Graduation date: 1995

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 76-93).

Sulfite mutants representing five complementation groups, previously derived from an ethyl methanesulfonate-treated haploid strain of Saccharomyces cerevisiae were studied. Although the wildtype S. cerevisiae strain used (isogenic to X2180-1 A) had a basal tolerance for sulfite (7 μM free H₂SO₃), the sensitive and resistant mutants were found to tolerate less than 3 to 5.5, or greater than 19 μM free H₂SO₃, respectively. No apparent correlation was found between the response to sulfite and generation time in rich (YEPD) or minimal media. Resistant mutant 11-1 had an extended lag phase relative to wildtype. Mutant and wildtype proteins were labeled with ³⁵S-methionine to determine differences in banding patterns due to sulfite-specific induction or disappearance of polypeptides. No obvious differences following SDS-PAGE and autoradiography were observed upon induction with 0.213 μM free H₂SO₃. No consistent correlations were found between the sulfite phenotypes and responses to other reducing agents. Sensitive mutant 35-2 appeared to be three to ten times more sensitive to dithiothreitol than wildtype and sensitive mutant 47-9 was three to four times more sensitive to sodium nitrite and three to seven times more sensitive to sodium thiosulfate than wildtype. Log phase cells of sensitive mutant 33-2 were found to have significantly less glutathione than wildtype. Wildtype contained 62.6 nmol min⁻¹ mg protein⁻¹ (62.6 mU mg protein⁻¹) glutathione reductase (GR) and 2.78 nmol min⁻¹ mg protein⁻¹ (2.78 mU mg protein⁻¹) glutathione S-transferase (GST). Log phase cells of one resistant mutant showed a significantly higher level of GR than wildtype, 135%. The resistant mutants as well as some of the sensitive mutants had reduced GST levels. Survival rates of the mutants in buffer in the presence of sulfite did not correlate with their sensitive or resistant phenotypes, suggesting that survival and growth in the presence of sulfite are not necessarily related functions. Relative to wildtype, survival upon prolonged storage at 4°C was markedly reduced for two of the four sensitive mutants, one of which was 33-2, and was enhanced for one resistant and another sensitive mutant. / Graduation date: 1992

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.

There is substantial evidence that oxidative stress causes negative outcomes in many cell and tissue types. This is especially true of skeletal muscle, as it is continually subjected to various sources of reactive oxygen species (ROS). Oxidative stress in muscle has been linked to several disease states as well as to the normal aging process. Oxidative stress has also been associated with increased apoptotic signalling. Furthermore, elevated apoptosis is consistently observed in aged skeletal muscle and is thought to be one of the mechanisms of age-related muscle atrophy. Due to its post-mitotic nature, skeletal muscle may be more susceptible to the harmful effects of oxidative stress in light of its limited regenerative capacity. As a protective measure, a sophisticated antioxidant system exists in muscle consisting of both enzymatic (superoxide dismutases (SOD’s), catalase, glutathione peroxidase) and non-enzymatic elements (glutathione: GSH). GSH is a ubiquitously expressed tripeptide essential to maintenance of the redox status of the cell. Its role in skeletal muscle apoptosis, especially in different muscle types, is currently unclear. To elucidate the potential role of GSH in skeletal muscle apoptosis and oxidative stress, L-buthionine-[S,R]-sulfoximine (BSO) was used to deplete GSH in young (34.85 ± 0.68 wks) and old (69.11 ± 3.61 wks) male Sprague-Dawley rats. Thiol levels (GSH, GSSG), ROS production, 4-hydroxy-2-nonenal (4HNE) levels, DNA fragmentation and apoptosis-related protein expression were examined in soleus (SOL) and white gastrocnemius (WG) muscle. BSO led to significant GSH depletion (89% in SOL, 96% in WG) compared to age-matched controls. Catalase upregulation, in the absence of change in SOD levels, was evident as a result of BSO treatment and advancing age in both muscle tissues. BSO treatment also resulted in increased DNA fragmentation in WG and SOL, with elevated ROS production in SOL only; both of these effects were independent of age. Advancing age resulted in elevated caspase activity and Hsp70 protein content, with a concomitant decrease in anti-apoptotic ARC in SOL but not WG. Additionally, ROS production, 4HNE content, DNA fragmentation and ARC levels were all significantly elevated in SOL compared to WG. These data indicate that SOL may be subjected to a state of elevated cellular stress. There is also some evidence that GSH depletion increases DNA fragmentation while age contributes to a degradative loss of glycolytic muscle.

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.

Magnetic resonance imaging (MRI) is one of the most powerful imaging techniques for research and clinical diagnosis. To expand upon the intrinsic capabilities of MRI, new contrast agents that can detect the presence of biomarkers in vivo are being developed. My Masters thesis research focuses on the design and synthesis of a new MRI contrast agent that can detect glutathione (GSH), a biomarker that has been implicated in a number of oxidative stress diseases. This new MRI contrast agent is based on chelated dysprosium (Dy), an inorganic metal, which provides negative contrast to surrounding tissue. Preliminary data has shown that attaching a poly(ethylene glycol) (PEG) chain to the Dy chelate, effectively increasing its molecular weight, enhances the contrast ability of Dy. Using this new information, the contrast agent was designed to have a large molecular weight PEG chain attached to the Dy chelate through a disulfide, creating a thiol-sensitive linkage. In the presence of a thiol-containing molecule such as GSH, the Dy will be dePEGylated through a disulfide exchange reaction, removing the molecular weight effect of the PEG, and allowing for the detection of GSH by MRI. This new MRI contrast agent could provide insight into the progression and diagnosis of oxidative stress pathologies associated with GSH.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 74-91).

Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Leaves vi, ix and 167 are blank. Typescript. Includes bibliographical references (leaves 173-174). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Leaves vi, ix and 167 are blank. Typescript. Includes bibliographical references (leaves 173-174). Also available on the Internet.