This report details the activities and inhibition of metal-substituted human carbonic anhydrase II (M-HCA-II). The traditional activities (hydrolysis of CO2 and para-nitrophenol acetate) in addition to new activities (oxidation of 2-aminophenol, disproportionation of H2O2, and disproportionation of superoxide) were investigated. Values reported for the relative hydrolytic activities of M-HCA-IIs are reported here for the first time, ranging from 47.5 % (plus or minus 0.6) to 86 % (plus or minus 4) for the hydrolysis of CO2 and from 0.299 % (plus or minus 0.012) to 4.72 % (plus or minus 0.015) for the hydrolysis of para-nitrophenol acetate. With respect to new activities, only the oxidation of 2-aminophenol was observed. Turnover was observed for Fe-HCA-II (kcat/KM = 3.6 plus or minus 1.3 mM-1 s-1) and Cu-HCA-II (kcat/KM = 8 plus or minus 2 mM-1 s-1). Inhibition of Zn-, (di-substituted) Cu2-, and Cu/Zn-HCA-II hydrolysis of CO2 and para-nitrophenol acetate by sulfanilamide, coumarin, and ortho-coumaric acid were investigated. Sulfanilamide was shown to inhibit: Zn-HCA-II, Cu2-HCA-II, and Cu/Zn-HCA-II - (with CO2) KM = 8.9 plus or minus 1.1 microM, 11 plus or minus 2 microM, 8.8 plus or minus 1.4 microM and (with p-nitrophenyl acetate) KM = 8.4 plus or minus 1.0 microM, (none), 8.4 plus or minus 1.4 microM, respectively. No inhibition was observed for coumarin or ortho-coumaric acid or its derivatives for any CAs studied.

No description available.

Amyotrophic Lateral Sclerosis (ALS) is an always lethal motor neuron disease with unknown pathogenesis. Inhibitors of the molecular chaperone heat shock protein 90 (Hsp90) have limited neuroprotection in some models of motor neuron degeneration. However the direct effect of Hsp90 inhibition on motor neurons is unknown. Here we show that Hsp90 inhibition induced motor neuron death through activation of the P2X7 receptor. Motor neuron death required phosphatase and tensein homolog (PTEN)-mediated inhibition of the PI3K/AKT pathway leading to Fas receptor activation and caspase dependent death. The relevance of Hsp90 for motor neuron survival was investigated in mutant Cu/Zn superoxide dismutase (SOD) transgenic animal models for ALS. Nitrated Hsp90, a posttranslational modification known to induce cell death (Franco, Ye et al. 2013), was present in motor neurons after intracellular release of zinc deficient (Zn, D83S) and the SOD in which copper binding site was genetically ablated (Q) but not after copper deficient (Cu) wild type SOD. Zn deficient and Q mutant SOD induced motor neuron death in a peroxynitrite mediated and copper dependent mechanism. Nitrated Hsp90 was not detected in the spinal cord of transgenic animals for ALS-mutant SOD animal models until disease onset. Increased nitrated Hsp90 concentrations correlated with disease progression. Addition of Zn or Q SOD to nontransgenic brain homogenate treated with peroxynitrite led to an increase level of nitrotyrosine in comparison to wild type controls. However, in the same samples there was a 2 to 10 time increase in Hsp90 nitration as compared to nitrotyrosine. The selective increase is likely due to the binding of Hsp90 to Zn deficient and Q SOD as oppose to wild type SOD. These results suggest that Hsp90 nitration facilitated by mutant SOD may cause motor neuron degeneration in ALS. Targeted inhibition of nitrated Hsp90 may be a novel therapeutic approach for ALS. An alternative therapeutic strategy is to target the production of survival factors by glial cells. Riluzole is the only FDA approved drug for the treatment of ALS and it shows a small but significant increase in patient lifespan. Our results show that acute riluzole treatment stimulated trophic factor production by astrocytes and Schwann cells. However long-term exposure reversed and even inhibited the production of trophic factors, an observation that may explain the modest increase in patient survival in clinical trials. Discontinuous riluzole treatment can maintain elevated trophic factor levels and prevent trophic factor reduction in spinal cords of nontransgenic animals. These results suggest that discontinuous riluzole administration may improve ALS patient survival. In summary, we demonstrated that Hsp90 has an essential function in the regulation of motor neuron survival. We have also shown that Hsp90 was nitrated in the presence of mutant SOD and was present during symptom onset and increases as disease progresses, which may explain the toxic gain of function of mutant SOD. Finally we demonstrate a biphasic effect of riluzole on trophic factor production and propose changes in administration to improve effects in ALS patients.

(1) Background: Canine distemper virus (CDV)-induced demyelinating leukoencephalitis (CDV-DL) in dogs and Theiler’s murine encephalomyelitis (TME) virus (TMEV)-induced demyelinating leukomyelitis (TMEV-DL) are virus-induced demyelinating conditions mimicking Multiple Sclerosis (MS). Reactive oxygen species (ROS) can induce the degradation of lipids and nucleic acids to characteristic metabolites such as oxidized lipids, malondialdehyde, and 8-hydroxyguanosine. The hypothesis of this study is that ROS are key eector molecules in the pathogenesis of myelin membrane breakdown in CDV-DL and TMEV-DL. (2) Methods: ROS metabolites and antioxidative enzymes were assessed using immunofluorescence in cerebellar lesions of naturally CDV-infected dogs and spinal cord tissue of TMEV-infected mice. The transcription of selected genes involved in ROS generation and detoxification was analyzed using gene-expression microarrays in CDV-DL and TMEV-DL. (3) Results: Immunofluorescence revealed increased amounts of oxidized lipids, malondialdehyde, and 8-hydroxyguanosine in CDV-DL while TMEV-infected mice did not reveal marked changes. In contrast, microarray-analysis showed an upregulated gene expression associated with ROS generation in both diseases. (4) Conclusion: In summary, the present study demonstrates a similar upregulation of gene-expression of ROS generation in CDV-DL and TMEV-DL. However, immunofluorescence revealed increased accumulation of ROS metabolites exclusively in CDV-DL. These results suggest dierences in the pathogenesis of demyelination in these two animal models.

The three insecticides of interest were lindane (an organochlorine), malathion (an organophosphate) and piperonyl butoxide (PBO; a synergist). Based on minimum cytotoxicity (> LC25), the following concentrations were chosen for the pesticide mixture studies: 70μM lindane (Lind), 50μM malathion (Mal) and 55μM PBO. In the AlamarBlue cytotoxicity assay, individual pesticide and mixtures of malathion/PBO (MP) and malathion/lindane (ML) prompted cytotoxicity with varying intensities (Mal 18.8%, Lind 20.4%, PBO 23.5%, ML 53.6% and MP 64.9%). Cytopathological analysis revealed apoptotic features in treated cells and the DNA Ladder Assay confirmed the presence of DNA fragments. The specific mode of cell death was examined via the 7-aminoactinomycin D (7-AAD) Staining Assay. Apoptosis was detected in each treatment (Mal 6.5%, Lind 12.0%, PBO 13.2%, ML 19.3% and MP 23.4%). Furthermore, 7-AAD staining in combination with fluorescent-labeled monoclonal antibodies, PE-CD45RB/220 and FITC-CD90, was performed. B-cells were more susceptible to Mal and PBO treatments than were T-cells. The pro-oxidant activity of the pesticides was monitored via the Dichlorofluorescin Diacetate assay. Exposure to pesticides for 15 minutes increased H2O2 production above the controls, Mal 21.1%; Lind 10.8%; PBO 25.9%; ML 26.8%; MP 37.8%. The activities of antioxidant enzymes, glutathione peroxidase (GSH-Px) and glutathione reductase (GR) were altered by these treatments. GR was significantly reduced for the pesticide mixtures only (control: 51.7; Mal: 48.2; Lind: 50; PBO: 52.3; ML: 40.5; MP: 42 Units/mg). GSH-Px activity was severely reduced for all the pesticide treatments (control: 44.9; Mal: 30.2; Lind: 30.6; PBO: 32.4; ML: 21.1; MP: 21.1 Units/mg). These results indicate that exposure to these pesticide and pesticide mixtures induces apoptosis and oxidative stress. / Master of Science

<p>Women with pregestational diabetes have a 2-5 fold increased risk of giving birth to malformed babies compared with non-diabetic women. Diabetes-induced oxidative stress in maternal and embryonic tissues has been implicated in the teratogenic process. The malformations are likely to be induced before the seventh week of pregnancy, when the yolk sac is partly responsible for the transfer of metabolites to the embryo, and the uterine blood flow to the implantation site determines the net amount of nutrients available to the conceptus. We aimed to evaluate the effect on embryogenesis caused by a diabetes-induced disturbance in yolk sac morphology, uterine blood flow or altered maternal antioxidative status in conjunction with a varied severity of the maternal diabetic state.</p><p>We investigated to which extent maternal diabetes with or without folic acid (FA) supplementation affects mRNA levels and protein distribution of ROS scavenging enzymes (SOD, CAT, GPX), vascular endothelial growth factor-A (Vegf-A), folate binding protein-1 (Folbp-1), and apoptosis associated proteins (Bax, Bcl-2, Caspase-3) in the yolk sacs of rat embryos on gestational days 10 and 11. We found that maternal diabetes impairs, and that FA supplementation restores, yolk sac vessel morphology, and that maternal diabetes is associated with increased apoptotic rate in embryos and yolk sacs, as well as impaired SOD gene expression. We assessed uterine blood flow with a laser-Doppler-flow-meter and found increased blood flow to implantation sites of diabetic rats compared with controls. Furthermore, resorbed and malformed offspring showed increased and decreased blood flow to their implantation sites, respectively. In mice with genetically altered CuZnSOD levels, maternal diabetes increased embryonic dysmorphogenesis irrespective of CuZnSOD expression. We thus found the maternal diabetic state to be a major determinant of diabetic embryopathy and that the CuZnSOD status exerts a partial protection for the embryo in diabetic pregnancy. </p>

Cerca de 20 % dos casos familiares de esclerose lateral amiotrófica (ELAf) são causados por mutações na enzima Cu,Zn-superóxido dismutase (SOD1). Inicialmente se supôs que as enzimas mutantes teriam a atividade SOD comprometida, entretanto isto não foi comprovado. Atualmente, considera-se que as enzimas mutantes adquiram propriedades tóxicas. Quais seriam estas propriedades e como levariam à degeneração do neurônio motor são questões ainda não respondidas. Neste trabalho, comparamos neuroblastomas humanos transfectados com SOD1 G93A associada à ELAf (SH-SY5YG93A), e SOD1 selvagem (SH-SY5YWT) com células parentais (SH-SY5Y) em relação ao crescimento, viabilidade, produção basal de oxidantes, atividades SOD e peroxidásica e modificações estruturais da SOD. As células transfectadas apresentaram aumento na taxa de crescimento e na produção basal de oxidantes. As células SH-SY5YWT e SH-SY5YG93A mantiveram a expressão de SOD1 e atividade consistente com o aumento esperado de duas vezes, em estágios iniciais de cultura. A atividade peroxidásica do homogenato da célula SH-SY5YG93A foi maior. Após quatro semanas, a linhagem SH-SY5YG93A manteve a expressão de SOD1, mas as atividades dismutásica e peroxidásica diminuíram. A expressão de SOD1 aumentou a proporção de formas alteradas de SOD1, como enzima reduzida, multímeros formados por ponte dissulfeto e formas insolúveis em detergente, particularmente na linhagem SH-SY5YG93A. Entre estas formas insolúveis, identificamos um dímero covalente de SOD. Estas formas alteradas provavelmente são responsáveis pela ativação do proteassomo e estresse do retículo endoplasmático, verificados nas células transfectadas. Concluindo, a superexpressão da SOD1 foi suficiente para elevar as formas imaturas e oligomerizadas de SOD1 e a oxidação basal, e a mutação G93A ressaltou estes processos. / Some familial ALS (fALS) are caused by mutations in the Cu,Zn-superoxide dismutase enzyme (SOD1). It was thought that the mutated enzymes would have impaired SOD activity, but this has not been corroborated so far. Presently, it is more accepted that the mutated enzymes acquire a new toxic function. What this new toxic function is and how it relates to the degeneration of motor neurons remains debatable. Here, we compared human neuroblastoma cells transfected with fALS mutant G93A (SH-SY5YG93A) or wild-type SOD1 (SH-SY5YWT) with parent cells (SH-SY5Y) in regard to growth, viability, basal oxidant production, SOD and peroxidase activities, and SOD forms. Transfected cells presented increased growth rate and basal oxidant production. SH-SY5YWT and SH-SY5YG93A cells in early culture stage showed SOD expression and activity consistent with the expected two-fold increase; SH-SY5YWT homogenates showed increased peroxidase activity. After four weeks, SH-SY5YG93A maintained SOD1 expression levels but peroxidase and dismutase activities were lower. SOD1 expression increased the levels of altered SOD1 forms such as the reduced enzyme, disulfide multimers and detergent-insoluble forms, particularly in SH-SY5YG93A cells. Among the insoluble forms a covalent SOD dimer was identified. These altered SOD forms are probably responsible for proteasome activation and endoplasmatic reticulum stress response verified in transfected cells. In conclusion, SOD1 over-expression was sufficient to increase intracellular immature and oligomerized SOD1 forms and basal oxidation and the G93A mutation enhanced these processes.

A redução univalente de oxigênio molecular nas células produz intermediários altamente reativos tais como o íon radical Superóxido (O-&#8226;2), o radical hidroxil (HO&#8226;) e o peróxido de hidrogênio (H2O2). Níveis muito altos ou baixos de tais espécies representam séria ameaça ao metabolismo celular. Diferentes estados patológicos têm sido relacionados com níveis anormais destas espécies em eritrócitos e plaquetas. A proteção biológica contra efeitos tóxicos associados com níveis excessivos de espécies ativadas de oxigênio, tem sido atribuída a três enzimas principais presentes em eritrócitos e outras células: superóxido dismutase, catalase e glutationa peroxidase. Neste trabalho, foram feitas dosagens dessas enzimas protetoras em eritrócitos de indivíduos portadores de porfiria aguda intermitente. Esta doença caracteriza-se por um defeito na biossíntese de heme e clinicamente manifesta-se por dor abdominal intensa, paralisia e distúrbios neuropsíquicos. Foram encontrados nos pacientes em crise aguda atividades elevadas de superóxido dismutase e de glutationa peroxidase enquanto que a atividade de catalase permaneceu normal. Tais resultados apontam a possibilidade de que as manifestações clínicas de IAP relacionadas com toxicidade por oxigênio. Estejam Comparou-se também a atividade destas três enzimas em eritrócitos de residentes na cidade de são Paulo com o de moradores de Vila Parisi, conhecida pelo seu altíssimo nível de poluiçao atmosférica. Os níveis de superóxido dismutase e de glutationa peroxidase são bem mais elevados (cerca de 1,5 vezes) nos indivíduos residentes em Vila Parisi. A elevação da concentração intracelular destas enzimas pode ser sua resposta ao aumento da taxa de oxidação da oxihemoglobina, a qual é conhecida fonte de íons superóxido. Estes dados demonstram profundas diferenças no metabolismo de 02 entre esses dois grupos e talvez uma possível adaptação metabólica dos moradores de Vila Parisi aos altos níveis de poluição a que estão expostos. / The univalent reduction of molecular oxygen in cells produces very reactive species such as the superoxide anion (O-&#8226;2), the hydroxyl radical (HO&#8226;) and hydrogen peroxide (H2O2). Abnormally high or low concentrations of such species may represent a serious threat to the cellular metabolism. Indeed, several disorders have been associated with abnormal levels of these species in erythrocytes and platelets.The biological protection against toxic effects due to excessive levels of activated species of molecular oxygen has been attributed mainly to three enzymes occurring in erythrocytes and other cells: superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase. In the present work, we report the activities of SOD, GSH-Px and catalase in the erythrocytes of patients with intermittent acute porphyria (IAP), an inborn error in the heme biosynthetic pathway. The clinical manifestations of IAP include abdominal pain and neuropsychiatric symptons. In patients undergoing acute attack, we found increased levels of SOD and GSH-Px, while that of catalase remains unchanged. These results point to the possibility that the clinical manifestations of IAP are related to oxygen toxicity. We have also compared the activities of these enzymes in residents of the city of são Paulo (Brazil) and in subjects living in Vila Parisi (Cubatão, SP, Brazil), a village submitted to high levels of atmospheric pollution. The erythrocyte SOD and GSH-Px levels were found to be ca. 1.5 fold higher in residents of Vila Parisi. These high intracellular enzyme concentrations may reflect a biological defense against an increase in the rate of oxyhemoglobin oxidation, known to be a source of superoxide species. Our results point to important differences between the two populations with respect to the metabolism of oxygen and, perhaps, a metabolic adaptation in the residents of Vila Parisi to the dramatically high levels of atmospheric pollution.

Made available in DSpace on 2016-08-10T10:38:40Z (GMT). No. of bitstreams: 1 ALEANDRO GERALDO ALVES.pdf: 687501 bytes, checksum: 815caf3ef15e76a3ef410c769760c097 (MD5) Previous issue date: 2011-08-11 / Amyotrophic lateral sclerosis (ALS) is a multifactorial disease that affects motor neurons. In most cases, the disease is sporadic, however, 5 to 10% of patients have a familial history (FALS). Among patients with FALS, 12 to 23% present with mutations in the SOD1 gene. Objectives: To present a systematic review about the mutations described in SOD1 gene in patients with FALS. Methods: The databases used in this study included PubMed, ISI Web of Science and Cochrane Library Virtual Health. After reading the abstracts, 71 articles were selected and systematically reviewed on this study. Results: The largest number of publications was found in 1997, and Japan was the country with the majority of published studies on the subject, with 23 articles. The majority of the mutations were described in éxons four and five of SOD1 gene, and A4V, I113T, I144F, D90A and L38V were the most commonly mutation described. More than 156 mutations in the SOD1 gene have been cataloged in patients with ALS-F and these data are deposited in ALS GENETICS ONLINE DATABASE, a database that contains specific information on mutations associated with amyotrophic lateral sclerosis. However, the articles reviewed in this study described 103 mutations. Conclusions: Several mutations in the SOD1 gene have been described in patients with ALS-F, however, the relationship between such mutations and the pathogenesis of ALS-F remains unclear, as well as the relationship between mutations and disease progression. Further studies are necessary in order to better explain such relationship. / A esclerose amiotrófica lateral (EAL) é uma doença multifatorial que afeta os neurônios motores. Na maioria dos casos, a doença é esporádica, entretanto, 5 a 10% dos pacientes apresentam história familiar (EAL-F). Dentre os pacientes com EAL-F, 12 a 23% apresentam mutações no gene SOD1. O objetivo deste trabalho foi realizar uma revisão sistemática acerca das mutações descritas no gene SOD1 em pacientes com EAL-F. As bases de dados consultadas incluíram Pubmed, ISI Web of Science e Cochrane Biblioteca Virtual em Saúde. Após a revisão dos resumos, 71 artigos foram selecionados descrevendo mutações no gene SOD1 em pacientes com EAL-F. O ano que apresentou o maior número de publicações foi 1997 e o Japão foi o país que mais publicou sobre o assunto, aparecendo em 23 artigos. O maior número de mutações foi descrito nos éxons 4 e 5 do gene SOD1 e as mutações A4V, I113T, I144F, D90A e L38V foram as mais comumente citadas. Até o momento 156 mutações no gene SOD1 já foram catalogadas em pacientes com EAL-F e esses dados encontram-se depositados no ALS ONLINE GENETICS DATABASE, um banco de dados que contém informações específicas sobre mutações associadas à esclerose amiotrófica lateral. Entretanto, os artigos revisados neste estudo descrevem 103 destas mutações. As causas relacionadas às mutações no gene SOD1 permanecem incertas, assim como a relação entre tais mutações e a evolução da doença, portanto, muito ainda deve ser estudado acerca desse tema.

Made available in DSpace on 2017-07-21T19:59:47Z (GMT). No. of bitstreams: 1 Tatiane Dobrzanski.pdf: 2071508 bytes, checksum: e3c3a02783ca0c1929ed8b2a83555230 (MD5) Previous issue date: 2015-02-25 / Excessive use of herbicides for weed control in agriculture causes a selective pressure on soil microbiota and waters near application area, changing environmental balance. Some microorganisms have developed metabolic pathways for degradation of these xenobiotics, although tolerance and degradation processes can generate free radicals and affect survival. This study aimed to analyze the system of responses from soil and water strains, submitted to selective pressure by the herbicide Callisto®. Strains CCT7729 and CCT7730, isolated from soil and water, respectively, were identified as Bacillus sp., and showed different degradation routes, with different metabolites, already described in the literature. Mesotrione and its metabolites, and especially its commercial product Callisto, affected cell viability and altered cell membrane lipids from the tested strains, however, Bacillus sp. CCT7729 presented a more efficient system of responses to oxidative stress. This strain exhibited a greater efficiency to degrade mesotrione, lower rates of peroxide, lower rates of MDA, SOD high activity and low activity of catalase, when compared to Bacillus sp. CCT7730. Changes in membrane lipids can be considered as a defense against oxidative stress strategy. These results indicated the existence from a variety of metabolic pathways for mesotrione degradation to Bacillus sp. Probably metabolites induce different levels of toxicity in bacteria, and Bacillus sp. CCT7730 possibly degraded mesotrione in even harmful compounds, unlike the water line. It is possible that these different responses are related to the home environments of each strain, suggesting plasticity responses of Bacillus sp. for adaptation to toxic substances in different environmental contexts. / O uso intenso de herbicidas para controle de ervas daninhas na agricultura provoca uma pressão seletiva na microbiota do solo e de águas próximas à área de aplicação, alterando o equilíbrio ambiental. Alguns microrganismos apresentam vias metabólicas de degradação desses xenobióticos, entretanto, a tolerância e os processos de degradação podem gerar radicais livres capazes de afetar a sobrevivência. Este trabalho teve como objetivo analisar o sistema de respostas de linhagens provenientes de solo e água, e que foram submetidas a pressão seletiva pelo herbicida Callisto. Uma linhagem isolada de cada um destes ambientes, identificadas respectivamente, como Bacillus sp. CCT7729 e Bacillus sp. CCT7730, apresentaram rotas de degradação diferenciadas, com metabólitos diferentes dos já descritos na literatura. O mesotrione, seus metabólitos, e principalmente o Callisto, afetaram a viabilidade celular das linhagens deste estudo e alteraram os lipídios de membrana celular, no entanto, Bacillus sp. CCT7729 apresentou um sistema de respostas ao estresse oxidativo mais eficiente. Esta linhagem exibiu uma maior eficiência em degradar o mesotrione, menores taxas de peróxido, menores taxas de MDA, atividade SOD elevada e uma baixa atividade da catalase, ao contrário de Bacillus sp. CCT7730. Modificações nos lipídios de membrana podem ser consideradas como uma estratégia de defesa contra estresse oxidativo. Os resultados também indicaram uma diversidade de vias metabólicas nas duas linhagens de Bacillus sp. para a degradação do mesotrione. Provavelmente os metabólitos induziram diferentes níveis de toxicidade nas bactérias, sendo que Bacillus sp. CCT7730 possivelmente degradou o mesotrione em compostos ainda nocivos, ao contrário da linhagem de água. É possível que essas diferentes respostas estejam relacionadas com os ambientes de origem de cada linhagem, sugerindo uma plasticidade de respostas apresentadas por linhagens Bacillus sp. para adaptação a substâncias tóxicas em diferentes contextos ambientais.