Cytochrome P450 (CYP, P450) and uridine 5’-diphospho-glucuronosyltransferase (UGT) play crucial roles in drug metabolism phase I and II, respectively. Our previous studies suggest that there are functional interactions between P450 and UGT. We previously established a co-expression system featuring CYP3A4 and UGT2B7 using baculovirus-infected insect cells. Commercial microsomes are available that individually express CYP3A4/NADPH P450 reductase (CPR) or UGT2B7. It would be much easier if we could evaluate the functional interaction of CYP3A4 and UGT2B7 by simply mixing the microsomes. To address this issue, we presently compared our established co-expression system with a simple microsome mixing system. Co-expressed UGT2B7 suppressed CYP3A4 activity. On the contrary, adding UGT2B7 microsomes to CYP3A4/CPR microsomes significantly enhanced CYP3A4 activity. The enhancement was systematic and strongly dependent on UGT2B7 microsomes, and was abrogated by detergent treatment. The collective results suggested that the enhancement of CYP3A4 activity resulted from a non-physiological interaction between CYP3A4 and UGT2B7, which were both expressed on different membranes. The phenomenon was distinguishable and hardly ever reflected the physiological interaction. This pitfall can be avoided by not using simple mixing procedures. In selecting experimental materials and methods depending on the subject of the study, a co-expression system should be applied in the analysis of functional P450-UGT interaction.

Lysosome-associated membrane protein-1 (LAMP-1) is a type I membrane glycoprotein with a COOH-terminal cytoplasmic tail (CT) containing a lysosome-targeting signal of GYQTI³⁸²-COOH. This sequence is categorized as a tyrosine-based motif of GYXXΦ where Φ is a bulky hydrophobic amino acid residue. Lysosomal localization of LAMP-1 varies by changing the COOH-terminal amino acid residues. Adaptor protein (AP) complexes are reported to recognize the tyrosine-based signal peptide for efficient lysosomal transport of LAMP-1. In order to better understand the role of APs in lysosomal transport of LAMP-1, we have studied interactions of wild-type (WT) and mutated CTs of LAMP-1 with medium (μ) subunits of the four APs by a yeast two-hybrid (Y2H) system and subsequent computer-based molecular modeling. Among the μ subunits of AP-1, AP-2, AP-3 and AP-4, called μ1, μ2, μ3A, and μ4, respectively, the WT-CT significantly interacted with μ3Α in the Y2H system. The degree of interaction of the WT and mutated CTs with μ3A from the Y2H analyses correlated with that of their dissociation constants determined by computer-based molecular modeling, and also with that of the late endosomal and lysosomal amount of WT and the similar mutants of LAMP-1. These results reinforce the notion that AP-3 makes a major contribution to the vesicular transport of LAMP-1 to late endosomes and lysosomes via a direct intracellular route.

A key event in prion diseases is the conformational conversion of a cellular normal form of the prion protein (PrP^C) to disease-associated conformers (PrP^Sc). Polymorphisms or mutations in the human prion protein gene (PRNP) are associated with acquired or hereditary human prion diseases. When human prion protein (PrP) variants are characterized by cDNA expression systems in cultured cells, the endogenous prion protein may affect the behavior of ectopically expressed prion variants. To eliminate this issue, we herein created a PRNP-disrupted mutant cell line from human neuroblastoma BE(2)-M17 cells. No morphological differences were observed between PRNP-null and parental cells. We stably introduced cDNAs encoding E196K-PrP and E200K-PrP (both of which are Creutzfeldt-Jakob disease (CJD)-related mutants), a non-pathogenic E219K-PrP, and wild-type control PrP into PRNP-null mutant cells using the PiggyBac system, and found that the glycosylation pattern of E196K-PrP differed from that of other PrPs. We also found that non-glycosylated PrPs for E196K- and E200K-PrPs had distinct electrophoretic mobilities from that of WT-PrP, and E219K-PrPs exhibited slightly different mobilities in polyacrylamide gel electrophoresis under endogenous PrP-null conditions, demonstrating that the PRNP-disrupted human neuroblastoma cell line serves as a useful tool for examining PrPs with mutations or polymorphisms.

The Cockcroft–Gault (C–G) equation is widely used for drug dose settings in Japan. However, several reports have questioned its accuracy. In previous decades, estimation equations of creatinine clearance (CCr), such as the Orita–Horio equation, have been established based on the Japanese population. We previously built the fitted C–G and fitted Orita–Horio equations by fitting the coefficients of the estimation equations to the study population. However, the usefulness of these equations for drug dose settings remains unclear. Our preliminary study verifies the accuracy of these equations by comparing the predictive performance of the initial vancomycin (VCM) trough value between four equations: the conventional C–G (as control), conventional Orita–Horio, fitted C–G, and fitted Orita–Horio equations. Patients receiving VCM intravenously between January 2015 and March 2019 at Hokkaido University Hospital were enrolled. Overall, 308 patients were included. As initial dose setting methods, we selected two therapeutic drug monitoring (TDM) analysis software: SHIONOGI-VCM-TDM ver.2009 (VCM-TDM) and Vancomycin MEEK TDM analysis software Ver2.0 (MEEK). Predictive performances were evaluated by calculating mean prediction error and mean absolute prediction error (MAE). The lowest MAE was obtained with the conventional C–G equation using VCM-TDM, indicating high predictive performance. However, contrasting result was obtained with MEEK, where the highest MAE was obtained using conventional C–G equation. Moreover, no significant differences were observed in MAE between the other three equations, suggesting that accurate dose settings are not always achieved, despite using accurate CCr equations based on the Japanese population.

This study proposes and evaluates a precise and labor-saving method for quantifying phthalic-acid esters (PAEs) in indoor air based on solid-phase extraction. Three different adsorbents were evaluated; i.e., two types of octadecyl silica (ODS) filter and a styrene–divinylbenzene (SDB) copolymer cartridge. Calibration curves for five PAEs [diethyl phthalate (DEP), diisobutyl phthalate, di-n-butyl phthalate (DBP), benzyl butyl phthalate (BBP), and di(2-ethylhexyl) phthalate (DEHP)] were created using an internal standard (DBP-d₄). Values of the coefficient of determination (R²) indicated good linearity of the calibration curves (R² > 0.9953). Among the three adsorbents, the SDB cartridge was easiest to handle because it can be used without cleaning and has the lowest blank value. The recovery of deuterated PAEs (DEP-d₄, DBP-d₄, BBP-d₄, and DEHP-d₄) did not differ significantly among the three adsorbents; values were consistently > 89.7% for an air volume of 2.88 m³. During simultaneous indoor air sampling, PAE concentrations were very similar for the three adsorbents. Interlaboratory validation studies were conducted in five laboratories to validate the proposed method for two PAEs (DBP and DEHP). The mean recoveries of the two PAEs added to two types of adsorbent were 91.3–99.9%, the reproducibility relative standard deviations (RSD_R) were 5.1–13.1%, and the Horwitz ratio (HorRat) values were 0.31–0.79. The proposed method using solid-phase extraction with two types of adsorbents provides accurate estimates of PAEs in ambient air.