The neuroprotective effects of AF4, a flavonoid-enriched extract derived from the peel of Northern Spy apples (containing quercetin-3-O-glucoside, quercetin-3-O-galactoside, quercetin-3-O-rhamnoside, quercetin-3-O-rutinoside, epicatechin, and cyanidin-3-O-galactoside) were examined by assessing neuronal loss and motor impairment resulting from hypoxic-ischemic (HI) brain injury in adult C57BL/6 mice. Relative to vehicle treatment (water, 10mL/kg/day), oral administration of AF4 (50 mg/kg/day) for 3 days reduces HI-induced neuronal loss in the striatum and hippocampus, motor impairments, and reduces the ability of LPS to stimulate the production of TNF-alpha in whole blood. Pretreatment with AF4 (1 ug/mL) decreased the death of mouse primary cortical neurons subjected to oxygen glucose deprivation (12 hours) in comparison to vehicle (DMSO) or the same concentration of quercetin or its metabolites. Taken together these findings indicate that AF4 reduces HI-induced brain injury and motor deficits by increasing the resistance of vulnerable neurons to ischemic cell death and decreasing the production of inflammatory cytokines.

Oxidative stress resulting from an imbalance between free radicals and antioxidants is considered to be an important etiological factor in the development and maintenance of hypertension. Angiotensin II (Ang II) has been shown to be an important regulator of blood pressure and acts to elevate blood pressure by its pressor effects. The pressor effects of Ang II are well documented but recent evidence has suggested another possible role of Ang II in elevating blood pressure, whereby it acts via an independent mechanism that is directly linked with oxidative stress. The spontaneously hypertensive rat (SHR) is a widely used model in the investigation of the pathophysiological mechanisms involved in hypertension. This study was therefore undertaken to determine whether Ang II acts as a causative factor via oxidative stress in the development and maintenance of hypertension in the SHR. This was elucidated by evaluating the role of both the endogenous in vivo levels of Ang II as well as an exogenous sub-pressor dose of Ang II, on oxidative stress and its associated parameters. The parameters evaluated included, the antioxidant status of the model on the basis of the levels of the major antioxidant enzymes viz. SOD, GPx and catalase; the free radical generating capacity, by assessing the activity of the membrane bound enzyme NADPH oxidase and the levels of H2O2. The study also evaluated the levels of the endogenous vasodilator nitric oxide (NO), remodelling of the vasculature and the level of tissue oxidative stress in the kidney. The results show that the SHR has an elevated plasma Ang II level and an elevated level of oxidative stress, thus showing that in this model there is an intimate link between oxidative stress and Ang II. The SHR also shows depleted levels of NO and thus a decreased vasodilatory capacity and increased remodelling of the vasculature. The kidney showed an increased level of lipid peroxidation, which was due to the elevated levels of oxidative stress. All of these pathophysiological changes contribute to the elevation in blood pressure in this model. The long term infusion of the sub-pressor dose of Ang II affected the SHR to a greater extent than the Wistar. Although the dose of Ang II elevated the blood pressure in both models, the degree of the pathophysiological changes associated with the elevation in blood pressure was greater in the SHR. The Ang II infusion in both these models demonstrated that in the SHR which is genetically predisposed to hypertension, adjustments are made to the antioxidant system, that result in an elevated level of protection against oxidative stress. These results show that Ang II acts as a causative factor in the pathogenesis of hypertension in the SHR via its well documented pressor effects, as well as via a multitude of independent mechanisms that are linked to oxidative stress. This is substantiated by the significant decrease in NO that is caused by the elevated oxidative stress, as well as the previously described pathophysiological changes. This study has therefore shown that Ang II has an intimate causative link with oxidative stress that results in parallel mechanisms that work concomitantly with each other in hypertension in this model. / Thesis (Ph.D.)-University of KwaZulu-Natal, Westville, 2011.

Type 2 diabetes is now recognized as a worldwide epidemic. Pancreatic beta-cell decompensation in the presence of insulin resistance is a major mechanism for the development of type 2 diabetes and may be triggered by mitochondrial dysfunction. Alcoholism is a known risk factor for type 2 diabetes. Excessive or chronic alcohol consumption leads to increased oxidative stress and mitochondrial dysfunction in beta-cells. Prohibitin is a multifunctional protein that also regulates mitochondrial biogenesis and function. Although it has anti-oxidant effects in some cell types, its role in pancreatic beta-cells is not known. This study has investigated the effects of prohibitin in ethanol treated pancreatic beta-cells using RINm5F and INS-1E cell lines. Prohibitin was found to be expressed in pancreatic beta-cells with localization to the nucleus and the perinuclear area. Ethanol increased the expression of prohibitin and induced its translocation from the nucleus to the mitochondria. Ethanol, through its metabolism by alcohol dehydrogenase (ADH), increased oxidative stress and altered mitochondrial membrane potential, decreased the activity of mitochondrial respiratory complexes I and IV, and uncoupled energy production with resulting reduction in ATP production. This was associated with activation of the proinflammatory enzyme c-Jun N-terminal kinase and proapoptotic proteins Bax and caspase-3, leading to beta-cell apoptosis. Ethanol also reduced glucose induced insulin secretion without alteration of the beta-cell transcription factors PDX-1 and MafA. Treatment with exogenous prohibitin or cellular overexpression of endogenous prohibitin attenuated ADH activity, prevented the deleterious effects of ethanol on mitochondrial function and reduced apoptosis, whereas prohibitin knockdown enhanced ethanol-induced apoptosis. In addition, prohibitin per se increased PDX-1 and MafA levels. Through the above mechanisms, prohibitin restored glucose induced insulin secretion in ethanol exposed beta-cells. In brief, ethanol causes mitochondrial dysfunction and induces apoptosis in beta-cells, which result in a reduction of insulin secretion; whereas prohibitin prevents mitochondrial dysfunction, apoptosis, and -cell failure by stabilizing mitochondrial complexes I and IV and inhibiting ADH activity during ethanol metabolism. In addition, prohibitin in itself increases the levels of beta-cell transcription factors. As a consequence, prohibitin maintains normal pancreatic beta-cell function and could be useful in diabetes prevention and treatment.

Converging evidence indicates that SOD1 aggregation is a common feature of mutant SOD1 (mSOD1)-linked FALS, and seems to be directly related to the gain-of-function toxic property. However, the mechanisms of protein aggregation are not fully understood. To study the contribution of modification on cysteine residues in SOD1 aggregation, we systematically examined the redox state of SOD1 cysteine residues in the G37R transgenic mouse at different stages of ALS and under oxidative stress induced by H2O2. Our data showed that under normal circumstances, cysteine 111 in SOD1 is free. Under oxidative stress, it is prone to oxidative modification by providing the thiolate anion (S-). With the progression of ALS, increased levels of oxidative insults facilitated the oxidation of thiol groups of cysteine residues. Human mutant SOD1 could generate an upper shifted band in SDS-PAGE, which turned out to be a Cys111-peroxidized SOD1 species. We also found that at different stages of ALS, accumulated oxidative stress facilitated the aggregates formation, which were not mediated by disulfide bond. The oxidative modification of cysteine 111 may promote the formation of disulfide bond-independent SOD1 aggregates. In addition, we investigated the correlation between nitrosative stress and S-nitrosylation of protein disulfide isomerase (PDI) in the mechanism of aggregates formation. Our data showed that up-regulated inducible nitric oxide synthase (iNOS) generated high levels of nitric oxide (NO), which induced S-nitrosylation of PDI with the progression of ALS in the spinal cords of mSOD1 transgenic mice. This correlation was confirmed by treating SH-SY5Y cells with NO donor SNOC to trigger the formation of S-nitrosylated PDI (SNO-PDI). When mSOD1 was overexpressed in SH-SY5Y cells, iNOS expression was up-regulated, NO generation was increased consequently. Furthermore, both SNO-PDI and mSOD1 aggregates were detected in these cells. Blocking NO generation with NOS inhibitor N-nitro-L-arginine (NNA) attenuated the S-nitrosylation of PDI; the formation of mSOD1 aggregates was inhibited as well. We conclude that NO-mediated S-nitrosylation of PDI is highly linked to the accumulation of mSOD1 aggregates in ALS.

Lycopene is a antioxidant found in natural and processed foods. The connection between antioxidants and heart disease has been explored in several observational studies1-4, yet very few investigators have examined the impact of dietary antioxidants in patient with advanced heart disease such as heart failure (HF). A novel strategy for preventing or delaying the complications of HF related to inflammation and oxidative stress may be to increase dietary lycopene. The purpose of this dissertation was to test the impact of dietary intervention consisting of lycopene (V8® juice) on biomarkers of inflammation and oxidative stress in patients with HF; Prior to testing the dietary intervention, preliminary work was conducted: 1) a review of the literature on dietary lycopene interventions in patients with HF and 2) a longitudinal study to examine whether lycopene and sodium intake interact to produce an effect on event-free survival in patients with HF. Forty patients with HF were randomly assigned to one of two treatment groups (intervention and usual care). The intervention group received 24 mg of lycopene by drinking 11.5 ounces of V8®100% vegetable juice daily for 30 days. The usual care group continued their usual diet. Serum levels of uric acid and C-reactive protein were obtained to determine the impact of the lycopene dietary intervention. Patients in the intervention group had higher levels of plasma lycopene after one month drinking V8® juice. We also found a significant decrease in plasma CRP levels among women in the intervention group, while there was no change in CRP levels among men in the intervention group. This dissertation has provided insight about lycopene as a potential nutritional intervention for patients with HF, aimed at reducing inflammation and oxidative stress. This dietary intervention is practical, easy to replicate, cost effective and is safe for patients with HF. Additional research is needed to determine the effects of long-term outcomes of dietary antioxidants in patients with HF.

The work described in this thesis investigates the mechanisms by which copper complexes catalyse the generation of reactive oxygen species (ROS), including the highly reactive hydroxyl radical (.OH), and induce oxidative damage to DNA. An ESR study into the copper-Fenton reaction revealed that, in the presence of buffers and other copper chelators, .OH is generated. In contrast, it is suggested that a Cu(III) species may be formed in the reaction of aqueous, unchelated copper ions. The generation of .OH in the copper-Fenton reaction, under biomimetic conditions, was confirmed by analysis of the products formed following the incubation of DNA components with this system. Preferential binding of Cu(II) to guanosine over the other nucleosides was determined and copper redox cycling at GC sites was found to be more facile than at AT sites. Stability constants for the copper complexes with several other biochemically important ligands such as glutathione (GSH), Quin2 and 1,10-phenanthroline (OP) were also measured. The ease of redox cycling for the copper complexes was found to be of the order: OP ~ Quin2 > GSH. However, OP enhanced both the copper-Fenton reaction and copper-induced DNA damage while both GSH and Quin2 were inhibitory. Gel chromatography studies suggested that ternary complex formation occurs between Cu(I)-DNA and both Quin2 and OP. This implies that the ternary complex with OP is more redox active than that with Quin2. Whilst cysteine enhanced copper-mediated DNA damage at early incubation times, it was more protective than GSH and homocysteine at later stages. The effects at early incubation times are attributed to the ease of copper redox cycling in the presence of thiols while the effects over prolonged incubations reflect Cu(II) stabilisation by the respective disulphides or similar products.

No description available.

No description available.

Type 2 diabetes is now recognized as a worldwide epidemic. Pancreatic beta-cell decompensation in the presence of insulin resistance is a major mechanism for the development of type 2 diabetes and may be triggered by mitochondrial dysfunction. Alcoholism is a known risk factor for type 2 diabetes. Excessive or chronic alcohol consumption leads to increased oxidative stress and mitochondrial dysfunction in beta-cells. Prohibitin is a multifunctional protein that also regulates mitochondrial biogenesis and function. Although it has anti-oxidant effects in some cell types, its role in pancreatic beta-cells is not known. This study has investigated the effects of prohibitin in ethanol treated pancreatic beta-cells using RINm5F and INS-1E cell lines. Prohibitin was found to be expressed in pancreatic beta-cells with localization to the nucleus and the perinuclear area. Ethanol increased the expression of prohibitin and induced its translocation from the nucleus to the mitochondria. Ethanol, through its metabolism by alcohol dehydrogenase (ADH), increased oxidative stress and altered mitochondrial membrane potential, decreased the activity of mitochondrial respiratory complexes I and IV, and uncoupled energy production with resulting reduction in ATP production. This was associated with activation of the proinflammatory enzyme c-Jun N-terminal kinase and proapoptotic proteins Bax and caspase-3, leading to beta-cell apoptosis. Ethanol also reduced glucose induced insulin secretion without alteration of the beta-cell transcription factors PDX-1 and MafA. Treatment with exogenous prohibitin or cellular overexpression of endogenous prohibitin attenuated ADH activity, prevented the deleterious effects of ethanol on mitochondrial function and reduced apoptosis, whereas prohibitin knockdown enhanced ethanol-induced apoptosis. In addition, prohibitin per se increased PDX-1 and MafA levels. Through the above mechanisms, prohibitin restored glucose induced insulin secretion in ethanol exposed beta-cells. In brief, ethanol causes mitochondrial dysfunction and induces apoptosis in beta-cells, which result in a reduction of insulin secretion; whereas prohibitin prevents mitochondrial dysfunction, apoptosis, and -cell failure by stabilizing mitochondrial complexes I and IV and inhibiting ADH activity during ethanol metabolism. In addition, prohibitin in itself increases the levels of beta-cell transcription factors. As a consequence, prohibitin maintains normal pancreatic beta-cell function and could be useful in diabetes prevention and treatment.

Converging evidence indicates that SOD1 aggregation is a common feature of mutant SOD1 (mSOD1)-linked FALS, and seems to be directly related to the gain-of-function toxic property. However, the mechanisms of protein aggregation are not fully understood. To study the contribution of modification on cysteine residues in SOD1 aggregation, we systematically examined the redox state of SOD1 cysteine residues in the G37R transgenic mouse at different stages of ALS and under oxidative stress induced by H2O2. Our data showed that under normal circumstances, cysteine 111 in SOD1 is free. Under oxidative stress, it is prone to oxidative modification by providing the thiolate anion (S-). With the progression of ALS, increased levels of oxidative insults facilitated the oxidation of thiol groups of cysteine residues. Human mutant SOD1 could generate an upper shifted band in SDS-PAGE, which turned out to be a Cys111-peroxidized SOD1 species. We also found that at different stages of ALS, accumulated oxidative stress facilitated the aggregates formation, which were not mediated by disulfide bond. The oxidative modification of cysteine 111 may promote the formation of disulfide bond-independent SOD1 aggregates. In addition, we investigated the correlation between nitrosative stress and S-nitrosylation of protein disulfide isomerase (PDI) in the mechanism of aggregates formation. Our data showed that up-regulated inducible nitric oxide synthase (iNOS) generated high levels of nitric oxide (NO), which induced S-nitrosylation of PDI with the progression of ALS in the spinal cords of mSOD1 transgenic mice. This correlation was confirmed by treating SH-SY5Y cells with NO donor SNOC to trigger the formation of S-nitrosylated PDI (SNO-PDI). When mSOD1 was overexpressed in SH-SY5Y cells, iNOS expression was up-regulated, NO generation was increased consequently. Furthermore, both SNO-PDI and mSOD1 aggregates were detected in these cells. Blocking NO generation with NOS inhibitor N-nitro-L-arginine (NNA) attenuated the S-nitrosylation of PDI; the formation of mSOD1 aggregates was inhibited as well. We conclude that NO-mediated S-nitrosylation of PDI is highly linked to the accumulation of mSOD1 aggregates in ALS.