Cytotoxicity and oxidative stress induced by the glyceraldehyde-related Maillard reaction products for HL-60 cells

We demonstrated the cytotoxicity of glyceraldehyde-related Maillard reaction products for HL-60 cells. Glyceraldehyde-modified bovine serum albumin and glyceraldehyde-modified casein inhibited the proliferation of HL-60 cells. The reaction products formed from glyceraldehyde and Nalpha-acetyllysine had also a cytotoxic effect on HL-60 cells. The cytotoxic effect was prevented by N-acetylcysteine or pyrrolidinedithiocarbamate as the antioxidants. In addition, the reaction products depressed the intracellular glutathione level, and induced the reactive oxygen species (ROS) production. These results suggested that the glyceraldehyde-related advanced glycation end products (AGEs) induced the cytotoxicity and the oxidative stress.We previously reported that the glyceraldehyde-related AGE was identified as 1-(5-acetylamino-5-carboxypentyl)-3-hydroxy-5-hydroxymethyl-pyridinium, named GLAP (glyceraldehyde-derived pyridinium compound), formed from glyceraldehyde and Nalpha-acetyllysine (Biosci. Biotechnol. Biochem., 67, 930-932 (2003)). In this study, GLAP inhibited the proliferation of HL-60 cells, and the inhibitory effect was prevented by the antioxidants. Furthermore, GLAP depressed the intracellular glutathione level, and induced the ROS production.This work indicated the possibility that the cytotoxicity and the oxidative stress in the progression of diabetic complications and chronic renal disease might be induced by GLAP.     

Identification of a collagen production-promoting factor from an extract of royal jelly and its possible mechanism

We have previously shown that royal jelly (RJ) promoted collagen production by skin fibroblasts in the presence of ascorbic acid-2-O-alpha-glucoside (AA-2G). In this study, we purified the honeybee RJ-derived collagen production-promoting factor (HBRJ-CPF) from an alkali-solubilized fraction of RJ by C18 reverse-phase column chromatography. The elution profile by the C18 column chromatography and the molecular mass of the purified HBRJ-CPF material coincided with those of 10-hydroxy-2-decenoic acid (10H2DA). We then examined the collagen production-promoting activities of several commercially available fatty acids contained in RJ. We found that 10H2DA and 10-hydroxydecanoic acid increased the collagen production in a dose-dependent manner. Furthermore, 10H2DA induced the fibroblast cell line, NHDF, to produce transforming growth factor-beta 1 (TGF-beta 1) which is an important factor for collagen production. As expected, the collagen production-promoting activity of 10H2DA was neutralized by the anti-TGF-beta 1 antibody. These result suggest that HBRJ-CPF identified as 10H2DA promoted the collagen production of AA-2G-treated fibroblasts by inducing TGF-beta 1 production.     

In vitro reconstitution of rice anthranilate synthase: distinct functional properties of the alpha subunits OASA1 and OASA2

Anthranilate synthase (AS) is a key enzyme in the biosynthesis of various indole compounds including tryptophan. AS consists of two subunits, alpha and beta, and converts chorismate to anthranilate. Two or more AS alpha-subunit genes have been identified and characterized in several land plants. Although alpha subunits of AS induced by elicitation have been suggested to play significant roles in secondary metabolism, the biochemical and precise functional properties of individual AS isozymes have remained unclear. We have previously identified and characterized two AS alpha-subunit genes (OASA1 and OASA2) in rice (Oryza sativa ). To provide further insight into the enzymatic functions of AS isozymes in rice, we have now isolated rice cDNAs encoding the AS beta subunits OASB1 and OASB2 and reconstituted AS isozymes in vitro with the wheat germ cell-free system for protein expression. Both OASB subunits conferred glutamine-dependent AS activity on either OASA1 or OASA2, indicating the absence of a marked functional difference between the two beta subunits in terms of amidotransferase activity. Furthermore, both OASA subunits required assembly with a beta subunit to achieve maximal enzymatic activity even with NH(4)(+) as the amino donor. The V (max) and K (i) for tryptophan of the OASA1-OASB1 isozyme with glutamine as the amino donor, however, were 2.4 and 7.5 times, respectively, those of OASA2-OASB1, suggesting that AS isozymes containing OASA1 possess a higher activity and are less sensitive to feedback inhibition than those containing OASA2. Our biochemical characterization of reconstituted AS isozymes has thus revealed distinct functional properties of these isozymes in rice.     

Synthesis and evaluation of substituted 4-alkoxy-2-aminopyridines as novel neuropeptide Y1 receptor antagonists

A series of substituted 4-alkoxy-2-aminopyridines 2, which were formally derived from neuropeptide Y1 antagonist 1 by replacing the morpholino portion with alkoxy groups, were synthesized and evaluated as neuropeptide Y Y1 receptor antagonists. Primary structure-activity relationships and identification of potent 4-alkoxy derivatives are described.     

Involvement of putative SNF2 chromatin remodeling protein DRD1 in RNA-directed DNA methylation

In plants, the mechanism by which RNA can induce de novo cytosine methylation of homologous DNA is poorly understood. Cytosines in all sequence contexts become modified in response to RNA signals. Recent work has implicated the de novo DNA methyltransferases (DMTases), DRM1 and DRM2, in establishing RNA-directed methylation of the constitutive nopaline synthase promoter, as well as the DMTase MET1 and the putative histone deacetylase HDA6 in maintaining or enhancing CpG methylation induced by RNA. Despite the identification of enzymes that catalyze epigenetic modifications in response to RNA signals, it is unclear how RNA targets DNA for methylation. A screen for mutants defective in RNA-directed DNA methylation identified a novel putative chromatin-remodeling protein, DRD1. This protein belongs to a previously undefined, plant-specific subfamily of SWI2/SNF2-like proteins most similar to the RAD54/ATRX subfamily. In drd1 mutants, RNA-induced non-CpG methylation is almost eliminated at a target promoter, resulting in reactivation, whereas methylation of centromeric and rDNA repeats is unaffected. Thus, unlike the SNF2-like proteins DDM1/Lsh1 and ATRX, which regulate methylation of repetitive sequences, DRD1 is not a global regulator of cytosine methylation. DRD1 is the first SNF2-like protein implicated in an RNA-guided, epigenetic modification of the genome.     

Superoxide activates uncoupling proteins by generating carbon-centered radicals and initiating lipid peroxidation: studies using a mitochondria-targeted spin trap derived from alpha-phenyl-N-tert-butylnitrone

Although the physiological role of uncoupling proteins (UCPs) 2 and 3 is uncertain, their activation by superoxide and by lipid peroxidation products suggest that UCPs are central to the mitochondrial response to reactive oxygen species. We examined whether superoxide and lipid peroxidation products such as 4-hydroxy-2-trans-nonenal act independently to activate UCPs, or if they share a common pathway, perhaps by superoxide exposure leading to the formation of lipid peroxidation products. This possibility can be tested by blocking the putative reactive oxygen species cascade with selective antioxidants and then reactivating UCPs with distal cascade components. We synthesized a mitochondria-targeted derivative of the spin trap alpha-phenyl-N-tert-butylnitrone, which reacts rapidly with carbon-centered radicals but is unreactive with superoxide and lipid peroxidation products. [4-[4-[[(1,1-Dimethylethyl)-oxidoimino]methyl]phenoxy]butyl]triphenylphosphonium bromide (MitoPBN) prevented the activation of UCPs by superoxide but did not block activation by hydroxynonenal. This was not due to MitoPBN reacting with superoxide or the hydroxyl radical or by acting as a chain-breaking antioxidant. MitoPBN did react with carbon-centered radicals and also prevented lipid peroxidation by the carbon-centered radical generator 2,2'-azobis(2-methyl propionamidine) dihydrochloride (AAPH). Furthermore, AAPH activated UCPs, and this was blocked by MitoPBN. These data suggest that superoxide and lipid peroxidation products share a common pathway for the activation of UCPs. Superoxide releases iron from iron-sulfur center proteins, which then generates carbon-centered radicals that initiate lipid peroxidation, yielding breakdown products that activate UCPs.