As proteínas são consideradas importantes alvos para os oxidantes, devido à abundância em sistemas biológicos e às altas constantes de reações com estas espécies. Adicionalmente, têmse demonstrado que o triptofano (W) é um aminoácido extremamente susceptível a oxidação, inclusive pelo oxigênio singlete (1O2). A reação do W com o 1O2 tem despertado o interesse de diversos pesquisadores. Recentemente, esta reação tem atraído mais atenção, uma vez que produtos de oxidação do W tais como N-formilquinurenina (FMK) e quinurenina (kn) têm sido associados com algumas condições patológicas, tais como o desenvolvimento de catarata e a formação de agregados covalentes da superóxido dismutase envolvidos na esclerose lateral amiotrófica. Entretanto, há poucos trabalhos enfocando detalhadamente as reações, com estudos de estabilidade, identificação de subprodutos e propostas mecanísticas. Desta forma, pretendemos com este trabalho contribuir no esclarecimento do mecanismo de oxidação do triptofano pelo 1O2, através da análise e caracterização de produtos de oxidação gerados. Com este intuito, dois hidroperóxidos de triptofano isômeros (WOOH cis e trans, em relação ao grupamento carboxila) foram completamente caracterizados por análises de HPLC/espectrometria de massa e RMN como os principais produtos de oxidação do W pelo 1O2. Estes hidroperóxidos demonstraram ser relativamente estáveis às temperaturas ambiente e fisiológica, decompondo lentamente para os alcoóis correspondentes. O aumento do pH e/ou o aquecimento das soluções contendo os WOOH leva a decomposição quimiluminescente dos WOOH à FMK. Utilizando hidroperóxidos isotopicamente marcados com [18O] (W18O18OH) foi possível confirmar que a FMK formada durante esta decomposição era marcada com dois átomos de oxigênio. Este resultado demonstra que os dois átomos da FMK são derivados do grupamento hidroperóxido. Em adição, estas reações são quimiluminescentes, sugerindo o envolvimento de um intermediário dioxetano. Este mecanismo foi confirmado uma vez que o espectro de quimiluminescência da decomposição dos WOOH pode ser sobreposto ao espectro de fluorescência da FMK, inequivocamente identificado a FMK como a espécie emissora. Dioxindoilalaninas diastereoisoméricas também foram caracterizadas como produtos de oxidação do triptofano pelo 102; uma possível via radicalar foi excluída. Em suma, este estudo contribuiu na elucidação das bases químicas envolvidas na oxidação do triptofano por 102, através da caracterização dos produtos formados e da investigação detalhada dos mecanismos de decomposição destes produtos. / Proteins have been considered important targets for reactive oxygen species. Indeed, tryptophan (W) has been shown to be a highly susceptible amino acid to many oxidizing agents, including singlet molecular oxygen (1O2). The reaction of 1O2 with W has long been a matter of concern, and has recently attracted considerably more attention because W-derived oxidation products such as N-formylkynurenine (FMK) and kynurenine (kn) have been associated with some pathological conditions such as the development of cataracts and the formation of covalent aggregates of superoxide dismutase, which has been implicated in amyotrophic lateral sclerosis. Despite the intense interest in the mechanism of W oxidation, there are a lot of gaps that remains to be elucidated. In this context, the current study was undertaken to investigate the chemical basis involved in W oxidation by 1O2. We are concerned about the chemistry of the initially formed hydroperoxides, their stability, further reactions and the mechanism leading to FMK conversion. With this purpose, two cis and trans tryptophan hydroperoxide (WOOH) isomers were completely characterized by HPLC/mass spectrometry and NMR analyses as the major W-oxidation photoproducts. Also, they were shown to be relatively stable at ambient and physiological temperatures, leading to a slow decomposition to the corresponding alcohols. Increasing the pH or heating the solutions gives rise to a luminescent decomposition of the WOOH to FMK. Using 18O-labeled hydroperoxides (W18O18OH), it was possible to confirm the formation of a two-oxygen-labeled FMK molecule derived from W18O18OH decomposition. This result shows that both oxygen atoms in FMK are derived from the hydroperoxide group. In addition, these reactions are chemiluminescent (CL), indicating a dioxetane cleavage pathway. This mechanism was confirmed since the CL spectrum of the WOOH decomposition matched the FMK fluorescence spectrum, unequivocally identifying FMK as the emitting species. Diastereoisomeric dioxindoyalanine were also characterized as oxidation products derived from the reaction of W with 1O2. The involvement of radicals in this reaction was excluded. In summary, this work offers further insights into the chemistry involved in W-oxidation, through the characterization of photoproducts and the detailed investigation about the decomposition mechanism of these products.

No description available.

The hydrophobic effect and hydrogen bonding interactions have long been considered to be the dominant forces in protein folding. However, the contribution of hydrogen bonds to stabilizing proteins has been difficult to clarify. As the intramolecular hydrogen bonds are formed in place of hydrogen bonds with solvent during folding, measures of stability fail to give a significant change in free energy. Previous studies on hydrogen bonding interactions have shown that they are only marginally important. Three long-range side chain-main chain hydrogen bonds have been found in the alpha subunit of tryptophan synthase (αTS), a (βα)8TIM barrel protein. These long-range noncovalent interactions connect either the N-terminus of one β-strand with the C-terminus of the succeeding and anti-parallel α-helix (F19-D46 and I97-D124) or the N-terminus of an α-helix with the C-terminus of the succeeding β-strand (A103-D130). By analogy, these interactions are designated as βα- or αβ-hairpin clamps. Surprisingly, the removal of any one of these clamp interactions, by replacement of the aspartic acid with alanine, results in significantly decreased thermodynamic stability for the native state and a substantial loss of secondary structure. When compared to several other side chain-side chain and short-range side chain-main chain interactions in αTS, these hairpin clamps clearly play a unique role in the structure and stability of αTS. The generality of these observations for βα-hairpin clamps in TIM barrel proteins was tested by experimental analysis of the clamps in a pair of homologous indole-3-glycerol phosphate synthase (IGPS) TIM barrels of low sequence identity. The results suggest that only the subset of conserved βα-hairpin clamps with hydrogen bond length less than 2.80 Å make substantive contributions to stability and/or structure. Those clamps with longer hydrogen bonds make modest contributions to stability and structure, similar to other types of side chain-main chain or side chain-side chain hydrogen bonds. The role of these clamps in defining the structures of the super-family of TIM barrel proteins was examined by a survey of 71 TIM barrel proteins from the structural database. Conserved features of βα-hairpin clamps are consistent with a 4-fold symmetry, with a predominance of main chain amide hydrogen bond donors near the N-terminus of the odd-number β-strands and side chain hydrogen bond acceptors in the loops between the subsequent α-helices and even-numbered β-strands. In this configuration, the clamps provide an N-terminal cap to odd-number β- strands in the β-barrel. Taken together, these findings suggest that βα-hairpin clamps are a vestigial signature of the fundamental βαβ building block for the (βα)8 motif and an integral part of the basic TIM barrel architecture. The relative paucity of βα-hairpin clamps remaining in TIM barrel structures and their variable contributions to stability imply that other determinants for structure and stability of the barrel have evolved to render a subset of the clamp interactions redundant. Distinct sequence preferences for the partners in the βα-hairpin clamps and the neighboring segments may be useful in enhancing algorithms for structure prediction and for engineering stability in TIM barrel proteins.

Amino acids derived from the degradation of casein in cheese serve as precursors for the generation of key flavor compounds. Microbial degradation of tryptophan (Trp) is thought to promote formation of aromatic compounds that impart putrid fecal or unclean flavors in cheese, but pathways for their production have not been established. This study investigated tryptophan catabolism by Lactobacillus casei LC301 and LC202 and Lactobacillus helveticus CNRZ32 and LH212 cheese flavor adjuncts in carbohydrate starvation (pH 6.5, 30 or 37°C, no sugar) and cheese-like conditions (pH 5.2, 4% NaCl, 15°C, no sugar). Enzyme assays of cell-free extracts revealed both species of Lactobacillus catabolized tryptophan to indole lactic acid via indole pyruvic acid through transamination followed by dehydrogenation. Micellar electrokinetic capillary chromatography of culture supernatants showed these enzymes also catalyzed the reverse reactions, i.e., conversion of indole lactic acid to tryptophan. Tryptophan decarboxylase activity was detected in Lactobacillus cell-free extracts, but tryptamine was not detected in culture supernatants. Analysis of culture supernatants showed that tryptophan metabolism in Lactobacillus casei did not differ between the two conditions of incubation as it did in Lactobacillus helveticus LH212 and CNRZ32. Lactobacillus helveticus LH212, for example, did not catabolize Trp in carbohydrate starvation but did in cheese-like conditions. While cells of L. helveticus CNRZ32 did not catabolize Trp in either condition, they catabolized indole pyruvic acid to only Trp in carbohydrate starvation and to both Trp and indole lactic acid in cheese-like conditions. Micellar electrokinetic capillary chromatography of culture supernatants incubated under either starvation or cheese-like conditions showed Lactobacillus casei strains produced more indole lactic acid, and Lactobacillus helveticus strains favored tryptophan anabolic reactions. Based on the results obtained in this study, a putative pathway for the catabolism of tryptophan by lactobacilli in cheese is proposed.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21428号 / 農博第2306号 / 学位論文||H30||N5156(農学部図書室) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 近藤 直, 准教授 小川 雄一, 教授 飯田 訓久 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

No description available.

This research uses Regression analysis of fluorescence spectroscopy results to correlate with Biochemical Oxygen Demand (BOD5). Fluorescence spectroscopy was applied to samples taken from seven sample sites in the Provo and Orem waste water treatment plants found in Utah County. A total of 161 samples were collected for this research. 23 samples each were taken from four sites in the Provo waste water treatment plant namely Provo head works, aeration basin, primary filter settlement basin and the Provo effluent basin. The Orem head works, the clarifier and the Orem effluent basin were the three sample sites in the Orem waste water treatment plant where 23 samples each were collected to carry out the analysis. The fluorescent characteristics of the samples were determined using fluorescence spectrometry. These intensities were correlated with standard five day Biochemical Oxygen Demand (BOD5) values which were used as a measure of the amount of biodegradable organic material present. Chemical oxygen demand (COD) data were also taken from these treatment plants for correlation purposes. Three different correlation analyses were made which were the correlation of fluorescence spectroscopy excitation-emission matrix (EEM) against (1) individual sites BOD and COD values (2) Provo only and Orem only BOD and COD values (3) combined Provo and Orem BOD and COD values. The correlation of Individual site EEMs against BOD and COD values produced the best results. There was a higher correlation of EEM with BOD data than COD data. The R-squared for the combined Provo and Orem BOD data was 0.756 and that for COD was 0.729. Very high R-squared was obtained for Provo Influent data and Orem Influent data which were 0.955 and 0.946 respectively. This method can be used by wastewater stakeholders in deriving quick results in determining potential pollution events within a shorter time frame. This research demonstrates that there is a correlation between EEM and BOD/COD.

Neurons depend upon neurotransmitter release through regulated exocytosis to accomplish the immense processing performed within the central nervous system. The SNARE hypothesis points to a family of proteins that are thought to enable the membrane fusion that leads to exocytosis. The secondary structure of SNAP-25 is unique among SNARE proteins in that it has two alpha helical SNARE motifs and a cysteine rich (C85, C88, C90, C92) membrane interacting region but notransmembrane domain. The cysteines may be modified by palmitoylation or oxidation but the role of these modifications in vivo is not well understood. Our goal is to elucidate possible regulatory roles of SNAP-25 that relate to its unique structure and these reversible modifications. However, the study of SNAP-25 in reconstituted systems is hampered by a lack of readily available palmitoylated SNAP-25. A method for in vitro palmitoylation of SNAP-25 by HIP14, a neuronal acyltransferase, is described along with the application of a biotinylation streptavidin assay to verify palmitoylation. Palmitoylation increases the extent to which SNAP-25 interacts with lipids as observed with an environment sensitive trpytophan fluorescence assay. Palmitoylation also alters the phase transition of DPPC lipids differently than unpalmitoylated SNAP-25.This effect on the membrane may influence fusion events. Oxidation of the cysteine residues may be responsible for the sensitivity of SNAP-25 to reactive oxygen species. Our data suggests that, when oxidized, SNAP-25 does not interact with membranes to the same extent as palmitoylated SNAP-25. This may provide a mechanism for reducing exocytosis during oxidative stress. Also, oxidized SNAP-25 is not susceptible to Botulinum Neurotoxin E. The effects of oxidation and palmitoylation on the protein interactions of SNAP-25 may shed light on its role in the SNARE complex and membrane fusion.

Prenatal stress may increase the risk for depression in offspring and it has been suggested that this could be linked to alterations in tryptophan metabolism, leading to serotonergic changes. Dietary patterns based on the Mediterranean (Med) diet, which includes foods rich in nutrients involved in the tryptophan-serotonin pathway, have been linked to depressive symptom improvements when used as an intervention. This thesis examined, in a mouse model, whether a Med-based diet normalized depressive-like behaviour and changes in the serotonin system in the colon and hippocampus resulting from a repeated physical restraint stressor administered during the second trimester in adult C57BL/6N female and male offspring. The Med-based diet modulated behaviour and hippocampal serotonin receptors primarily in females and changed the enzyme involved in the colonic serotonergic pathway in males. These results suggest that a Med-based diet may help improve behavioural disturbances stemming from prenatal stress in a sex-specific way, perhaps through its actions on the gut-brain serotonin system.

No description available.