No description available.

No description available.

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

Les effets d'un concentré de melon, SODB®, particulièrement riche en SOD, ont été étudiés en mode curatif sur un modèle animal d'obésité nutritionnellement induite. La question du mécanisme d'action de ce concentré de melon a également été posée dans ce travail.Dans la première partie, des tests in vitro ont permis d'évaluer et de mieux caractériser le pouvoir antioxydant de SODB®. Il a également été montré, grâce à une étude sur rats sains, que SODB® n'était pas toxique aux doses utilisées.Dans une deuxième partie, des effets bénéfiques de SODB® sur le foie et le tissu adipeux d'animaux obèses ont été démontrés.Dans une dernière partie, le mécanisme d'action de SODB® a été discuté. Pour cela, les différents résultats obtenus sur SODB® ont été comparés avec les données d'une revue sur les effets de l'administration de SODs. Grâce à ce travail, des hypothèses de mécanisme d'action ont pu être proposées. En effet, SODB® semblerait agir en augmentant l'expression des enzymes antioxydantes endogènes. L'induction de la défense antioxydante pourrait passer par l'activation de la voie du Nrf2/ARE, à la suite d'une cascade d'évènements initiée au niveau de la barrière intestinale. La compréhension totale du mécanisme d'action de ce concentré de melon nécessite cependant plus d'investigations. / The effects of a curative supplementation of a melon concentrate, SODB®, particularly rich in SOD, have been studied on a diet-induced obesity animal model. In this work, we also wondered what mechanisms of action are involved in SODB® efficacy.First, we evaluated and characterized the antioxidant capacity of SODB® with in vitro tests. The safety of several doses used for SODB® supplementation has also been demonstrated on healthy rats.Then, we showed beneficial effects of SODB® on the liver and the adipose tissue of obese animals.Finally, we discussed the mechanism of action of SODB®, comparing all results obtained after SODB® supplementation with a review on SODs administration effects.This work allowed to propose hypothesis of mechanism of action. Indeed, SODB® seems to exert its antioxidant properties inducing increased endogenous antioxidant enzymes expression. The Nrf2/ARE pathway could be involved in this induction, after cascade events from intestinal barrier. More investigations are needed to determine the complete mechanism of action of this melon concentrate.

Aldeídos de colesterol (Secosterol A e Secosterol B) têm sido detectados em amostras de cérebro humano e investigados em modelos de doenças neurodegenerativas como possíveis marcadores e intermediários do processo patológico. Estes oxisteróis constituem uma classe de eletrófilos derivados de lipídeos que podem modificar e induzir agregação de proteínas. A esclerose lateral amiotrófica (ELA) é um distúrbio neurodegenerativo associado ao acúmulo de agregados imunorreativos de superóxido dismutase (Cu, Zn-SOD, SOD1). O objetivo deste trabalho foi avaliar a presença de aldeídos de colesterol em ratos modelo ELA e sua capacidade de induzir a formação de agregados de SOD1 in vitro. Aldeídos de colesterol foram analisados no plasma, medula espinhal e córtex motor de ratos ELA. Uma quantidade elevada de Secosterol B foi detectada no córtex motor desses ratos em comparação com animais controle. Adicionalmente, os experimentos in vitro mostraram que Secosterol B e Secosterol A induziram a agregação da SOD1 em uma forma amiloidogênica que se liga à tioflavina T. Esta agregação não foi observada com o colesterol e os seus hidroperóxidos. Usando aldeídos de colesterol marcados com grupo alquinil e um ensaio de click chemistry, foi observado que os agregados de SOD1 estão ligados covalentemente aos aldeídos. A modificação covalente da proteína foi confirmada por análise de MALDI-TOF, que mostrou a adição de até cinco moléculas de aldeídos de colesterol à proteína por base de Schiff. Curiosamente, a análise comparativa com outros eletrófilos derivados de lipídeos (e.g. HHE e HNE) demonstrou que a agregação de SOD1 aumentou proporcionalmente à hidrofobicidade dos aldeídos, observando-se a maioragregação com aldeídos de colesterol. Os sítios de modificação da SOD1 foram caracterizados por nanoLC-MS/MS após digestão da proteína com tripsina, onde foram identificadas lisinas como o principal aminoácido modificado. Em geral, nossos dados mostram que a oxidação do colesterol que leva à produção de aldeídos de colesterol é aumentada no cérebro de ratos ELA e que os aldeídos altamente hidrofóbicos derivados de colesterol podem promover eficientemente modificação e agregação de SOD1. / Secosterol aldehydes (Secosterol B and Secosterol A) have been detected in human brain samples and investigated in models of neurodegenerative diseases as possible markers and intermediates of the pathological process. These oxysterols constitute a class of lipid-derived electrophiles that can modify and induce aggregation of proteins. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder associated with the accumulation of immunoreactive aggregates of superoxide dismutase (Cu, Zn-SOD, SOD1). The objective of this work is to evaluate the presence of secosterol aldehydes in ALS rats and their ability to induce formation of SOD1 aggregates in vitro. Secosterol aldehydes were analyzed in plasma, spinal cord and motor cortex of ALS rats. A higher amount of Secosterol B was detected in the motor cortex of these rats compared to control animals. In addition, in vitro experiments have shown that Secosterol B and Secosterol A induce aggregation of SOD1 into an amyloidogenic form that binds to thioflavin T. This aggregation was not apparent in incubations with cholesterol and its hydroperoxides. Using alkynyl-labeled secosterol aldehydes and a click chemistry assay, it was found that the SOD1 aggregates are covalently linked to the aldehydes. Covalent modification of the protein was confirmed by MALDI-TOF analysis, which showed the addition of up to five molecules of secosterol aldehydes to the protein by Schiff base formation. Interestingly, the comparative analysis with other lipid-derived electrophiles (e.g. HHE and HNE) demonstrated that the aggregation of SOD1 increased according to the hydrophobicity of the aldehydes. Compared to the other electrophiles, a higher SOD1 aggregation was observed with secosterol aldehydes. SOD1 modification sites were characterized by nanoLC-MS/MS afterprotein digestion with trypsin, revealing lysine as the major amino acid modified in these experiments. Collectively, our data show that cholesterol oxidation leads to the production of secosterol aldehydes, which are increased in the brain of ALS rats, and that these highly hydrophobic aldehydes can efficiently promote the modification and aggregation of SOD1.

A emergência rápida e uniforme de plântulas em campo constituem pré-­ requisitos essenciais para o sucesso da produção comercial de sementes de hortaliças. Um dos principais problemas enfrentados por produtores de pimentão refere-­se à germinação lenta e estabelecimento de estande desuniforme, o que ressalta a importância do desenvolvimento de técnicas que visem aprimorar o desempenho de lotes de sementes. Com o objetivo de verificar a eficiência do condicionamento fisiológico de sementes de pimentão associado a biorreguladores sobre o potencial fisiológico das sementes, foi desenvolvida esta pesquisa utilizando dois cultivares, AF-6 e AF-7, representados por três e quatro lotes de sementes, respectivamente. Foram investigados possíveis efeitos do condicionamento com 24- epibrassinolídeo, ácido giberélico ou o bioestimulante Stimulate&reg;. As sementes foram avaliadas quanto à germinação e vigor, incluindo observações de atividade enzimática das sementes e análises de parâmetros de crescimento de plântulas utilizando o software SVIS&reg;. Por fim, o tratamento considerado promissor, condicionamento com 24-epibrassinolídeo na concentração de 10-8 M, foi avaliado quanto ao crescimento radicular das plantas, eficiência pela utilização do condicionamento em sistema de tambor, e comportamento das sementes durante o armazenamento. Diversos benefícios foram verificados após o condicionamento utilizando 24-epibrassinolídeo a 10-8 M, sobretudo a velocidade de germinação das sementes, atividade de enzimas antioxidantes e hidrolíticas, crescimento e uniformidade de desenvolvimento das plântulas. O condicionamento com 24- epibrassinolídeo a 10-8 M também favoreceu o desempenho dos lotes de sementes durante o armazenamento. A inclusão desse biorregulador no condicionamento em sistema de tambor é eficiente para o tratamento comercial de sementes de pimentão. / Rapid and uniform seedling emergence in field are essential for successful commercial production of vegetables. One of the main problems faced by bell pepper growers is the slow germination and uneven stand establishment, which highlights the importance of developing techniques that improve the performance of seed lots. In order to evaluate the efficiency of seed priming with bioregulators on physiological potential of bell pepper seeds, this research was developed using two cultivars, AF-6 and AF-7, represented by three and four seed lots, respectively. Possible effects of seed priming using 24-epibrassinolide were compared to gibberellic acid, and Stimulate&reg;. Germination and vigor evaluations were carried out, including analysis of seed enzymatic activity and parameters of seedlings growth using the SVIS&reg; software. At the end, the most promising treatment, seed priming with 24- epibrassinolide of 10-8 M was evaluated regarding to plant root growth, drum priming and seed performance during storage. Several benefits were verified after seed priming with 24-epibrassinolide, especially on germination speed, antioxidant and hydrolytic enzymes as well as seedling growth and uniformity development. Primed seeds with 24-epibrassinolide also showed better performance during seed storage. This bioregulador can be used during drum priming for commercial bell pepper seed treatment.