Search

Abstract: The conformations of poly(l-glutamic acid) [P(Glu)] in solutions of the bipolar amphiphile 1,20-icosanediylbis(alkylammonium chloride) [C20(RA)2], where RA includes trimethylammonium (TMA), dimethylammonium (DMA), or methylammonium (MA), were investigated with measurements of the circular dichroism spectra at 10–35 °C. All C20(RA)2 induced an α-helix of P(Glu) in the aqueous solutions. The residue molar ellipticity at 222 nm showed a similar dependence on the amphiphile concentration (C s) below 0.5 of the ratio of 2C s to the residue concentration (C p) of P(Glu), but it separated into three directions at 2C s/C p>0.5. C20(MA)2 induced an α-helix of P(Glu) at 2C s/C p<0.5 followed by a helix aggregate at 2C s/C p>0.5. C20(DMA)2 and C20(TMA)2 also induced an α-helix, but a helix aggregate. C20(TMA)2 indicated a strong temperature dependence and did not induce a complete α-helix at 35 °C.

For preparation of an affinity ligand, an N-fatty acyl moiety of galactosylceramide (GalCer) was chemically replaced with omega-amino-fatty acid including amino-n-hexanoic acid or amino-n-dodecanoic acid to obtain omega-aminoGalCer. For the synthesis of the compound, galactosylsphingosine (GalSph) was coupled with N-trifluoroacetyl omega-amino-fatty acid which was prepared by a reaction with S-ethyltrifluorothioacetate. After removal of the N-trifluoroacetyl group in a mild alkaline solution, in which an N-fatty acyl group was retained, aminoGalCer composed of an N-hexanoyl or an N-dodecanoyl group was obtained with an overall yield of 90%. Their chemical structures were confirmed by proton nuclear magnetic resonance and fast atom bombardment-mass spectrometries. These aminoGalCers and GalSph as well as immobilized aminoGalCer were sulfated by a glycolipid sulfotransferase from rat kidney. Furthermore, immobilized aminoGalCer on gel matrix was used for affinity chromatography of the sulfotransferase, resulting in an excellent increase in the purification (14,000-fold) with a recovery rate of 40%.

A cell line (SMKT-R3) established from human renal cell carcinoma was characterized for the presence of sulfolipids and glycolipid sulfotransferases. Sulfolipids were found to constitute a large part of the acidic glycolipid fraction in SMKT-R3 cells. These findings were confirmed by metabolic labelling with 35S-sulfate. These sulfolipids were expressed at the surface of SMKT-R3 cells as ascertained by cytofluorometry using a monoclonal antibody directed to sulfolipids. Furthermore, markedly high activity levels of glycolipid sulfotransferases were observed in SMKT-R3 cells compared with other cell lines. These results suggest that the increased synthesis of sulfolipids in renal cell carcinoma tissue (Sakakibara et al., 1989. Cancer Res., 49, 335-339) is due to the elevation of the sulfotransferase activities of renal carcinoma cells themselves.

Lyso-GM3 and -GM1 gangliosides were prepared from the corresponding N,N'-dideacylated gangliosides using N-trifluoroacetylation at the sphingosine moiety, followed by N-acetylation and mild saponification. The blocking reaction was performed using a water-ether bilayer system at alkaline medium, in which the N-trifluoroacetylation occurred predominantly at the lipid moiety. Through the procedure, lysoGM3 and lysoGM1 were obtained with higher yields from the corresponding dideacylated gangliosides than through the previous method using 9-fluorenylmethoxycarbonyl chloride as a blocking group or of direct N-acetylation of it on liposomes containing starting ganglioside and other lipid. Chemical structures of the lysogangliosides and the synthetic intermediates were confirmed by the proton nuclear magnetic resonance spectrometry and negative fast atom bombardment-mass spectrometry.

Purpose: Frequently used anti-cancer drug irinotecan(CPT11), inhibitor of DNA synthesis, is recently known as to have apoptosis effect, though little is known yet. This study investigated this apoptosis mechanism in human hepatocelluar carcinoma cell lines (Huh7).Methods: The cells were cultured with SN38, active metabolite of CPT11, for 24 h. The cells were analyzed to investigate expression of p53 protein and apoptosis related protein by Western Blotting. In addition to this, another group of cells were cultured with SN38 after p53 antisense(AS) pre-treatment for 24 h and were analyzed by immunocytochemistry for apoptosis related protein evaluation.Results: SN38 decreased cell survival, and increased expression of p53, caspase-3 and CAD (caspase-activated DNase). Expression of Bcl-xL was suppressed by SN38. On the other hand, in p53 AS pre-treated cells, SN38 decreased expression of p53 protein and apoptosis was suppressed. Caspase-3 expression was induced by SN38 while suppressed by p53 AS+SN38 treatment. Anti-apoptotic protein Bcl-xL was suppressed by SN38 while not by p53 AS+SN38.Conclusion: This study proved active metabolite SN38 increased p53, apoptosis inducing protein caspase-3, and decreased anti-apoptotic protein Bcl-xL, while these changes were reversed by p53 AS pre-treatment. these results suggest that p53-mediated apoptosis is important mechanism for CPT11 anti-tumor effect on human hepatocellular carcinoma.Clinical Pharmacology & Therapeutics (2004) 75, P60–P60; doi: 10.1016/j.clpt.2003.11.228

Objectives It has been reported that lipophilic drug bioavailability was changed by grapefruit juice intake with its inhibitory effect on CYP3A4, or on efflux transporter P-glycoprotein. On the other hand, pravastatin(PRV) as hydrophilic HMG-CoA reductase inhibitor may be absorbed via intestinal OATP (organic anion transporter protein) and MRP2 (multidrug resistance associsted protein 2), hydrophilic membrance transporters. Dresser et al suggested orange juice (OJ) as the inhibitor of OATPs by showing the reduction of bioavailability of antihistaminic drug fexofenadin in humans. Thus, we studied the effect of OJ on the pharmacokinetics (PK) of PRV when co-administrated with OJ.Results and Methods Male Wistar rats were used. Rat was gived 100mg/kg (P.O) single dose PRV with water (control) or OJ, and the PK of PRV was investigated. Serum concentrations of PRV were quantified by HPLC in this study. OJ increased significantly peak serum concentration and AUC of PRV (153%,141%),respectively, whereas the elimination half-life was not changed significantly.Discussion Our data suggested that OJ increased oral PRV bioavailability, while not on clearance. This may be explained by altered intestinal pH which can change the OATP function, or by the direct effect on the transporters with ingredients of OJ.Clinical Pharmacology & Therapeutics (2004) 75, P77–P77; doi: 10.1016/j.clpt.2003.11.293

Purpose: CYP3A7 gene expression is increased in some adults as those with hepatocellular carcinoma, although this P450 is dominant with fetus or neonate, while CYP3A4 dominant with normal adult. We hypothesized the discrepancy of promoter sequence in up-stream between these P450 genes play important role, and attempted to investigate this in the cell line, especially focusing on p53.Method: (1) HepG2 and Huh7 (human hepatocellular carcinoma), Huh6 (hepatoblastoma), and other cancer cell lines were purchased and cultured to examine the expression of CYP3A7 mRNA and p53 protein. (2) p53 sense (SE) and p53 antisense (AS) were designed by the human p53 cDNA sequence report (GenBank), and of those influence in HepG2 cells on expression of CYP3A7 mRNA, CYP3A4 mRNA with RT-PCR method, and also of p53 protein with Western blot analysis were analyzed, respectively. Results: (1) HepG2 and Huh7 cell line expressed increased CYP3A7 mRNA and p53 protein, while other cell lines did not remarkably. (2) p53 AS decreased CYP3A7 mRNA and p53, while not with p53 SE in HepG2 cells.Discussion: There is p53 recognizing region in up-stream of 5` promoter region of CYP3A7 gene (-328 to -337bp), though not of CYP3A4 gene. This fact and the present results will suggest the importance of p53 in CYP3A7 regulation in liver tissue.Clinical Pharmacology & Therapeutics (2004) 75, P17–P17; doi: 10.1016/j.clpt.2003.11.063