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

<p>Diss. (sammanfattning) Umeå : Umeå universitet, 1983, härtill 5 uppsatser</p> / digitalisering@umu

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Aconitase is an enzyme of the Krebs cycle that catalyzes the isomerization of citrate to isocitrate. In addition to its enzymatic activity, Aco1 has been reported to bind to mitochondrial DNA (mtDNA) and mediate its maintenance in the budding yeast S. cerevisiae. In the absence of Aco1, cells rapidly lose mtDNA and become “petite” mutants. The purpose of this study is to uncover the mechanism behind mtDNA loss due to an aco1 deletion mutation. We found that an aco1 mutation activates the mitochondria-to-nucleus retrograde (RTG) signaling pathway, resulting in increased expression of citrate synthases (CIT) through the activation of two transcription factors Rtg1 and Rtg3. Increased activity of CIT leads to increased iron accumulation in cells, which is known to raise reactive oxygen species (ROS). By deleting RTG1, RTG3, genes encoding citrate synthases, orMRS3 and MRS4, encoding two irontransporters in the mitochondrial inner membranes, mtDNA loss can be prevented in aco1 deletion mutant cells. We further show that the loss of SOD1, encoding the cytoplasmic isoform of superoxide dismutase, but not SOD2, encoding the mitochondrial isoform of superoxide dismutase, prevents mtDNA loss in aco1 mutant cells. Altogether, our data suggest that mtDNA loss in aco1 mutant cells is caused by the activation of the RTG pathway and subsequent iron accumulation and toxicity in the mitochondria.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting the upper and lower motor neurons causing muscle atrophy and paralysis followed by death. Aggregates containing superoxide dismutase (SOD1) are found as pathological hallmark in diseased ALS patients. Consequently ALS is regarded as a protein misfolding disorder like Alzheimer’s disease and Parkinson’s disease. So far, little is known about the cause and mechanism behind SOD1 aggregation but the inherent property of all polypeptide chains to form stable aggregated structures indicates that the protein misfolding diseases share a common mechanism. Our results show that SOD1 aggregation starts from the globally unfolded state, since fibrillation is fastest at full occupancy of denatured protein induced either by chemical denaturation or mutation. Even so, the fibrillation rate shows a surprisingly weak dependence on the concentration of globally unfolded SOD1 indicating fibril fragmentation as the dominant mechanism for aggregate formation. This is further supported by the observation that the SOD1 sample has to be mechanically agitated for fibrillation to occur.  Interestingly, we observe a similar SOD1 aggregation behaviour in vivo, where the survival times of ALS transgenic mice correlates with mutant stability, and aggregate growth depends weekly on the concentration of unfolded monomer. Additionally, in-cell NMR measurements reveal that in live cells the thermodynamic equilibrium is shifted towards the unfolded state of SOD1, which is also more fully extended than in vitro. This suggests that the globally unfolded aggregation competent protein is more abundant in the crowded environment in vivo than dilute in vitro conditions. Finally, antibody analysis of aggregates from ALS transgenic mice reveals the existence of aggregate strains involving different parts of the protein depending on mutation, which may offer an explanation for the various disease phenotypes observed in ALS. Altogether these findings provide important clues for understanding SOD1 aggregation with implications for ALS, as well as other protein misfolding diseases.

O estresse oxidativo reflete um desequilíbrio na manutenção do estado redox intracelular que pode resultar no acúmulo de espécies oxidantes. Nestas condições, estas espécies podem gerar danos na estrutura/função do DNA, disfunções mitocondriais, alterações no enovelamento de proteínas, peroxidação de lipídeos, dentre outros danos. Assim, o estresse oxidativo pode contribuir para diversas condições patológicas, como por exemplo, câncer, desordens neurológicas, aterosclerose, diabetes e asma. A infecção persistente por papilomavírus humano (HPV) de alto risco está etiologicamente associada ao câncer de colo uterino, uma das principais causas de morte por câncer em mulheres no mundo todo. Além disso, esses vírus estão associados a uma porcentagem relevante de cânceres de pênis, vulva, ânus e cabeça e pescoço. Em células infectadas por HPV de alto risco oncogênico, o estresse oxidativo resultante do metabolismo anormal dos queratinócitos e a resposta inflamatória crônica não efetiva, podem contribuir no processo de transformação celular. Além disso, a presença das oncoproteínas E6 e E7 interfere nos mecanismos de reparo de lesões em DNA, favorecendo o acúmulo de mutações. Ainda mais, a instabilidade genômica promovida pelo estresse oxidativo, pode favorecer a integração do HPV no genoma das células infectadas, principal causa da persistência viral e progressão de lesões ao câncer. A proteína superóxido dismutase 2 (SOD2) contribui com a homeostase celular ao catalisar a dismutação de radicais ânion superóxido em oxigênio e peróxido de hidrogênio, prevenindo a inativação direta de biomoléculas. Em estudos anteriores, observamos que o aumento do transcrito de SOD2 está associado à resistência ao efeito antiproliferativo do fator de necrose tumoral (TNF) em células imortalizadas por HPV. Além disso, mostramos que existe uma correlação direta entre o aumento dos níveis da proteína SOD2 e a severidade de lesões da cérvice uterina. Finalmente, identificamos SOD2 como um marcador preditivo independente de metástases linfonodais inguinais em pacientes com carcinoma de pênis. No entanto, o papel desta proteína na patogênese associada ao HPV não tem sido estudado em profundidade. O presente estudo visa determinar o envolvimento de SOD2 no processo de transformação celular mediado por HPV e seu valor como marcador preditivo de patologias associadas a este vírus / Oxidative stress reflects a redox imbalance in the cell in favor of oxidant species, which may result in DNA damage, mitochondrial dysfunction, protein misfolding and lipid peroxidation, among others. As a result, oxidative stress is thought to contribute to diverse pathologies such as cancer, neurological disorders, atherosclerosis, diabetes and asthma. Persistent infection with high risk human papillomavirus (HPV) types is etiologically associated with cervical cancer, one of the leading causes of cancer related female death worldwide. Moreover, HPV infection is associated with a significant percentage of penile, vulvar, anal and head and neck carcinomas. In cells infected with high-oncogenic risk HPV types the oxidative stress generated by the abnormal keratinocytes metabolism and non-efficient chronic inflammatory response may contribute to the cellular transformation process. Furthermore, expression of E6 and E7 oncoproteins interferes with several DNA repair mechanisms, favoring the accumulation of mutations. Moreover, genomic instability promoted by oxidative stress may favor HPV integration in host cell genome, the main cause of viral persistence and of precursor lesions progression to cancer. The protein superoxide dismutase 2 (SOD2) contributes to cellular homeostasis by catalyzing radical super anion dismutation in oxygen and oxygen peroxide preventing the direct inactivation of biomolecules. In previous studies we observed that SOD2 mRNA upregulation is associated with the resistance to tumor necrosis factor (TNF) antiproliferative effect in HPV-immortalized cells. Besides, we showed the existence of a direct correlation between increased SOD2 protein expression and cervical lesions severity. Finally, we identified SOD2 as an independent predictive marker of inguinal lymph node metastases in patients with penile carcinomas. However, the role of this protein in HPV-associated pathologies has not been investigated in depth. The goal of this study is to determine the involvement of SOD2 in HPV-mediated cell transformation and its value as a predictive marker in pathologies caused by this virus