Produção de oxido nitrico pela oxido nitrico sintase, nitrato redutase e uma atividade mitocondrial redutora de nitrito na resposta de defesa de Arabidopsis thaliana a Pseudomonas syringae

Orientador: Ione SalgadoTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A origem do óxido nítrico (?NO) em plantas tem sido controversa e uma enzima do tipo óxido nítrico sintase (NOS), bem como nitrato redutase (NR) são consideradas possíveis fontes deste radical livre. Contudo, ainda não há nenhuma evidência direta da formação de ?NO a partir da NR durante interações planta-patógeno. Nos estudos aqui descritos foram utilizadas plantas de Arabidopsis thaliana deficientes na enzima NR para investigar o mecanismo de produção de ?NO em resposta à inoculação da bactéria Pseudomonas syringae pv. maculicola (Psm). Aumento na síntese de L-citrulina a partir de L-arginina foi observado em homogenatos de folhas de plantas do tipo selvagem e daquelas deficientes na síntese de NR, após inoculação com Psm. Experimentos de ressonância paramagnética eletrônica, contudo, mostraram uma produção muito maior de ?NO dependente de NO2-, mas não de L-arginina ou NO3-, síntese esta inibida por alta concentração de L-arginina. A inibição da cadeia de transporte de elétrons da mitocôndria aboliu ou inibiu parcialmente esta atividade em plantas mutantes ou selvagens, respectivamente. A utilização do marcador fluorescente 4,5 diaminofluoresceína diacetato (DAF-2DA) mostrou que a inoculação com Psm aumentou substancialmente a produção de ?NO in situ em plantas selvagens, mas não em mutantes deficientes na síntese de NR. No entanto, a infiltração de L-arginina ou NO2- causou uma produção de ?NO in vivo nas plantas mutantes. Plantas de A. thaliana deficientes na síntese de NR mostraram suscetibilidade à P. syringae que foi prevenida pela co-infiltração de NO2-. O conjunto de resultados sugere que NOS, NR e uma importante atividade mitocondrial de redução de NO2- podem cooperar para produzir ?NO em quantidade suficiente para prevenir a disseminação de Psm em plantas de A. thalianaAbstract: The origin of nitric oxide (?NO) in plants has been controversial and nitric oxide synthase (NOS)-like enzyme and nitrate reductase (NR) are claimed as potential sources. However, there is still no direct evidence for ?NO formation from NR during plant-pathogen interactions. Here, we used NR-defective double mutants of Arabidopsis thaliana to investigate the mechanism of ?NO production following infection by an avirulent strain of Pseudomonas syringae pv. maculicola (Psm). Increased formation of L-citrulline from L-arginine was observed in leaf homogenates of both wild-type and nia1 nia2 mutant plants. Electron paramagnetic resonance experiments, however, showed a much higher ?NO production that was dependent on NO2- rather than on L-arginine or NO3- and was also inhibited at high L-arginine concentrations. Inhibition of mitochondrial electron transport abolished and inhibited partially this activity in nia1 nia2 mutants and wild-types, respectively. In situ ?NO production was substantially increased in wild type-plants but not in mutants, following Psm challenge, as measured with the fluorescent probe 4,5-diaminofluorescein diacetate. However, in vivo ·NO production in NR-deficient mutants was visualized only after L-arginine or NO2- infiltration into the leaves. A. thaliana plants defective in NR showed susceptibility to P. syringae that was prevented by infiltration of NO2-. The overall results suggest that NOS, NR, and an important mitochondrial-dependent nitrite-reducing activity cooperate to produce ?NO in amounts enough to prevent Psm dissemination in A. thaliana plantsDoutoradoBioquimicaDoutor em Biologia Funcional e Molecula

Sialic acids: from the comprehension of their involvement in biological processes to antiinfluenza drug design

Sialic acids are nine-carbon carbohydrates that occur widely in nature and occupy the terminal portions of some glycoproteins and glycolipids of cell membranes. These carbohydrates are closely involved in cell-cell interactions and in processes such as microbial infection, inflammation, etc. Studies on the participation of sialic acids in biological processes have provided comprehension about their role in the infection by the influenza virus, the causal agent of flu. In this article, we present an overview of the importance of sialic acids in the influenza virus infection and how the knowledge of their involvement in this process has allowed the development of selective and efficient drugs against the virus.30631

Quinolines: microwave-assisted synthesis and their antifungal, anticancer and radical scavenger properties

An efficient method for the synthesis of quinolines using microwave irradiation was developed providing 28 quinolines with good yields. The reaction procedures are environmentally friendly, convenient, mild and of easy work-up. Quinolines were evaluated for their antifungal, anticancer and antioxidant properties and exhibited high activities in all tests performed.Centro de Investigación y Desarrollo en Ciencias Aplicada

Produção de flavonoides e atividade oxido nitrico sintase na resposta de defesa de soja ao fungo causador do cancro da haste

Orientador: Ione SalgadoDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: O presente trabalho analisou a participação da enzima óxido nítrico sintase (NOS) na produção de fitoalexinas, como parte do mecanismo de defesa de soja ao ataque do fungo Diaporthe phaseolorum f. sp. meridionalis (Dpm), causador da doença do cancro da haste. A produção de fitoalexinas e a atividade NOS em cotilédones de soja eliciados com extrato do fungo Dpm foram comparadas com aquela induzida por nitroprussiato de sódio (SNP), uma molécula capaz de liberar óxido nítrico (NO). Os flavonóides produzidos foram identificados e quantificados utilizando Cromatografia Líquida de Alta Eficiência (CLAE) e padrões autênticos. A atividade NOS foi determinada pela síntese de L-[U_14C] citrulina a partir de L-[U-14C] arginina. A eliciação dos cotilédones de soja com o extrato de Dpm ou com SN P induziu intensa produção de fitoalexinas. Não foi observada produção de fitoalexinas quando o SNP foi substituído por ferricianeto que difere estruturalmente do primeiro apenas por não conter o NO. A produção de fitoalexinas induzida pelo extrato de Dpm, mas não aquela induzida por SNP, foi inibida quando os cotilédones de soja foram pré-tratados com inibidores característicos da enzima NOS de origem animal. A eliciação com Dpm induziu também atividade NOS nos tecidos em contato com o eliciador, atividade não observada nos tecidos eliciados por SNP. A atividade NOS induzida mostrou ser dependente de Ca2+ e NADPH e foi inibida por L-NAME [NG-nitro-L-arginina metil éster], AMG [Aminoguanidina] e L-NIL [L-N6-(1-iminoetil) lisina], inibidores característicos da NOS de origem animal. A indução de atividade NOS precedeu a ativação da produção de flavonóides nos cotilédones eliciados por Dpm. O acúmulo destes metabólitos foi mais precoce quando os cotilédones foram eliciados com SNP. O conjunto de resultados sugeriu que na eliciação por Dpm ocorreu a ativação de uma enzima do tipo NOS produzindo NO que foi capaz então de induzir a produção de flavonóides que apresentam atividade antimicrobiana. Não foram observadas diferenças significativas na produção de fitoalexinas pelos cotilédones entre os cultivares IAC-18 e IAC-14, resistente e suscetível ao Dpm, respectivamente. Estes resultados indicaram que ambos os cultivares seriam capazes de se defender ao ataque do patógeno nos estádios iniciais de desenvolvimento da plantaAbstract: The formation of phytoalexin is part of the defense mechanism of soybeans against the fungus Diaporthe phaseolorum f. sp. meridionalis (Dpm), the causal agent of stem canker disease. In this work, the involvement of nitric oxide synthase (NOS) in phytoalexin production was examined. Phytoalexin formation and NOS activity in soybean cotyledons stimulated with a Dpm extract were compared with the levels in cotyledons exposed to sodium nitroprusside (SNP), a nitric oxide (NO) donor. The flavonoids were identified and quantified by High Performance Liquid Chromatography (HPLC) using authentic standards. NOS activity was determined by the biosynthesis of L-[U_14C] citrulline from L-[U_14C] arginine. Exposure of soybean cotyledons to the Dpm extract or SNP resulted in a high accumulation of phytoalexin, but this was not observed when SNP was replaced by ferricyanide, a structural analog of SNP devoid of the NO moiety. Phytoalexin production induced by the Dpm extract, but not by SNP, was prevented when the cotyledons were pretreated with NOS inhibitors. The Dpm extract also induced NOS activity in soybean cotyledon tissue proximal to the site of extract application. No such activity was observed with SNP. The induced NOS activity was Ca2+- and NADPH-dependent and was sensitive to the NOS inhibitors L-NAME [NGnitro-L-arginine methyl ester], AMG [aminoguanidine] and L-NIL [L-N6-(iminoethyl) Iysine]. The induction of NOS activity preceded the maximal accumulation of flavonoids in cotyledons exposed to the Dpm extract. The accumulation of these metabolites was faster in SNP than in Dpm treated cotyledons. These results suggest that Dpm extract induce NOS activity, with the formation of NO subsequently stimulating biosynthesis of flavonoids. There was no significant difference in the phytoalexin accumulation between cotyledons from cultivars IAC-18 and IAC-14 which are resistant and susceptible to Dpm, respectively. Thus, both cultivars should be able to defend themselves against attacks by pathogens in the earlier stages of plant developmentMestradoBioquimicaMestre em Biologia Funcional e Molecula

Método Para Aumentar A Produção De Daidzeìna E Genisteìna Em Soja

MÉTODO PARA AUMENTAR A PRODUÇÃO DE DAIDZEÍNA E GENISTEÍNA EM SOJA. No qual são empregados compostos que liberam óxido nítrico (NO) ou solução saturada de NO gás para induzir o aumento da quantidade de daidzeína e genisteína além dos níveis pré-existentes em plantas de soja. A produção de daidzeína e genisteína pela adição de liberadores de NO em cultura líquida de células de soja, em calos, em sementes ou em cortes feitos nas superfícies de cotilédones da referida planta incrementa os níveis destas isoflavonas em torno de 60 a 120 vezes quando comparados aos experimentos realizados sem a adição de liberadores de NO. De uma maneira geral, as metodologias já descritas para a obtenção de extratos enriquecidos com daidzeína e genisteína baseiam-se apenas na extração do conteúdo destas isoflavonas inerentes de leguminosas. Estas isoflavonas têm reconhecida atividade antioxidante, antitumoral e são utilizadas no tratamento de reposição hormonal sendo conhecidas como fitoestrógenos.BR0304014 (A)A01H3/04C12P17/06C12P17/06A01H3/04BR20030304014A01H3/04C12P17/06C12P17/06A01H3/0

Salinity-induced accumulation of endogenous H2S and NO is associated with modulation of the antioxidant and redox defense systems in Nicotiana tabacum L. cv. Havana

Salinity is one of the abiotic factors that most affect crop growth and production. This study focused on the effect of high salinity on the endogenous levels of the signaling molecules hydrogen sulfite (H2S) and nitric oxide (NO) in Nicotiana tabacum leaves and the extent of these for the biochemically-driven plant tolerance to such abiotic stress. The NaCl treatment for 10 days led to an expressive augment of H2S and NO levels. This increase was correlated with the raise of l-Cys and l-Arg and the induction of l-cysteine desulfhydrase, cyanoalanine synthase, cysteine synthase, nitrate reductase and arginase, enzymes known to be involved in the biosynthesis of H2S or NO. The enzymatic antioxidant system (superoxide dismutase and catalase activity) was boosted and the non-enzymatic antioxidant glutathione was intensively oxidized in leaves upon stress allowing plants to cope with oxidative stress. Lower stomatal conductance was observed in stressed plants in comparison with control ones. Moreover, the high activity of antioxidant enzymes and high rate of glutathione oxidation following salt stress were considerably decreased upon NO or H2S scavenging. Thus, increment in NO and H2S levels and their interplay, along with metabolic and physiological changes, contributed to tobacco survival to extreme salinity conditions

Nitric Oxide Synthase-Mediated Phytoalexin Accumulation in Soybean Cotyledons in Response to the Diaporthe phaseolorum f. sp. meridionalis Elicitor

Phytoalexin biosynthesis is part of the defense mechanism of soybean (Glycine max) plants against attack by the fungus Diaporthe phaseolorum f. sp. meridionalis (Dpm), the causal agent of stem canker disease. The treatment of soybean cotyledons with Dpm elicitor or with sodium nitroprusside (SNP), a nitric oxide (NO) donor, resulted in a high accumulation of phytoalexins. This response did not occur when SNP was replaced by ferricyanide, a structural analog of SNP devoid of the NO moiety. Phytoalexin accumulation induced by the fungal elicitor, but not by SNP, was prevented when cotyledons were pretreated with NO synthase (NOS) inhibitors. The Dpm elicitor also induced NOS activity in soybean tissues proximal to the site of inoculation. The induced NOS activity was Ca(2+)- and NADPH-dependent and was sensitive to the NOS inhibitors N(G)-nitro-l-arginine methyl ester, aminoguanidine, and l-N(6)-(iminoethyl) lysine. NOS activity was not observed in SNP-elicited tissues. An antibody to brain NOS labeled a 166-kD protein in elicited and nonelicited cotyledons. Isoflavones (daidzein and genistein), pterocarpans (glyceollins), and flavones (apigenin and luteolin) were identified after exposure to the elicitor or SNP, although the accumulation of glyceollins and apigenin was limited in SNP-elicited compared with fungal-elicited cotyledons. NOS activity preceded the accumulation of these flavonoids in tissues treated with the Dpm elicitor. The accumulation of these metabolites was faster in SNP-elicited than in fungal-elicited cotyledons. We conclude that the response of soybean cotyledons to Dpm elicitor involves NO formation via a constitutive NOS-like enzyme that triggers the biosynthesis of antimicrobial flavonoids