Oxidative stress induced by a dihydropyrazine derivative.

The Maillard reaction contributes to the complications of diabetes and normal aging. Dihydropyrazines (DHPs), which are produced during the Maillard reaction, generate radicals and possess DNA strand-cleaving activities in vitro. In the present study, we evaluated the genotoxic and cytotoxic potentials of a DHP derivative, cyclohexyl-DHP, which is obtained as a mixture of two isomers, 2,3,5,6,7,8-hexahydroquinoxaline (endo-type) and 1,2,3,5,6,7-hexahydroquinoxaline (exo-type), fused with a cyclohexyl ring. Cyclohexyl-DHP caused DNA strand breaks in plasmid pUC18, especially in the presence of Cu(2+). By using Escherichia coli mutant strains, we observed that cyclohexyl-DHP exposure strongly reduced the survival rate of a cytosolic sodium dodecyl sulfate (SOD)-deficient strain (sodA sodB), significantly reduced the survival rates of DNA repair-deficient strains (recA and uvrB) and mildly reduced the survival rate of a catalase-deficient strain (katE katG) compared with the survival rate of the wild-type strain. Addition of Cu(2+) enhanced the cell killing ability of cyclohexyl-DHP. The frequency of mutations induced by cyclohexyl-DHP increased dose-dependently in the sodA sodB strain. Assays with the highly water-soluble tetrazolium salt WST-1 revealed that cyclohexyl-DHP strongly generated superoxide anions. Moreover, cyclohexyl-DHP elevated the protein carbonyl levels in E. coli. These findings indicate that cyclohexyl-DHP could potentially generate superoxide anions, and cause not only breakage of chromosomal DNA leading to mutagenic lesions but also induce damage to cellular proteins.

Growth suppression of human cancer cells by polyphenolics from sweetpotato (Ipomoea batatas L.) leaves.

Sweetpotato leaves (Ipomoea batatas L.) contain a high content of polyphenolics that consist of caffeic acid, chlorogenic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid, and 3,4,5-tri-O-caffeoylquinic acid. We investigated the suppression of the proliferation of selected human cancer cells by phenolic compounds isolated from sweetpotato leaf. The human cancer cells used in this research included a stomach cancer (Kato III), a colon cancer (DLD-1), and a promyelocytic leukemia cell (HL-60). Caffeic acid and di- and tricaffeoylquinic acids dose-dependently depressed cancer cell proliferation, and the difference in sensitivity between caffeoylquinic acid derivatives and each kind of cancer cell was observed. Specifically, 3,4,5-tri-O-caffeoylquinic acid effectively depressed the growth of three kinds of cancer cells, and caffeic acid had an exceptionally higher effect against HL-60 cells than other di- and tricaffeoylquinic acids. In attempting to clarify the mechanism of growth suppression with the addition of the apoptotic inhibitor N-ethylmaleimide, we observed that the nuclear granulation in 3,4,5-tri-O-caffeoylquinic acid-treated HL-60 cells suggested apoptosis induction. This effect was confirmed by DNA fragmentation, an increase of caspase-3 activity, and expression of c-Jun. Growth suppression of HL-60 cells by 3,4,5-tri-O-caffeoylquinic acid was determined to be the result of apoptotic death of the cells. These results indicate that 3,4,5-tri-O-caffeoylquinic acid may have potential for cancer prevention.

Mutation spectrum induced by dihydropyrazines in Escherichia coli.

Dihydropyrazine (DHP), which induces mutagenesis in E. coli, was investigated. From analyzing mutations in the chromosomal rpoB gene, the mutation spectrum in uvrB strain revealed the different behavior on exposure to two DHP derivatives 3-hydro-2,2,5,6-tetramethylpyrazine (HTMP), and 2,3-dihydro-5,6-dimethylpyrazine (DHDMP). A higher level of DHP-induced mutation was observed, with base substitutions at G : C pairs predominant. HTMP and DHDMP increased the frequency of G : C to T : A transversions. HTMP increased the frequency of G : C to A : T transitions, than did DHDMP. These findings suggest that DHPs prefer to attack the G : C pair and that different DHP derivatives may prefer distinct mutagenic base pairs; and further, that nucleotide excision repair may be involved in the repair of DHP-induced mutations.

Pipecolic acid induces apoptosis in neuronal cells.

Pipecolic acid, a lysine metabolite, is thought to be a factor responsible for hepatic encephalopathy; however, the underlying mechanism is far from understood. Twenty minutes treatment with D-, L-, and DL-pipecolic acid at concentrations ranging from 1 to 100 microM, except for 1 microM L-pipecolic acid, had no inhibitory effect on excitatory postsynaptic responses in the dentate gyrus of rat hippocampal slices. In a whole-cell voltage-clamp configuration, DL-pipecolic acid (10 and 100 microM) did not affect voltage-sensitive Na(+) channel currents and K(+) channel currents, but it potentiated voltage-sensitive Ca(2+) channel currents, but to a lesser extent, in cultured rat cortical neurons and Neuro-2A cells, a mouse neuroblastoma cell line. Notably, 72-h treatment with D-, L-, and DL-pipecolic acid reduced Neuro-2A cell viability in a dose-dependent manner at concentrations ranging from 1 to 100 microM in a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, in parallel with reactions to propidium iodide, a marker of cell death, and Hoechst 33,342, a marker of apoptosis in a fluorescent microscopic study, with DL-pipecolic acid being the most potent. The results of the present study suggest that pipecolic acid could cause hepatic encephalopathy by inducing neuronal cell death, perhaps apoptosis, rather than by depressing neurotransmissions.

Chicken HDAC2 down-regulates IgM light chain gene promoter activity.

In a chicken B cell line, DT40, the disruption of HDAC2 (chHDAC2) gene causes an alteration of several gene expressions including chicken IgM light chain (chIgM-L) gene by 2D-PAGE analysis. To investigate the transcriptional function of chHDAC2, we employed the chIgM-L promoter reporter plasmid. We found that chHDAC2 represses activated chIgM-L promoter activity. In transient expression experiments in NIH 3T3 cell, the specific histone deacetylase inhibitor tricostatin A (TSA) increased transactivation of chIgM-L promoter activity mediated by chicken Oct-1 and OBF-1 proteins. In transient coexpression of the three class I chicken histone deacetylases (chHDAC1-3) tested, only chHDAC2 repressed the activated chIgM-L promoter activity. These findings suggest that chHDAC2 might be recruited to the chIgM-L promoter and specifically repress chIgM-L transcription.

A rare combination of sites of involvement by Mycobacterium intracellulare in a hemodialysis patient: multifocal synovitis, spondylitis, and multiple skin lesions.

PURPOSE: Atypical mycobacterial infection is a rare but serious hazard in immunocompromised patients including those undergoing maintenance hemodialysis and immunosuppressive therapy. Recognition of unusual involvement patterns is important. METHODS: We describe an extremely rare combination of complications caused by such an organism in a patient with end-stage renal disease: spinal osteolysis and multiple skin lesions associated with synovitis. RESULTS: The patient had received a renal allograft 18 years previously but developed infection with Mycobacterium avium-M. intracellulare complex including dermatologic manifestations, spondylitis, and synovitis involving the wrist and lateral malleolus after initiation of hemodialysis when the transplanted kidney failed. An empirical antibiotic regimen failed to alleviate skin lesions or fevers, or to lower an elevated C-reactive protein concentration, until the patient's dose of methylprednisolone was increased to treat mild adrenal insufficiency. The increase resulted in rapid resolution of skin lesions. A compression fracture 6 months later was attributed to spondylitis caused by the same organism. CONCLUSIONS: We suspect that spondylitis represented the primary focus of M. intracellulare infection.

Cloning and characterization of the chick Oct binding factor OBF-1.

We have cloned the chicken homolog of OBF-1, chOBF-1, which comprises 256 amino acids, and exhibits only 65% overall identity to the human and mouse OBF-1 proteins. Amino acid sequence alignment revealed the putative Oct-binding sequence, RPYQGVRVKEPVKELL(K/R)RKRG, which is conserved among chicken, mouse and human. chOBF-1 protein was demonstrated to bind chicken Oct-1 protein by the in vitro immunoprecipitation experiment, and chOBF-1 was shown to functionally activate the chicken immunoglobulin (Ig) light chain promoter in the NIH 3T3 cell. Taken together, these data indicate that the Ig gene transcription machinery, including Oct-1 and OBF-1, has been highly conserved in vertebrate evolution.

Spectroscopic evidence that salicylic acid converts a temporally inactivated form of horseradish peroxidase (compound III) to the irreversibly inactivated verdohemoprotein (P-670).

We obtained spectroscopic evidence in support of salicylate-dependent inactivation of horseradish peroxidase-C. Addition of salicylate to the enzyme arrested at a temporal inactive state (Compound III) in the presence of H2O2, resulted in rapid and irreversible inactivation of the enzyme yielding verdohemoproteins (P-670). Multiple roles for salicylate in peroxidase-catalyzed reactions are discussed.

Spectroscopic evidence in support of horseradish peroxidase compound II-catalyzed oxidation of salicylic acid but not of phenylethylamine.

Salicylic acid and phenylethylamine are putative substrates for naturally occurring reactions for generation of reactive oxygen species, which are catalyzed by plant peroxidases. Here, we used commercially available highly purified horseradish peroxidase-C (HRP-C) as a model enzyme for spectroscopic analysis, and obtained data suggesting that the Compound II form of HRP-C does not utilize phenylethylamine as substrate. In contrast, addition of salicylic acid to Compound II resulted in rapid conversion of Compound II to the native form.