Estudos epidemiológicos têm indicado que a vacinação com BCG pode induzir uma resposta imune protetora que está além daquela obtida contra tuberculose. Esta vacina está associada à indução de resposta imune Th1, aumento de anticorpos a antígenos não relacionados, menos atopia, menor anergia e redução da mortalidade infantil. No entanto, os mecanismos que sustentam estes efeitos não-específicos permanecem não esclarecidos. Nós investigamos os efeitos de diferentes preparações de BCG (BCG viável e inativados - EFD BCG e HK BCG) sobre o sistema NADPH oxidase, a expressão do TLR-2 e a modulação da produção de citocinas (IL-12p40, TNF-a, IFN-g e IL-10) por leucócitos humanos in vitro. Os resultados demonstram que diferentes preparações de BCG induzem diferentes respostas, tanto em relação à atividade do sistema NADPH oxidase como à modulação da produção de citocinas por leucócitos humanos in vitro. Além disso, demonstramos que em condições inflamatórias como a asma, a modulação da produção de citocinas pelos BCG é diferente daquela observada em sujeitos saudáveis. / Epidemiological studies have indicated that the BCG vaccination may induce a protective immune response that can go beyond what is obtained against tuberculosis. This vaccine is associated to the induction of Th1 immune response, increase antibodies response to unrelated antigen, less atopy, less anergy and a reduction in childhood mortality. Nevertheless, the mechanisms that sustain these non-specific effects remain unclear. We investigated the effects of different BCG preparations (viable BCG and two inactivated BCG EFD BCG and HK BCG) over the NADPH oxidase, the TLR-2 gene expression and over the modulation of the cytokine production (IL-12p40, TNF-a, IFN-g and IL-10) by human leukocytes in vitro. The results demonstrated that different BCG preparations induce different responses, as in respect to the activity of the NADPH oxidase system as in relation to the modulation of cytokine production by human leukocytes in vitro. Beyond this we demonstrated that in inflammatory conditions as asthma, the modulation of cytokine production by the BCG is different of that observed in healthy individuals.

Linfomas são as neoplasias hematopoiéticas mais comumente relatadas em cães, podendo ocorrer em qualquer idade. A etiologia ainda é desconhecida, porém se aceita que seja multifatorial. São classificadas de acordo com sua localização anatômica, estadio clínico da Organização Mundial de Saúde (OMS), critérios cito-histológicos e imunofenotípicos. A anemia é a anormalidade hematológica mais observada em pacientes com câncer e manifesta-se em cerca dois terços dos cães com linfoma. Os radicais livres de oxigênio produzidos pelas células tumorais aumentam o estresse oxidativo, sendo este demonstrado por mudanças na atividade das enzimas antioxidantes, podendo ocorrer oxidação de proteínas de DNA, aumento da peroxidação de lipídeos e alterações da fragilidade osmótica eritrocitária, acarretando a diminuição da meia vida das hemácias e, sendo essa diminuição um dos fatores que pode levar à anemia nos casos de linfoma. A superoxido dismutase (SOD) é uma enzima antioxidante que catalisa a dismutação do radical superóxido em oxigênio e peróxido de hidrogênio, protegendo as células dos danos induzidos por estes radicais livres. O objetivo deste estudo foi determinar as concentrações eritrocitárias de superóxido dismutase, além de substâncias reativas ao ácido tiobarbitúrico (MDA), o estado antioxidante total (TAS) e a fragilidade osmótica eritrocitária, em cães hígidos e com linfoma multicêntrico com e sem anemia, para avaliar a influência desses mecanismos no desenvolvimento das anemias associadas a essa neoplasia. Amostras de sangue foram obtidas de 24 cães com linfoma multicêntrico, sendo 10 sem anemia e 14 com anemia, e 20 cães saudáveis. Foi observada diferença significante no estado antioxidante total entre os grupos controle, grupos experimentais com linfoma multicêntrico com anemia e sem anemia (p < 0,0001). Não houve diferença significante quando os cães com linfoma com anemia e sem anemia foram comparados, assim como na avaliação das concentrações plasmáticas do MDA entre o grupo controle, grupo experimental com linfoma multicêntrico com anemia e grupo experimental com linfoma multicêntrico sem anemia (p = 0,823). A diferença também não foi significante quando se comparou o grupo controle e o grupo dos animais doentes (p=0,671). A diferença não foi significante na avaliação das concentrações eritrocitárias de superoxido dismutase entre o grupo controle, grupo experimental com linfoma multicêntrico com anemia e grupo com linfoma multicêntrico sem anemia (p=0,0748.). Entretanto, foi observada diferença significante entre o grupo controle e o grupo experimental com linfoma multicêntrico (p=0,0168). Não foi observada diferença na fragilidade osmótica eritrocitária dos cães com linfoma multicêntrico com anemia e sem anemia, as concentrações de NaCl em que observou-se 50% de hemólise estavam dentro dos valores postulados por Jain, 1963. Os resultados obtidos indicam um aumento do estresse oxidativo em cães com linfoma e que a anemia observada nos pacientes com linfoma não esta relacionada diretamente a diminuição das defesas antioxidantes eritrocitárias ou à alterações nas propriedades de membrana secundarias a peroxidação lipídica, que poderiam interferir na fragilidade do eritrócito / Lymphoma is hematopoietic malignancies most commonly reported in dogs can occur at any age. The etiology is still unknown, but is accepted to be multifactorial. They are classified according to their anatomic location, clinical stage of the World Health Organization (WHO), cytological and histological criteria and immunophenotype. Anemia is the most commonly observed hematologic abnormalities in patients with cancer and manifests itself in about two thirds of dogs with lymphoma. The oxygen free radicals produced by tumor cells increases oxidative stress, which is demonstrated by changes in antioxidant enzyme activities, protein and DNA oxidation, increased lipid peroxidation and alterations of erythrocyte osmotic fragility, leading to a decrease in half life of red blood cells and this decrease is one factor that can lead to anemia in cases of lymphoma. The superoxide dismutase (SOD) is an antioxidant enzyme that catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide, protecting cells from damage induced by these free radicals. The aim of this study was to determine erythrocyte superoxide dismutase, and thiobarbituric acid reactive substances (MDA), total antioxidant status (TAS) and erythrocyte osmotic fragility, and in healthy dogs with multicentric lymphoma with and without anemia, to evaluate the influence of these mechanisms in the development of anemia associated with this neoplasia. Blood samples were obtained from 24 dogs with multicentric lymphoma, 10 and 14 with anemia without anemia and 20 healthy dogs. Significant difference was observed in total antioxidant status between the control group, experimental groups with multicentric lymphoma with anemia and without anemia (p <0.0001). There was no significant difference when dogs with lymphoma with anemia and without anemia were compared no significant difference in assessment of plasma concentrations of MDA between the control group, experimental group with multicentric lymphoma with anemia and experimental group with multicentric lymphoma without anemia (p=0.823). The difference was not significant when comparing the control group and the group of sick animals (p = 0.671). The difference was not significant in the evaluation of erythrocyte superoxide dismutase concentrations between the control group, experimental group with multicentric lymphoma with anemia and patients with multicentric lymphoma without anemia (p = 0.0748).. However, significant difference was observed between the control and experimental group with multicentric lymphoma (p = 0.0168). No difference was observed in erythrocyte osmotic fragility of dogs with multicentric lymphoma with anemia and without anemia, the concentrations of NaCl in which there was 50% hemolysis were within the range postulated by Jain, 1963. The results indicate an increased oxidative stress in dogs with lymphoma and that the anemia observed in patients with lymphoma is not directly related to the decrease in erythrocyte antioxidant defenses or the changes in properties of secondary membrane lipid peroxidation, which could interfere with the fragility of erythrocyte

Iron/Manganese superoxide dismutases (Fe/Mn-SODs) are metalloenzymes with highly conserved protein folds, active sites, and dimer interfaces. They protect cells against oxidative stress by catalyzing the conversion of the cytotoxic free radical superoxide to molecular oxygen and hydrogen peroxide. The majority are highly specific for the type of metal (iron or manganese) present within the active site. However, there are many key aspects of metal specificity and catalytic activity that lack a structural explanation. Computational analyses suggested that several residues are important for fine-tuning the redox potential of the metal in the active site and thereby the catalytic activity. The main objective of this thesis is to evaluate the influence of several point mutations (M27V, G73A, H75I, L80F, D150G and Q172D) and one double mutation (Q149G+G74Q)) in terms of metal specificity, catalytic activity and three-dimensional structure using the superoxide dismutase from Trichoderma reesei (TrSOD) as a model system. The corresponding genes were cloned, expressed and the resulting proteins characterized by X-ray crystallography, electron paramagnetic resonance (EPR), atomic absorption spectroscopy (AAS), dynamic light scattering (DLS) and their enzymatic activity determined. The native protein was shown to be able to use either Mn or Fe (5000 units/mg and 500 units/mg, respectively) for catalysis suggesting it to be properly classified as cambialistic. Structures for native TrSOD and the Mn-G73A, Fe-H75I, Mn-L80F, Fe-D150G and Fe-M27V, Mn-M27V mutants were solved at 2.3 &Aring;, 2.0 &Aring;, 2.03 &Aring;, 2.0 &Aring;, 1.85 &Aring;, 1.4 &Aring; and 1.6 &Aring; resolution, respectively. The H75I, L80F and M27V mutations are easily accommodated by small local structural changes to the three-dimensional structure. On the other hand, the G73A mutation destabilize one of the dimer-dimer interfaces of the tetramer making it possible for two distorted tetramers to interact forming an octamer. This enzyme also lost all catalytic activity probably due to resulting exposure of the active site consistent with the observation of a sixth ligand (solvent molecule) bound to the metal in one subunit. The D150G mutant remained tetrameric but with reduced symmetry related to the rearrangement of the last helix (H9). Our results show that a large impact on activity and oligomerization of TrSOD can be generated by a single amino acids substitution in some cases and provide some insights into our understanding of the structural details associated with the metal ion specificity and oligomerization in superoxide dismutases. / Superóxido dismutases de ferro e manganês (Fe/Mn-SODs) são metaloenzimas com enovelamentos, sítios ativos e interfaces diméricas altamente conservados. Estas enzimas protegem as células contra o estresse oxidativo pela conversão do ânion superóxido em oxigênio molecular e peróxido de hidrogênio. A maioria são altamente específicas pelo tipo de metal (ferro ou manganês) presente no sítio ativo. Entretanto, existem vários aspectos críticos sobre a especificidade pelo metal e da atividade catalítica que ainda não foram explicados em termos estruturais. Análises computacionais sugerem que vários resíduos são importantes para o ajuste do potencial redox do metal no sitio ativo e, portanto, a atividade catalítica. O objetivo principal deste trabalho é avaliar a influência de mutações simples (TrSOD) (M27V, G73A, H75I, L80F, D150G e Q172D) e dupla (Q149G + G74Q) em superóxido dismutases de Trichoderma reesei em termos de especificidade pelo metal, atividade catalítica e estrutura. Os genes correspondentes foram clonados, expressos e as proteínas resultantes caracterizadas por cristalografia de raios-X, ressonância paramagnética electrónica (EPR), espectroscopia de absorção atómica (AAS), dispersão de luz dinâmica (DLS), e a atividade enzimática foi determinada. Foi mostrado que a proteína nativa é capaz de usar tanto Mn quanto Fe (5000units/mg e 500units/mg, respectivamente) para catálise sugerindo que deveria ser a classificada como enzima cambialistica. Estruturas da enzima nativa e mutantes (Mn-G73A, Fe-H75I, Mn-L80F, Fe-D150G, Fe-M27V e Mn-M27V) foram resolvidas a resoluções de 2.3 &Aring;, 2.0 &Aring;, 2.03 &Aring;, 2.0 &Aring;, 1.85 &Aring;, 1.4 &Aring; e 1.6 &Aring; respetivamente. As mutações H75I, L80F e M27V são acomodadas facilmente por reajustes locais na estrutura tridimensional. Por outro lado, a mutação G73A desestabiliza uma das interfaces dímero-dímero do tetrâmero levando à formação de um octâmero feito por dois tetrâmeros distorcidos. Esta enzima também perde atividade provavelmente devido a um aumento na acessibilidade do sítio ativo, coerente com a observação de um sexto ligante (molécula de solvente) coordenando o metal em uma das subunidades. O mutante D150G continuou tetramérica mas com simetria reduzida relacionado com o rearranjo da última hélice (H9). Estes resultados mostram que, em alguns casos, uma mutação simples pode ter um impacto significativo no estado oligomérico e atividade catalítica da proteína TrSOD e fornece conhecimentos para a nossa compreensão dos detalhes estruturais associados com a especificidade de íons metálicos e oligomerização em superóxido dismutases.

Linfomas são as neoplasias hematopoiéticas mais comumente relatadas em cães, podendo ocorrer em qualquer idade. A etiologia ainda é desconhecida, porém se aceita que seja multifatorial. São classificadas de acordo com sua localização anatômica, estadio clínico da Organização Mundial de Saúde (OMS), critérios cito-histológicos e imunofenotípicos. A anemia é a anormalidade hematológica mais observada em pacientes com câncer e manifesta-se em cerca dois terços dos cães com linfoma. Os radicais livres de oxigênio produzidos pelas células tumorais aumentam o estresse oxidativo, sendo este demonstrado por mudanças na atividade das enzimas antioxidantes, podendo ocorrer oxidação de proteínas de DNA, aumento da peroxidação de lipídeos e alterações da fragilidade osmótica eritrocitária, acarretando a diminuição da meia vida das hemácias e, sendo essa diminuição um dos fatores que pode levar à anemia nos casos de linfoma. A superoxido dismutase (SOD) é uma enzima antioxidante que catalisa a dismutação do radical superóxido em oxigênio e peróxido de hidrogênio, protegendo as células dos danos induzidos por estes radicais livres. O objetivo deste estudo foi determinar as concentrações eritrocitárias de superóxido dismutase, além de substâncias reativas ao ácido tiobarbitúrico (MDA), o estado antioxidante total (TAS) e a fragilidade osmótica eritrocitária, em cães hígidos e com linfoma multicêntrico com e sem anemia, para avaliar a influência desses mecanismos no desenvolvimento das anemias associadas a essa neoplasia. Amostras de sangue foram obtidas de 24 cães com linfoma multicêntrico, sendo 10 sem anemia e 14 com anemia, e 20 cães saudáveis. Foi observada diferença significante no estado antioxidante total entre os grupos controle, grupos experimentais com linfoma multicêntrico com anemia e sem anemia (p < 0,0001). Não houve diferença significante quando os cães com linfoma com anemia e sem anemia foram comparados, assim como na avaliação das concentrações plasmáticas do MDA entre o grupo controle, grupo experimental com linfoma multicêntrico com anemia e grupo experimental com linfoma multicêntrico sem anemia (p = 0,823). A diferença também não foi significante quando se comparou o grupo controle e o grupo dos animais doentes (p=0,671). A diferença não foi significante na avaliação das concentrações eritrocitárias de superoxido dismutase entre o grupo controle, grupo experimental com linfoma multicêntrico com anemia e grupo com linfoma multicêntrico sem anemia (p=0,0748.). Entretanto, foi observada diferença significante entre o grupo controle e o grupo experimental com linfoma multicêntrico (p=0,0168). Não foi observada diferença na fragilidade osmótica eritrocitária dos cães com linfoma multicêntrico com anemia e sem anemia, as concentrações de NaCl em que observou-se 50% de hemólise estavam dentro dos valores postulados por Jain, 1963. Os resultados obtidos indicam um aumento do estresse oxidativo em cães com linfoma e que a anemia observada nos pacientes com linfoma não esta relacionada diretamente a diminuição das defesas antioxidantes eritrocitárias ou à alterações nas propriedades de membrana secundarias a peroxidação lipídica, que poderiam interferir na fragilidade do eritrócito / Lymphoma is hematopoietic malignancies most commonly reported in dogs can occur at any age. The etiology is still unknown, but is accepted to be multifactorial. They are classified according to their anatomic location, clinical stage of the World Health Organization (WHO), cytological and histological criteria and immunophenotype. Anemia is the most commonly observed hematologic abnormalities in patients with cancer and manifests itself in about two thirds of dogs with lymphoma. The oxygen free radicals produced by tumor cells increases oxidative stress, which is demonstrated by changes in antioxidant enzyme activities, protein and DNA oxidation, increased lipid peroxidation and alterations of erythrocyte osmotic fragility, leading to a decrease in half life of red blood cells and this decrease is one factor that can lead to anemia in cases of lymphoma. The superoxide dismutase (SOD) is an antioxidant enzyme that catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide, protecting cells from damage induced by these free radicals. The aim of this study was to determine erythrocyte superoxide dismutase, and thiobarbituric acid reactive substances (MDA), total antioxidant status (TAS) and erythrocyte osmotic fragility, and in healthy dogs with multicentric lymphoma with and without anemia, to evaluate the influence of these mechanisms in the development of anemia associated with this neoplasia. Blood samples were obtained from 24 dogs with multicentric lymphoma, 10 and 14 with anemia without anemia and 20 healthy dogs. Significant difference was observed in total antioxidant status between the control group, experimental groups with multicentric lymphoma with anemia and without anemia (p <0.0001). There was no significant difference when dogs with lymphoma with anemia and without anemia were compared no significant difference in assessment of plasma concentrations of MDA between the control group, experimental group with multicentric lymphoma with anemia and experimental group with multicentric lymphoma without anemia (p=0.823). The difference was not significant when comparing the control group and the group of sick animals (p = 0.671). The difference was not significant in the evaluation of erythrocyte superoxide dismutase concentrations between the control group, experimental group with multicentric lymphoma with anemia and patients with multicentric lymphoma without anemia (p = 0.0748).. However, significant difference was observed between the control and experimental group with multicentric lymphoma (p = 0.0168). No difference was observed in erythrocyte osmotic fragility of dogs with multicentric lymphoma with anemia and without anemia, the concentrations of NaCl in which there was 50% hemolysis were within the range postulated by Jain, 1963. The results indicate an increased oxidative stress in dogs with lymphoma and that the anemia observed in patients with lymphoma is not directly related to the decrease in erythrocyte antioxidant defenses or the changes in properties of secondary membrane lipid peroxidation, which could interfere with the fragility of erythrocyte

A atividade de catalase em Candida albicans tem sido sugerida como um mecanismo de resistência ao antifúngico anfotericina B. Neste contexto, poucos são os estudos de enzimas como catalase e superóxido dismutase em leveduras do gênero Candida expostas a diferentes situações. Assim, este estudo teve por objetivo investigar o efeito da exposição de Candida a antineoplásicos, íons metálicos e antifúngicos como fluconazol e anfotericina B sobre a atividade dessas enzimas. Os resultados apontaram que o antineoplásico metotrexato aumentou a atividade da catalase em C. albicans, que íons metálicos como cobre, zinco, manganês e ferro produzem um efeito variável na atividade de superóxido dismutase, bem como, um efeito variável de um íon no acúmulo de outro. Também verificamos através de nossos resultados que a indução de resistência ao fluconazol e anfotericina B aumentam a atividade de catalase e superóxido dismutase em C. albicans e C. dubliniensis. Esses resultados sugerem que o antineoplásico metotrexato e a indução de resistência a anfotericina B e fluconazol podem gerar um estresse oxidativo em leveduras do gênero Candida que possivelmente se adaptam a esse estresse aumentando seus mecanismos de defesa antioxidante. Esse efeito pode induzir a uma maior resistência desses organismos ao ataque de células fagocíticas do hospedeiro. / Catalase is an enzyme that has been suggested to be involved in resistance mechanisms to antifungal drug such as amphotericin B. There are few studies focusing on catalase and superoxide dismutase in yeasts, such as Candida, exposed to different situations. Thereby, the aim of the present study was to investigate the effect of exposing Candida to antineoplastic drugs, metallic ions and antifungial drugs, namely fluconazole and amphotericin B, on catalase and superoxide dismutase activities. Results show that methotrexate induced catalase activity in C. albicans and that metallic ions, such as copper, zinc, manganese and iron produced a variable effect on superoxide dismutase activity of C. albicans, as well as a variable effect in the uptake of one ion on another. We also showed that fluconazole and amphotericin B resistance increased catalase and superoxide dismutase activity in C. albicans and C. dubliniensis. These results suggest that methotrexate as well as the induction of fluconazole- and amphotericin B-resistance may induce oxidative stress in yeasts such as Candida, which may adapt by increasing antioxidant defense mechanisms. This effect may induce a major resistance of this yeast to phagocytic cell attack.

O objetivo desse trabalho foi avaliar o estado nutricional de mulheres obesas em relação ao zinco (Zn) e a associação com marcadores do estresse oxidativo e com polimorfismos em genes das enzimas antioxidantes SOD1 e SOD3. A amostra foi composta por 60 mulheres obesas (OB) e por um grupo controle composto por 55 mulheres eutróficas (CON). Amostras de sangue e urina de 24 horas foram coletadas para análise de zinco, malondialdeído (MDA), 8-isoprostano urinário, atividade antioxidante das enzimas superóxido dismutase (SOD) e glutationa peroxidase (GPx). Foram avaliados dois polimorfismos de nucleotídeo único (SNP) presentes nas enzimas SOD1 e SOD3, o +35A/C e Arg213Gli. Foram aplicados três recordatórios alimentares de 24 horas, incluindo um dia de final de semana. As comparações entre os grupos foram feitas pelos testes qui-quadrado, t-Student, ANOVA e Mann-Whitney. As associações entre as variáveis quantitativas foram realizadas por meio do coeficiente de correlação r de Pearson. Modelo de regressão linear multivariada backward foi feito a fim de se analisar as regressões das variáveis desfecho zinco plasmático, MDA e 8-isoprostano. O equilíbrio de Hardy-Weinberg foi calculado pelo teste do qui-quadrado. A média da ingestão de zinco foi de 6,8 mg/dia e 7,4 mg/dia no grupo CON e OB, respectivamente. As concentrações de Zn plasmático e eritrocitário e a atividade das enzimas SOD e GPx não apresentaram diferença entre os grupos. Ambos os grupos apresentaram deficiência no Zn plasmático. As concentrações de MDA e 8-isoprostano foram maiores no grupo CON, enquanto as concentrações de creatinina e Zn urinários foram maiores no grupo OB. No grupo CON, foram verificadas associações negativas entre Zn plasmático e MDA (p=0,002) e glicemia (p<0,0001). A regressão multivariada mostrou correlação negativa entre o Zn plasmático e o MDA. O 8-isoprostano sofreu influência negativa da creatinina urinária e positiva da atividade física. Já a glicemia e o consumo de energia e de lipídio apresentaram correlação positiva com o MDA. Na análise dos SNPs, foram encontradas apenas duas participantes heterozigotas para o SNP +35A/C e nenhuma participante com o alelo variante para o SNP Arg213Gli. Concluiu-se que os indivíduos obesos e eutróficos apresentaram deficiência de Zn, o que pode estar relacionado ao estresse oxidativo. O estresse oxidativo foi mais intenso para o grupo controle e não foi observada associação entre o IMC e os marcadores MDA e 8-isoprostano. Não foi possível fazer correlação dos polimorfismos com os parâmetros avaliados / The aim of this study was to assess the nutritional zinc status and its association with oxidative stress markers and with polymorphisms in genes of SOD1 and SOD3 in obese women. The sample consisted of 60 obese women (OB) and a control group of 55 normal-weight women (CON). Blood samples and 24-hour urine were collected for analysis of zinc, malondialdehyde (MDA), urinary 8-isoprostane, antioxidant activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx). Two single nucleotide polymorphisms (SNP) were evaluated in the genes of SOD1 and SOD3 enzymes, +35A/C and Arg213Gly. Three food 24-hour recalls, including one weekend day were applied. Comparisons between groups were made by chi-square test, Student\'s t - test, ANOVA and Mann-Whitney. Associations between means of quantitative variables were performed by the correlation coefficient r of Pearson. Backward multivariate linear regression was done in order to analyze the regressions of outcome variables plasma zinc, MDA and 8-isoprostane. The Hardy-Weinberg equilibrium was calculated by chi-square. The mean zinc intake was 6.8 mg/day and 7.4 mg/day in CON and OB groups, respectively. Concentrations of plasma and erythrocyte Zn and activity of SOD and GPx enzymes showed no difference between groups. Both groups showed a deficiency in plasma Zn. The concentrations of MDA and 8-isoprostane were higher in the CON group, whereas the concentrations of creatinine and urinary Zn were higher in the OB group. In the CON group, negative associations between plasma Zn and MDA (p = 0.002) and glucose (p < 0.0001) were observed. Multivariate regression analysis showed a negative correlation between plasma Zn and MDA. The 8-isoprostane suffered negative influence of urinary creatinine and positive influence of physical activity. Blood sugar and energy and lipid consumption showed a positive correlation with MDA. In the analysis of SNPs, were found only two heterozygous participants for the SNP +35A/C and no participant with the variant allele for the SNP Arg213Gly. It was concluded that obese and normal subjects showed Zn deficiency, which may be related to oxidative stress. Oxidative stress was higher in the control group and no association was observed between BMI and MDA and 8-isoprostane markers. It was not possible to correlate polymorphisms with evaluated parameters.

A scientific and cultural revolution occurred with the sequencing of the human genome. The information provided by this accomplishment has provided tools for researchers to test new ideas in silico and on the bench. In redox biology many of the genes, transcripts, proteins, and redox active species have been well characterized. However, the vast majority have not been quantitated in an absolute manner. This is a necessary step to provide the tools for mathematical modeling and systems biology approaches for predicting changes in the cellular redox environment and the biochemical and biological consequences. Here we demonstrate techniques for the absolute quantitation of human catalase, glutathione peroxidase, peroxiredoxin, thioredoxin, and superoxide dismutase within cells. These techniques can be parsed into two groups: detection of activity and detection of total amount of species. Methods for the absolute quantitation of active catalase, peroxiredoxins, and superoxide dismutase have been developed by utilizing specific characteristics of each enzyme. Catalase generates oxygen in the presence of hydrogen peroxide that can easily be detected with a Clark electrode (oxygen monitor); the data are fit to a single-exponential to determine the observed pseudo-first-order rate constant. From this the effective number of fully active catalase enzymes in the sample can be determined. Peroxiredoxin in the disulfide state can be reduced by thioredoxin; thioredoxin from E. coli loses fluorescence upon oxidation. The loss of fluorescence over time is mathematically fit to a single-exponential to determine the observed pseudo first-order rate constant from which the number of active enzymes can be determined. Using an inhibition assay to detect superoxide dismutase activity along with the rate constants at which superoxide reacts with the dismutase and the competing superoxide-reacting-indicator-molecule, the concentration of active superoxide dismutase can be determined. To detect the total amount of protein of an enzyme in a biological sample, an immunoassay was first implemented. This method utilized Bio-Plex® beads from Bio-Rad; however, it was problematic because the antibodies applied did not perform satisfactorily not allowing sufficient signal-to-noise to be deployed. Quantitative mass spectrometry was then implemented to detect total catalase, glutathione peroxidase 1, peroxiredoxin 2, and thioredoxin 1 in human red blood cells. With the absolute concentration of these enzymes and proteins along with data for oxygen consumption rates and peroxisomal hydrogen peroxide concentration for several cell lines, we hypothesize that a reasonable model of hydrogen peroxide and superoxide flux can be constructed. Quantitative data such as these provide the foundation for the new redox biology of the 21st century. Presented here is a roadmap for the obligatory first steps to dissect quantitatively the cellular and tissue metabolic pathways and redox networks that are the basis of all of biology.

Macropinocytosis and phagocytosis, two actin-dependent and clathrin independent events of endocytosis, enable the cells such as macrophages and neutrophils to either internalize pathogens and initiates the human innate immune response or serve as a direct entry route for productive infection of pathogen. Dictyostelium discoideum, soil-living amoeba, a unicellular eukaryote that could professionally internalize fluid phase or particles several folds more than that of macrophages and neutrophils. Additionally, multiple key signaling pathways are conserved between Dictyostelium and mammalian cells, including pathways affecting small GTPases Ras and Rac and their downstream effectors, and F-Actin remodeling. All these traits makes Dictyostelium an excellent model organism to study the process pf macropinocytosis and phagocytosis. Upon internalization of the prey, these macropinocytes and phagocytes are often in an environment of increased production of superoxide radicals in the prey-containing vesicles, which helps stimulates the downstream signaling pathways to digest the prey inside. However, the mechanism of how superoxide regulates the process of macropinocytosis and phagocytosis is not fully understood. We had previously reported that Dictyostelium cells lacking Superoxide dismutase C (SodC) exhibited aberrantly high level of active RasG, high basal level of Phosphatidylinositol-3,4,5-triphosphate (PIP3), and severe chemotaxis defects. Now we report that sodC- cells displayed aberrant endosomal vesicle trafficking, significantly compromised particle uptake and defective cell to substratum matrix adhesion compared to that of wild type cells. By using high resolution live imaging microscope we also show that sodC- cells have defects in F-Actin remodeling at the phagocytic rim extension and F-Actin depolymerization of the nascent phagosome. Interestingly, the introduction of overexpressing of cytoplasmic superoxide dismutase (SodA), redox insensitive RasG (C118A) or treatment of PI3K inhibitor LY294002 in sodC- cells significantly rescued the defects of endosomal vesicle trafficking, particle uptake and adhesion. This project suggests that superoxide dismutase C regulates the endosomal vesicle trafficking, phagocytosis and cell to substratum matrix adhesion through the RasG/PI3K signaling axis in Dictyostelium cells.

Eukaryotic gene expression is a complex process and can be controlled at the level of transcription, post-transcription or translation and post-translation. In recent years, there is a growing interest in understanding the role of 3'-untranslated region (UTR) in regulating mRNA turnover and translation. The 3'-UTR harbors the poly(A) signal and post-transcriptional regulatory sequences like miRNA and AU-rich elements (AREs). The presence of multiple poly(A) sites often results in multiple transcripts; shorter transcripts correlating with more protein abundance. Manganese superoxide dismutase (MnSOD) is a nuclear encoded and mitochondrial matrix localized antioxidant enzyme that converts mitochondrial generated superoxide to hydrogen peroxide. Human MnSOD has two poly(A) sites resulting in two transcripts: 1.5 and 4.2 kb. We hypothesize that the 3'-UTR of MnSOD regulates its mRNA and protein levels as well as activity in response to growth states and environmental stress. Results from a Q-RT-PCR assay showed a preferential accumulation of the shorter MnSOD transcript during quiescence, which correlated with an increase in MnSOD activity. The accumulation of the longer MnSOD transcript during proliferation was associated with a decrease in MnSOD activity. Log transformed expression ratio of the longer to shorter transcript was also higher in proliferating epithelial non-cancerous (mammary: MCF-10A) and cancer cells (mammary: MB-231, SUM 159; oral squamous: SQ20B, FaDu, Cal27; and lung: A549, H292), suggesting that the abundance of the longer transcript is independent of cellular transformation status, instead it is dependent on cellular growth state. Interestingly, the abundance of the longer transcript directly correlated with percent S-phase (R2=0.86). The shorter transcript was enriched in irradiated MB-231 cells. MCF-10A cells exposed to 2-(4-chlorophenyl)benzo-1,4-quinone (4-Cl-BQ), a metabolite of the environmental pollutant polychlorinated biphenyl 3, showed a significant decrease in the abundance of the 4.2 kb transcript due to a faster mRNA turnover, 14 h compared to 20 h in untreated control cells. The decrease in the 4.2 kb transcript levels was associated with a corresponding decrease in MnSOD protein levels and activity, which resulted in a significant inhibition of quiescent cells entry into the proliferative cycle. Deletion and reporter assays showed: (a) a significant decrease in reporter activity in constructs carrying multiple AREs that are present in the 3'-UTR of the longer MnSOD transcript; (b) irradiation increased the reporter activity of the constructs carrying the 3'-UTR sequence of the shorter MnSOD transcript and (c) N-acetyl-cysteine increased the reporter activity of constructs carrying multiple AREs. Because the longer transcript carries AREs, our results identified redox sensitive AREs as novel regulators of MnSOD transcript levels. We conclude that MnSOD 3'-UTR is a novel molecular sensor regulating MnSOD mRNA levels in response to different growth states and environmental stress. A better understanding of the 3'-UTR regulating gene expression could lead to the development of new molecular biology-based redox therapy designed to treat proliferative disorders.

Multiple myeloma (MM) is a prevalent B-cell neoplasm that remains incurable with currently available chemotherapeutic drugs. Existing drug regimens result in initial disease remission but MM often relapses with an aggressive, drug resistant phenotype with uniform mortality. Bortezomib (BTZ, proteasome inhibitor) is a frontline anti-MM drug that is used for treatment of newly diagnosed and relapsed MM. However both intrinsic and acquired BTZ resistance is observed. Hence, gaining a mechanistic understanding of BTZ-resistance can provide novel targets to increase and restore BTZ cytotoxicity in MM. Studies show that BTZ-mediated proteasome inhibition generates oxidative stress therefore, BTZ resistance can be caused by an increase in cellular antioxidant capacity of MM cells. Antioxidants like superoxide dismutases (SODs), glutathione (GSH), and glutathione peroxidases (GPxs) can maintain cellular redox homeostasis and confer resistance to oxidative stress. Additionally, an increased glucose metabolism can assist in maintaining low reactive oxygen species (ROS) levels formed as by-products of endogenous or therapy induced oxidative stress. This led us to test the hypothesis that BTZ resistance in MM is linked to redox regulation via the antioxidant network and generation of reducing equivalents. Retrospective analysis of clinically annotated MM dataset shows a correlation between SOD1 gene expression, MM progression, and poor overall and event free survival. In a MM cell line model with intrinsic or acquired BTZ resistance, our results show a correlation between half maximal inhibitory concentration (IC50) of BTZ and CuZnSOD activity. Upon inhibition of CuZnSOD activity with a clinically approved drug, disulfiram (DSF, Antabuse), BTZ cytotoxicity was increased. Furthermore, enforced overexpression of CuZnSOD conferred BTZ resistance in an otherwise BTZ sensitive MM cell line. MM cell lines with differential intrinsic BTZ cytotoxicity displayed a correlation between BTZ IC50 and GSH levels as well as GPx-1 activity. Gene expression profiling data from patients showed that poor prognosis associates with increased glycolytic gene expression in MM. Also, MM cell lines with intrinsic resistance toward BTZ exhibited increased glucose uptake, increased mRNA expression and activity of glucose-6-phosphate dehydrogenase (G6PD) with increased cytotoxicity with glucose deprivation or 2-deoxyglucose (2-DG) treatment. In conclusion, our results provide a rationale for utilizing redox-based combination protocols of clinically approved drugs (i.e. DSF and 2-DG) with BTZ to improve MM therapy responses.