Browsing by Subject "Amino acid"
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Item Amino acids, polyamines, and nitric oxide synthesis in the ovine conceptus(Texas A&M University, 2005-08-29) Kwon, Hyuk JungThe objective of this study was to determine concentrations of amino acids and polyamines as well as nitric oxide (NO) and polyamine synthesis in the ovine conceptus (embryo/fetal and associated placental membrane). Ewes were hysterectomized on Days 30, 40, 60, 80, 100, 120, or 140 of gestation to obtain allantoic and amniotic fluids, intercotyledonary placenta, placentomes and uterine endometrium for the analyses. Alanine, citrulline plus glutamine accounted for about 80% of total α-amino acids in allantoic fluid during early gestation. Serine (16.5 mM) contributed about 60% of total α-amino acids in allantoic fluid on Day 140 of gestation. Maximal ornithine decarboxylase (ODC) and arginase activities and highest rates of polyamine and NO synthesis occured in all tissues on Day 40 of gestation. In ovine allantoic and amniotic fluids, polyamines were most abundant during early (Days 40-60) and late (Days 100-140) gestation, respectively. Activity of guanosine 5??-triphosphate-cyclohydrolase I (GTP-CH), and concentrations of NOS cofactors, tetrahydrobiopterin (BH4) and NADPH (nicotinamide adenine dinucleotide), peaked on Day 40 of gestation in placental and endometrial tissues. In these tissues, NO synthesis was positively correlated with total NOS activity, GTP-CH activity, and concentrations of BH4 and NADPH. The physiological significance of these changes was manifested by undernutrition-induced intrauterine growth retardation (IUGR). Maternal undernutrition (50% of National Research Council nutrient requirements) reduced concentrations of total α-amino acids in fetal plasma and fluids, and retarded fetal growth at both mid (Day 78) and late (Day 135) gestation. Concentrations of polyamines in fetal fluids were lower in underfed ewes than in control-fed ewes. Realimentation of underfed ewes between Days 78 and 135 of gestation increased concentrations of total α-amino acids and polyamines in fetal plasma and fluids, when compared with non-realimented ewes. Results of these studies demonstrate metabolic coordination among the several integrated pathways to enable high rates of polyamine and NO synthesis in the placenta and endometrium during early pregnancy. Collectively, our findings may have important implications for both IUGR and fetal origins of adult disease.Item Expanding the genetic code in mammalian cells(2011-08) Xiang, Liang; Zhang, Zhiwen Jonathan; Georgiou, George; Roy, Krishnendu; Ren, Pengyu; Yin, WhitneyProteins are diverse polymers of covalently linked amino acids. They play a role in almost every biological process that occurs within an organism. Twenty different amino acids are genetically encoded by mammalian cells to build proteins. The sequence of these amino acids determines the protein’s final shape, structure, and function. Modern molecular cloning techniques allow for the genetic encoding and expression of mutant proteins that have one or more amino acids replaced with one of the others. The roles of individual amino acids in a protein can therefore be studied. Proteins with novel functions have also been designed or evolved using this technology. However, the genetic code is limited to the twenty natural amino acids. Nonnatural amino acids have unique side groups that not found on any of the twenty natural amino acids. They can be site-specifically incorporated using a mutant orthogonal suppressor tRNA/aminoacyl-tRNA synthetase (aaRS) pair. Each pair only allows for one type of nonnatural amino acid to be genetically encoded. This technology has resulted in the incorporation of over fifty different types of nonnatural amino acids into proteins in prokaryotic and eukaryotic cells. Unfortunately, most of these pairs are not orthogonal outside of prokaryotic systems and only a few have been developed for mammalian cells. To create more mammalian pairs a nonnatural aaRS has to be evolved and screened in a cumbersome process. In this dissertation an approach is outlined that can be used to change the orthogonality of existing nonnatural suppressor tRNA/aaRS pairs. As a result of the orthogonality change many previously unavailable pairs can be shuttled into mammalian cells. The ability to genetically encode a 21st amino acid is a powerful tool in the study and engineering of proteins.Item Single channel analysis of thiol binding to a putative site of alcohol action on the glycine receptor(2009-08) Goldstein, Beth Erlichman; Mihic, S. JohnAn alcohol and anesthetic binding pocket is hypothesized to exist among transmembrane domains of the α1 glycine receptor (GlyR). Prior work has shown that amino acid residue serine-267 plays a significant role in the enhancing effects of alcohol and anesthetics and is theorized to form part of an alcohol and anesthetic binding cavity among subunit transmembrane domains. Propyl methanethiosulfonate (PMTS), an alcohol-like thiol, was previously shown to bind to a cysteine residue introduced at position 267 (S267C) and this resulted in permanent enhancement of GlyR function. If ethanol is binding to residue 267 in wildtype GlyR to potentiate receptor function then we hypothesized that covalent thiol labeling would produce receptor enhancement by the same mechanisms as ethanol. Using outside-out patch single channel electrophysiology we determined the open and closed dwell-times and burst properties of S267C GlyR in the absence and presence of PMTS. The primary consequence of PMTS binding to S267C GlyR was an increase in the lengths of burst durations, paralleling the main effect of ethanol on wildtype GlyR. Our findings thus provide a new line of evidence suggesting that ethanol is exerting its enhancing effects on the GlyR through its interactions with amino acid residue 267 in the second transmembrane domain.