide (NO) is a key small molecule that orchestrates plant growth, development and immune function. The chief mechanism for the transfer of NO bioactivity is thought to be S-nitrosylation, the addition of an NO moiety to a protein cysteine thiol to form an S-nitrosothiol (SNO). The enzyme S-nitrosoglutathione reductase (GSNOR) indirectly controls the total levels of cellular S-nitrosylation, by turning over S-nitrosoglutathione (GSNO), the major cellular NO donor. In tomato (Solanum lycopersicum. L) a decrease in GSNOR expression, which is expected to increase the extent of cellular SNO formation, resulted in morphological phenotypes and disabled disease resistance. In contrast, increased GSNOR activity enhanced protection against an ordinarily virulent bacterial pathogen. Collectively, these results are similar to previous findings using the reference plant, Arabidopsis thaliana. Thus, the role of GSNOR may be highly conserved across the plant kingdom and manipulating the function of this protein may control important agricultural traits in crop plants.

Changes in cellular redox status are implicated in the regulation of developmental and defence-related responses. The absence of S-nitrosoglutathione reductase (GSNOR) function in Arabidopsis leads to an accumulation of cellular S-nitrosoglutathione (GSNO), a mobile reservoir of nitric oxide (NO) which impacts the cellular redox tone. Consequently, the GSNOR knockout mutant, atgsnor1-3 displays defects in growth, time to flowering and pathogen resistance. Although it is now well established that GSNO is a key redox signalling molecule, the molecular mechanisms that underpin GSNO function remains largely unknown. RAP1 (REDOX-ASSOCIATED PROTEIN 1) was identified based on its dynamic changes of expression in atgsnor1-3 and sid2 plants upon avirulent Pseudomonas syringae pv. tomato (Pst) DC3000 (avrB) challenge. Pathogen-induced RAP1 expression was shown to be independent of the plant hormones salicylic acid, jasmonic acid, abscisic acid and ethylene. Recombinant RAP1 protein was shown to exhibit E3 ligase activity in vitro. Application of the NO donors (GSNO and Cysteine-NO (CysNO)) reduced the E3 ligase activity of RAP1 significantly. Biotinswitch analysis showed that RAP1 was S-nitrosylated and site-directed mutagenesis of RAP1 suggested that the S-nitrosylated site is the cysteine residue C325. The rap1 line does not show obvious developmental phenotypes, however, overexpressing RAP1 enhanced lateral root branching in young seedlings. Overexpression of a truncated RAP1 (RAP1ΔRING) led to a loss of apical dominance. In addition, rap1/rap2 double mutants showed delayed flowering, suggesting RAP1 might be involved in the regulation of plant growth and development. RAP1 may also be involved in plant defence, as rap1, rap2 and rap1/rap2 mutants exhibited increased susceptibility to PstDC3000 and Arabidopsis powdery mildew. Interestingly, rap1 plants showed enhanced resistance to methyl viologen (MV), which is in line with the phenotype of atgsnor mutants. Also, expression of RAP1 was rapidly inducible by ultraviolet-B (UV-B) light. As RAP1 expression and RAP1 E3 ligase activity are redox-related, it is speculated that RAP1 may be involved in redoxmediated regulation of a broad range of physiological responses.

Most S-nitrosothiols (RSNO) are unstable in aqueous solution and decompose to release nitric oxide. This is catalysed by copper ions. A mechanistic study of NO formation from S-nitrosoglutathione (GSNO), the nitroso derivative of the most abundant thiol in the body, was carried out. This relatively stable S-nitrosothiol decomposed in the presence of reduced glutathione and copper ions. The products were identified as nitric oxide and oxidised glutathione, observed as a complex of copper. The role of reduced glutathione was two fold. Firstly, it reduced Cu(^2+) to produce the active Cu(^+) catalytic species and regenerated the catalyst from the oxidised glutathione complex. Spectrophotometric kinetic measurements, under pseudo first order conditions ([GSNO]»[Cu(^2+)]) showed no conventional order with respect to nitrosothiol. This was attributed to the inconstant concentration of available copper during reaction due to the ability of reduced glutathione to complex Cu(^+) and oxidised glutathione to complex Cu(^2+). An investigation of thiol induced decomposition of GSNO and other more stable nitrosothiols showed the reaction to be first order with respect to thiol and nitrosothiol. The rate equation was determined and has been explained in terms of a radical mechanism involving rate limiting attack of the thiyl radical on the RS-NO bond. The decomposition of S-nitrosothiols via the mercuric ion was also investigated. Reactions were stoichiometric rather than catalytic, and the products determined to be H(_2)NO(_2)(^+) and RSHg(^+). The rate equation was established and the reaction found to be first order in S-nitrosothiol and Hg(^2+). Second order rate constants obtained for a variety of nitrosothiols showed no dependency of the rate of the reaction on the structure of R. A mechanism was proposed involving direct attact of the Hg(^2+) ion on S of the nitroso group, reflecting the high affinity of the metal ion for the sulfur atom. Similarly, an investigation into Ag(^+) mediated S-nitrosothiol decomposition showed the reaction to be stoichiometric. Nitric oxide was not a product of this reaction.

Cells are constantly exposed to different stresses – one being redox stress, which is induced by metal, reactive oxygen species and reactive nitrogen species. S-nitrosoglutathione reductase (GSNOR) helps modulate redox stress by two different mechanisms – either by reducing S-nitrosoglutathione (GSNO) to oxidized glutathione (GSSG) or by oxidizing hydroxymethyl glutathione (HMGSH), a biproduct of glutathione and formaldehyde, to formic acid. GSNO has the potential to posttranslational modify proteins in two different manners, either by S-nitrosation or by S-glutathionylation. Interestingly, GSNOR can be modified by its substrate GSNO, either by S-nitrosation, which has previously been reported, or, as discussed in this thesis, by S-glutathionylation. As S-glutathionylation has been reported to occur through intermediate species, the S-glutathionylation of GSNOR appears to occur though the S-nitrosated intermediate, instead of the most common route of an oxidation pathway. It is hypothesized that the S-glutathionylation, and the overall presence of glutathione, can act as a buffer to regulate the amount of nitrosation that GSNOR experiences, and thus the enzymatic activity. It is has reported that the S-nitrosation occurs on three different non-structural, non-catalytic, solvent-accessible cysteine residues. Experimentation was conducted using Saccharomyces cerevisiae as a model organism to determine how those three cysteine residues of the GSNOR homolog Sensitive to Formaldehyde 1 (SFA1) participate in the indirect detoxification of formaldehyde, through the hydroxymethyl glutathione pathway. It has been determined that cysteine 370 is not as important as previously thought, but the other one or two cysteines (either cysteine 10 or 271) do indeed play a role in the detoxification, but further analysis needs to be conducted.

Orientador: Ione Salgado / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T17:34:18Z (GMT). No. of bitstreams: 1 Frungillo_Lucas_M.pdf: 2003193 bytes, checksum: 004553c47da2f38ee16eaf4e64b34cda (MD5) Previous issue date: 2011 / Resumo: Embora o radical óxido nítrico (NO) seja um importante sinalizador em plantas, pouco se conhece sobre os mecanismos que controlam sua homeostase na célula. Acreditase que a enzima S-nitrosoglutationa redutase (GSNOR) tenha um papel relevante no metabolismo de S-nitrosotióis (SNO), e consequentemente na homeostase do NO, através do catabolismo da S-nitrosoglutationa (GSNO). Apesar de a mitocôndria ser um importante alvo do NO, o papel da GSNOR na funcionalidade de mitocôndrias vegetais ainda não foi descrito. Este trabalho teve como objetivo caracterizar mitocôndrias isoladas a partir de cultura celular liquida de Arabidopsis thaliana transgênicas com maior (L1) e menor (L5) expressão da GSNOR em relação ao tipo selvagem. O conteúdo de S-nitrosotióis e peróxido de hidrogênio e a emissão de NO, determinados espectrofotometricamente e fluorimetricamente com DAF-2, respectivamente, foram comparados entre células nas fases de crescimento linear (5 dias de cultivo) e estacionária (10 dias de cultivo; estresse nutricional). O consumo de oxigênio e a degradação de NO por mitocôndrias isoladas nas diferentes fases de cultivo celular foram determinados com eletrodos específicos. Na fase linear o L1 apresentou menor (81%) e o L5 maior (162%) conteúdo de S-nitrosotióis, em relação ao tipo selvagem. Na fase estacionária o conteúdo de S-nitrosotióis foi reduzido e o padrão foi invertido. A emissão de NO pelas células após 5 dias de cultivo foi maior no L5 e não diferiu estatisticamente entre o L1 e o selvagem. Após 10 dias de cultivo os três genótipos apresentaram incremento na emissão de NO, porém o L5 apresentou menor emissão que os outros genótipos. Após 5 dias de cultivo microcalos dos transgênicos L1 e L5 apresentaram menor conteúdo de peróxido de hidrogênio que o tipo selvagem. Porém, em uma condição de estresse nutricional o conteúdo de peróxido de hidrogênio foi estatisticamente igual para todos os genótipos. Ensaios com mitocôndrias isoladas mostraram que o transgênico L1 foi o único incapaz de aumentar a atividade da oxidase alternativa (AOX) e teve as atividades do complexo I e da NADH desidrogenase externa inibidas na situação de estresse. O L5 apresentou maior atividade da NADH desidrogenase externa de modo constitutivo e da proteína desacopladora (UCP) no décimo dia. Ainda, na situação de estresse a capacidade de degradação de NO foi aumentada nos transgênicos L1 e L5. Entretanto, o L5 apresentou maior resistência à inibição da respiração provocada pelo NO, provavelmente devido a maior atividade da AOX. O conjunto dos resultados sugere um importante papel da GSNOR em controlar as alterações funcionais de mitocôndrias de A. thaliana mediadas por NO / Abstract: Although the radical nitric oxide (NO) is an important sign in plants, little is known about the mechanisms that control it's homeostasis in cell. It is believed that the enzyme Snitrosoglutathione reductase (GSNOR) has an important role in the metabolism of Snitrosothiols (SNO), and consequently of NO homeostasis through catabolism of Snitrosoglutathione (GSNO). Although mitochondria are an important target of NO, the role of GSNOR on plant mitochondria functionality has not been described yet. This study aimed to characterize mitochondria isolated from liquid cell culture of transgenic Arabidopsis thaliana with higher (L1) and lower (L5) GSNOR expression relative to wild type. The content of S-nitrosothiols and hydrogen peroxide and the NO emissions, determined spectrophotometrically and fluorimetric with DAF-2, respectively, were compared between cells in the linear (5 days culture) and stationary (10 days culture, nutritional stress) growth phases. Oxygen uptake and NO degradation by mitochondria isolated at different stages of cell culture were determined with specific electrodes. In the linear phase L1 showed lower (81%) and L5 increased (162%) content of S-nitrosothiols compared to wild type. At stationary phase S-nitrosothiols contents has been reduced and the pattern was reversed. The emission of NO by the cells after 5 days of culture was higher in L5 and do not statistically different between the L1 and wild type. At 10 days culture the genotypes showed an increase in the NO emission, but L5 showed lower emissions than the other genotypes. At 5 culture transgenic lines L1 and L5 showed a lower content of hydrogen peroxide than the wild type. However, in a condition of nutritional stress, the content of hydrogen peroxide was statistically the same for all genotypes. Tests with isolated mitochondria showed that transgenic L1 was the only one unable to increase the activity of alternative oxidase (AOX) and had the activities of complex I and NADH dehydrogenase at stress. The L5 showed a constitutive higher activity of the external NADH dehydrogenase and uncoupling protein (UCP) activity at the tenth day. Furthermore, NO degradation capability by mitochondria at nutritional stress situation of NO was increased in transgenic L1 and L5. However, L5 mitochondria showed greater resistance to respiration inhibition caused by NO, probably due to increased activity of AOX. The overall results suggest an important GSNOR role in controlling the mitochondria functional changes of A. thaliana mediated by NO / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

Les maladies inflammatoires chroniques de l’intestin (MICI) représentent un problème de santé publique majeur touchant de jeunes patients. Aux récidives inflammatoires, à la mauvaise qualité de vie et à l’espérance de vie réduite des patients viennent s’ajouter la durée, l’efficacité parfois limitée et le coût des traitements actuellement proposés. La recherche de nouvelles stratégies permettant de prévenir les récidives inflammatoires est primordiale. Ainsi, le rôle du S-nitrosoglutathion (GSNO, donneur et forme de stockage naturelle de NO) dans le maintien de l’intégrité de la barrière intestinale est étudié dans cette thèse, en décrivant : i) l’effet concentration-dépendante du GSNO sur la perméabilité intestinale (modèle de chambre de Ussing, expression des protéines de jonctions cellulaires…), ii) des formulations innovantes de GSNO (nanoparticules composites) à base de nanoparticules d’Eudragit®RL, elles-mêmes encapsulées dans une matrice polymérique à base d’alginate. Différents procédés de formulation ont été testés. Une caractérisation physicochimique des objets développés et des études de perméabilité sur des modèles cellulaires (Caco-2) ont conduit à l’obtention de trois formulations, adaptées à la voie orale et permettant de délivrer de façon prolongée le GSNO. Cependant, la prise en charge du GSNO libre ou formulé, l’identification des cibles du NO au niveau intestinal, mais aussi les doses et durées du traitement in vivo restent encore à définir avant de proposer ce donneur de NO comme candidat pour un traitement préventif des MICI / Inflammatory bowel diseases (IBD) (Crohn’s disease, ulcerative colitis…), are disabling pathologies affecting young patients and presenting the particularity that most of the current agents act by down regulating chronic inflammation in the intestine mucosa and cannot cure the disease. As the pivotal role of S-nitrosoglutathione (GSNO) in preventing intestinal inflammation and gut barrier failure has been clearly pointed out, therapies based on pharmaceutical technology to limit chronic inflammation and prevent relapses of barrier failure by an optimized dosage form of GSNO appears as an interesting challenge. The objective of this project relies on i) studying GSNO concentration/response functions in intestine by using Ussing chamber ex vivo model and following protein cell junction expression, ii) developing polymer nanocomposite particles encapsulating GSNO and adapted to the oral route, with GSNO protection, controlled delivery and local effect in intestine as major requirements. GSNO loaded polymeric (Eudragit® RL) nanoparticles were included in polymer matrix based on alginate; different processes were tested and particles were characterized: high GSNO loading, GSNO sustained release and local retention for 4 h in cellulo study (Caco-2) were obtained. Thus, therapies based on GSNO administration should represent a novel strategy to limit chronic inflammation and prevent relapses of barrier failure in IBD patients

<p>Nitric oxide (NO) is an important chemical factor that controls vascular tone in the cardiovascular system. NO is a vasodilatory molecule that plays a role in blood pressure and blood flow regulation as well as vessel formation and tissue cell proliferation. NO influences the flow by which nutrients and other metabolites required for growth are transported to the tissues. The aim of this study was to investigate if NO, through mediation by the NO donors Sodium Nitroprusside (SNP) and S-Nitrosoglutathione (GSNO) affect growth and oxygen consumption of prenatal broiler chicken. The results indicate that, although the treatments did not have clear significant effects on the embryos or the organs examined, a slight delay in development can be observed in the GSNO treatment embryos. The study could not conclude, however, if this was due to effects of NO donors</p>

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2013-11-06T17:30:24Z No. of bitstreams: 1 Felipe Miranda Santos Potencial terapeutico das S-nitroglutationa-.pdf: 5390094 bytes, checksum: 048e66640081905e53e87d3353d7c25c (MD5) / Made available in DSpace on 2013-11-06T17:30:24Z (GMT). No. of bitstreams: 1 Felipe Miranda Santos Potencial terapeutico das S-nitroglutationa-.pdf: 5390094 bytes, checksum: 048e66640081905e53e87d3353d7c25c (MD5) Previous issue date: 2012 / Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / Universidade Federal da Bahia. Faculdade de Farmácia. Salvador, BA, Brasil / A intoxicação pelo paracetamol é a principal causa de insuficiência hepática aguda (IHA) em vários países do ocidente. A hepatotoxicidade é mediada por um metabólito intermediário reativo que depleta as reservas do antioxidante endógeno glutationa (GSH). O tratamento precoce com n-acetilcisteína (NAC) é recomendado para restabelecer a concentração fisiológica de GSH. A snitrosoglutationa (GSNO) é uma molécula antioxidante derivada do GSH capaz de reduzir o estresse oxidativo em diversos sistemas celulares e modelos experimentais. OBJETIVO: Avaliar se GSNO é capaz de reduzir a taxa de mortalidade, extensão da necrose hepática, manifestações bioquímicas e comparar sua eficácia com NAC e GSH no tratamento da IHA experimental induzida por paracetamol. METODOLOGIA: Camundongos isogênicos machos da linhagem C57Bl/6 foram tratados por três semanas com água suplementada com etanol a 10%. Os animais foram divididos em cinco grupos. O grupo 1 (controle negativo) recebeu solução salina 0,9%. Os demais grupos receberam 300 mg/Kg de paracetamol para indução de IHA. Após 3 horas, o grupo 2 (controle positivo) foi tratado com salina tamponada com fosfato (PBS) e os grupos 3, 4 e 5 foram tratados, respectivamente, com 600 Umol/kg de NAC, GSH e GSNO. A eutanásia foi feita 12 horas após a indução de IHA. A extensão da necrose hepática foi avaliada por morfometria através do software IMAGEPRO-PLUS. Os níveis séricos de transaminases e fosfatase alcalina foram avaliados como marcadores bioquímicos de lesão hepática. A taxa de mortalidade foi avaliada em um experimento independente, após uma dose de 350 mg/Kg de paracetamol. RESULTADOS: O tratamento com GSNO 600 Umol/kg aumentou a taxa de sobrevida em relação aos grupos tratados com NAC ou PBS. Entretanto, não houve diferença de mortalidade entre os grupos GSNO e GSH. A avaliação morfométrica revelou menor extensão de necrose hepática nos animais tratados com GSNO em comparação com NAC e PBS. Houve redução de atividade sérica de ALT, mas não de AST no grupo GSNO em comparação com PBS e NAC. Os níveis séricos de fosfatase alcalina, albumina, ureia e creatinina não apresentaram diferenças entre os diversos grupos. CONCLUSÃO: O tratamento com GSNO aumenta a taxa de sobrevida e reduz a extensão de necrose hepática na IHA experimental por paracetamol. O GSNO apresenta eficácia superior à NAC e idêntica ao GSH em dose equimolar. Estes achados sugerem que o efeito protetor do GSNO parece independer da porção nitroso da molécula. Possíveis mecanismos de proteção extra-hepáticos merecem ser investigados / Paracetamol overdose is the main cause of acute liver failure (ALF) in western countries. The hepatotoxicity is mediated by a reactive metabolite that depletes the pool of glutathione (GSH), an endogenous antioxidant molecule. Early treatment with n-acetylcysteine (NAC) is recommended to replenish the pool of GSH. S-nitrosoglutathione (GSNO) is a potent antioxidant molecule that reduces oxidative stress in several cellular systems and experimental models. OBJECTIVE: To evaluate if GSNO reduces the mortality rate, the hepatocelular necrosis extension and to compare its therapeutic efficacy with NAC and GSH in experimental ALF induced by paracetamol. METHODS: Male mice were treated for three weeks with alcohol 10% orally. The animals were divided in five groups. Group 1 (negative control) received saline 0.9%. All the other groups received 300 mg/Kg paracetamol for induction of ALF. After 3 hours, group 2 (positive control) received phosphate buffered saline (PBS) and groups 3, 4 and 5 were treated respectively with 600 Umol/kg of NAC, GSH and GSNO. The animals were sacrificed after 12 hours of induction of ALF. The area of liver necrosis was evaluated by morphometric analysis with the software IMAGEPRO. Transaminases and alkaline phosfatase were determined as markers of liver injury. Mortality rate was evaluated in an independent experiment after a dose of 350 mg/Kg of paracetamol. RESULTS: GSNO treatment (600 Umol/kg) significantly improved the survival rate compared to PBS and NAC treatments. There was no statistical difference in survival rate between GSNO and GSH groups. In addition, GSNO attenuated the area of liver necrosis in comparison to NAC and PBS, but not to GSH. GSNO reduced the serum ALT, but not AST activity in comparison to PBS and NAC. There was no statistical difference in alkaline phosphatase, urea, creatinine and albumin among the groups that received paracetamol. CONCLUSION: GSNO treatment augmented survival rate and reduced the area of liver necrosis in comparison to NAC, but was equally as effective as GSH. These findings suggest that the hepatoprotector effect of GSNO is independent of the nitroso moiety of the molecule. Potential extra-hepatic mechanisms remain to be evaluated.

Nitric oxide (NO) is an important chemical factor that controls vascular tone in the cardiovascular system. NO is a vasodilatory molecule that plays a role in blood pressure and blood flow regulation as well as vessel formation and tissue cell proliferation. NO influences the flow by which nutrients and other metabolites required for growth are transported to the tissues. The aim of this study was to investigate if NO, through mediation by the NO donors Sodium Nitroprusside (SNP) and S-Nitrosoglutathione (GSNO) affect growth and oxygen consumption of prenatal broiler chicken. The results indicate that, although the treatments did not have clear significant effects on the embryos or the organs examined, a slight delay in development can be observed in the GSNO treatment embryos. The study could not conclude, however, if this was due to effects of NO donors

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