Research projects

1. The relationship between the primary structure and the enzyme activity of Bacillus cereus sphingomyelinase was investigated by site-directed mutagenesis, converting Asp and His residues to Gly and Ala residues, respectively, During the study, the structural similarity between bacterial sphingomyelinase and bovine pancreatic DNase I was established by 3D-1D method. Taken together, we concluded that His151, His296 and Asp295 of B.cereus sphingomyelinase are essential for the catalytic and the hemolytic activities and that Asp233 affects the enzyme activity by interacting with His296 whereas Asp126 and Asp156 are involved in substrate recognition by the enzyme. 2. The structure of Penicillium notatum phospholipase B was characterized by us as one of the GPI-anchored proteins. Also, we succeeded in the expression of phospholipase B gene in Escherichia coli as an inert form. We demonstrated that this enzyme was released from the sphaeroplast membrane of P.notatum into the culture medium by the autocatalytic cleavage of GPI anchor.13EA03 : 3. For the study of the structure-activity relationship on Bacillus thuringiensis phosphatidylinositol-specific phospholipase C,we established mass-production system of this phospholipase, using the host-vector system of Bacillus brevis which yielded 500 fold that amount of the enzyme produced by B.thuringiensis.

To explore the physiological roles of drug-metabolizing enzymes in peripheral blood cells, we examined which isoforms of cytochrome P450 (CYP) and conjugation enzymes (sulfotransferases (SULTs), UDP-glucuronyl transferases (UGT) and glutathione S-transferases (GST)) families were expressed in human myeloid leukemia cell lines (U937, HL-60 and K562) and lymphoid cell lines (BALL-1, MOLT-4 and Jurkat) by RT-PCR. We observed relatively high expression of CYP1A1, CYP1B1, CYP2A6, CYP2A7, CYP2D6, and CYP2E1 in all cell types, but CYP2A13 and CYP2C9 expression was not detected. Expressions of aryl hydrocarbon (Ah) receptor and Ah receptor nuclear translocater (ARNT), which mediate induction of the CYP1 family, were also detected in all cell types. Cell-type specific expression of CYP3A4 and CYP3A5 was observed in MOLT-4 and K562 cells. Weak, but significant, expression of CYP3A7 was detected in K562 cells. We detected expression of SULT1A1, 1A3 and 1C2 in all cell lines although cell-type specific expressions were observed for SULT1B1 (U937 and Jurkat) and SULT2A1 (K562). Expression of UGT1A family, which is known to metabolize many drugs and xenobiotics, was exclusively observed in MOLT-4 and BALL-1. BALL-1 expressed 8 sub families of UGT1A and MOLT-4 expressed 2 UGT1A sub families. All cell lines expressed GSTA4, P1 and T1, however, GSTM1 expression, which is supposed to detoxify carcinogens in cigarette smoke, was detected only in MOLT-4 cells. Oxidative stress (hydroperoxide) induced CYP3A4 and GSTP1 expressions in K562 cells. Chemical inducers (p-naphthoflavone and 3-methylcholanthren) induced CYP1A1 expression in MOLT-4 cells. The profile of drug-metablizing enzymes in the culture cells reported here provides information that furthers our understanding of the physiological roles of drug metabolizing enzymes in human blood cells.

It has been reported that habitual coffee intake reduces the risk of developing lifestyle-related diseases associated with obesity and inflammation. We found that the coffee bean roasting ingredient suppresses the differentiation of adipocytes, a risk factor of obesity, by promoting the degradation of the adapter molecule IRS1 of the insulin signaling pathway, using the preadipocyte 3T3-L1 . Regarding the anti-inflammatory effect of coffee, we found that the coffee bean roasting ingredient suppresses the inflammatory response of LPS-induced mouse macrophage cells, and the effect was due to the activation of the transcription factor Nrf2 by promoting the degradation of Keap1. The above results lead to the elucidation of the effect of prevention of lifestyle diseases caused by drinking of coffee on the molecular bases.

We found that components in roasting coffee strongly inhibit the sulfate conjugation of estrogen in the cell model of human gastrointestinal tract, Caco-2. We hypothesized that this inhibitory effect of coffee on the estrogen sulfation is correlated to the preventive effect of daily coffee consumption on lifestyle-related diseases. To demonstrate this hypothesis, we tried to purify and identify the inhibitory components of coffee. From 1kg decaffeinated instant coffee, we succeeded in purification and identification of the component. The component has a novel nitrogen-containing aromatic structure(MW. 205, C_<11> H_<11> NO_3). We also found that components in coffee affect gene expression of proteins involved in estrogen metabolism(SULT1E1, sulfatase, and BCRP), and analyzed the mechanism underlying the phenomenon.

Coffee is one of the world’s most commonly consumed beverages. Recent epidemiological studies show that coffee consumption has been associated with a lower risk of several life-style related disorders such as diabetes, obesity, cancers and dementia. On the other hand, dysfunction of metabolism of steroids such as stress hormone, estrogens and androgens is closely linked to the risk of incidence of these disorders. We investigated effects of coffee on the metabolism of these steroid hormones in human cancer cells such as colon, breast, prostate cancers and glioma. We obtained results suggesting protective effects of coffee on colon and breast cancers but not to prostate cancer. In addition, our result suggest that coffee may increase production of neurosteroids that act to lower the risk of dementia.