Structural Evidence for a Copper-Bound Carbonate Intermediate in the Peroxidase and Dismutase Activities of Superoxide Dismutase

Copper-zinc superoxide dismutase (SOD) is of fundamental importance to our understanding of oxidative damage. Its primary function is catalysing the dismutation of superoxide to O2 and H2O2. SOD also reacts with H2O2, leading to the formation of a strong copper-bound oxidant species that can either inactivate the enzyme or oxidise other substrates. In the presence of bicarbonate (or CO2) and H2O2, this peroxidase activity is enhanced and produces the carbonate radical. This freely diffusible reactive oxygen species is proposed as the agent for oxidation of large substrates that are too bulky to enter the active site. Here, we provide direct structural evidence, from a 2.15 Å resolution crystal structure, of (bi)carbonate captured at the active site of reduced SOD, consistent with the view that a bound carbonate intermediate could be formed, producing a diffusible carbonate radical upon reoxidation of copper. The bound carbonate blocks direct access of substrates to Cu(I), suggesting that an adjunct to the accepted mechanism of SOD catalysed dismutation of superoxide operates, with Cu(I) oxidation by superoxide being driven via a proton-coupled electron transfer mechanism involving the bound carbonate rather than the solvent. Carbonate is captured in a different site when SOD is oxidised, being located in the active site channel adjacent to the catalytically important Arg143. This is the probable route of diffusion from the active site following reoxidation of the copper. In this position, the carbonate is poised for re-entry into the active site and binding to the reduced copper. © 2012 Strange et al

Kinetic Characterization of Sulfenic Acid Reduction in 1-Cys Peroxiredoxins by Ascorbate

Peroxiredoxins (Prxs) are Cys-based peroxidases with\ud remarkable catalytic efficiency that can be divided into 1-Cys or 2-\ud Cys, depending on the number of Cys residues involved in\ud catalysis. Initially, reduction of Prx was described to be strictly\ud dependent on thiols, but later we showed that ascorbate can also\ud reduce the sulfenic intermediate of 1-Cys Prx (1-Cys Prx-SOH) in\ud various organisms [1]. Here, the kinetic characterization of 1-Cys\ud Prx-SOH reduction by ascorbate is described. Reduction of 1-Cys\ud Prx-SOH by ascorbate was initially analyzed using an enzyme\ud from A. fumigatus (AfPrxA) that is 37% similar to PRDX6 (human\ud 1-Cys Prx). H2O2 levels were determined by means of a specific\ud electrode (Free Radical Analyzer 4100), using a steady-state bisubstrate approach. AfPrxA decomposed H2O2 with good\ud efficiency (kcat/KM = 7.4 x103\ud M1\ud .s1\ud ), through a Bi-Bi Ping-Pong\ud mechanism. To further support these findings, a second,\ud independent approach was also employed: competition between\ud dichlorophenolindophenol (DCPIP) and AfPrxA-SOH for\ud ascorbate. DCPIP is a redox sensor, whose blue color is lost\ud when reduced and its second-order rate constant with ascorbate\ud is 718 M1\ud .s1\ud , enabling the determination of the rate constant of the\ud reaction between AfPrxA-SOH and ascorbate: 1.5 x 103\ud M1\ud .s1\ud .\ud Therefore, by two independent approaches, we showed that\ud ascorbate efficiently reduced AfPrxA-SOH. Next, the reductions of\ud 1-Cys Prx SOH in other organisms (bacteria, yeast and plant)\ud were also investigated and again the constants were in the\ud 103\ud M1\ud .s1 range. We conclude that the reduction of 1-Cys PrxSOH\ud by ascorbate is probably relevant in the subcellular\ud compartments in which this reductant is present at millimolar\ud levels. We are currently studying the reduction of 1-Cys Prx-SOH\ud by ascorbate in other proteins, which could open new\ud perspectives in cellular redox processes in vivo.\ud [1] Proc.Natl.Acad.Sci. USA. 2007 104:4886-9

Plinia trunciflora extract administration prevents HI-induced oxidative stress, inflammatory response, behavioral impairments, and tissue damage in rats

The disruption of redox homeostasis and neuroinflammation are key mechanisms in the pathogenesis of brain hypoxia–ischemia (HI); medicinal plants have been studied as a therapeutic strategy, generally associated with the prevention of oxidative stress and inflammatory response. This study evaluates the neuroprotective role of the Plinia trunciflora fruit extract (PTE) in neonatal rats submitted to experimental HI. The HI insult provoked a marked increase in the lipoperoxidation levels and glutathione peroxidase (GPx) activity, accompanied by a decrease in the brain concentration of glutathione (GSH). Interestingly, PTE was able to prevent most of the HI-induced pro-oxidant effects. It was also observed that HI increased the levels of interleukin-1β in the hippocampus, and that PTE-treatment prevented this effect. Furthermore, PTE was able to prevent neuronal loss and astrocyte reactivity induced by HI, as demonstrated by NeuN and GFAP staining, respectively. PTE also attenuated the anxiety-like behavior and prevented the spatial memory impairment caused by HI. Finally, PTE prevented neural tissue loss in the brain hemisphere, the hippocampus, cerebral cortex, and the striatum ipsilateral to the HI. Taken together our results provide good evidence that the PTE extract has the potential to be investigated as an adjunctive therapy in the treatment of brain insult caused by neonatal hypoxia–ischemia

Caracterização do pão regional do distrito de Viseu e de Pão São

Dissertação de Mestrado em Qualidade e Tecnologia AlimentarO objectivo deste estudo visa a caracterização de diferentes amostras de pão Regional e pão São, procurando encontrar semelhanças e identificar os elementos diferenciadores dos mesmos. As amostras de pão Regional foram recolhidas no distrito de Viseu: Carregal do Sal, Cabanas de Viriato, Santar, Viseu, Mangualde, São Pedro do Sul e São Cipriano. No caso do pão São as amostras foram fornecidas pela empresa produtora, Fábrica do Pão localizada em Seia. Procedeu-se à caracterização física, química e sensorial, tendo sido avaliadas as diferentes propriedades em termos de média e desvio padrão das réplicas efectuadas. Posteriormente, procedeu-se a uma análise estatística, para comparação de médias e avaliação da significância das diferenças, tendo para tal sido usado o teste de Tuckey HSD. Foram ainda calculados os coeficientes de correlação de Pearson. Os resultados foram analisados recorrendo ao software SPSS para o Windows versão 19.0 e Statistic versão 6., sendo realizado o teste de análise de variância "one-way" (ANOVA). Numa fase posterior, os valores obtidos para as características do pão foram sujeitos a uma análise de componentes principais (PCA) e uma análise de Cluster. Em relação aos parâmetros químicos analisados no pão Regional, as amostras apresentaram algumas semelhanças e algumas diferenças, assim como também a nível físico e sensorial. No entanto, no que respeita à análise sensorial, as amostras foram, de modo geral, percepcionadas da mesma forma pelos provadores, não sendo detectadas diferenças significativas. Ainda assim, em termos de apreciação global a amostra preferida foi a da pastelaria Flor de Cabanas. Em relação aos parâmetros químicos, físicose sensoriais do pão São, as amostras mostraram-se semelhantes entre si, o que pode ser explicado por terem basicamente a mesma constituição em termos de ingredientes. Contudo, será de assinalar algumas diferenças em termos de fibra, hidratos de carbono, proteína e cloretos, devido à presença de farinha de tremoço numa das amostras. Quando comparadas as amostras de pão Regional com as de pão São, verificou-se que estas se apresentaram bastante diferentes, quer em termos químicos, físicos ou nível sensorial. Foi possível observar que ao nível sensorial os pães Regionais foram os preferidos pelos provadores.ABSTRACT: The aim of this study is the characterization of different samples of regional bread and healthy bread, trying to identify the similarities and contrasting elements in them. The regional bread samples were collected in the District of Viseu: Carregal do Sal, Cabanas de Viriato, Santar, Viseu, São Pedro do Sul and São Cipriano. The healthy bread samples were provided by the production company "Fábrica do Pão", located in Seia. The physical, chemical and sensory characterization was undertaken, having been avaluted the different proprerties in terms of mean value and standard deviation of the replicas made. Later, we proceeded to a statistical analysis for comparison of means and assessing the significance of the differences, having been used for this the Tukey HSD test. We also calculated the Pearson correlation coefficients. The results were analyzaed using the SPSS software version 19.0 for Windows and Statistica version 6, having been performed an analysis of variance "one way"(ANOVA). At a later stage, the values obtained for the characteristics of bread were subjected to principal components analysis (PCA) and cluster analysis. Regardind the chemical parameters analyzed in the regional bread, the samples showed some similarities and differences, as well as regarding the physical and sensory parameters. However, concerning the sensory analysis, the samples were generally perceived by the panelists in the same way, and no significant differences were observed. Still, in terms of the same way preferred by the panelists the sample from pastry "Flor de Cabanas". With respect to chemical, physical and sensory parameters of the healthy bread, the samples were similar, which can be explained by having basically the same constitution in terms of ingredients. However, there can be noted some differences in terms of fiber, carbohydrates, protein and chlorides, due to the presence of lupine flour in one sample. When comparing the regional bread samples with the healthy bread ones, it was found that they quite different, either at the chemical, physical or sensory levels. It was observed that at the sensory level the regional breads were those preferred by the tasters

Caracterização dos estilos de vida dos sem-abrigo da cidade do Porto

O fenómeno dos sem-abrigo está em constante crescimento nos centros urbanos, na cidade no Porto o mesmo acontece, sendo esta uma realidade ainda pouco conhecida. Têm sido realizados alguns estudos sobre esta problemática, no entanto poucos incidem sobre a população portuguesa, pouco se sabe sobre como vivem estes indivíduos e sobre o que define o seu estilo de vida. Os estilos de vida têm vindo a ser uma área de crescente interesse para estudo, visto que afecta a nossa saúde e a longo prazo tem influência nos padrões de morbilidade e mortalidade. Com este estudo procurou-se caracterizar os sem-abrigo da cidade do Porto e os comportamentos que tipificam o seu estilo de vida, bem como verificar se existem variáveis dos estilos de vida que se encontram correlacionadas com a presença de sintomatologia psicopatológica. Para este efeito, foi realizado um inquérito por questionário a 30 pessoas que vivem na condição de sem-abrigo na cidade do Porto, através da administração de um questionário de estilos de vida e da Brief Psychiatric Rating Scale (BPRS). Concluímos que os sem-abrigo se caracterizam por ser do sexo masculino, solteiros de nacionalidade portuguesa e baixa escolaridade. Constatamos que a maioria apresenta comportamentos pouco saudáveis como fumar e não praticar exercício físico, contudo têm cuidado com a higiene pessoal, apresentam uma boa higiene do sono e manifestam poucos comportamentos sexuais de risco. Verificamos que a ansiedade se encontra correlacionada negativamente com o stress, higiene do sono, insight e alimentação. As perturbações somáticas mostraram uma correlação com a higiene do sono, o humor depressivo com a higiene do sono e insight, o retraimento emocional com a socialização e falta de cooperação com a socialização, sendo todas estas correlações negativas. Encontramos ainda uma correlação positiva entre a lentificação e o consumo de substâncias e uma correlação negativa entre a lentificação e higiene do sono. Considerando os resultados obtidos pensamos ser fundamental prosseguir com estudos de investigação nesta área, para que de futuro as intervenções junto desta população consigam dar uma melhor resposta ao seu estado de saúde.The phenomenon of homelessness is steadily increasing in urban centers, including in city of Porto, witch is a reality not well know. Some studies have been conducted on this issue, although few focus on the population of Portugal, little is known about how these persons live and what defines their lifestyle. The lifestyle have been an area of growing interest to study, since it is known that affects ours health and long term influences on patterns of morbidity and mortality. The purpose of this study is to characterize the lifestyles of the homeless in the city of Porto, realizing who are they and what are the behaviors that characterize their lifestyle, as well as verifying if there are lifestyles variables correlated with psychopathological symptomatology. We studied 30 persons who live in the condition of homelessness in the city of Porto, by using a lifestyle questionnaire and the Brief Psychiatric Rating Scale (BPRS). We conclude that homeless are characterized by being male, single, of Portuguese nationality and low. We find that most have unhealthy behaviors such as smoking and physical exercise, but be careful with personal hygiene, have good sleep hygiene and exhibit few sexual risk behaviors. We found that anxiety is correlated negatively with stress, sleep hygiene, insight and nutrition. The somatic concern showed a correlation with sleep hygiene, the depressed mood with sleep hygiene and insight, emotional withdrawal with socialization and lack of cooperation with socialization, which are all negative correlations. We also found a positive correlation between motor retardation and substance use and a negative correlation between motor retardation and sleep hygiene. Considering these results we think it vital to continue with research studies in this area, so that in future interventions with this population are able to better respond to your health

Investigation on solubilization protocols in the refolding of the thioredoxin TsnC from Xylella fastidiosa by high hydrostatic pressure approach

The lack of efficient refolding methodologies must be overcome to take full advantage of the fact that bacteria express high levels of aggregated recombinant proteins. High hydrostatic pressure (HHP) impairs intermolecular hydrophobic and electrostatic interactions, dissociating aggregates, which makes HHP a useful tool to solubilize proteins for subsequent refolding. A process of refolding was set up by using as a model TsnC, a thioredoxin that catalyzes the disulfide reduction to a dithiol, a useful indication of biological activity. The inclusion bodies (IB) were dissociated at 2.4 kbar. The effect of incubation of IB suspensions at 1–800 bar, the guanidine hydrochloride concentration, the oxidized/reduced glutathione (GSH/GSSG) ratios, and the additives in the refolding buffer were analyzed. To assess the yields of fully biologically active protein obtained for each tested condition, it was crucial to analyze both the TsnC solubilization yield and its enzymatic activity. Application of 2.4 kbar to the IB suspension in the presence of 9 mM GSH, 1 mM GSSG, 0.75 M guanidine hydrochloride, and 0.5 M arginine with subsequent incubation at 1 bar furnished high refolding yield (81%). The experience gained in this study shall help to establish efficient HHP-based protein refolding processes for other proteins.This work was supported by grants from the State of São Paulo Research Foundation – FAPESP (Process 10/13353-0) and National Council for Scientific and Technological Development – CNPq (Process 479816/2007-7) and fellowship 134597/2010-9 from National Council for Scientific and Technological Development – CNPq

Redox regulation of the proteasome via S-glutathionylation

The proteasome is a multimeric and multicatalytic intracellular protease responsible for the degradation of proteins involved in cell cycle control, various signaling processes, antigen presentation, and control of protein synthesis. The central catalytic complex of the proteasome is called the 20S core particle. The majority of these are flanked on one or both sides by regulatory units. Most common among these units is the 19S regulatory unit. When coupled to the 19S unit, the complex is termed the asymmetric or symmetric 26S proteasome depending on whether one or both sides are coupled to the 19S unit, respectively. The 26S proteasome recognizes poly-ubiquitinylated substrates targeted for proteolysis. Targeted proteins interact with the 19S unit where they are deubiquitinylated, unfolded, and translocated to the 20S catalytic chamber for degradation. The 26S proteasome is responsible for the degradation of major proteins involved in the regulation of the cellular cycle, antigen presentation and control of protein synthesis. Alternatively, the proteasome is also active when dissociated from regulatory units. This free pool of 20S proteasome is described in yeast to mammalian cells. The free 20S proteasome degrades proteins by a process independent of poly-ubiquitinylation and ATP consumption. Oxidatively modified proteins and other substrates are degraded in this manner. The 20S proteasome comprises two central heptamers (β-rings) where the catalytic sites are located and two external heptamers (α-rings) that are responsible for proteasomal gating. Because the 20S proteasome lacks regulatory units, it is unclear what mechanisms regulate the gating of α-rings between open and closed forms. In the present review, we discuss 20S proteasomal gating modulation through a redox mechanism, namely, S-glutathionylation of cysteine residues located in the α-rings, and the consequence of this post-translational modification on 20S proteasomal function.FAPESP (Fundação de Amparo a Pesquisa do Estado de São Paulo)Instituto Nacional de Ciência, Tecnologia e Inovação de Processos Redox em BioMedicinaConselho Nacional de Ciência e Tecnologia (CNPq)CAPE

Applications of Site-Specific Labeling to Study HAMLET, a Tumoricidal Complex of α-Lactalbumin and Oleic Acid

umor cells), and its tumoricidal activity has been well established.-acetylgalactosaminyltransferase II (ppGalNAc-T2) and further conjugated with aminooxy-derivatives of fluoroprobe or biotin molecules.We found that the molten globule form of hLA and αD-hLA proteins, with or without C-terminal extension, and with and without the conjugated fluoroprobe or biotin molecule, readily form a complex with OA and exhibits tumoricidal activity similar to HAMLET made with full-length hLA protein. The confocal microscopy studies with fluoroprobe-labeled samples show that these proteins are internalized into the cells and found even in the nucleus only when they are complexed with OA. The HAMLET conjugated with a single biotin molecule will be a useful tool to identify the cellular components that are involved with it in the tumoricidal activity

Identification of Thioredoxin Glutathione Reductase Inhibitors That Kill Cestode and Trematode Parasites

Parasitic flatworms are responsible for serious infectious diseases that affect humans as well as livestock animals in vast regions of the world. Yet, the drug armamentarium available for treatment of these infections is limited: praziquantel is the single drug currently available for 200 million people infected with Schistosoma spp. and there is justified concern about emergence of drug resistance. Thioredoxin glutathione reductase (TGR) is an essential core enzyme for redox homeostasis in flatworm parasites. In this work, we searched for flatworm TGR inhibitors testing compounds belonging to various families known to inhibit thioredoxin reductase or TGR and also additional electrophilic compounds. Several furoxans and one thiadiazole potently inhibited TGRs from both classes of parasitic flatworms: cestoda (tapeworms) and trematoda (flukes), while several benzofuroxans and a quinoxaline moderately inhibited TGRs. Remarkably, five active compounds from diverse families possessed a phenylsulfonyl group, strongly suggesting that this moiety is a new pharmacophore. The most active inhibitors were further characterized and displayed slow and nearly irreversible binding to TGR. These compounds efficiently killed Echinococcus granulosus larval worms and Fasciola hepatica newly excysted juveniles in vitro at a 20 µM concentration. Our results support the concept that the redox metabolism of flatworm parasites is precarious and particularly susceptible to destabilization, show that furoxans can be used to target both flukes and tapeworms, and identified phenylsulfonyl as a new drug-hit moiety for both classes of flatworm parasites

Additive Contributions of Two Manganese-Cored Superoxide Dismutases (MnSODs) to Antioxidation, UV Tolerance and Virulence of Beauveria bassiana

The biocontrol potential of entomopathogenic fungi against arthropod pests depends on not only their virulence to target pests but tolerance to outdoor high temperature and solar UV irradiation. Two Beauveria bassiana superoxide dismutases (SODs), BbSod2 and BbSod3, were characterized as cytosolic and mitochondrial manganese-cored isoenzymes (MnSODs) dominating the total SOD activity of the fungal entomopathogen under normal growth conditions. To probe their effects on the biocontrol potential of B. bassiana, ΔBbSod2, ΔBbSod3, and three hairpin RNA-interfered (RNAi) mutants with the transcripts of both BbSod2 and BbSod3 being suppressed by 91–97% were constructed and assayed for various phenotypic parameters in conjunction with ΔBbSod2/BbSod2, ΔBbSod3/BbSod3 and wild-type (control strains). In normal cultures, the knockout and RNAi mutants showed significant phenotypic alterations, including delayed sporulation, reduced conidial yields, and impaired conidial quality, but little change in colony morphology. Their mycelia or conidia became much more sensitive to menadione or H2O2-induced oxidative stress but had little change in sensitivity to the hyperosmolarity of NaCl and the high temperature of 45°C. Accompanied with the decreased antioxidative capability, conidial tolerances to UV-A and UV-B irradiations were reduced by 16.8% and 45.4% for ΔBbSod2, 18.7% and 44.7% for ΔBbSod3, and ∼33.7% and ∼63.8% for the RNAi mutants, respectively. Their median lethal times (LT50s) against Myzus persicae apterae, which were topically inoculated under a standardized spray, were delayed by 18.8%, 14.5% and 37.1%, respectively. Remarkably, the effects of cytosolic BbSod2 and mitochondrial BbSod3 on the phenotypic parameters important for the fungal bioncontrol potential were additive, well in accordance with the decreased SOD activities and the increased superoxide levels in the knockout and RNAi mutants. Our findings highlight for the first time that the two MnSODs co-contribute to the biocontrol potential of B. bassiana by mediating cellular antioxidative response