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The effects of hydroxylation at the C8 of a deoxyguanosine residue in DNA were studied by NMR analysis of a self-complementary dodecanucleotide, d(C1-G2-C3-oh8G4-A5-A6-T7-T8-C9-G10-C11-G12), which has an 8-hydroxy-2'-deoxyguanosine (oh8dG) residue at the 4th position. NMR data indicate that the 8-hydroxyguanine (oh8G) base takes a 6,8-diketo tautomeric form and is base-paired to C with Watson-Crick type hydrogen bonds in a B-form structure. The thermal stability of the duplex is reduced, but the overall structure is much the same as that of the unmodified d(CGCGAATTCGCG) duplex. The structural changes caused by 8-hydroxylation of the deoxyguanosine, if any, are localized near the modification site.

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Checkpoints, which monitor DNA damage and regulate cell cycle progression, ensure genomic integrity and prevent the propagation of transformed cells. DNA damage activates the p53-dependent checkpoint pathway that induces expression of p21

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S U M M A R Y We have previously reported the TLR4 expression in human intestinal lymphatic vessels. In the study here, microarray analysis showed the expression of the TLR4, MD-2, CD14, MyD88, TIRAP, TRAM, IRAK1, and TRAF6 genes in cultured human neonatal dermal lymphatic microvascular endothelial cells (LEC). The microarray analysis also showed that LEC expressed genes of IL-6, IL-8, VCAM-1, and ICAM-1, and the real-time quantitative PCR analysis showed that mRNA production was increased by lipopolysaccharide (LPS). The LPS-induced IL-6, IL-8, VCAM-1, and ICAM-1 production in LEC was suppressed by the introduction of TLR4-specific small interfering RNA, and also by anti-TLR4, nobiletin, and CAPE pretreatment. These findings suggest that LEC has TLR4-mediated LPS recognition mechanisms that involve at least activation of NF-kB, resulting in increased expression of IL-6, IL-8, VCAM-1, and ICAM-1. Both the LPS effect on the gene expression and also the suppression by nobiletin and CAPE pretreatment on the protein production were larger in IL-6 and in VCAM-1 than in IL-8 and in ICAM-1 in LEC. The signal transduction of NF-kB and AP-1-dependent pathway may be more critical for the expression of IL-6 and VCAM-1 than that of IL-8 and ICAM-1 in LEC. LYMPHATIC VESSELS express not only platelet-endothelial adhesion molecule-1 (PECAM-1) but also the intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in inflamed and uninflamed human small intestine and tongue, and lymphatic endothelium enhances VCAM-1 and ICAM-1 production with tumor necrosis factor-a (TNF-a) (Sawa et al. 1999(Sawa et al. ,2007Ebata et al. 2001). It is well established that VCAM-1 and ICAM-1 induction needs a signaling pathway that involves protein kinase C and p38 mitogenactivated protein kinases (MAPKs)-mediated activation of transcription factors nuclear factor-kappaB (NF-kB) and activator protein 1 (AP-1) (Voraberger et al. 1991;Ahmad et al. 1998;Ishizuka et al. 1998;Oertli et al. 1998;Kobuchi et al. 1999;Lawson et al. 1999;Roebuck 1999). It is thought that lymphatic endothelium has signal transduction pathways to induce leukocyte adhesion molecule expression through receptors for inflammatory cytokines. However, it has not been established whether lymphatic endothelium has the expression mechanisms of immunological functional molecules independent of the inflammatory cytokines.The toll-like receptor (TLR) initiates a series of innate immune mechanisms against various microorganism infections by sensing the presence of pathogen-associated molecular patterns (PAMPs) like lipopolysaccharide (LPS), which is the major component of the outer surface of Gram-negative bacteria. The LPS stimulates leukocyte and blood endothelium through the LPS recognition systems, binding with CD14 and transferring to TLR4 and MD-2 complex, followed by a myleoid differentiation primary response protein (MyD88)-dependent or -independent pathway that culminates in the early-or late-phase activation of the IkB kinase (IKK) complex and MAPK...

An oligodeoxyribonucleotide containing 8-hydroxyadenine (OH8Ade) was chemically synthesized and single- and double-stranded c-Ha-ras gene fragments with OH8Ade at the second position of codon 61 were prepared. The single-stranded ras gene fragment was used as a template for in vitro DNA synthesis with the Klenow fragment of Escherichia coli DNA polymerase I, Taq DNA polymerase, rat DNA polymerase beta and mouse DNA polymerase alpha. The former two enzymes exclusively incorporated dTMP opposite OH8Ade. The DNA polymerases alpha and beta misinserted dGMP, and dAMP and dGMP, respectively. The c-Ha-ras gene was constructed using the double-stranded ras gene fragment containing OH8Ade and was transfected into NIH 3T3 cells. The gene with OH8Ade induced focus formation, indicating that OH8Ade elicited point mutations in cells. When c-Ha-ras genes present in transformed cells were analyzed, an A-->G transition and an A-->C transversion were detected. These results indicate that OH8Ade induced misincorporation in in vitro DNA synthesis and mutations in mammalian cells.

Adsorption and reactions of 2-methylpropene (isobutene) on mordenite were investigated between 150 and 300 K by infrared (IR) spectroscopy. The methyl groups of isobutene interacted with the acidic OH groups of mordenite below 160 K, which was converted to the strongly interacted π-bonded isobutene to the acidic OH groups (π-complex). Above 200 K, the π-bonded isobutene dimerized even under evacuation condition. The stable 2,4,4-trimethyl-2-pentoxy species was identified by FT-IR by comparison with the spectra of the adsorbed C8 olefins. The expected monomeric reaction intermediate, the 2-methyl-2-propoxy (tert-butoxy) species, was not observed. This is probably due to the rapid migration and reaction of the adsorbed isobutene molecules.