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Since the genetic code first was determined, many have claimed that it is organized adaptively, so as to assign similar codons to similar amino acids. This claim has proved difficult to establish due to the absence of relevant comparative data on alternative primordial codes and of objective measures of amino acid exchangeability. Here we use a recently developed measure of exchangeability to evaluate a null hypothesis and two alternative hypotheses about the adaptiveness of the genetic code. The null hypothesis that there is no tendency for exchangeable amino acids to be assigned to similar codons can be excluded here as expected from earlier work. The first alternative hypothesis is that any such correlation between codon distance and amino acid distance is due to incremental mechanisms of code evolution, and not to adaptation to reduce deleterious effects of future mutations. More specifically, new codon assignments that occur by ambiguity reduction or by codon capture will tend to give rise to correlations, whether due to the condition of amino acid ambiguity, or to the condition of similarity between a new tRNA synthetase (or tRNA) and its parent. The second alternative hypothesis, the adaptive hypothesis, then may be defined as an excess relative to what may be expected given the incremental nature of evolution, reflecting true adaptation for robustness rather than an incidental effect. The results reported here indicate that most of the nonrandomness in the amino acids to codon assignments can be explained by incremental code evolution, with a small residue of orderliness that may reflect code adaptation.

Amino acids are the building blocks of protein, but also play important cellular signaling roles. The mechanisms through which altered levels of many amino acids are sensed and the signals transmitted are still largely unknown. Increasing evidence is showing that these signals may influence the aging process. In this regard, methionine restriction appears to be an evolutionary conserved mechanism to delay aging and supplementation with glycine can mimic methionine restriction to extend lifespan in rodents. Tryptophan restriction may also activate specific anti-aging pathways, but it could interfere with cognitive function. With exercise the consumption of dietary branched chain amino acids (BCAAs) may be beneficial in building muscle mass, but high levels of BCAAs as well as tyrosine and phenylalanine in the bloodstream are associated with metabolic disease such as insulin resistance. Individual supplementation or restriction of several different amino acids has shown promise in the treatment of insulin resistance in rodents. Much progress regarding the effects of amino acids on longevity has been made using yeast, nematodes, and fruit flies. Clearly, much more research is needed to understand the signaling pathways activated by amino acid imbalance before efficacious and well-tolerated dietary interventions can be developed for human aging and aging-related disorders. In this review the mechanisms through which altered dietary and cellular levels of the twenty proteogenic amino acids affect aging or aging-related disorders are discussed.

The B family of DNA-dependent DNA polymerases (pols) are uniquely sensitive to inhibition by N2-(p-n-butylphenyl)deoxyguanosine 5'-triphosphate (BuPdGTP). The affinity of members of the B family for BuPdGTP varies greatly, as does the ability of certain members to use the modified nucleotide as a substrate. For example, eukaryotic pol α has high affinity for the nucleotide, but incorporates it into DNA poorly, while T4 pol has lower affinity, but incorporates the nucleotide well. This thesis addresses two questions: 1) What are the amino acid residue(s) that impart sensitivity to BuPdGTP? and 2) is incorporation of BuPdGTP required for inhibition? To answer the first question, molecular modeling was used with the crystal structure of RB69 pol [Wang et al., 1997], an enzyme closely related to T4 pol [Wang et al., 1995]. This modeling identified a structural pocket adjacent to the polymerase active site that could serve as the butylphenyl "receptor". Based upon this modeling, a chimeric T4 pol containing the residues corresponding to the butylphenyl receptor from human pol α was designed and engineered for expression. The chimera was hypothesized to have a pol α-like phenotype with respect to its response to BuPdGTP (higher sensitivity/ lost ability to incorporate). The chimera was found to be unstable during purification, leaving the hypothesis unresolved. To answer the second question, non-substrate derivatives of BuPdGTP were in which the α,β anhydride oxygen of the triphosphate were replaced with either a CH2 or NH. The ability of the latter derivatives to inhibit polymerase activity and to serve as substrates was measured on T4 pol, RB69 pol and human pol α. Both derivatives retained high potency, but were not substrates under the conditions tested. These compounds were potent, selective inhibitors of B family pol that should be useful in the formation of a stable complex of enzyme:DNA:inhibitor for crystallization trials.

It has been hypothesized that an increase in the ratio of plasma tryptophan (TRP) to branched-chain amino acid (BCAA) concentrations may mediate an increase in cerebral serotonin synthesis, through an increased cerebral tryptophan uptake. It is postulated that the increased brain serotonin content may induce central fatigue during prolonged exercise. Until present, this postulate had not been subject to rigorous scientific testing during prolonged exercise. Therefore the aim of this study was to investigate whether ingesting a BCAA supplement during prolonged exercise improves physical performance and central fatigue. The use of such a supplement during prolonged exercise could then be expected to have a large effect on performance. Eight trained cyclists (VO₂ max= 61.9 ± 4.3 ml 02/kg/min) ingested, in random order, a drink containing either 10% carbohydrate (CHO), 10% CHO and 0.16% branched-chain amino acid (BCAA) or 0.16% BCAA. Every hour, for the duration of the exercise (4 hours, 55% VO₂ max) blood samples were analysed for amino acids, ammonia, free fatty acids, glycerol, glucose and insulin concentrations. Urine was analysed for urea and creatinine concentrations. Heart rate, oxygen consumption (VO₂), respiratory exchange ratio (RER) and rating of perceived exertion were also analysed. Thereafter, subject's 40km time trial performance and RPE was assessed on a Velodyne windtrainer. Central fatigue following the time trial was quantified using the Sternberg reaction-time paradigm. The serum concentration of the BCAA's declined as a result of the exercise, in the BCAA only trial. Tryptophan concentration, however, did not change during the exercise. The serum TRP:BCAA ratio increased (0.16 ± 0.06 to 0.20 ± 0.10; p≤0.05) in the CHO trial only. The BCAA trial differed from the two trials in which CHO was ingested because plasma ammonia and glucose concentrations did not increase, while free fatty acids (FF A's) and glycerol concentrations increased significantly (p≤0.05). The lower RER in the BCAA trials suggests a higher proportion of fat was oxidised in these trials, compared to the other two trials. Cycling performance, over a 40km time trial, (CHO= 68.59 ± 6.02; CHO+ BCAA = 68.00 ± 3.01; BCAA = 69.43 ± 5.35 min/sec), ratings of perceived exertion, submaximal or maximal heart rates, and mental performance were not different between trials. Data from this study appears to refute the thesis hypothesis that an increase in serum TRP:BCAA decreases physical performance and central fatigue, during prolonged exercise.

Thesis (M.S.)--Massachusetts Institute of Technology, Dept. of Nutrition and Food Science, 1981. / Bibliography: leaves 100-104. / by Tina Talvikki Kangas. / M.S.

Indiana University-Purdue University Indianapolis (IUPUI) / Chlamydia trachomatis is an obligate intracellular bacterial pathogen that can infect the eyes, genital tract, and disseminate to lymph nodes in humans. Many C. trachomatis infections are clinically asymptomatic and can become chronic if left untreated. When humans are infected with C. trachomatis, a cytokine that is produced is interferon-gamma (IFN-γ). In vitro, IFN-γ stimulates expression of the host enzyme indoleamine 2,3-dioxygenase. This enzyme converts free intracellular tryptophan to N-formylkynurenine. Tryptophan starvation induces C. trachomatis to enter a viable-but-nonculturable state termed persistence, which has been proposed to play a key role in chronic Chlamydial disease. To circumvent host induced tryptophan depletion, urogenital strains of C. trachomatis encode a functional tryptophan synthase (TS). TS synthesizes tryptophan from indole and serine, allowing Chlamydia to reactivate from persistence. Transcriptomic analysis revealed C. trachomatis differentially regulates hundreds of genes in response to tryptophan starvation. However, genes that mediate entry, survival, and reactivation from persistence remain largely unknown. Using a forward genetic screen, we identified six Susceptible to IFN-γ mediated Persistence (Sip) mutants that have diminished capacities to reactivate from persistence with indole. Mapping the deleterious persistence alleles in three of the Sip mutants revealed that only one of the mutants had a mutation in TS. The two other Sip mutants mapped had mutations in CTL0225, a putative integral membrane protein, and CTL0694, a putative oxidoreductase. Neither of these genes plays a known role in tryptophan synthesis. However, amino acid (AA) competitive inhibition assays suggest that CTL0225 may be involved in the transport of leucine, isoleucine, valine, cysteine, alanine, and serine. Additionally, metabolomics analysis indicates that all free amino acids are depleted in response to IFN-γ, making this amino acid transporter essential during persistence. Taken together we have identified two new chlamydial persistence genes that may play a role in chronic chlamydial disease.

Sinefungin is an antibiotic and a close structural analog of S-adenosylmethionine, a co-factor for a vast number of enzymes. Sinefungin is one of a class of natural products containing both nucleoside and amino acid moieties. Sinefungin is a conformationally complex molecule. Because the sinefungin molecule consists of a planar adenine ring capable of both syn and anti orientation with respect to a conformationally mobile ribose ring and a flexible amino acid side chain, it has a wide range of conformational features that may be related to its bioactivity. Both lD and 2D F1NMR methods were used to evaluate vicinal proton coupling constants in order to determine probable dihedral angles about the C(2')-C(3') bond of the ribose ring and the C(α)-C(β), C(β)-C(γ), C(γ)-C(δ) and C(4') and C(5') dihedral angles of the amino acid side chain for proton spectra taken above pH 9.4. At this pH sinefungin is conformationally less flexible than S-adenosylmethionine. Overlapping resonances a detailed conformational analysis of sinefungin below pH 9.4. Most interesting was the discovery that the H(5') and H(5") resonances have the same chemical shift in acidic solutions so that valuable coupling constant information is lost. MM2 calculations were consistent with NMR results.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19601号 / 医博第4108号 / 新制||医||1014(附属図書館) / 32637 / 京都大学大学院医学研究科医学専攻 / (主査)教授 岩井 一宏, 教授 柳田 素子, 教授 山下 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM