Friedreich Ataxia (FRDA) is an autosomal recessive degenerative disorder. It is caused by an abnormal expansion of GAA trinucleotide repeats in the first intron of the gene encoding frataxin. Since rates of cell division have been linked to oxidative stress, we have examined several parameters of oxidative stress in a FRDA primary fibroblast cell line that had a dramatically different growth rate. In the FRDA fibroblasts, the high level of reactive oxygen species (ROS) indicated elevated oxidative stress. The elevated glutathione peroxidase (Gpx) activity in the ROS defense system may represent an adaptive response to the high oxidative stress. The increased mitochondrial membrane potential (MMP) likely contributed to increased oxidant production, which could be contributed by elevated ROS. This increased oxidant production might be responsible for increased rate of progression through the cell cycle.<p> Furthermore, the elevated oxidative stress is also associated with progressive neural pathology of FRDA. In FRDA, pathology is first seen in the dorsal root ganglia and the dorsal columns of the spinal cord. Due to the abnormal metal distribution seen in the FRDA spinal cord and medulla, we hypothesized that metal binding proteins were abnormally distributed in FRDA. In our FRDA samples, we observed the well established histopathology of FRDA and examined the distribution of some metal binding proteins (frataxin, ferritin and metallothionein) through immunohistochemistry. Our results showed demyelination and loss of axons in the degeneration areas of the two FRDA cases. In addition, we found that the metal binding proteins were abnormally distributed in the FRDA spinal cord and the medulla. The abnormal distributions of the metal binding proteins were characterized by low expressions of iron binding proteins, especially frataxin and cytosolic ferritin, and undetectable expression of the copper and zinc binding protein, metallothionein. In summary, the rapid cell growth is a feature of FRDA fibroblast cell lines. We also tested Gpx activity, measured oxidant levels and determined the MMP in a FRDA primary fibroblast cell line that had a dramatically fast growth rate. The FRDA histopathology studies showed the metal binding proteins including frataxin, ferritin and metallothionein were abnormally distributed in the spinal cord and the medulla.

Damage to cardiac contractile proteins such as myosin light chain 1 (MLC1), during oxidative stress is mediated by reactive oxygen species such as peroxynitrite (ONOO-), resulting in impairment of cardiac systolic function. The purpose of this study is to investigate the effects of the increased level of ONOO- on MLC1 degradation by the proteolytic enzyme matrix metalloproteinase-2 (MMP-2) during oxidative stress which ultimately decreases cardiac function.<p> In the present study two distinct models were utilized to demonstrate the mechanism by which MLC1 is modified by ONOO- and how these post-translational modifications lead to its increased degradation by MMP-2. In a model of newborn hypoxia-reoxygenation in piglets we demonstrated that ONOO--induced nitration and nitrosylation of tyrosine and cysteine residues of MLC1 increase its degradation by MMP-2. Furthermore, we found nitration of a tyrosine residue located adjacent to the cleavage site for MMP-2. We verified these results by using a model of isolated rat heart myocytes to determine that the same mechanism responsible for cardiac dysfunction in newborn piglets occurs in isolated myocytes and that the MMP-2 involved in degradation of MLC1 is located within the myocytes. Moreover, we were able to determine that this mechanism occurs during ischemia itself before the onset of reperfusion. Furthermore, we have found that pharmacological intervention aimed at inhibition of MLC1 nitration/nitrosylation during ischemia by the ONOO- scavenger FeTPPS (5,10,15,20-tetrakis-[4-sulfonatophenyl]-porphyrinato-iron[III]), or inhition of MMP-2 activity with phenanthroline, provides an effective protection of cardiomyocyte contractility. The work presented here provides new evidence on the mechanisms of regulation of contractile proteins during the development of contractile dysfunction.

Several studies have found a positive correlation between hypertension and hyperhomocysteinemia. Increasing evidence implicates oxidative stress as one of the initiating events closely linked to the homocysteines ability to damage endothelium, subsequently causing vascular dysfunction. We previously found that heparin protects cultured endothelial cells from free radical injury and oral heparin at 1 mg/kg/48h prevents venous thrombosis in a rat model in vivo. Our objective was to study the protective effects of oral heparin in a rat model with elevated plasma homocysteine (Hcy) concentrations, and begin to elucidate whether the pathophysiological effects of Hcy are mediated through an oxidative mechanism causing endothelial dysfunction.<p> Elevated plasma Hcy levels were induced by feeding male Wistar Kyoto rats a diet containing an additional 1.7% methionine for 8 weeks. Groups included rats fed additional methionine, methionine plus oral heparin (1 mg/kg/48h by gastric feeding tube), and age-matched controls fed normal rat chow. At the end of 8 weeks of treatment, rats were anesthetized using 1.5% isoflurane in 100% oxygen. Hemodynamics parameters were assessed by inserting a Millar Mikro Tip pressure transducer into the left ventricular chamber and the thoracic aorta. Fasting plasma total Hcy levels were measured using a Hcy immunoassay kit with an Abbott IMx instrument. Malondialdehyde (MDA) concentrations, a lipid peroxidation product and marker for oxidative stress, was measured by a spectrophotometric method in serum and tissue samples. Glutathione (GSH) concentrations, an important antioxidant for low-level oxidative stress was measured by HPLC in plasma and tissues samples. Lastly, tissue samples from each experimental group were stained with the TUNEL method to assess their respective percentage of apoptotic endothelial cells. Results were expressed as mean ± S.E. Unpaired Students two-tailed t-test was employed to assess the difference between groups with p < 0.05 considered significant.<p> Plasma Hcy was significantly elevated after 8 weeks in the methionine (7.17 ± 0.46 umol/L) and methionine plus heparin treated rats (7.02 ± 0.40 umol/L) compared to control (5.46 ± 0.36 umol/L). All measures of arterial pressure, systolic (SP) and diastolic pressure (DP) and mean arterial pressure (MAP), were significantly elevated in rats fed the methionine diet without heparin (119.9 ± 3.9 mmHg; 90.3 ± 3.5 mmHg; 97.7 ± 2.9 mmHg, respectively) compared to controls (107.8 ± 2.5 mmHg; 79.2 ± 2.1 mmHg; 88.8 ± 2.2 mmHg, respectively) but not compared to heparin (114.7 ± 3.3 mmHg; 83.4 ± 2.4 mmHg; 93.8 ± 2.7 mmHg, respectively). Left ventricular end diastolic pressure (LVEDP) was significantly elevated with the methionine diet without heparin (14.2 ± 2.5 mmHg) but not with heparin treatment (8.4 ± 1.9 mmHg) versus controls (7.1 ± 1.1 mmHg). Also, left ventricular systolic pressure (LVSP) was significantly elevated in the methionine fed rats after 8 weeks (122.6 ± 3.2 mmHg) compared to controls (112.3. ± 2.9 mmHg). Heparin treatment had no effect on LVSP (119.9 ± 3.2 mmHg). <p> Additionally, the results of this study showed that oral heparin treatment significantly decreased liver MDA concentrations (2.42 ± 0.28 nmol/mg protein) compared to the methionine treated group (5.10 ± 0.96 nmol/mg protein) and methionine treatment alone significantly reduced MDA concentrations in kidney tissue (1.59 ± 0.12 nmol/mg protein) compared with controls (3.26 ± 0.66 nmol/mg protein). Methionine diet significantly decreased GSH concentrations in plasma (0.59 ± 0.59 µmol/L) compared with controls (4.24 ± 0.94 µmol/L) and oral heparin treatment significantly attenuated the decrease in GSH concentrations in left ventricle tissue samples (0.0229 ± 0.0023 µmol/mg protein) compared with methionine treatment alone (0.0135 ± 0.0016 µmol/mg protein). <p> Elevated plasma homocysteine levels, induced by methionine diet feeding significantly increased the percent of apoptotic endothelial cells in the aortas (17.04 ± 3.74%) and superior mesenteric arteries (17.99 ± 1.90%) of WKY rats compared with control aortas and mesenteric arteries (6.08 ± 3.24%; 7.43 ±1.62%, respectively) and compared to oral heparin treated mesenteric arteries (7.31 ± 1.18%). <p> The results of this study showed that elevated plasma levels of Hcy correlate with the development of hypertension, defined as significantly increased arterial pressure. Oral heparin treatment prevented the significant increase in arterial pressures and LVEDP, decreased MDA concentrations and therefore the oxidative stress on the liver, attenuated the decrease caused by elevated plasma Hcy in left ventricle GSH concentrations, and significantly reduced the number of apoptotic endothelial cells in the superior mesenteric artery of high methionine fed rats. We conclude that elevated levels of plasma Hcy contributes to the development of hypertension and furthermore towards the onset of heart failure likely through an oxidative mechanism and that oral heparin reduces the overall oxidative stress in specific physiological environments, preventing Hcy mediated endothelial cell apoptosis.

Chronic inflammation is considered strongly influence the morbidity and mortality of patients with end-stage renal disease (ESRD) through its multiple pathogenic roles, in association with oxidative stress, accelerated aging, endothelial dysfunction and atherosclerosis, malnutrition, dialysis-related amyloidosis, anaemia, and immune dysfunction . Hemodialysis ¡]HD¡^ is widely used for kidney failure patients,it is a method for removing waste products such as creatinine and urea. However, at present it is well known the course of hemodialysis can create obvious inflammation condition and oxidation pressure. The oxidation stress of HD can arise from the osmosis pressure and oxidative environment of dialysis tube. The oxidative stresses will finally modify proteins which turn out initiate the short term and long term complications related to renal diseases of HD patients. We identified oxidated proteins in the hemodialysis tube of 16 HD patients. The protein oxidation level was determined by Oxyblot assay. The oxidation proteins were further identified by LC/MS detection. Many serum proteins were detected to be oxidized including albumin, apoA, immunoglobin,beta-globin, hemoglobin, etc. It has been well documented that albumin is quite vulnerable to ROS and elevated levels of carbonyl groups of albumin have been reported in plasma of dialysis patients. Inflammatory effects were further tested. The oxidation proteins of HD patients induce pro-inflammatory factor TNF-alpha expression of HEK293T and HEK293 cells. These results indicate HD induce protein oxidation, and inflammatory response which may responsible for complications of End-stage kidney disease (ESRD). Keywords¡GHemodialysis, oxidative stress, ESRD, HEK293, inflammatory

Duchenne muscular dystrophy (DMD) is the most devastating form of muscular dystrophy caused by a mutation in the dystrophin gene. Defects in the dystrophin gene in DMD, are homologous to that found in mdx mice, and result in profound muscle damage, inflammation and weakness in diaphragm and limb muscles. Dystrophin, a scaffolding protein located in the sarcolemmal cytoskeleton, helps cells to maintain their structural integrity and associates with critical cell signaling molecules that regulate cell growth and repair (e.g., nNOS). While the contributing mechanisms leading to DMD-induced degenerative muscle function and damage are multi-factorial, elevated oxidative stress has been proposed as a central mechanism. In contrast, antioxidants can attenuate muscle damage as well as improve contractile function in dystrophin-deficient muscles. However, it is unknown if oxidative stress is a causal factor in dystrophin-deficient diaphragm muscle pathology and specifically targeted antioxidant (e.g., EUK-134) treated early in the course of the disease (3-4 weeks) can modulate oxidative stress, functional damage and weakness in mdx diaphragm. Therefore, the purpose of this study was to determine the effects of catalase/superoxide dismutase mimetic EUK-134 on damage, inflammation, and contractile function of the diaphragm muscle in mdx mice. We hypothesized that (a) EUK-134 would attenuate muscle damage and oxidative stress in mdx diaphragm, (b) EUK-134 would reduce inflammatory cells and an important transcription factor including nuclear factor-kappaB (NF-kB) in mdx diaphragm and (c) EUK-134 would restore proteins that attach to dystrophin such as nNOS and cytoskeletal proteins back to sarcolemmal region and improve muscle contractility in mdx diaphragm. C57BL/10ScSn wild type and mdx mice were given EUK-134 (30mg/kg, i.p., injection) beginning at 20 days of age for 8 days. The mice were euthanized and the diaphragm muscle was harvested at 4 weeks of age, the time of peak inflammation, and analyzed to measure myofiber inflammation, NF-kB activation, cytoskeletal proteins and oxidative stress markers using Western immunoblotting, ELISA, immunofluoresence, and immunohistochemistry. We found that EUK-134 ameliorated muscle damage and oxidative stress in mdx diaphragm. EUK-134 protected against inflammation by decreasing NF-kB activation in the nucleosome fraction of mdx diaphragm. Further, EUK-134 partially rescued nNOS and k-1 syntrophin back to sarcolemmal membranes and recovered force generation even in acute application in vitro in mdx diaphragm. These results are the first to demonstrate a causal relationship between oxidative stress and pathology caused by dystrophin-deficient diaphragm muscle. Moreover, the data indicate that EUK-134 has a protective effect against muscle damage, inflammation, and contractility in mdx diaphragm. We believe that the results from our investigation will provide clinical significance, as we expect to elucidate mechanisms by which oxidative stress contribute to tissue damage and weakness in dystrophic diaphragm.

This study has investigated the gene expression of ubiquitin¡B20S proteasome beta subunit type 1 (20s£]1)¡Bubiquitin-conjugating enzyme e2 (ucee2)¡BATP-dependent caseinolytic protease regulatory subunit (clpC) in the marine macroalga Ulva fasciata Delile in relation to the hypersalinity-induced oxidative stress and protein oxidation. During the early stage (0-1 h), the water contents and TTC (2,3,5-tripheny tetrazolium chloride) reduction ability maintained unchanged but recovery ability and photosynthetic ability (PS II activity as indicated by Fv/Fm) were decreased along with accumulated H2O2, suggesting the occurrence of oxidative stress. Only ubiquitin expressed at this stage. During 1-3 h, water lost (approximately 33% of the control) with a further decrease in recovery ability, TTC reduction ability¡BPS II activity but more H2O2 accumulation and protein carbonyl compound. The transcripts of 20s£]1 and clpC and caseinolytic protease activity increased at this stage with the maximum of clpC at hour 3. In the 6-48 h, water lost seriously with high accumulated free amino acid at 6-12 h but low recovery ability. The transcript amounts of ubiquitin¡B20s£]1 and ucee2 increased marked during this stage, in which these might be related to programmed cell death caused by long-term exposure to hypersalinity. Reactive oxygen species (ROS) scavengers inhibited H2O2 accumulation, caseinolytic proteolytic activity increase, carbonyl compound formation and gene expression of ubiquitin¡B20s£]1¡Bucee2¡BclpC, indicating a role of ROS in the regulation of protease genes. A role of polyamines in the regulation of protease gene expression was tested. Spermidine and spermine inhibited the gene expression of ubiquitin¡B20s£]1 and ucee2, the oxidation of proteins (carbonyl groups) and the induction of caseinolytic protease activity in 90‰-treated thalli, whereas putrescine inhibited clpC expression, the oxidation of proteins and caseinolytic protease activity but enhanced the gene expression of ubiquitin¡B20s£]1 and ucee2. In conclusion, the results of the present investigation show that the degradation of oxidatively damaged proteins under hypersalinity conditions by increased caseinolytic protease activity is driven by the up-regulation of clpC gene expression via ROS and polyamines. It seems likely that the induction of ubiquitin¡B20s£]1 and ucee2 gene expression might be associated with the hypersalinity-mediated programmed cell death.

The E. coli ArsA is involved in arsenic detoxification but the role of yArsA (ArsA homologue of Saccharomyces cerevisiae, encoded by YDL100C ORF) in yeast is still undefined. Disruption of YDL100C ORF is not lethal. To study the role of yArsA in oxidative tolerance, wild type and knock out strain were grown in presence or absence of 1 mM H2O2 and assayed the expression of anti-oxidation machanisms . The results show that molecular oxidation is higher and catalase activity is lower in KO compared with WT. It suggests that increased ROS and decreased catalase activity are the cause of cell death. Further analysis of the expression of ROS defense mechanisms by RT-PCR show that there is no significant difference in TRR1, GSH1, and SOD1 expression in WT and KO grown in presence of 1 mM H2O2 but the CTT1, TPS1, NTH1 expression in KO are less than WT grown under oxidative stress. GSH contents is consistent with the result of RT-PCR, and trehalose contents is higher in KO strain under oxidative stress. Loss of catalase activity and decreased efficiency of degrading trehalose suggest that the deficiency in activation of general stress response in KO when grown in the presence of H2O2. Therefore, yArsA would be involved in expressing the general stress response in oxidative tolerance.

Thesis (M. S. in Chemistry)--Vanderbilt University, Dec. 2009. / Title from title screen. Includes bibliographical references.

Dissertation (Ph.D.)--Auburn University, 2009. / Abstract. Includes bibliographic records (p.71-89).

Evidence shows that consumption of a Mediterranean diet can lower the risk of all-cause and cause-specific mortality suggesting that this diet has an overall effect on health. Antioxidants found within olive oil, the primary source of fat in the Mediterranean diet, may be leading contributors to the decreased disease risk. More specifically, hydroxytyrosol (HT), one of the most active and powerful antioxidants found in olive oil, has the ability to increase total antioxidant status and lower levels of lipid peroxidation. In addition to a healthy diet, physical activity decreases the risk of cardiovascular morbidity and mortality; however, aerobic exercise of sufficient intensity or duration can induce oxidative stress. Therefore, the purpose of this study was to investigate the effects of 6 weeks of HT supplementation on antioxidant status and markers of inflammation in healthy, recreationally active males before and throughout acute aerobic exercise bouts. Using a randomized, double-blind, repeated-measures, placebo-controlled design, sixty-one (n=61) participants were randomly assigned to consume a placebo (PLA), low dose of HT (LHT, 50 mg/day), or high dose of HT (HHT, 150 mg/day). Throughout the course of the study, the participants performed four time trial rides (TT1-TT4) on cycle ergometers. TT1 occurred before supplementation, TT2 halfway through the supplementation period, and TT3 and TT4 occurred in the sixth week and final two days of supplementation. Blood was drawn prior to (pre) and just before termination (end) of each time trial to measure markers of antioxidant status and inflammation during exercise. We did not observe significant main effects for treatment on any of the markers for antioxidant status (TEAC) or for markers of inflammation (oxLDL, CRP, 8IP, TNFα, IL-6, IL-10, IL-1β, or IL-1ra). Significant treatment-by-time interactions occurred for CRP, 8IP, and IL-6 although significant treatment differences in these measures were not detected. We conclude that chronic and acute HT supplementation does not improve antioxidant status nor decrease markers of inflammation in this population at rest, during, or following exercise. / text