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Catalase and superoxide dismutase, but not peroxidase activity was detected in cell-free extracts of Mycobacterium avium, M. intracellulare and M. scrofulaceum (MAIS). The M. scrofulaceum isolates had the highest catalase activity, while both M. avium and M. intracellulare had significantly lower activities. The percentage of catalase activity remaining, after exposing cell-free extracts from late log grown cells to 53°C for 50 minutes allowed differentiation among all three species. Polyacrylamide gel electrophoresis of crude extracts demonstrated two bands of catalase activity in both M. avium and M. intracellulare extracts and four bands of activity in M. scrofulaceum extracts. These bands differed in their susceptibility to heat inactivation and inhibition by 3-amino-1,2,4-triazole. M. scrofulaceum strains, but not M. avium and M. intracellulare, demonstrated extracellular catalase activity. The susceptibility to H₂O₂ of 6 M. avium strains, differing in catalase activity and cell permeability, were tested. At a concentration of 0.02% H₂O₂, all M. avium strains were resistant, while differences in susceptibility were seen at 0.08% H₂O₂. Strains of low extract catalase activity and high H₂O₂ permeability were most susceptible. The superoxide dismutase activities of the MAIS strains tested were similar and no species-specific differences could be discerned. Electrophoresis of crude extracts demonstrated a single band of activity for each of the MAIS strains. Extracellular superoxide dismutase activity was detected in four of six MAIS strains. The metal type of MAIS superoxide dismutase was indirectly determined by inactivation with KCN, NaN₃ and H₂O₂. / M.S.

A gene coding for the cambialistic superoxide dismutase (SOD) was isolated from a LambdaGEM-11 genomic library of <i>Bacteroides fragilis</i>. In order to generate a complete genomic library, <i>B. fragilis</i> genomic DNA was partially digested with the restriction endonuclease Sau3AI and was ligated to cloning vector, LambdaGEM-11. After in vitro packaging, DNA was used to infect <i>E. coli</i> KW 251. The genomic library was finally established in the plaque population. Recombinant phage DNAs containing the SOD gene were detected by a ³²P-labelled synthetic oligonucleotide with 17 bases. The sequence of this oligonucleotide was deduced from the N-terminal amino acid sequence of <i>B. fragilis</i> FeSOD. Two recombinant phage DNAs were selected based on he results of plaque hybridization. Further analysis with restriction mapping and DNA sequencing revealed that only one recombinant phage DNA contained the SOD gene. Southern hybridization and restriction mapping located the SOD gene in the SalI-BamHI fragment (2.1 kb). Sequence analysis identified the orientation and open reading frame (ORF) of the gene. Translation of ORF revealed that SOD consists of 193 amino acid residues. The size of the deduced polypeptide is consistent with the molecular weight of SOD subunit (MW 21,000). The B. fragilis SOD sequence was compared with those of other SODs. The amino acid residues contributing metal ligands, the hydrophobic shell of the active site, and amino acids at the subunit contact are almost fully conserved in B. fragilis SOD. Expression of SalI-BamHI fragment in E. coli SOD double mutant (sodA, sodB), QC1799, produced an active SOD whose activity zymogram was identical to that of purified B. fragilis SOD. In addition, Western analysis of the expressed protein separated on SDS acrylamide gel also displayed a band identical to the subunit of B. fragilis SOD. However, a larger molecular weight band was also detected. This band migrated closely to the subunit of B. fragilis SOD. This larger peptide may be the product of gene translation from an ATG 21 bases upstream of the ATG start codon of B. fragilis gene. The cambialistic feature of SOD gene product was also confirmed from in vitro and in vivo metal substitution. / Ph. D.

Mn-salophen complex with superoxide-scavenging activity was prepared from manganese(III) acetate dihydrate and salophen in ethanol. Visible absorption spectrum of the red-brown solution exhibited a broad absorption band at 430 - 450 nm with two shoulders between 500 and 600 nm which were absent with either salophen or manganic acetate alone. Titration of salophen with manganese(III) was consistent with a 1:1 Mn to salophen stoichiometry of the complex based on changes in the absorbance at 500 nm or of superoxide scavenging activity. The SOD-like activity of the complex in the xanthine-xanthine oxidase/cytochrome <i>c</i> assay was 1450 units/mg salophen. The SOD activity of the complex was suppressed 50% in the presence of EDTA (1 mM), but was not altered in the presence of bovine serum albumin (1 mg/ml) or crude protein extract of <i>E. coli</i> QC779 <i>sodA sodB</i> (1 mg/ml). <i>E. coli</i> QC779 <i>sodA sodB</i> grew scantily after an 8 hour lag phase in aerobic M63 glucose minimal medium. / Ph. D.

The superoxide dismutase from a radiation-resistant bacterium Deinococcus radiodurans has been purified to electrophoretic homogeneity. The superoxide dismutase has a specific activity of 3300 units/mg and an apparent molecular mass of 43,000 daltons. The enzyme contains 1.5 gram-atom of manganese per mol dimer, and is composed of two identical subunits of 23,500 daltons. The enzyme rapidly loses its catalytic activity and metal content upon dialysis in denaturing reagent, guanidine hydrochloride, and the metal ion chelator 8-hydroxyquinoline. The denatured apoprotein was renatured upon removal of the denaturant by dialysis. The renatured apoprotein assumed a gross conformation similar to the native enzyme as indicated by fluorescence spectroscopy. The renatured apoprotein was reconstituted to the native specific activity upon addition of manganese in the absence of denaturant. The manganese econstituted enzyme contained 1.7 gram-atom of manganese per mol dimer, and had a specific activity of 3650 units/mg. Kinetic studies revealed that the reconstitution with manganese was pH-dependent, and was inhibited by competing metal ions (iron and zinc). / Ph. D.

A rabbit lateral meniscectomy model was used to induce osteoarthritis. Separate studies were conducted to evaluate the progression of osteoarthritis and to identify possible biological markers. First, 21 male, New Zealand White rabbits were divided into 3 groups (n = 7 / group). A randomly selected left or right stifle underwent a lateral meniscectomy. The 3 groups were: corticosteroid administration, forced exercise and surgical control. An open field maze was used to assess mobility weekly. The rabbits were euthanitized 47 days after surgery. Histopathologic examination found that the lateral meniscectomy induced more severe lesions than in the non-surgical contralateral stifle. It also showed a significant sparing effect on erosion of cartilage in the corticosteroid group. The corticosteroid group, but not the exercise group, caused a significant increase in mobility (p = 0.008) compared to the surgical control. Secondly, synovial fluid was harvested from the 12 rabbits on days 0, 6, 26, 40, and 57 with surgery occurring on day 12. Trypan blue was used in the lavage fluid to estimate the volume of harvested synovial fluid. There was a significant increase in the volume harvested on day 26 (p < 0.001). Superoxide dismutase concentration in synovial fluid increased after surgery, although not significantly. These studies verify that the lateral meniscectomy model produce histopathologic lesions consistent with osteoarthritis. Furthermore, use of trypan blue appears to be a reliable concentration marker in a lavage sample to measure harvested synovial fluid. / Master of Science

Yes / Quercetin is reported to reduce blood pressure in hypertensive but not normotensive humans, but the role of endothelial redox signaling in this phenomenon has not been assessed. This study investigated the effects of physiologically obtainable quercetin concentrations in a human primary cell model of endothelial dysfunction in order to elucidate the mechanism of action of its antihypertensive effects. Angiotensin II (100 nM, 8 h) induced dysfunction, characterized by suppressed nitric oxide availability (85 ± 4% p<0.05) and increased superoxide production (136 ± 5 %, p<0.001). These effects were ablated by an NADPH oxidase inhibitor. Quercetin (3 μM, 8 h) prevented angiotensin II induced changes in nitric oxide and superoxide levels, but no effect upon nitric oxide or superoxide in control cells. The NADPH oxidase subunit p47(phox) was increased at the mRNA and protein levels in angiotensin II-treated cells (130 ± 14% of control, p<0.05), which was ablated by quercetin co-treatment. Protein kinase C activity was increased after angiotensin II treatment (136 ± 51%), however this was unaffected by quercetin co-treatment. Physiologically obtainable quercetin concentrations are capable of ameliorating angiotensin II-induced endothelial nitric oxide and superoxide imbalance via protein kinase C-independent restoration of p47(phox) gene and protein expression. / Innovate UK and Boots Pharmaceuticals

Superoxide, the product of the one-electron reduction of dioxygen, is a molecule of enormous importance. It participates in a variety of critical physiological processes and is also an important component of fuel cells where it is an intermediate in the cathodic reaction. However, the electrochemical behavior of superoxide, mainly its reduction, is not well understood. Here, the electrochemical behavior of superoxide has been investigated in acetonitrile on glassy carbon electrodes, through cyclic voltammetry experiments. By stabilizing the electrogenerated superoxide, aprotic solvents afford an opportunity to study its electrochemical reactions further. Superoxide was generated electrochemically from dioxygen at the first voltammetric peak. In the presence of hydrogen-bond donors (water, methanol, 2-propanol), the superoxide forms a complex with the donor resulting in a positive shift in the formal potential which can be analyzed to obtain formation constants for these complexes. Stronger acids (2,2,2- trifluoroethanol, 4-tert-butylphenol) result in protonation of superoxide followed by reduction to produce HO₂-. On scanning to more negative potentials a second peak is observed which is irreversible and extremely drawn out along the potential axis indicating a small value of the transfer coefficient α. Addition of hydrogenbond donors, HA, brings about a positive shift in this peak, without a noticeable change in shape. The reaction occurring at the second peak is a concerted proton-coupled electron transfer (PCET) in which the electron is transferred to superoxide and a proton is transferred from HA to superoxide forming HO₂- and A- in a concerted process. We estimate the standard potential for this reaction for the case of water as the donor. This value suggests that the reaction at the second peak occurs at very high driving forces. Kinetic simulations using both Butler-Volmer and Marcusian schemes were performed to estimate the kinetic parameters. The unusually low rate constants obtained suggest high nonadiabaticity for this PCET reaction. The reaction was also found to proceed with an unusually large reorganization energy. Consistent with a PCET, a kinetic isotope effect, HA vs. DA, was detected for the three hydrogen-bond donors.

Using gentian and petunia as the experimental systems, potential alternative post-harvest treatments for cut flowers were explored in this project. Pulsing with GA₃ (1 to 100 µM) or sucrose (3%, w/v) solutions delayed the rate of senescence of flowers on cut gentian stems. The retardation of flower senescence by GA₃ in both single flower and half petal systems was accompanied by a delay in petal discoloration. The delay in ion leakage increase or fresh weight loss was observed following treatment with 5 or 10 µM GA₃ of the flowers at the unopen bud stage. Ultrastructural analysis showed that in the cells of the lower part of a petal around the vein region, appearance of senescence-associated features such as degradation of cell membranes, cytoplasm and organelles was faster in water control than in GA₃ treatment. In particular, degeneration of chloroplasts including thylakoids and chloroplast envelope was retarded in response to GA₃ treatment. In the cells of the top part of a petal, more carotenoids-containing chromoplasts were found after GA₃ application than in water control. In petunia, treatment with 6% of ethanol or 0.3 mM of STS during the flower opening stage was effective to delay senescence of detached flowers. The longevity of isolated petunia petals treated with 6% ethanol was nearly twice as long as when they were held in water. Senescence-associated petal membrane damage, weight decline, ovary growth and decrease in protein and total RNA levels were counteracted in ethanol-treated petals. The accumulation of ROS, particularly superoxide and hydrogen peroxide, was also inhibited or delayed by ethanol application. Anti-senescence mechanisms, particularly the changes of oxidative / antioxidant metabolism involved in petal senescence, were investigated. In gentian, activities of AP and SOD but not POD in the GA₃-treated petals were significantly higher than those of the control. In isolated petunia petals, the decreased trends of antioxidative SOD and AP activities during senescence were apparently prevented in response to ethanol treatment although the levels of ascorbate and photo-protective carotenoids were not affected. Furthermore, by optimizing a range of critical PCR parameters such as primer combinations, cDNA concentrations and annealing temperatures, a reliable protocol has been established for quantifying the expression level of Cu-Zn SOD gene in petunia petals using SYBR Green I based real-time RT-PCR. A 228 bp gene fragment of Cu-Zn SOD was isolated from petunia (var. 'hurrah') using RT-PCR. It was found that the mRNA level (relative to 18S rRNA level) of Cu-Zn SOD decreased significantly after 6 days in water. However, there was about a 55-fold increase in Cu-Zn mRNA level after 6 days of ethanol treatment when compared to water-treated petals. Similarly, down-regulation of the mRNA level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also observed during senescence of petunia petals. Increased vase life of petunia petals by ethanol treatment was correlated with promotion of GAPDH expression by a factor of about 16 on day 6. Taking together, the anti-senescence effects of GA₃ and ethanol are at least partially associated with an increased efficiency of petal system utilizing ROS since the selected antioxidants were significantly maintained when compared to the corresponding values for the control.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Introduction: manganese dependent superoxide dismutase (SOD2), is an important antioxidant enzyme, superoxide dismutase to anion produced in mitochondria in hydrogen peroxide, which in turn is catalyzed by glutathione peroxidase (GPX) into water and oxygen. Although be crucial for healthy cell, the role of SOD2 in cancer is highly controversial because in some kinds of cancers this enzyme exhibits a marked antitumor activity, while in others have a pro-tumor role. Previous investigations involving a polymorphism in codon 16 of SOD2 gene in which there is an exchange of a valine with an alanine (Val16Ala-SOD2) have associated increased efficiency of enzyme SOD2 at high risk of some cancers. However, in certain types of tumors, such as colorectal cancer are conflicting results. Studies suggest that high levels in tumor cells SOD2 colorectal cancers are associated with tumor progression. Perhaps this difficulty in defining the role of SOD2 in colorectal cancer biology is linked to great influence of environmental factors on the gastrointestinal system, especially the diet. For this reason, the development of an unbalance pharmacological model to investigate the role of superoxide-anion imbalance hydrogen peroxide (Superoxide Anion Hydrogen Peroxide imbalance, AS-HP) in colorectal cancer may be relevant. Objective: This study investigated the in vitro effect of drug-AS-HP imbalance caused by exposure to paraquat and the porphyrin in the viability and proliferative rate of commercial line of colorectal cancer cells (HT-29) and the response of these cells to chemotherapy oxaliplatin. The study also assessed the effect of AS-HP unbalance in the modulation of the expression of apoptotic genes, cell cycle and oxidative in HT-29 cells. Methods. HT- 29 obtained from American Type Cell Culture Collection (ATCC) were grown in DMEM, 10% fetal bovine serum, 1% antibiotic and antifungal in an oven with 5% CO2 and 37 ° C temperature. After 24 h the transfer of cells to 96-well plates at a concentration of 10 5 cells per well were exposed to these concentrations of 0.1 uM paraquat which is a superoxide-generating molecule or porphyrin which is a molecule with a similar effect SOD2 enzyme. Part of the cells was treated with oxaliplatin at a concentration of 20um and the other not. The effect on the viability, cell proliferation, cell cycle, apoptosis and modulation of genes of the cell cycle, apoptosis and oxidative metabolism (SOD1, SOD2, CAT, GPX, Caspase 3, Caspase 8, BAX, BCL-2 and P53colocar the name gene) was also evaluated. Assays were done in triplicate and compared by analysis of variance followed via test post hoc Tukey. Results: pharmacological imbalance AS-HP obtained via exposure of colorectal cancer cells to paraquat and porphyrin changed the standard of viability, cell cycle and in the modulation of gene expression. Both paraquat as nna porphyrin concentration 0.1 uM reduced the viability and proliferation rate of HT-29 cells. However, this effect was more pronounced in cells exposed to paraquat. The action of oxaliplatin was enhanced by the presence of paraquat when analyzed, the mortality rate, apoptosis, cell proliferation rate. Paraquat tamém induced cell cycle interruption phases S and G2 / M Any paraquat as porphyrin were able to modulate differentially markers of oxidative metabolism and expression of genes investigated. However, the results were quite heterogeneous. This heterogeneity may be associated with chromosomal instability in cancer cells that have high levels, and varied mutational. Conclusion: The results confirm the hypothesis that the AS-HP unbalance acts on the biology of colorectal cancer, and in particular increased levels of superoxide, not only increase the mortality rate but also inhibit cell proliferation enhancing so antitumor action of oxaliplatin. These results may be clinically relevant in the construction of pharmaceutical and / or nutritional strategies as the use of vitamins and other dietary supplements which operate in AS-HP sheet and to assist in the successful chemotherapeutic treatment of disease. / Introdução: a superóxido dismutase dependente de manganês (SOD2), é uma importante enzima antioxidante, que dismuta o ânion superóxido produzido na mitocôndria em peróxido de hidrogênio, que por sua vez é catalisado pela glutationa peroxidase (GPX) em água e oxigênio. Apesar de ser crucial para a célula saudável, o papel da SOD2 no câncer é bastante controverso, pois em alguns tipos de neoplasias apresenta uma clara ação antitumoral, enquanto que em outras tem um papel pró tumoral. Investigações prévias envolvendo um polimorfismo no códon 16 do gene da SOD2 no qual ocorre uma troca de uma valina por uma alanina (Val16Ala-SOD2), têm associado maior eficiência da enzima SOD2 com risco elevado de alguns tipos de câncer. Entretanto, em certos tipos de tumores, como o câncer colorretal os resultados são conflitantes. Estudos sugerem que os níveis elevados de SOD2 em células de tumores colorretais estão associados com a progressão do tumor. Possivelmente esta dificuldade em definir o papel da SOD2 na biologia do câncer colorretal esteja vinculado a grande influência de fatores ambientais sobre o sistema gastrointestinal, com destaque a dieta. Por este motivo, o desenvolvimento de um modelo farmacológico de desbalanço para investigar o papel do desbalanço ânion superóxido-peroxido de hidrogênio (Superoxide Anion Hydrogen Peroxide imbalance, AS-HP) no câncer colorretal pode ser considerado relevante. Objetivo: investigar o efeito in vitro do desbalanço farmacológico do AS-HP causado pela exposição ao paraquat e a porfirina na viabilidade e taxa proliferativa da linhagem comercial de células de câncer colorretal (HT-29) e na resposta destas células ao quimioterápico oxaliplatina. O estudo também avaliou o efeito do desbalanço AS-HP na modulação da expressão de genes apoptóticos, do ciclo celular e oxidativos nas células HT-29. Métodos. Células HT-29 obtidas da American Type Culture Collection (ATCC) foram cultivadas em meio DMEM, 10% de soro bovino fetal, 1% de antibióticos e antifúngicos em estufa com CO2 a 5% e temperatura de 37oC. Após 24 h da transferência das células para placas de 96 poços na concentração de 10 5 células por poço, estas foram expostas a concentração de 0,1 uM de paraquat, que é uma molécula geradora de superóxido, ou de porfirina, que é uma molécula com efeito similar a enzima SOD2. Parte das células foi tratada com oxaliplatina na concentração de 20uM e outra não. O efeito na viabilidade, proliferação celular, ciclo celular, apoptose, e na modulação dos genes do ciclo celular, apoptose e metabolismo oxidativo (β-actina, SOD1, SOD2, CAT, GPX, Caspase 3, Caspase 8, BAX, BCL-2 e P53) também foram avaliados. Os ensaios foram realizados em triplicatas e comparados por análise de variância de uma via seguido de teste post hoc de Tukey. Resultados: o desbalanço farmacológico AS-HP obtido via exposição das células de câncer colorretal ao paraquat e porfirina alterou o padrão de viabilidade, ciclo celular e na modulação da expressão gênica. Tanto o paraquat quanto a porfirina na concentração 0,1 uM diminuíram a viabilidade e a taxa de proliferação das células HT-29. No entanto, este efeito foi mais pronunciado em células expostas ao paraquat. A ação da oxaliplatina foi potencializada pela presença do paraquat quando foram analisadas a taxa de mortalidade, apoptose, taxa de proliferação celular. O paraquat também induziu interrupção do ciclo celular nas fases S e G2 / M. Tanto o paraquat quanto a porfirina foram capazes de modular diferencialmente marcadores do metabolismo oxidativo e a expressão dos genes investigados. Entretanto, os resultados foram bastante heterogêneos. Esta heterogeneidade pode estar relacionada com a instabilidade cromossômica de células tumorais que apresentam níveis mutacionais altos e variados. Conclusão: os resultados obtidos corroboram a hipótese de que o desbalanço AS-HP age sobre a biologia do câncer colorretal, e que em especial o aumento nos níveis de superóxido, não só aumentam a taxa de mortalidade mas também inibem a proliferação celular, potencializando assim, a ação antitumoral da oxaliplatina. Estes resultados podem ser clinicamente relevantes na construção de estratégias farmacológicas e/ou nutricionais, como um adjuvante ao tratamento o uso de vitaminas ou outros suplementos dietéticos, que atuem no balanço AS-HP e que auxiliem no sucesso do tratamento quimioterápico da doença.