Browsing by Subject "Nitric oxide"
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Item The catalytic mechanism of dimethylarginine dimethylaminohydrolase (DDAH) from pseudomonas aeruginosa(2006) Stone, Everett Monroe; Fast, WalterDimethylarginine dimethylaminohydrolase (DDAH) catalyzes the hydrolysis of Nw–methyl–L–arginine (NMMA) and Nw,Nw –methyl–L–arginine (ADMA) to L-citrulline and methylamine or dimethylamine, respectively. ADMA and NMMA are endogenous inhibitors of nitric oxide synthase (NOS) in mammals. DDAH therefore partially regulates NOS activity, making it an attractive therapeutic target in disease states involving overproduction of nitric oxide. Understanding the mechanism of DDAH is important to inhibitor design and elucidating its physiological function. DDAH is a member of the amidinotransferase superfamily, and has conserved active–site residues including cysteine, histidine, and glutamate/aspartate that are integral to catalysis. In DDAH from Pseudomonas aeruginosa, the active–site Cys249 is activated as a nucleophile upon binding substrate, and forms a covalent intermediate concomitant with loss of the alkylamine leaving group. The active–site His162 has a dual role, first as a general acid in protonating the alkylamine leaving group and second as a general base in generating a hydroxide for attack on the covalent intermediate. The active–site Glu114 is essential for properly orienting and ionizing His162. The use of a substrate analog, S–methyl-L-thiocitrulline (SMTC), enabled development of a new method of continuously monitoring DDAH activity, allowing facile screening of inhibitors. Using this method, a haloacetamidine was identified as an active–site directed inactivator motif for DDAH, and other members of the amidinotransferase superfamily.Item Cutaneous and cerebral microvascular response to the ingestion of flavanols in young and older humans : role of nitric oxide(2014-08) Harrison, Michelle Lorraine; Brothers, Robert MatthewThese studies explored interactions between flavanols and nitric oxide in order to investigate implications for vascular health. Study 1 investigated acute effects of flavanol consumption on cutaneous microvascular endothelial function in young and older individuals along with chronic exposure in older individuals. This was accomplished by assessing skin blood flow response to local heating (thermal reactivity, TR); skin was clamped at 34°C and 40°C and values were normalized to those attained at 43°C. Older individuals demonstrated an attenuated TR at baseline during the entire local heating phase (58.4 ± 2.5% versus 49.3 ± 2.6%, p<0.05). Acutely following flavanol ingestion there was a significant increase in TR (52.4 ± 2.1% versus 56.1 ± 2.0%, p=0.05) that was not different with age. There was no effect of chronic flavanol exposure on TR in older individuals; however, there was a significant decrease in mean arterial pressure (95 ± 3 mmHg versus 91 ± 3 mmHg, p<0.001). These results contribute to research regarding flavanols increasing NO bioavailability; acutely via an improvement in cutaneous microvascular endothelial function and chronically via a reduction in blood pressure. Study 2 investigated the acute effects of flavanol consumption on cerebrovascular endothelial function in young and older individuals along with chronic flavanol exposure in older individuals. This was accomplished by assessing basal cerebral blood flow indices (cerebral vascular conductance index, CVCi) and CBF response to hypercapnia (cerebral vasomotor reactivity; CVMR). At baseline older individuals demonstrated a reduced CVCi (0.85 ± 0.04 cm/s*mmHg versus 0.55 ± 0.04 cm/s*mmHg p=0.001) and CVMR (8.6 ± 0.6 versus 6.9 ± 0.4, p=0.05). An unexpected finding was that flavanol ingestion led to an acute decrease in CVCi (0.71 ± 0.04 cm/s*mmHg versus 0.62 ± 0.04 cm/s*mmHg p<0.05) and CVMR (8.6 ± 0.6 versus 6.1 ± 0.5, p=0.001) that was not different with age. In older individuals, chronic exposure led to a significant increase in CVCi (0.60 ± 0.05 cm/s*mmHg versus 0.72 ± 0.06 cm/s*mmHg, p<0.05) but had no effect on CVMR. These data provide evidence for an improvement in cerebral hemodynamics following chronic exposure in older individuals.Item Effects of Hypoxia Exposure on Hepatic Cytochrome P450 1A (CYP1A) Expression in Atlantic Croaker: Molecular Mechanisms of CYP1A Down-Regulation(Public Library of Science, 2012-07-16) Rahman, Md. Saydur; Thomas, PeterHypoxia-inducible factor-α (HIF-α) and cytochrome P450 1A (CYP1A) are biomarkers of environmental exposure to hypoxia and organic xenobiotic chemicals that act through the aryl hydrocarbon receptor, respectively. Many aquatic environments heavily contaminated with organic chemicals, such as harbors, are also hypoxic. Recently, we and other scientists reported HIF-α genes are upregulated by hypoxia exposure in aquatic organisms, but the molecular mechanisms of hypoxia regulation of CYP1A expression have not been investigated in teleost fishes. As a first step in understanding the molecular mechanisms of hypoxia modulation of CYP1A expression in fish, we characterized CYP1A cDNA from croaker liver. Hypoxia exposure (dissolved oxygen, DO: 1.7 mg/L for 2 to 4 weeks) caused significant decreases in hepatic CYP1A mRNA and protein levels compared to CYP1A levels in fish held in normoxic conditions. In vivo studies showed that the nitric oxide (NO)-donor, S-nitroso-N-acetyl-DL-penicillamine, significantly decreased CYP1A expression in croaker livers, whereas the competitive inhibitor of NO synthase (NOS), Nω-nitro-L-arginine methyl ester, restored CYP1A mRNA and protein levels in hypoxia-exposed (1.7 mg DO/L for 4 weeks) fish. In vivo hypoxia exposure also markedly increased interleukin-1β (IL-1β, a cytokine), HIF-2α mRNA and endothelial NOS (eNOS) protein levels in croaker livers. Pharmacological treatment with vitamin E, an antioxidant, lowered the IL-1β, HIF-2α mRNA and eNOS protein levels in hypoxia-exposed fish and completely reversed the down-regulation of hepatic CYP1A mRNA and protein levels in response to hypoxia exposure. These results suggest that hypoxia-induced down-regulation of CYP1A is due to alterations of NO and oxidant status, and cellular IL-1β and HIF-α levels. Moreover, the present study provides the first evidence of a role for antioxidants in hepatic eNOS and IL-1β regulation in aquatic vertebrates during hypoxic stress.Item Estrogen and the aging brain of male rats(2016-12) Nutsch, Victoria Lynn; Dominguez, Juan M.; Gore, Andrea C., 1964-; Hofmann, Hans; Cummings, Molly; Gonzales, RuebenGonadal steroid hormones exert an influence on many aspects of neurobiology in men, including memory, learning and sexual dysfunction. Though testosterone is the main circulating gonadal steroid hormone in males, estradiol is also important, and together these hormones play complementary roles. While the specific roles of estrogen have been studied to some extent in young adults, little is known during aging, when sexual behavior can become impaired. I used a rodent model to examine estradiol’s role in sexual behavior and gene expression in 3 regions, selected for their importance in behavioral neuroendocrine functions and high concentrations of estrogen receptors: the medial preoptic area (mPOA), medial amygdala (MeA), and bed nucleus of the stria terminalis (BnST). My studies focused first on how age and sexual experience affects expression and activation of estrogen receptor α (ERα) and androgen receptor (AR) after sexual behavior in aging intact males. Quantification of neurons expressing hormone receptors in the mPOA revealed that neither ERα nor androgen receptor (AR) showed an age-related change in expression in the mPOA. While both ERα and AR were activated after copulation, the age-related changes were specific to ERα in the central mPOA. There were only mild deficits in sexual behavior. Serum estradiol was also elevated in both aged and copulating animals, but estradiol concentrations only correlated with sexual behavior in aged animals. In a second study, I determined how hormone deprivation (castration) and replacement with estradiol caused changes to gene expression in the mPOA, BnST and MeA. Each region had unique patterns of gene expression in response to aging and estradiol treatment. The mPOA only had changes in expression as a result of hormone administration, while the BnST had primarily age-related changes. The MeA had the greatest number of affected genes, mainly interactions between estradiol treatment and aging. These studies emphasize the importance of estradiol in aging males, and the need for continued study on its role in neuroendocrine and sexual function.Item An investigation of the irreversible inhibition of human N[superscript ω], N[superscript ω]- dimethylarginine dimethylaminohydrolase (DDAH1)(2014-08) Burstein, Gayle Diane; Fast, Walter L.; Whitman, Christian; Zhang, Yan; Iverson, Brent; Hoffman , DavidNitric oxide synthases (NOS) are responsible for the production of nitric oxide (NO), an essential cell-signaling molecule, in mammals. There are three isoforms of NOS with widely different tissue distribution. The overproduction of NO is marked in many human disease states and cancers, however due to the similarities of the enzyme isoforms, targeting NOS for inhibition has proven challenging. Endogenously, the methylated arginines, N[superscript ω]-monomethyl-L-arginine (NMMA) and asymmetric N[superscript ω], N[superscript ω]-dimethyl-L-arginine (ADMA), inhibit NOS. N[superscript ω], N[superscript ω]-Dimethylarginine dimethylaminohydrolase (DDAH1) metabolizes these methylated arginines and thus relieves NOS inhibition. The role of DDAH1 in the regulation of diseases such as cancer and septic shock is still being elucidated. It is thought that targeting DDAH1 for inhibition rather than NOS may circumvent many of the current problems with the treatment of NO overproduction such as isoform selectivity. My PhD studies focus on the synthesis of a series of irreversible inhibitors of DDAH1, an extensive study of their in vitro mode of inhibition, a comparison of analytical fitting methods, and the viability and efficacy of the inactivators in a human cell line. I also studied a potential endogenous inactivator of DDAH1, nitroxyl (HNO), a one-electron reduction product of NO.Item Mechanisms of cutaneous microvascular endothelial dysfunction in young black Americans(2016-12) Kim, Kiyoung, active 2013; Tanaka, Hirofumi, Ph. D.; Farrar, Roger P.; Castelli, Darla M.; Brothers, Robert Matthew; Davis, Scott L.Black Americans have an increased risk for developing a variety of cardiovascular disease (CVD) when compared to white Americans and other populations in the United States. It has also been demonstrated that the underlying impairments in black Americans manifest during early adulthood prior to any overt signs of risk, which leads to higher rates of CVD related morbidity and mortality in black Americans than other populations. Study 1 was designed to investigate the potential mechanisms of cutaneous microvascular dysfunction in young college-age black Americans. This was assessed by measuring the skin blood flow response to local heating while various vasoactive substances were delivered into the cutaneous interstitial space by intradermal microdialysis. We demonstrated that an attenuated nitric oxide (NO) mediated vasodilation due in part to a relative deficit of L-arginine in the endothelial cells is one mechanism by which microvascular dysfunction occurs in young black Americans. Study 2 conducted to investigate the effects of acute cocoa flavanol intake on cutaneous microvascular function in young black Americans. This was assessed by measuring the skin blood flow response to local heating and delivery of vasoactive substances (as described above) before and after consumption of a beverage high in flavanol content. Study 2 demonstrated that acute flavanol intake improved cutaneous microvascular function in response to local heating in young black Americans relative to young white Americans. Study 3 was designed to investigate the effects of acute flavonal intake on endothelium-dependent microvascular dilation in response to exogenous administration of methacholine (MCh) in young black Americans. This was assessed by skin blood flow responses to incremental dose of MCh, which was delivered by intradermal microdialysis, before and after consumption of a beverage high in polyphenol content. Study 3 identified that acute flavanol intake did not alter the dose-response curve of MCh-induced cutaneous vasodilation in either racial groups. Overall, the series of studies in this dissertation may provide evidence that young black Americans have attenuated microvascular function relative to young white Americans, and that a potential mechanism of decreased microvascular function is a decrease in NO bioavailability and/or NO mediated vasodilation, which is related to a deficit of L-arginine in the endothelial cells in young black Americans. Furthermore, our findings may provide evidence that the consumption of cocoa flavanols is an effective therapeutic strategy to prevent and/or delay the development of CVD at least in young black Americans.Item Proving 2-aminobiphenyl nitric oxide probes in cells and designing sequence-defined oligomers for sequencing(2021-05-06) Escamilla, Pedro Rogelio; Anslyn, Eric V., 1960-; Shear, Jason B; Liu, Hung-wen; Humphrey, Simon M; Sessler, Jonathan LThe simple diatom radical nitric oxide (NO) has numerous roles in biology. In organisms, healthy nanomolar NO levels are highly regulated. Deficiencies in NO can lead to atherosclerosis, diabetes, glaucoma, and many other conditions. Yet organisms also purposefully increase NO concentrations to micromolar levels in combatting pathogens. However, these high levels can backfire and damage cells and tissues, as found for Parkinson’s disease and ischemia, among others. Fluorescent NO probes enable live cell, tissue, and sometimes whole-animal imaging with minimal perturbation of their biological environment. 2-aminobiphenyl-based probe NO550 brought about a novel mechanism of detection in which NO-surrogate nitrosonium cation is assimilated into the nascent cinnoline fluorophore, resulting in low background and increased sensitivity. A family of second-generation 2-aminobiphenyl based probes were designed, producing four top performers in abiotic conditions. Chapter 1 describes the synthesis of these top candidates and their evaluation in cells. Two emerged as promising options for NO researchers. Chapter 2 describes the design and synthesis of sequence-defined non-natural oligomers that are sequenceable. Biological polymers peptides and DNA/RNA’s exquisitely complex properties are dictated by their sequence; changing the sequence sufficiently may have detrimental effects on their performance. Both their synthesis and sequencing continue being optimized; a human genome that once required thirteen years to sequence now takes one day. Not as developed but growing rapidly is the field of non-natural sequence-defined polymers. Without the constraints of biology, limitless options exist for backbone structures and sidechains. Such diversity discourages the focused development of sequencing technologies on par with those for biological polymers. Consequently, tandem MS is the favored form of analysis. We sought to change the approach to polymer design by factoring in the sequencing of the polymer in addition to its synthesis. Oligourethanes based on β-aminoalcohols were designed so that the terminal unit could be derivatized to sequence by cyclizing upon itself and thereby releasing the remainder of the oligomer. Several derivatizations were investigated, to find that the oligomer sequenced itself without any terminal unit transformation, rather by self-immolation under conditions that slowed the “unzipping” of the polymer sufficiently to be able to detect the intermediate sequences.Item The synthesis of novel conducting polymers and oligomers for use in electrical devices, drug delivery systems, and energy dynamics studies(2010-05) Villa, Monica Irais, 1982-; Holliday, Bradley J.; Jones, Richard A., 1954-Described herein are three projects centered on the synthesis of conducting polymer derivatives for various applications. The first is the novel synthesis of 9,9-dioctylfluorene-co-benzothiadiazole [F8BT] oligomers through solid phase synthesis for the study of the thermodynamics and kinetics of electron transfer in the polymer. The second endeavor involves the synthesis of a series of 4”,3’’’-dialkyl-2,2’:5’,2”:5”,2’’’:5’’’,2’’’’:5’’’’,2’’’’’-sexithiophenes for the studies on crystal packing and surface deposition of organic p-type semiconducting materials. Lastly is described the development of a conducting metallopolymer based on the ligand 2,6-Bis(4-(2,2’-bithiophen-5-yl)-1H-pyrazol-1-yl)pyridine for use in a drug delivery system.