b, Detection of mRNA for P16 by RT-PCR analysis. These results strongly suggest that the production of P21 and P16 was timely induced by alkanes at a transcription level. Because fatty acid, triacylglycerol, DCPK, and paraquat were no efficient inducer of P21 and P16 production, it is plausible that

alkane molecules directly Angiogenesis inhibitor or indirectly control the transcriptional regulation of P21 and P16 genes. Amino acid sequence of P24 The N-terminal amino acid sequence of P24 was determined to be PFELPALPYPYDALEP (P24-N). This sequence was completely matched with that of superoxide dismutase (SOD) from strains in the genus Geobacillus. Cloning and sequencing of the entire gene encoding P24 revealed that it is a Mn-dependent type SOD of 204 amino acid residues, and showed 99.0% identical to Mn-SOD of G. kaustophilus HTA426 (YP_148310) or G. stearothermophilus (P00449) and 96% identical to G. thermodenitrificans NG80-2 (YP_001126490). The amino acid residues responsible for Mn binding, 76-GGXXXHXXE-84 and 49-QD-50,

were completely conserved in P24. Detection of enzyme activities responsible for eliminating reactive oxygen molecules SOD detoxifies superoxide anion to hydrogen peroxide, which in turn is generally broken down to water by the function of catalase or peroxidase. The B23 cells grown in the presence or absence of alkanes were tested for SOD, catalase, and peroxidase activity staining methods. The SOD activity of the B23 cells grown in the presence of alkane was slightly higher than that of the cells grown in the absence of alkanes as expected Ro 61-8048 datasheet (Fig. 6a). It was found that catalase activity was detectable Phosphoribosylglycinamide formyltransferase in the B23 cells only when they were grown on alkanes (Fig. 6b). When 0.5% glucose or glycerol was used as carbon source in the culture, the activities of SOD and catalase remained low. This observation indicates that these enzymes responsible for oxidative stress tolerance were produced as a result of not nutritional starvation (shift from nutrient L-broth to LBM mineral salts medium) but of alkane metabolisms. On the other hand, neither the SOD nor catalase was induced by alkanes in the G. thermoleovorans

LEH-1 cells. Although it has been reported that LEH-1 showed relatively high peroxidase activity irrespective of the presence and absence of alkane in the media [18], this enzyme activity was not detectable level for both the B23 and H41 cells (figure not shown). Interestingly, SOD activity in LEH-1 cells with alkanes was disappeared in the presence of alkanes. This would have been occurred because SOD inducible oxygen molecules were mostly consumed by alkane degradation enzymes including acyl-CoA dehydrogenase and by regeneration of NAD+. Figure 6 Activity staining of SOD (a) and catalase (b). Crude cell extracts of G. thermoleovorans B23 and LEH-1 grown for 14 days on alkanes (+) and on 0.5% glucose (-) were separated on 7.5% native polyacrylamide gel. Arrows indicate respective enzyme activities.