Enhancing effects of heterocyclic amines and beta-carbolines on the induction of chromosome aberrations in cultured mammalian cells.

The effects of post-treatment with heterocyclic amines and beta-carbolines on the induction of chromosome aberrations were studied in Chinese hamster CHO K-1 cells and SV40-transformed excision repair-deficient human XP2OSSV cells. The number of chromosome aberrations induced by UV and MMC were increased by post-treatment with Trp-P-1 and Trp-P-2, in both the presence and the absence of S9 mix. A alpha C, MeA alpha C, Glu-P-1, Glu-P-2, IQ, MeIQ, harman and harmine increased chromosome aberrations only in the presence of S9 mix. Glu-P-2, IQ, MeIQ, harman, and harmine did not induce chromosome aberrations by themselves at the concentrations used in this study. Trp-P-1, Trp-P-2, A alpha C, MeA alpha C and Glu-P-1 were weak clastogens by themselves, but at much higher concentrations than those at which they increased the induction of chromosome aberrations in cells pretreated with UV or MMC. Therefore, the increases in chromosome aberrations were not considered to be additive.     

Metabolic fate of luteolin and its functional activity at focal site

Luteolin has been shown to possess potent antioxidant and anti-inflammatory/anti-allergic activities. In order to evaluate a chemopreventive role of luteolin in inflammatory responses involved in the pathogenesis of atherosclerosis and cancer etc., the metabolic fate of luteolin in rats and humans was investigated by HPLC analysis, and its effect on cell surface expression of adhesion molecules in human umbilical vein endothelial cells(HUVECs) was examined by ELISA. Luteolin monoglucuronide, which was a main metabolite, and free luteolin were detected in rat plasma and human serum. Luteolin monoglucuronide was hydrolyzed to free luteolin by beta-glucuronidase released from neutrophils stimulated with lonomycin and Cytocharasine B. Luteolin suppressed the TNF-alpha induced ICAM-1 expression significantly. Among nine flavonoids (40 microM) examined, chrysin, apigenine, quercetin and galangin also demonstrated suppressive effct on it. These results suggest the posssibility that deconjugation of luteolin monoglucuronide occurs and that free luteolin showed functional acyivities such as suppression of TNF-alpha induced ICAM- 1 expression at inflammation site.     

Identification of genes required for growth under ethanol stress using transposon mutagenesis in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

The yeast Saccharomyces cerevisiae exhibits high ethanol tolerance compared with other microorganisms. The mechanism of ethanol tolerance in yeast is thought to be regulated by many genes. To identify some of these genes, we screened for ethanol-sensitive S. cerevisiae strains among a collection of mutants obtained using transposon mutagenesis. Five ethanol-sensitive (ets) mutants were isolated from approximately 7,000 mutants created by transforming yeast cells with a transposon (mTn-lacZ/LEU2)-mutagenized genomic library. Although these mutants grew normally in a rich medium, they could not grow in the same medium containing 6% ethanol. Sequence analysis of the ets mutants revealed that the transposon was inserted in the coding regions of BEM2, PAT1, ROM2, VPS34 and ADA2. We constructed deletion mutants for these genes by a PCR-directed disruption method and confirmed that the disruptants, like the ets mutants, were ethanol sensitive. Thus, these five genes are indeed required for growth under ethanol stress. These mutants were also more sensitive than normal cells to Calcofluor white, a drug that affects cell wall architecture, and Zymolyase, a yeast lytic enzyme containing mainly beta-1,3- glucanase, indicating that the integrity of the cell wall plays an important role in ethanol tolerance in S. cerevisiae.     

Retention of Saccharomyces cerevisiae cell wall proteins through a phosphodiester-linked {beta}-1,3-/{beta}-l,6-glucan heteropolymer

Yeast cell wall proteins, including Cwp1p and      

Enzymatic in situ determination of stereospecificity of NAD-dependent dehydrogenases

Amino acid racemases inherently catalyze the exchange of alpha-hydrogen of amino acids with deuterium during racemization in 2H2O. When the reactions catalyzed by alanine racemase (EC 5.1.1.1) and L-alanine dehydrogenase (EC 1.4.1.1), which is pro-R specific for the C-4 hydrogen transfer of NADH, are coupled in 2H2O, [4R-2H]NADH is exclusively produced. Similarly, [4S-2H]NADH is made in 2H2O with amino-acid racemase with low substrate specificity (EC 5.1.1.10) and L-leucine dehydrogenase (EC 1.4.1.9), which is pro-S specific. We have established a simple procedure for the in situ analysis of stereospecificity of C-4 hydrogen transfer of NADH by an NAD-dependent dehydrogenase by combination with either of the above two couples of enzymes in the same reaction mixture. When the C-4 hydrogen of NAD+ is fully retained after sufficient incubation, the stereospecificity of hydrogen transfer by a dehydrogenase is the same as that of alanine dehydrogenase or leucine dehydrogenase. However, when the C-4 hydrogen of NAD+ is exchanged with deuterium, the enzyme to be examined shows the different stereospecificity from alanine dehydrogenase or leucine dehydrogenase. Thus, we can readily determine the stereospecificity by 1H NMR measurement without isolation of the coenzymes and products.     

Ethanol-induced death in yeast exhibits features of apoptosis mediated by mitochondrial fission pathway

Cell death in yeast (Saccharomyces cerevisiae) involves several apoptotic processes. Here, we report the first evidence of the following processes, which are also characteristic of apoptosis, in ethanol-induced cell death in yeast: chromatin condensation and fragmentation, DNA cleavage, and a requirement for de novo protein synthesis. Mitochondrial fission protein, Fis1, appears to mediate ethanol-induced apoptosis and ethanol-induced mitochondrial fragmentation. However, mitochondrial fragmentation in response to elevated ethanol levels was not correlated with cell death. Further, in the presence of ethanol, generation of reactive oxygen species was elevated in mutant fis1Delta cells. Our characterization of ethanol-induced cell death in yeast as being Fis1-mediated apoptosis is likely to pave the way to overcoming limitations in large-scale fermentation processes, such as those employed in the production of alcoholic beverages and ethanol-based biofuels.     

Global gene expression analysis of yeast cells during sake brewing

During the brewing of Japanese sake, Saccharomyces cerevisiae cells produce a high concentration of ethanol compared with other ethanol fermentation methods. We analyzed the gene expression profiles of yeast cells during sake brewing using DNA microarray analysis. This analysis revealed some characteristics of yeast gene expression during sake brewing and provided a scaffold for a molecular level understanding of the sake brewing process.     

Elucidation of amidating reaction mechanism by frog amidating enzyme, peptidylglycine alpha-hydroxylating monooxygenase, expressed in insect cell culture.

A frog 'peptidylglycine alpha-amidating monooxygenase (PAM, EC 1.14.17.3)' was expressed in cultured insect cells by using the baculovirus expression vector system. The enzyme, recovered in the culture medium, was purified to homogeneity. Its apparent molecular mass (43 kd), estimated by both SDS-PAGE and molecular sieving, was higher than the value (39 kd) for the 'PAM' (AE-I) purified from frog skin. N-terminal sequence analysis indicated that cleavage of signal sequence had occurred but the propeptide still remained at the N terminus. The glycine-extended model peptide X-Gly (mean = Ala-Ile-Gly-Val-Gly-Ala-Pro) was used as substrate for the purified enzyme. The reaction product formed at pH 5.4 was isolated and characterized by amino acid sequence analysis, FAB-MASS and 1H-NMR. It was shown that the purified enzyme had converted the model peptide to the C-terminal alpha-hydroxyglycine-extended peptide [X-Gly(OH)] instead of the amidated product (X-NH2), indicating that the enzyme widely known as 'PAM' should be called 'peptidylglycine alpha-hydroxylating monooxygenase'. A novel enzyme, present in the insect cell culture medium and separable from the expressed monooxygenase, could convert the alpha-hydroxyglycine-extended peptide to the amidated product at physiological pH values. It is concluded that the alpha-amidation of glycine-extended peptides is a two-step process catalyzed by the monooxygenase and the novel enzyme.