Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

<p>Glycine, a lipophilic non-essential amino acid biosynthesized from L-serine, L-alanine and L-threonine, plays a role in the biosynthesis of proteins, nucleotides, and glutathione. It is a neurotransmitter in the central nervous system and acts as a co-agonist at the N-methyl-D aspartate (NMDA) receptor. Oral administration of glycine is helpful in the management of schizophrenia. While long-term oral treatment with glycine is considered to exert a cardio-vascular protective role by overcoming endothelial dysfunction and oxidative stress, there are no systematic studies examining the cardiovascular effects of glycine. Recently, we showed that the precursor/metabolite of glycine, L-serine, evoked endothelium-dependent vasodilatation in rat mesenteric arterioles. Acute intravenous administration of L-serine produced a rapid, dose-dependent fall in blood pressure (BP) in both normotensive and hypertensive rats. These responses were abolished in the combined presence of the Ca²⁺ activated small and intermediate conductance K⁺ channel inhibitors, apamin and Tram-34/charybdotoxin. In contrast, intravenous administration of glycine evoked a fall in BP in normotensive Wistar-Kyoto (WKY) rats and an elevation of BP in spontaneously hypertensive rats (SHR), and in WKY rats subjected to chronic nitric oxide (NO) synthase (NOS) inhibition by oral treatment with NOS inhibitor, L-NAME (N^G-nitro-L-arginine-methylester). Therefore, <i>in vivo</i> and <i>in vitro</i> studies were designed to address the mechanisms that contribute to the opposite effects of glycine in normotensive <i>vs</i>. hypertensive rats.</p> <p>Experiments were performed using 14 weeks old male WKY, chronic L-NAME treated WKY and SHR strains. <i>In vivo</i> studies involved examination of changes in systemic hemodynamic parameters such as mean arterial pressure (MAP), heart rate (HR), total peripheral resistance (TPR) and cardiac output (CO) as well as regional hemodynamic parameters of changes in blood flow and vascular resistance in major organs/tissues following acute intravenous administration of glycine using fluorescent microsphere distribution technique. Parallel complementary <i>in vitro</i> studies were conducted to examine the effects of glycine on changes in basal tone and phenylephrine (PE) constricted tone in aortic rings with endothelium-intact and endothelium-denuded states after isolation from WKY and SHR strains. All these studies were conducted in the presence and absence of two NMDA antagonists, MK-801 and memantine.</p> <p>In normotensive WKY rats, glycine (1 mmol/L) administration decreased MAP (P<0.01), TPR (P<0.05) while it increased CO (P<0.01) and blood flow to brain (215%), kidney (190%) and heart (160%). In SHR and L-NAME treated WKY rats, glycine administration elevated MAP and TPR but reduced CO (P<0.01) and blood flow to brain, kidney and heart. These effects were abolished in animals pretreated with either MK-801 or memantine. These data are consistent with the likely expression of vascular NMDA receptors activated by glycine in brain and kidney. Glycine (0.5-3.0 mmol/L) <i>per se</i> increased basal tone (E_max1.4g) in aortic rings from all rats and it was absent following incubation with NMDA antagonist(s). The concentration-dependent vasodilatation (I_max43%) evoked by glycine in PE-constricted rings with intact endothelium of WKY rats was attenuated by either MK-801 or L-NAME. Such vasodilator responses to glycine could be abolished by pretreatment with either NMDA antagonist or L-NAME. In contrast, in PE-constricted rings of SHR strain, addition of glycine enhanced the tone and this was abolished in the presence of NMDA antagonist, MK-801.</p> <p>Taken together, these data suggest that NMDA receptors, likely present on both endothelium and vascular smooth muscle cells, predominantly in brain, kidney and coronary vascular beds as well as the conduit vessel, aorta, could contribute to the systemic and regional hemodynmic effects of glycine. In normotensive WKY rats, addition of glycine promotes endothelium/NO-dependent vasodilatation subsequent to Ca2+ mobilization evoked following endothelial NMDA receptor activation by glycine. Thus, glycine-induced dose-dependent, reversible fall in MAP and TPR in normotensive WKY rats is likely associated with NO-dependent/endothelium-mediated vasodilatation. However, in hypertensive rat models such as in the SHR model with endothelial dysfunction and reduced NO bioavailability or in chronic L-NAME treated rats, glycine administration promotes vascular NMDA receptor activation leading to enhanced vascular tone resulting in increased TPR and MAP.</p> <p>These observations are important and critical in terms of considering glycine as a possible antihypertensive agent as this was proposed by several earlier reports that did not explore the direct vascular effects of glycine in hypertensive animal models.</p>

<p>Glycine, a lipophilic non-essential amino acid biosynthesized from L-serine, L-alanine and L-threonine, plays a role in the biosynthesis of proteins, nucleotides, and glutathione. It is a neurotransmitter in the central nervous system and acts as a co-agonist at the N-methyl-D aspartate (NMDA) receptor. Oral administration of glycine is helpful in the management of schizophrenia. While long-term oral treatment with glycine is considered to exert a cardio-vascular protective role by overcoming endothelial dysfunction and oxidative stress, there are no systematic studies examining the cardiovascular effects of glycine. Recently, we showed that the precursor/metabolite of glycine, L-serine, evoked endothelium-dependent vasodilatation in rat mesenteric arterioles. Acute intravenous administration of L-serine produced a rapid, dose-dependent fall in blood pressure (BP) in both normotensive and hypertensive rats. These responses were abolished in the combined presence of the Ca²⁺ activated small and intermediate conductance K⁺ channel inhibitors, apamin and Tram-34/charybdotoxin. In contrast, intravenous administration of glycine evoked a fall in BP in normotensive Wistar-Kyoto (WKY) rats and an elevation of BP in spontaneously hypertensive rats (SHR), and in WKY rats subjected to chronic nitric oxide (NO) synthase (NOS) inhibition by oral treatment with NOS inhibitor, L-NAME (N^G-nitro-L-arginine-methylester). Therefore, <i>in vivo</i> and <i>in vitro</i> studies were designed to address the mechanisms that contribute to the opposite effects of glycine in normotensive <i>vs</i>. hypertensive rats.</p> <p>Experiments were performed using 14 weeks old male WKY, chronic L-NAME treated WKY and SHR strains. <i>In vivo</i> studies involved examination of changes in systemic hemodynamic parameters such as mean arterial pressure (MAP), heart rate (HR), total peripheral resistance (TPR) and cardiac output (CO) as well as regional hemodynamic parameters of changes in blood flow and vascular resistance in major organs/tissues following acute intravenous administration of glycine using fluorescent microsphere distribution technique. Parallel complementary <i>in vitro</i> studies were conducted to examine the effects of glycine on changes in basal tone and phenylephrine (PE) constricted tone in aortic rings with endothelium-intact and endothelium-denuded states after isolation from WKY and SHR strains. All these studies were conducted in the presence and absence of two NMDA antagonists, MK-801 and memantine.</p> <p>In normotensive WKY rats, glycine (1 mmol/L) administration decreased MAP (P<0.01), TPR (P<0.05) while it increased CO (P<0.01) and blood flow to brain (215%), kidney (190%) and heart (160%). In SHR and L-NAME treated WKY rats, glycine administration elevated MAP and TPR but reduced CO (P<0.01) and blood flow to brain, kidney and heart. These effects were abolished in animals pretreated with either MK-801 or memantine. These data are consistent with the likely expression of vascular NMDA receptors activated by glycine in brain and kidney. Glycine (0.5-3.0 mmol/L) <i>per se</i> increased basal tone (E_max1.4g) in aortic rings from all rats and it was absent following incubation with NMDA antagonist(s). The concentration-dependent vasodilatation (I_max43%) evoked by glycine in PE-constricted rings with intact endothelium of WKY rats was attenuated by either MK-801 or L-NAME. Such vasodilator responses to glycine could be abolished by pretreatment with either NMDA antagonist or L-NAME. In contrast, in PE-constricted rings of SHR strain, addition of glycine enhanced the tone and this was abolished in the presence of NMDA antagonist, MK-801.</p> <p>Taken together, these data suggest that NMDA receptors, likely present on both endothelium and vascular smooth muscle cells, predominantly in brain, kidney and coronary vascular beds as well as the conduit vessel, aorta, could contribute to the systemic and regional hemodynmic effects of glycine. In normotensive WKY rats, addition of glycine promotes endothelium/NO-dependent vasodilatation subsequent to Ca2+ mobilization evoked following endothelial NMDA receptor activation by glycine. Thus, glycine-induced dose-dependent, reversible fall in MAP and TPR in normotensive WKY rats is likely associated with NO-dependent/endothelium-mediated vasodilatation. However, in hypertensive rat models such as in the SHR model with endothelial dysfunction and reduced NO bioavailability or in chronic L-NAME treated rats, glycine administration promotes vascular NMDA receptor activation leading to enhanced vascular tone resulting in increased TPR and MAP.</p> <p>These observations are important and critical in terms of considering glycine as a possible antihypertensive agent as this was proposed by several earlier reports that did not explore the direct vascular effects of glycine in hypertensive animal models.</p>

Thesis (M.S.)--University of Louisville, 1998. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (M.S.)--University of Louisville, 1998. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Alcohol is abundantly consumed by society and general anesthetics are used everyday in operating suites throughout the world, yet the sites and mechanisms of action for these drugs are not completely understood. Glycine receptors (GlyRs) are pentameric ion channels expressed throughout the brain and spinal cord and have become increasingly popular targets in the study of alcohol action. Each GlyR subunit is composed of four alpha helical transmembrane segments (TM1-4), and although amino acids involved with alcohol action have been previously identified in TM1-4, the orientation of each of these residues with respect to a putative alcohol/anesthetic binding cavity remains controversial. In order to better characterize this binding cavity within the GlyR, we conducted a series of experiments using cysteine mutagenesis and biochemical cross-linking. In Aim 1, the participation of TM1 with TM3 in a common alcohol/anesthetic binding cavity was further investigated. We used two-electrode voltage clamp electrophysiology in Xenopus oocytes to demonstrate the ability of A288 in TM3 to form cross-links with I229 in TM1, which reduced the ability of both alcohol and anesthetics to modulate GlyR function. Aim 2 investigated whether TM3 could also participate in a binding cavity with TM4. We have shown that residues in TM4 are able to form cross-links with A288 in TM3, and found that cross-linking between TM3 and those residues in TM4 also reduced the ability of alcohol and anesthetics to enhance GlyR function. Aim 3 determined whether these cross-links are formed between residues within the same subunit (intra-subunit) or between subunits (inter-subunit), and ultimately whether these residues participate in a common alcohol/anesthetic binding cavity within or between GlyR subunits. GlyR protein, which measures about 50 kDa, was extracted from oocytes injected with the cysteine mutants, and immunoblotting was used with a GlyR-specific antibody to subsequently help quantify band ratios between cross-linked and uncross-linked conditions. We found an increase in the 100:50 kDa band ratio for the TM1-3 mutant only, but not TM3-4 mutant or the wild-type, which suggests TM1-3 may participate in an alcohol binding cavity between GlyR subunits while TM3-4 may contribute to a binding cavity within a subunit. / text

No description available.

A soja é uma cultura de grande importância na economia mundial. No Brasil, o complexo da soja (grão, farelo e óleo) é responsável por uma parcela significativa das exportações. No entanto, o sucesso da produção é dependente de muitos fatores bióticos e abióticos. A ocorrência de déficit hídrico talvez seja hoje, e mais ainda no futuro, o principal desafio das culturas produtoras de grãos. Sendo assim, a obtenção de cultivares de soja tolerantes à desidratação é de grande interesse. Por ser de caráter poligênico, a tolerância à seca é difícil de ser trabalhada no melhoramento genético clássico. Sendo assim, o uso da engenharia genética apresenta-se como caminho para alcançar este atributo. A regulação transcricional de genes endógenos e a busca da precisão no controle de transgenes são as bases das estratégias de engenharia genética, que apostam nos fatores de transcrição e nos promotores induzíveis para desenvolver plantas tolerantes a estresses abióticos. As proteínas WRKY formam uma grande família de fatores de transcrição que está envolvida em processos fisiológicos e bioquímicos importantes nos vegetais, entre eles, a resposta das plantas ao déficit hídrico. A determinação do perfil de expressão de genes da família WRKY em condição de seca e a avaliação da atividade de seus promotores são os objetivos deste trabalho. Dez genes WRKY induzidos em situação de seca foram selecionados para o estudo: Glyma15g11680, Glyma09g37470, Glyma01g31920, Glyma07g02630, Glyma08g23380, Glyma05g36970, Glyma05g20710, Glyma05g29310, Glyma09g41050, Glyma08g15050. A análise in silico da sequência promotora desses genes revelou a presença de cis-elementos relacionados à resposta da planta a estresses. O perfil de expressão determinado por RT-qPCR mostrou que os genes Glyma15g11680, Glyma07g02630, Glyma05g36970, Glyma08g15050 e Glyma08g23380 são expressos diferencialmente entre os genótipos tolerante e suscetível e também entre folhas e raízes. Para fins de caracterização funcional dos promotores, fragmentos de 500bp e 2000bp a montante do sítio de início da transcrição dos genes Glyma05g36970, Glyma08g15050 e Glyma15g11680 foram clonados em vetores apropriados para transformação genética de plantas. Foram obtidas plantas de tabaco transformadas pelo sistema Agrobacterium tumefaciens. / Soybean is a crop of great importance for the world economy. In Brazil, the soybean complex (beans, meal and oil) is responsible for a significant portion of exports. However, the successful production is influenced by biotic and abiotic factors. The occurrence of drought is today, and perhaps even more in the future, the main challenge of grain crops. Thus it is of great interest to obtain soybean cultivars tolerant to desiccation. The drought tolerance trait is difficult to obtain through classical breeding due to its polygenic basis. In this context, genetic engineering is presented as a way to achieve this attribute. The transcriptional regulation of endogenous genes and the precise control of transgenes are the foundations of genetic engineering strategies that use transcription factors and inducible promoters to develop plants tolerant to abiotic stresses. The WRKY proteins form a large family of transcription factors that are involved in important physiological and biochemical processes in plants, including the response to water deficit. The goals of this work are the determination of the expression profile of WRKY genes under drought conditions and the evaluation of its promoters activity. Ten WRKY genes which are induced by drought were selected for this study: Glyma15g11680, Glyma09g37470, Glyma01g31920, Glyma07g02630, Glyma08g23380, Glyma05g36970, Glyma05g20710, Glyma05g29310, Glyma09g41050 and Glyma08g15050. The in silico analysis of these gene promoter sequences revealed the presence of cis - elements related to plant stress responses. The expression pattern determined by RT- qPCR showed that Glyma15g11680, Glyma07g02630, Glyma05g36970, Glyma08g15050 and Glyma08g23380 genes are differentially expressed between tolerant and susceptible genotypes and between leaves and roots. For the functional characterization of the promoters, 500 and 2000 bp fragments upstream of the transcription start site of Glyma05g36970, Glyma08g15050 and Glyma15g11680 genes were cloned into appropriate vectors for plant genetic transformation. Tobacco plants transformed by Agrobacterium tumefaciens system were obtained.

There is a great need for new analgesics. The current problem in treatment of severe pain is that side effects limit the effectiveness of therapy. Glycine receptors are important in modulation of nociception, suggesting a novel class of analgesics. Previous studies in rats show that intrathecal administration of glycine agonists and amino acids structurally similar to glycine have antinociceptive effects. The effects of isovaline, a unique, non-proteogenic glycine-like aminoacid, have not been studied. Isovaline is absorbed from the gut and transported across the blood-brain-barrier. We examined the hypothesis that isovaline produces antinociception in mice. Administration of strychnine, an antagonist at glycine receptors, into the cisterna magna or lumbar intrathecal space resulted in allodynia, localized to the somatotopic distribution of the trigeminal and lumbar nerves. These findings provided a basis for models of lumbar and trigeminal neuralgia. Racemic isovaline blocked strychnine induced allodynia in both models without apparent side effects. We next investigated the antinociceptive effects of glycine-like amino acids in formalin foot assay, a conventional rodent model of acute and chronic pain. Antinociceptive effects were demonstrated on intrathecal administration of glycine, beta-alanine, and isovaline. Intravenous isovaline produced significant antinociceptive effects in the formalin foot model. The toxicity of isovaline and related amino acids were determined. Exploratory behavior, gait, and responses to stimuli were used to assess sedation. The rotarod test was used to examine central nervous system (CNS) and neuromuscular toxicities of intravenous isovaline. Lumbar administration of glycine and beta-alanine caused scratching and/or lower body weakness. Isovaline at 7-times intrathecal ED50 produced lower body weakness in some animals. None of the amino acids produced sedation comparable to morphine. At 6-times ED50, beta-alanine produced weakness. Both glycine (ED50) and beta-alanine (3x ED50) but not isovaline produced local nerve irritation. Intracisternal injection of glycine did not reverse allodynia and resulted in death. Neither R nor S enantiomers of isovaline impaired performance on the rotarod test. Isovaline has significant antinociceptive properties. Given the absence of apparent CNS or motor toxicity, isovaline has potential as a clinical analgesic.

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2002. / On t.p. "A̳" is subsript. Includes bibliographical references (leaves 160-177). Also available in electronic version. Access restricted to campus users.