To investigate the ability of the production of H(2)O(2) by polyphenols, we incubated various phenolic compounds and natural polyphenols under a quasi-physiological pH and temperature (pH 7.4, 37 degrees C), and then measured the formation of H(2)O(2) by the ferrous ion oxidation-xylenol orange assay. Pyrocatechol, hydroquinone, pyrogallol, 1,2,4-benzenetriol, and polyphenols such as catechins yielded a significant amount of H(2)O(2). We also examined the effects of a metal chelator, pH, and O(2) on the H(2)O(2)-generating property, and the generation of H(2)O(2) by the polyphenol-rich beverages, green tea, black tea, and coffee, was determined. The features of the H(2)O(2)-generating property of green tea, black tea, and coffee were in good agreement with that of phenolic compounds, suggesting that polyphenols are responsible for the generation of H(2)O(2) in beverages. From the results, the possible significances of the H(2)O(2)-generating property of polyphenols for biological systems are discussed.
Culturable leaf-associated bacteria inhabiting a plant have been considered as promising biological control agent (BCA) candidates because they can survive on the plant. We investigated the relationship between bacterial groups of culturable leaf-associated bacteria on greenhouse- and field-grown tomato leaves and their antifungal activities against tomato diseases in vitro and in vivo. In addition, the isolated bacteria were analyzed for N-acyl-homoserine lactone (AHL) and indole-3-acetic acid (IAA) production, which have been reported to associate with bacterial colonization, and resistance to a tomato alkaloid (alpha-tomatine). Leaf washings and subsequent leaf macerates were used to estimate the population size of epiphytic and more internal bacteria. Bacterial population sizes on leaves at the same position increased as the leaves aged under both greenhouse and field conditions. Field-grown tomatoes had significantly larger population sizes than greenhouse-grown tomatoes. Analysis of 16S rRNA gene (rDNA) sequencing using 887 culturable leaf-associated bacteria revealed a predominance of the Bacillus and Pseudomonas culturable leaf-associated bacterial groups on greenhouse- and field-grown tomatoes, respectively. Curtobacterium and Sphingomonas were frequently recovered from both locations. From the 2138 bacterial strains tested, we selected several strains having in vitro antifungal activity against three fungal pathogens of tomato: Botrytis cinerea, Fulvia fulva, and Alternaria solani. Among bacterial strains with strong in vitro antifungal activities, Bacillus and Pantoea tended to show strong antifungal activities, whereas Curtobacterium and Sphingomonas were not effective. The results indicated the differences in antifungal activity among predominant bacterial groups. Analysis of alpha-tomatine resistance revealed that most bacterial strains in the dominant groups exhibited moderate or high resistance to alpha-tomatine in growth medium. Furthermore, some Sphingomonas and Pantoea strains showed AHL and IAA production activities. Strain 125NP12 (Pantoea ananatis) showed particular alpha-tomatine resistance, and AHL and IAA production had the highest protective value (91.7) against gray mold. Thus, the differences of these physiological properties among dominant bacteria may be associated with the disease suppression ability of BCAs on tomato plants.
Wheat can cause severe IgE-mediated systematic reactions, but knowledge on relevant wheat allergens at the molecular level is scanty. The aim of the present study was to achieve a more detailed and comprehensive characterization of the wheat allergens involved in food allergy to wheat using proteomic strategies, referred to as "allergenomics". Whole flour proteins were separated by two-dimensional gel electrophoresis with isoelectric focusing and lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, IgE-binding proteins were detected by immunoblotting with sera of patients with a food allergy to wheat. After tryptic digestion, the peptides of IgE-binding proteins were analyzed by matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry. In this study, we identified four previously reported wheat allergens or their sequentially homologous proteins [serpin, alpha-amylase inhibitor, gamma-gliadin, and low molecular weight (LMW) glutenin] by a database search. As a result of the high resolution of two-dimensional gel electrophoresis, nine subunits of LMW glutenins were identified as the most predominant IgE-binding antigens. The two-dimensional allergen map can be beneficial in many ways. It could be used, for example, for precise diagnosis of wheat-allergic patients and assessment of wheat allergens in food. Additionally, we compared allergenomics to conventional biochemical methods and evaluated the usefulness of a proteomic strategy for identifying putative allergens to wheat allergy.
The levels of a-aminoadipic-d-semialdehyde residue, the oxidative deamination product of lysine residue, in plasma protein from streptozotocin-induced diabetic rats were evaluated. a-Aminoadipic-d-semialdehyde was converted to a bisphenol derivative by acid hydrolysis in the presence of phenol, and determined by high performance liquid chromatography. Analysis of plasma proteins revealed three times higher levels of a-aminoadipic-d-semialdehyde in diabetic subjects compared with normal controls. Furthermore, we explored the oxidative deamination via the Maillard reaction and demonstrated that the lysine residue of bovine serum albumin is oxidatively deaminated during the incubation with various carbohydrates in the presence of Cu 2+ at a physiological pH and temperature. This experiment showed that 3-deoxyglucosone and methylglyoxal are the most efficient oxidants of the lysine residue. When the reaction was initiated from glucose, a significant amount of a-aminoadipic-d-semialdehyde was also formed in the presence of Cu 2+ . The reaction was significantly inhibited by deoxygenation, catalase, and a hydroxyl radical scavenger. The mechanism we propose for the oxidative deamination is the Strecker-type reaction and the reactive oxygen speciesmediated oxidation. Based on these findings, we propose a novel mechanism for the oxidative modification of proteins in diabetes, namely the oxidative deamination of the lysine residue via the Maillard reaction.
Dopamine (DA) is an unstable neurotransmitter that readily oxidizes to the DA quinone and forms reactive oxygen species, such as superoxide and hydrogen peroxide. The oxidized dopamine also forms thiol conjugates with sulfhydryl groups on cysteine, glutathione, and proteins. In the present study, we determined the redox potential of the protein-bound DA and established a novel mechanism for the oxidative modification of the protein, in which the DA-cysteine adduct generated in the DA-modified protein causes oxidative modification of the DA-bound protein in the presence of Cu2+. Exposure of a sulfhydryl enzyme, glyceraldehyde-3-phosphate dehydrogenase, to DA resulted in a significant loss of sulfhydryl groups and the formation of the DA-cysteine adduct. When the DA-modified protein was incubated with Cu2+, we observed aggregation and degradation of the DA-bound protein and concomitant formation of a protein carbonyl, a marker of an oxidatively modified protein. Furthermore, we analyzed the carbonyl products generated during the Cu2+-catalyzed oxidation of the DA-modified protein and revealed the production of glutamic and aminoadipic semialdehydes, consisting of the protein carbonyls generated. The cysteinyl-DA residue generated in the DA-modified protein was suggested to represent a redox-active adduct, based on the observations that the cysteinyl-DA adduct, 5-S-cysteinyldopamine, produced by the reaction of cysteine with DA, gave rise to the oxidative modification of bovine serum albumin in the presence of Cu2+. These data suggest that the DA-modified protein may be involved in redox alteration under oxidative stress, whereby DA covalently binds to cysteine residues, generating the redox-active cysteinyl-DA adduct that causes the metal-catalyzed oxidation of protein.
Alpha-aminoadipic semialdehyde (AAS) and gamma-glutamic semialdehyde (GGS) are identified as the major carbonyl products in oxidized proteins. To elucidate the formation pathway of AAS and GGS in vivo, we developed and validated a new quantification method. AAS and GGS in proteins were derivatized by reductive amination with NaCNBH(3) and p-aminobenzoic acid, a fluorescent reagent, followed by acid hydrolysis. It is noteworthy that the fluorescent derivatives were completely stable during acid hydrolysis. The present method permitted the specific, accurate, and sensitive quantification of both semialdehydes by fluorometric high-performance liquid chromatography. Analysis of proteins oxidized by various oxidation systems revealed that AAS and GGS are notably generated by the reaction of proteins with (*)OH, which is produced by metal-catalyzed oxidation (MCO). Furthermore, exposure of transferrin and human plasma to ascorbic acid and H(2)O(2) significantly promoted the formation of AAS and GGS in vitro, suggesting that both semialdehydes can be generated by MCO in vivo. We also demonstrated their generation through oxidative stress induced by acute iron overload in vivo. In this paper, we describe this analytical technique for simple and precise measurement of AAS and GGS and discuss their formation mechanism in vivo.
Polyphenols in several oxidation systems gained amine oxidase-like activity, probably due to the formation of the corresponding quinones. In the presence of Cu(II), o-and p-phenolic compounds exhibited amine oxidase-like activity, whereas only the o-phenolic compounds showed the activity in the presence of 1,1-diphenyl-2-picrylhydrazyl radical. The activity was determined by measuring the conversion of benzylamine to benzaldehyde by HPLC. Moreover, gallic acid, chlorogenic acid, and caffeic acid, which are plant polyphenols, converted the lysine residue of bovine serum albumin to a-amino-adipic semialdehyde residue, indicating lysyl oxidase-like activity. We also characterized the activity of pyrocatechol, hydroquinone, and pyrogallol in the presence of Cu(II). The oxidative deamination was accelerated at a higher pH, and required O 2 and transition metal ions. Furthermore, EDTA markedly inhibited the reaction but not b-aminopropionitrile, which is a specific inhibitor of lysyl oxidase. Catalase significantly inhibited the oxidation, implying the participation of hydroxyl radical in the reaction, but superoxide dismutase stimulated the oxidation, probably due to its radical formation activity. We discussed the mechanism of the oxidative deamination by polyphenols and the possible significance of the activity for biological systems.
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