Oxidative stress (OS) and inflammation can be detrimental to exercise performance. Antioxidants such as curcumin are shown to reduce exercise-induced OS, inflammation, muscle damage, and soreness. The purpose of this study was to examine the effect of curcumin on biomarker markers of OS (MDA, TAC), inflammation (TNF-á), muscle damage (CK) and soreness. Participants performed an exercise-induced muscle damage protocol. Before and after supplementation, subjects were randomly assigned to curcumin (1.5 g/day) or placebo for 28 days. Blood was sampled immediately before and after exercise, as well as 60 min, 24, and 48 h after exercise. No significant differences were observed for biomarkers of OS or inflammation. There was a treatment X condition interaction for CK, where CK were significantly lower post supplementation in the curcumin group (p < .0.0001). Curcumin resulted in significantly lower muscle soreness compared to the placebo (p = 0.0120) overall. In conclusion, curcumin may reduce muscle damage, and soreness without affecting the natural OS and inflammatory response to exercise.

Iron is an essential element for living organisms. However, as it is a transition metal, it can participate in Fenton reaction resulting in generation of free radicals and oxidative damage to tissues. Antioxidants may prevent possible iron toxicity by chelating free iron or scavenging free radicals. Falvonoids are naturally occurring substances that are capable of formation of complexes with metals, including iron. T h e y have been show to possess antioxidant activity, which depends on molecular complexity of numerous types of flavonoids, e.g. quercetin and silibinin. Bisphosphonates are synthetic drugs used to treat various metabolic diseases of bones. Their principál effect is an inhibition of osteclast activity leading to a decreased bone resorption. Bisphosphonates have been however shown to exert some antioxidant activity in in vitro experiments, too. The aim of this PhD thesis was to investigate the role of iron in toxicity of other metals (cadmium) and the effect of flavonoids (quercetin and silibinin) and bisphosphonates (clodronate, etidronate and risedronate) on iron-induced oxidative damage in vivo. Experiments were performed in male mice (CD-1, Charles River, 25-35 body weight). Iron was administered intraperitoneally or in the diet. Cadmium was administered subcutaneously. Flavonoids and...

Studies of adaptive responses of the microaerophile Spirillum volutans to various stresses such as heat and peroxide shock have been hampered by an inability to obtain reliable colony counts of the organism by the spread plate method. Colony counts approaching direct microscopic counts (DMCs) were obtained by inoculating culture dilutions into a semisolid version of the medium and by supplementing the medium with pyruvate, which destroys hydrogen peroxide. Use of the new overlay/pyruvate method for colony counts revealed that exposure of S. volutans to 40 degrees C for 100 min results in a greater survival at 45 degrees C compared with cells having no prior exposure to 40 degrees C. Spirillum volutans is catalase-negative and is rapidly killed by levels of H2O2 greater than 10 micromolar. A mutant isolated by single step mutagenesis with diethyl sulfate was able to survive and grow after exposure to 40 micromolar H2O2 and was effective in eliminating H2O2 concentrations added to the medium. In addition, the mutant had high NADH peroxidase activity (0.072 I.U. mg-1) whereas the wild type had no detectable activity (<0.0002 I.U. mg-1). Nevertheless, the mutant was no more tolerant to O2 than the wild type. NADH peroxidase activity has not previously been reported in bacteria having a strictly respiratory type of metabolism. The peroxide-resistant mutant constitutively expresses a 21.5 kDa protein as determined by one and two-dimensional PAGE. This protein was undetectable and noninducible in the wild type cells. Part of the gene that encodes the protein was cloned by using amino acid sequence data obtained by both mass spectrometry and NH2-terminal sequencing. The deduced 158 amino acid polypeptide showed high similarity to rubrerythrin and nigerythrin previously described in the anaerobes Clostridium perfringens and Desulfovibrio vulgaris and to putative rubrerythrin proteins found in some anaerobic archeons. This is the first report of this type of protein in an organism that must respire with oxygen. This rubreythrin-like protein may play a role in the peroxide resistance of the mutant. The methodology may be useful for rapid cloning of genes in other bacteria. / Ph. D.

[Truncated abstract] Transition metals in the plant mitochondrion have dual roles in regulating the function of the organelle. While metals participate in mitochondrial respiratory metabolism as ligands in bioenergetic, detoxifying, and various other metabolic enzymes, a breakdown in metal homeostasis during oxidative stress can perpetuate the cycling of ROS by redox active metal ions. Large-scale studies into the duplicitous roles of metal ions in biological systems has been lacking and in this thesis, a combination of metallomics, database annotations, membrane proteomics, metal-protein interactomics, structural biology, functional assays and mass spectrometry were all used to gain a clearer insight into the involvement of metal ions in affecting plant mitochondrial function. The Arabidopsis mitochondrion was shown to contain the transition metals cobalt, copper, iron, manganese, molybdenum, and zinc. Interestingly, the redox active copper and iron represented 75% of the mitochondrial metallome and these metal species were revealed to be highly labile during oxidative stress suggesting a possible contribution of metal-catalysed oxidation (MCO) in the damage of biological macromolecules. Bioinformatic analysis of metalloproteins predicted and experimentally determined to be mitochondrially localised revealed that metal ion transporters are poorly characterised. An in-depth proteomic analysis of the membrane proteome was conducted on mitochondria isolated from unstressed and stressed cell cultures resulted in the identification of stress-responsive as well as potential metal ion transporters. Also, many of the annotated metalloproteins predicted to be mitochondrial lack experimental evidence for subcellular localisation. ... However, based on evidence in the literature, it was hypothesised that metal-interacting sites may be the targets for MCO due to their affinity for metal ions. Attempts were made to identify the site specificity of MCO on mitochondrial proteins but no carbonyl sites could be found owing to technical problems associated with non-specific binding of proteins to the enrichment resin and low abundance of the labelled protein carbonyls. The use of the model protein BSA showed that protein oxidation occurs in clusters and the use of model peptides demonstrated that the ability of amino acid residues to complex metal ions is important in dictating susceptibility to MCO. Further experimental verification for the site specificity of MCO is required to determine the consequences of MCO on mitochondrial protein function. Overall, this thesis provided a large-scale analysis of the contributions of metal ions to mitochondrial respiratory metabolism with an emphasis on metal ion induced toxicity. Using multi-facetted approaches, an insight into the dynamic nature of mitochondrial metal homeostasis, stress responsive transporters, the interactions of metal ions with mitochondrial proteins and the possible mechanism in which proteins are specifically oxidised by MCO has been uncovered paving the way for future focused studies characterising the consequences of oxidative stress on specific proteins and their function.

Thesis (MAppSc) -- Auckland University of Technology, 2004. / Also held in print (98 leaves, 30 cm.) in Wellesley Theses Collection. (T 613.70993 MIG)

published_or_final_version / Molecular Biology / Doctoral / Doctor of Philosophy

No description available.

No description available.

The anthocyanins show efficient antioxidant properties and free radical scavenging properties which result in various health-promoting benefits. This research investigated the ability of anthocyanidins to distribute into mitochondria and protect mitochondria from oxidative stress. In an in vitro study, the uptake of pure cyanidin and quercetin, and their 3-glucosylated forms into isolated rat liver mitochondria was tested, along with their effects on mitochondrial oxidative stress parameters. The absorption of cyanidin was significantly higher (67% uptake of 125 µM) than the other three flavonoids. Measurements indicated that the cyanidin was taken up into or tightly bound by mitochondria. Also, results suggested that cyanidin uptake was partially dependent on membrane potential. When incubated together (internally and externally) with mitochondria all tested flavonoids decreased reactive oxygen species (ROS) generation during mitochondrial respiration, and inhibited lipid peroxidation to different extents. Importantly, pre-loaded CY showed much stronger effects against oxidative stress in two analyses than other flavonoids. Due to its greater uptake by mitochondria, cyanidin may provide greater protection in vivo. In an in vivo study, cyanidin, quercetin and their 3-glucosides were administered into rat tail vein to give a dose of 7.6 µmol/Kg body weight. Cyanidin and its glucoside had greater affinity to liver and kidney than did quercetin and its glucoside; particularly, all test tissues contained a significantly higher amount of cyanidin than other test flavonoids. Also, cyanidin accumulated more in liver mitochondria than other flavonoids, and consistent with in vitro results was present in mitochondria to a much greater extent than cyanidin glucoside. However, delivery of the flavonoids at this dose did not significantly affect the liver mitochondria susceptibility to lipid peroxidation or the level of endogenous tissue oxidative damage. Altogether the results show that cyanidin can rapidly and efficiently accumulate in mitochondria, wherein it exhibits strong bio-antioxidant activity against oxidative stress and may help protect mitochondrial function and integrity. Also, the anthocyanidin and its 3-glucoside have greater ability than flavonols to accumulate in organs; especially cyanidin presented in liver mitochondria to a much greater extent. Cyanidin could be a potent natural antioxidant compound that is effective in mitochondria-protective therapies.

The central thesis of this project is that damage caused by reactive nitrogen species, e.g. 3-nitrotyrosine (Tyr-NO2), constitutes a marker of disease progression/severity. A new sensitive electrochemiluminescence ELISA was optimised and validated for Tyr-NO2 measurement, giving a lower limit of quantification of 0.04 nM BSA-NO2, intra- and inter-assay CVs of 6.5% and 11.3%, an average recovery of 106 ± 3% and average linearity 0.998 ± 0.001. Nitrative stress, carbonyl stress and C-reactive protein (CRP) concentrations were measured before and after major elective surgery. CRP measurements confirmed the induction of an inflammatory response. Median serum Tyr-NO2 levels increased post-surgery to a median (inter-quartile range) value of 0.97 (0 – 1.7) fmol nitrated BSA (BSA-NO2) equivalents/mg protein compared with a pre-surgery level of 0.59 (0 – 1.3) fmol BSA-NO2 equivalents/mg protein (p<0.05). Oxidative damage was confirmed by serum protein carbonyl levels (p<0.05). In a second pre-/post- surgery study, patients who developed sepsis postoperatively had significantly higher serum Tyr-NO2 levels one day prior to diagnosis (median (IQR) 4.5 (1.65 – 8.21) fmol BSA-NO2 equivalents/mg protein) compared to patients without sepsis (1.2 (0.74 – 5.97) fmol BSA-NO2 equivalents/mg protein; p<0.05). Tyr-NO2 levels have not previously been measured before clinical diagnosis. However, Tyr-NO2 did not improve upon CRP as a diagnostic marker (area under the curve: Tyr-NO2 0.69 versus CRP 0.88). Nitrate (NO3¯) supplementation in healthy smokers was also studied. Plasma Tyr-NO2 levels were unaltered by supplementation or smoking status. Salivary nitration was unaffected by smoking and decreased with NO3¯ supplementation: the median (IQR) pre-supplementation was 0.67 (0.31-1.14) and post-supplementation was 0.43 (0.12-0.61) pmol BSA-NO2 equivalents/mg protein. Ozone-based chemiluminescence was utilised for nitrite (NO2¯) and NO3¯ measurement as indicators of ˙NO production. Plasma and salivary NO2¯ and NO3¯ concentrations increased significantly with NO3¯ supplementation (p<0.05). In contrast to published studies, brain frontal lobe Tyr-NO2 levels were not higher in dementia: the median (IQR) levels in dementia were 0.29 (0.19-0.57) and in non-dementia controls were 0.3 (0.22-0.55) pmol BSA-NO2 equivalents/mg protein. However, the median brain tissue NO2¯ concentration was significantly higher in the Alzheimer’s disease group (p<0.05). Western blotting revealed that nitration was predominantly in a few select proteins, with TOF-MS/MS analysis suggesting haemoglobin is one of these proteins. Measurement of nitrative stress using ozone-based chemiluminescence and an electrochemiluminescence-based-ELISA overcomes earlier methodological flaws, such as low sensitivity. Detection of total Tyr-NO2 in different inflammatory states indicates that its measurement could have potential as a marker of disease, but measurement of nitration in specific proteins may be more informative than total Tyr-NO2.