Methanogens are a group of microorganisms that utilize simple compounds such as H₂ + CO₂, acetate and methanol for the production of methane, an end-product of their metabolism.  These obligate anaerobes belonging to the archaeal domain inhabit diverse anoxic environments such as rice paddy fields, human guts, rumen of ruminants, and hydrothermal vents.  In these habitats, methanogens are often exposed to O₂ and previous studies have shown that many methanogens are able to tolerate O2 exposure.  Hence, methanogens must have developed survival strategies to be able to live under oxidative stress conditions.  The anaerobic species that lived on Earth during the early oxygenation event were first to face oxidative stress.  Presumably some of the strategies employed by extant methanogens for combating oxidative stress were developed on early Earth.   Our laboratory is interested in studying the mechanism underlying the oxygen tolerance and oxidative stress responses in methanogenic archaea, which are obligate anaerobe.  Our research concerns two aspects of oxidative stress.  (i) Responses toward extracellular toxic species such as SO32-, that forms as a result of reactions of O₂ with reduced compounds in the environment.  These species are mostly seen in anaerobic environments upon O₂ exposure due to the abundance of reduced components therein.  (ii) Responses toward intracellular toxic species such as superoxide and hydrogen peroxide that are generated upon entry of O₂ and subsequent reaction of O₂ with reduced component inside the cell.  Aerobic microorganisms experience the second problem.  Since a large number of microorganisms of Earth are anaerobes and the oxidative defense mechanisms of anaerobes are relatively less studied, the research in our laboratory has focused on this area.  My thesis research covers two studies that fall in the above-mentioned two focus areas. In 2005-2007 our laboratory discovered that certain methanogens use an unusual sulfite reductase, named F420-dependent sulfite reductase (Fsr), for the detoxification of SO32- that is produced outside the cell from a reaction between oxygen and sulfide.  This reaction occurred during early oxygenation of Earth and continues to occur in deep-sea hydrothermal vents.  Fsr, a flavoprotein, carries out a 6-electron reduction of SO32- to S2-.  It is a chimeric protein where N- and C-terminal halves (Fsr-N and Fsr-C) are homologs of F420H2 dehydrogenase and dissimilatory sulfite reductase (Dsr), respectively.  We hypothesized that Fsr was developed in a methanogen from pre-existing parts.  To begin testing this hypothesis we have carried out bioinformatics analyses of methanogen genomes and found that both Fsr-N homologs and Fsr-C homologs are abundant in methanogens.  We called the Fsr-C homolog dissimilatory sulfite reductase-like protein (Dsr-LP).  Thus, Fsr was likely assembled from freestanding Fsr-N homologs and Dsr-like proteins (Dsr-LP) in methanogens.  During the course of this study, we also identified two new putative F420H2-dependent enzymes, namely F420H2-dependent glutamate synthase and assimilatory sulfite reductase. Another aspect of my research concerns the reactivation of proteins that are deactivated by the entry of oxygen inside the cell.  Here I focused specifically on the role of thioredoxin (Trx) in methanogens.  Trx, a small redox regulatory protein, is ubiquitous in all living cells.  In bacteria and eukarya, Trx regulates a wide variety of cellular processes including cell divison, biosynthesis and oxidative stress response.  Though some Trxs of methanogens have been structurally and biochemically characterized, their physiological roles in these organisms are unknown.  Our bioinformatics analysis suggested that Trx is ubiquitous in methanogens and the pattern of its distribution in various phylogenetic classes paralleled the respective evolutionary histories and metabolic versatilities.  Using a proteomics approach, we have identified 155 Trx targets in a hyperthermophilic phylogenetically deeply-rooted methanogen, Methanocaldococcus jannaschii.  Our analysis of two of these targets employing biochemical assays suggested that Trx is needed for reactivation of oxidatively deactivated enzymes in M. jannaschii.  To our knowledge, this is the first report on the role of Trx in an organism from the archaeal domain. During the course of our work on methanogen Trxs, we investigated the evolutionary histories of different Trx systems that are composed of Trxs and cognate Trx reductases.  In collaboration with other laboratories, we conducted bioinformatics analysis for the distribution of one of such systems, ferredoxin-dependent thioredoxin reductase (FTR), in all organisms.  We found that FTR was most likely originated in the phylogenetically deeply-rooted microaerophilic bacteria where it regulates CO₂ fixation via the reverse citric acid cycle. / Ph. D.

Yes / Age-related frailty is a significant health and social care burden, with limited treatment options. There is a lack of suitable cell culture model for screening large numbers of test compounds to identify those which promote healthy skeletal muscle function. This paper describes the characterization of reactive oxygen and nitrogen species (RONS) signalling changes in young and aged myoblasts and myotubes using C2C12 cells, and the application of aged cultures to assess the effect of dietary polyphenols on RONS signalling. Aged myoblasts and myotubes showed significantly increased reactive oxygen species (p < 0.01 and p < 0.001 respectively), nitric oxide (p < 0.05 for myoblasts and myotubes), and lipid peroxidation (p < 0.05 for myoblasts and myotubes). Nine polyphenols were assessed in aged myoblasts and myotubes using concentrations and incubation times consistent with known pharmacokinetic parameters for these compounds. Although several polyphenols were seen to reduce single markers of RONS signalling, only kaempferol and resveratrol significantly reduced multiple markers in both cell models. Modulation of enzymatic antioxidant activities was assessed as a possible mechanism of action, although superoxide dismutase and catalase activities were significantly reduced in aged (versus young) myotubes (p < 0.01 and p < 0.05 respectively), no effect of polyphenol treatment on these enzyme activities were observed. Overall, this research has shown the utility of the C2C12 model (myoblasts and myotubes) for screening compounds in aged muscle, and that resveratrol and kaempferol (using pharmacokinetically-informed exposures) can modulate RONS signalling in skeletal muscle cells after an acute exposure. / NH is supported by a University of Hull-funded PhD studentship.

No / Drinking water disinfection byproducts (DBPs) are generated by the chemical disinfection of water and may pose a hazard to the public health. Previously we demonstrated that iodoacetic acid was the most cytotoxic and genotoxic DBP analyzed in a mammalian cell system. Little is known of the mechanisms of its genotoxicity. The involvement of oxidative stress in the toxicity of iodoacetic acid was analyzed with the antioxidants catalase and butylated hydroxyanisole (BHA). Iodoacetic acid toxicity was quantitatively measured with and without antioxidants in Salmonella typhimurium strain TA100 and with Chinese hamster ovary (CHO) cells. The endpoints included cytotoxicity in S. typhimurium or in CHO cells, mutagenicity in S. typhimurium, and genotoxicity in CHO cells. Neither catalase nor BHA reduced the level of iodoacetic acid induced cytotoxicity in S. typhimurium. In CHO cells neither antioxidant caused a significant reduction in iodoacetic acid induced cytotoxicity. However, in S. typhimurium, BHA or catalase reduced the mutagenicity of iodoacetic acid by 33.5 and 26.8%, respectively. Likewise, BHA or catalase reduced iodoacetic acid induced genomic DNA damage by 86.5 and 42%, respectively. These results support the hypothesis that oxidative stress is involved in the induction of genotoxicity and mutagenicity by iodoacetic acid.

Yes / The current study was designed to investigate the influence of monofloral Indian mustard bee pollen (MIMBP) and processed monofloral Indian mustard bee pollen (PMIMBP) supplementation on chronic swimming exercise-induced oxidative stress implications in the gastrocnemius muscle of Wistar rats. MIMBP was processed with an edible lipid-surfactant mixture (Captex 355:Tween 80) to increase the extraction of polyphenols and flavonoid aglycones as analyzed by UV spectroscopy and high performance liquid chromatography-photo diode array. Wistar rats in different groups were fed with MIMBP or PMIMBP supplements at a dose of 100 mg/kg, 200 mg/kg and 300 mg/kg individually, while being subjected to chronic swimming exercise for 4 weeks (5 d/wk). Various biochemical [superoxide dismutase (SOD), glutathione (GSH), malonaldehyde (MDA), nitric oxide (NO), and total protein content], mitochondrial (Complex I, II, III, and IV enzyme activity), and molecular (myostatin mRNA expression) parameters were monitored in the gastrocnemius muscle of each group. Administration of both MIMBP (300 mg/kg) and PMIMBP (100 mg/kg, 200 mg/kg, and 300 mg/kg) wielded an antioxidant effect by significantly improving SOD, GSH, MDA, NO, and total protein levels. Further MIMBP (300 mg/kg) and PMIMBP (200 mg/kg and 300 mg/kg) significantly improved impaired mitochondrial Complex I, II, III, and IV enzyme activity. Significant down-regulation of myostatin mRNA expression by MIMBP (300 mg/kg) and PMIMBP (200 mg/kg and 300 mg/kg) indicates a muscle protectant role in oxidative stress conditions. The study establishes the antioxidant, mitochondrial upregulatory, and myostatin inhibitory effects of both MIMBP and PMIMBP in exercise-induced oxidative stress conditions, suggesting their usefulness in effective management of exercise-induced muscular stress. Further, processing of MIMBP with an edible lipid-surfactant mixture was found to improve the therapeutic efficiency of pollen.

Metabolic redox status may have important implications to cattle health and production. Antioxidants and biomarkers of oxidative stress were evaluated in cattle under three phases of management. Each phase stood alone as a treatment model, and managerial aspects during the phase were evaluated as potential moderators of redox balance. Yearling heifers were used to assess the impact of fescue toxicosis and heat stress on selected markers in study 1. Intravaginal temperatures, ADG, serum prolactin, plasma malondialdehyde, and whole blood Se, along with peripheral blood mononuclear cell glutathione peroxidase, glutathione reductase, and reduced:oxidized glutathione were determined during summer grazing. Results suggested that endophyte consumption does not promote oxidative stress in cattle. Heat stress may alter glutathione redox of white blood cells. In study 2, effects of gradual weaning strategies (anti-suckle nose clip and fenceline wean) and transport were evaluated in calves. Calf weights, Se and malondialdehyde in plasma, along with glutathione peroxidase and glutathione reductase in leukocytes were measured at -7, 0, 1, and 7 days surrounding weaning and transport. Little benefit of gradual weaning was detected, and oxidative stress may have been negligible. In study 3, the influences of grain- and forage-based diets were compared in finishing steers pre- and post-harvest. Total antioxidant capacity and malondialdehyde concentration of plasma, along with serum alpha-tocopherol, beta-carotene, and gamma-tocopherol were measured. Antioxidants and lipid oxidation were assessed in beef. Forages promoting plasma antioxidant capacity may protect cattle against oxidative stress. Antioxidants derived from forages inhibit lipid oxidation in pasture-finished beef. / Master of Science

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease affecting 1 in every 3500 boys. The disease is characterized by the absence of the dystrophin protein from the sarcolemma of muscle cells. Muscle cells lacking dystrophin go through cycles of degeneration and regeneration and are considered susceptible to contraction-induced injury 144. Eventually, the satellite cell proliferative capacity is exhausted and the muscle fibers are replaced by connective and adipose tissue that yields a progressive loss of force generating capability. DMD patients typically die by their early 20's, primarily due to respiratory or cardiac failure. The precise role of dystrophin is not presently known. However, its absence suggests that it may play a role in both cellular calcium regulation and oxidative stress 152. Recent studies suggest increased reactive oxygen species (ROS) may precede the initial wave of wasting that marks disease onset 49. Therefore, it is possible oxidative stress may contribute as a pathogenic mechanism of DMD. Strategies to reduce the deleterious effects of oxidative stress could be an effective therapeutic approach. Regular exercise is known to increase antioxidant capacity in humans and mice 146. Green tea extract (GTE) is a powerful antioxidant that is easily supplemented in the diet 83. The purpose of this study was to test the hypotheses that (1) voluntary endurance exercise alone, (2) a diet supplemented with 0.05% (wt/wt) GTE alone, or exercise and GTE combined will blunt the effects of ROS and improve muscle strength and endurance in young mdx mice. Male mdx mice at age 21-days were randomly divided into one of 4 treatment groups: Run Normal diet (RunNorm; n=8), Sedentary Normal diet (SedNorm; n=8), Run GTE diet (RunGTE; n=10), and Sedentary GTE diet (SedGTE; n=8). RunNorm and RunGTE mice were given free access to a running wheel for 3 weeks while SedNorm and SedGTE mice were restricted to normal cage movement. At the end of 3 weeks, mice in each treatment group were sacrificed and assessed for daily and weekly running distances, content of actin and myosin proteins and fiber type distribution (tibialis anterior), contractile/mechanical and fatigue properties (extensor digitorum longus), creatine kinase levels and antioxidant capacity (serum), lipid peroxidation (gastrocnemius), and citrate synthase and beta-hydroxyacyl-CoA dehydrogenase activities (quadriceps and soleus). The key findings of this study were: In normal diet running mice (RunNorm), average daily distance run was increased 300% (from 0.5 to 2.1 km/d, P<0.05) from week 1 to week 3. In GTE diet (RunGTE) compared to RunNorm mice, total distance over the 3 weeks was markedly improved 128% (61.2 vs. 26.8 km, P<0.0001). Running, independent of diet increased EDL muscle tetanic stress (18%), serum antioxidant capacity (22%), citrate synthase activity (35%), and beta-oxidation (37%; all P<0.05). GTE, independent of running decreased lipid peroxidation (gastrocnemius:-64%; heart: -29%) and serum creatine kinase (-36%), and increased citrate synthase activity (59%; all P<0.05). These findings in dystrophic mice suggest that voluntary endurance exercise with or without GTE supplementation blunted the deleterious effects of ROS. If similar positive effects are evident in human DMD patients, then these approaches may be beneficial therapies either alone or in combination. / Master of Science

Oxidative stress is the imbalance of oxidants and reductants; it affects different parts of the organisms. In methane-producing archaea, Methanosarcina acetivorans, a rubrerythrin-like protein methanogen rubrerythrin, is involved in the oxidative defense of the microorganism. It is a 45 kDa homodimer protein in the buffer solution. ICPMS and EXAFS analyses confirmed the presence of iron and zinc in this protein's structure. EPR confirmed the iron (Fe3+) of the rubredoxin center with the characteristic peak in the low field having a 9.35 g value and a diiron center peak in the high field region with a characteristic 2.01 g value. A BioSAXS analysis was conducted to investigate the structural changes in a mRbr under different conditions. The results revealed that the protein undergoes substantial structural modifications when exposed to either oxidation or reduction. When treated with H2O2, the protein sample displayed an Rg of 47Ao and a Dmax of 167 Ao, indicating a larger size than the reduced state. Conversely, when subjected to Na2S2O4, the protein sample had an Rg of 45 Ao and a Dmax of 45 Ao.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第25516号 / 生博第532号 / 新制||生||70(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 鈴木 淳, 教授 北島 智也, 教授 見学 美根子 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Hyperglycaemia stimulates a variety of biochemical abnormalities. The area of particular interest in this study is the influence of non-enzymatic glycation of proteins on iron homeostasis, and particularly on non-transferrin-bound iron (NTBI) and its possible relation to atherogenesis in both Type 2 diabetic and obese non diabetic subjects. The link between non-enzymatic glycation of proteins and iron homeostasis, and development of macrovascular disease may be mechanistically different in Type 2 diabetic and obese non diabetic subjects due to a difference in the protein glycation pattern. Because the following in vivo study required storage of samples for up to two years to complete the processing of all the samples, a storage study was carried out using different anticoagulants and addition of reduced glutathione (GSH) to samples to study the effects of storage, thawing and freezing of the samples on the level of malondialdehyde (MDA), a biomarker of lipid peroxidation. This storage study showed that EDTA attenuated the action of lipid oxidation compared with lithium heparin (LiH). A combination of GSH with either EDTA or LiH added more protection from lipid peroxidation in the first week of storage, but due to the thawing and freezing of the sample the action of GSH diminished through its autooxidation, meaning that addition of GSH to samples in the following in vivo study would be useless. An in vivo study was carried out on iron-related parameters in three subject groups: control (non-diabetic, non-obese), Type 2 diabetic and obese non diabetic. Glycated haemoglobin (HbA1C) was strongly correlated with NTBI in the diabetic group. Also the level of NTBI was significantly increased in Type 2 diabetic subjects compared with other groups while the level of total iron was significantly decreased. The study showed a strong positive correlation between NTBI and a biomarker of endothelium dysfunction (E-selectin) in all groups studied. Although it is not possible from the current data to know if there is a causal relationship between these two parameters, it remains a possibility that iron released from its binding sites could initiate oxidative damage to the endothelial cells and begin the process of atherogenesis. Positive correlation at the 90% confidence level between NTBI and a biomarker of inflammation, high sensitivity C-reactive protein, is another indicator in this study of a link between increases in NTBI, inflammation, endothelium dysfunction and atherosclerosis. This study also showed for first time that NTBI is present in higher levels in the plasma of obese subjects compared to controls despite the obese subjects having significantly lower total iron. An in vitro study found that glycation of transferrin half saturated with iron increased with increasing glucose concentration, leading to decreased capacity of transferrin to hold iron and increased release of free iron. Also co-incubation of transferrin half saturated with iron with low density lipoprotein (LDL) and glucose showed oxidation of LDL (measured as MDA). This may be explained by the effect of glycation, leading to release of free iron, which catalyses oxidation of LDL. In addition, glycation of LDL may enhance the oxidation of LDL catalysed by iron. Both studies indicate that the glycation of proteins has a major impact on iron homeostasis leading to release of non-enzymatic glycation and contributing to one of the most common complications of Type 2 diabetes, atherosclerosis.

Fresh isolated erythrocyte ghost membranes and erythrocyte suspensions were incubated with an organic hydroperoxide, tert-butyl hydroperoxide, to establish the in vitro oxidative stress models. Circulating erythrocytes from normal individuals were fractionated into subpopulations of different ages by ultracentrifugation and used as an in vivo model. In these models, membrane iPF2alpha-III content accumulation was proportional to oxidative stress and correlated with decreased membrane fluidity. In circulating erythrocytes, membrane iPF2alpha-III increased with age and inversely correlated with membrane fluidity only in the core region. / Oxidative stress is involved in the pathophysiology of a wide variety of human diseases. Isoprostanes, a family of prostaglandin derivatives, are mainly derived from free radical peroxidation of specific polyunsaturated fatty acids (PUFA). Measurement of F2-isoprostanes (F2-iPs) or one specific biologically active isomer (iPF2alpha-III) is considered to be a reliable lipid peroxidation marker in human diseases. However, the association observed between increased plasma/urine F2-iPs and diseases does not necessarily reflect tissue oxidative damages. Circulating erythrocytes, a tissue with limited biosynthetic capacity and poor repair mechanism, would offer a number of advantages for assessment of in vivo oxidative damages. In this thesis, human erythrocyte membrane iPF2alpha-III content was investigated as a new marker for in vivo oxidative stress assessment. Membrane fluidity was used as an indirect marker of cellular function. / To use membrane iPF2alpha-III in a human disease with known oxidative stress burden, 49 Chinese patients on long-term haemodialysis and 31 healthy Chinese subjects were recruited. Both plasma and membrane iPF 2alpha-III showed that haemodialysis patients had increased oxidative stress. Only membrane iPF2alpha-III, but not the conventional used plasma iPF2alpha-III, correlated with membrane fluidity. Furthermore, the significant inverse correlation between membrane iPF 2alpha-III and the core region of membrane fluidity was observed for this group of patients too. Since membrane iPF2alpha-III was shown to provide a link between oxidative stress and erythrocyte function, it would be considered as a new marker of in vivo erythrocyte oxidative stress assessment. (Abstract shortened by UMI.) / Yu Xiongwen. / "July 2005." / Advisers: Wai Kei Christopher Lam; Chung Shun Ho. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3724. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 198-223). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.