Cytochrome P450 (CYP) 2A6 catalyzes nicotine C-oxidation, leading to cotinine formation, a major metabolic pathway of nicotine in humans. There are genetic polymorphisms in the human CYP2A6 gene. Previously, we demonstrated that in vivo nicotine metabolism is impaired with the CYP2A6*4, CYP2A6*7, and CYP2A6*10 alleles in Japanese subjects and Korean subjects. An allele possessing a point mutation in the TATA box termed CYP2A6*9 (T-48G) has been reported to decrease the transcriptional activity in vitro as assessed by luciferase assay. In this study we investigated the effects of the CYP2A6*9 allele on in vivo enzymatic activity by evaluating nicotine metabolism. The mutation of T-48G was found only on the CYP2A6*1A allele but not on the CYP2A6*1B allele. Allele frequencies of CYP2A6*9 in Japanese subjects (n = 92) and Korean subjects (n = 209) were 21.3% and 22.3%, respectively. In Korean subjects the cotinine/nicotine ratios as an index of nicotine metabolism in the subjects with CYP2A6*9/CYP2A6*9 (4.3 +/- 2.4) were significantly lower than those in the subjects with CYP2A6*1A/CYP2A6*9 (7.7 +/- 5.6) and CYP2A6*1A/CYP2A6*1A (10.4 +/- 9.2) (P <.05 and P <.005, respectively). In Japanese subjects a similar result was observed, although it was not significant. Thus it is suggested that the mutation in the TATA box (CYP2A6*9 allele) caused the decreased in vivo enzymatic activity. With an in vitro study, it was shown that the expression levels of CYP2A6 messenger ribonucleic acid and coumarin 7-hydroxylase activity in human livers genotyped as CYP2A6*1/CYP2A6*9 and CYP2A6*9/CYP2A6*9 tended to be lower than those in human livers genotyped as CYP2A6*1/CYP2A6*1, although there was no significant difference because of the small number of samples. These in vitro data supported the in vivo data demonstrating that the CYP2A6*9 allele caused the decreased expression level and enzymatic activity of CYP2A6.

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A suspension cultured cell line was established from the cultivar of Rosa hybrida 'Charleston' as a study model to understand the response of the anthocyanin biosynthesis pathway to environmental cues. The major identified anthocyanin in cell cultures was cyanidin 3-glucoside (chrysanthemin). The anthocyanin yield was enhanced by culturing cells in the EM medium with added sucrose at high concentration under additional UV-B radiation to white light. Three cDNA fragments were cloned with degenerate primers by RT-PCR and the obtained sequences shared high homology with putative key enzymes (DFR, ANS, and UF3GT) of other species. The expression levels of these three genes were promoted under optimum conditions for anthocyanin accumulation. These results suggest that expression levels of these genes were closely correlated with a temporal buildup of anthocyanins in response to environmental factors.Key words: Anthocyanin, chrysanthemin, cyanin, Rosa hybrida, suspension cell culture.Plant Biotechnology 23, 379-385 (2006) Original PaperThis article can be found at http://www.jspcmb.jp/ pelargonidin, cyanidin and peonidin type anthocyanins (Biolley et al. 1994). Recently, tissue culturing has become an important biotechnological technique to produce various secondary metabolites including anthocyanins on a mass scale for industrial and academic interest (Hirner et al. 2001;Gantet and Memelink 2002). Despite the fact that some plant species have been shown to produce anthocyanins in cell cultures, little information of the cell culture techniques that are capable of producing in vitro anthocyanins has been reported in rose.The biosynthesis of anthocyanins in plant cell culture is stimulated by not only genetic factors but also environmental factors such as light, temperature, growth regulators and nutrition (Sato et al. 1996;Zhang et al. 1997;Zhang et al. 2002). The identity of the environmental stimuli that lead to the accumulation of anthocyanins in rose is of considerable interest. To date, Sato et al. (1996) has examined the intensity of white light that induces anthocyanin production in strawberry cell culture lines; however, no information was obtained about the effect of light quality on the production of anthocyanins in cultural cells. Therefore, the originality of this research is to investigate anthocyanin biosynthesis in cultural cells exposed to UV-B (290-320 nm) radiation.In this study, we used Rosa hybrida cultivar 'Charleston', a climbing type, originated in Australia. The open flowers of 'Charleston' undergo a striking color change from yellow to red over 10-12 days under natural daylight. We established a suspension cultured cell line from this cultivar of rose. Anthocyanin content was quantified by HPLC to determine the most efficient conditions, such as light including UV-B radiation and media, for continuous production of the pigments in the rose cell suspension. The expression levels of three genes encoding putative key enzymes (DFR, ANS, and UF3GT) for anthocyanin biosynthesis were ana...

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