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Masakiyo Hosokawa

Publications and source records attributed to Masakiyo Hosokawa.

5 recordsLinked to original sources
PubMed2003

Purification, molecular cloning, and functional expression of inducible liver acylcarnitine hydrolase in C57BL/6 mouse, belonging to the carboxylesterase multigene family.

To identify the peroxisome proliferator-inducible acylcarnitine hydrolase in C57BL/6 mice, acylcarnitine hydrolase was purified to homogeneity using column chromatography. The purified enzyme, named ACH M1, had a subunit molecular weight of 60kDa. ACH M1 could hydrolyze classical carboxylesterase (CES) substrates as well as palmitoyl-dl-carnitine and these activities were inhibited by anti-rat CES antibodies. The peptide fragments of ACH M1 were identical to those of the deduced amino acid sequence of mouse CES2 isozyme. These findings suggested that ACH M1 was a member of the CES2 family. The mouse CES2 cDNA, designated mCES2, was cloned from mouse liver. The recombinant mCES2 expressing in Sf9 cells showed high level of catalytic activity toward acylcarnitines. Furthermore, the biological characteristics of the expressed protein were identical with those of ACH M1 in many cases, suggesting that mCES2 encodes mouse liver ACH M1.

Amino Acid Sequence↗
PubMed2003

CYP3A4 inducible model for in vitro analysis of human drug metabolism using a bioartificial liver.

CYP3A is responsible for approximately 50% of the therapeutic drug-metabolizing activity in the liver. The present study was undertaken to establish the CYP3A4 inducible model for analysis of human drug metabolism using a bioartificial liver composed of the functional hepatocellular carcinoma cell (HCC) line FLC-5. A radial-flow bioreactor (RFB), which is a carrier-filled type bioreactor, was used for 3-dimensional perfusion culture of FLC-5 cells. The CYP3A4 messenger RNA (mRNA) expression level 48 hours after rifampicin treatment in the RBF was approximately 100 times higher than that in a monolayer culture. Western blot analysis also demonstrated an increase in expression of the CYP3A protein. When testosterone, a substrate for CYP3A4, was added to the rifampicin-treated cell culture, 6 beta-hydroxy testosterone as a metabolite was formed. Electrophoretic mobility shift assay (EMSA) with a CYP3A4 ER6 probe demonstrated that relatively high molecular weight complex containing pregnane X receptor (PXR)/retinoid X receptor alpha(RXR alpha), compared with that in the monolayer culture, is possibly generated in the RFB culture of FLC-5 treated with rifampicin. Similarly, the assay with a probe of HNF-4 alpha-binding motif indicated the formation of a large protein complex in the RFB culture. Because it is known that PXR transactivates CYP3A4 gene via its response element and expression of PXR is regulated by HNF-4 alpha, the large complexes binding to response elements of PXR or HNF-4 alpha in the RFB culture may contribute to up-regulation of CYP3A4 mRNA. In conclusion, the bioartificial liver composed of human functional HCC cell line was useful in studying drug interactions during induction of human CYP3A4.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗
PubMed2002

Effects of G169R and P34S substitutions produced by mutations of CYP2D6*14 on the functional properties of CYP2D6 expressed in V79 cells.

CYP2D6 is a polymorphic enzyme that catalyzes the oxidation of various drugs. At least 40-mutant alleles of CYP2D6 have been reported. CYP2D6*14, which is one of them found in Asian populations, causes deficient activity of CYP2D6. Four amino acid substitutions, P34S, G169R, R296C, and S486T, are present in the protein encoded by CYP2D6*14 (CYP2D6 14). Among them, G169R is thought to be a definitive substitution because it is unique to CYP2D6 14. However, a previous study showed that the activity of G169R-substituted CYP2D6 was about 40% of wild-type CYP2D6, suggesting that a combination of G169R and other substitutions may be required to abolish the activity of CYP2D6. In the present study, we examined the effects of combined substitutions of G169R and P34S on the functional properties of CYP2D6 and compared them with those of a single substitution of G169R or P34S using a cDNA expression system of V79 cells. The results showed that a combined substitution of G169R and P34S reduced the activities of CYP2D6 to less than the detection limit of our analytical method for bufuralol 1'-hydroxylation and dextromethorphan O-demethylation. However, these activities were not completely abolished by a single substitution of P34S or G169R. The findings suggest that simultaneous substitution of G169R and P34S is crucial for almost completely abolishing the activity of CYP2D6 at least in V79 cells, although whether the absence of metabolism is due to the absence of functional protein or catalytic incompetency remains unclear because the levels of CYP2D6 protein expressed in V79 cells were too low to be determined by difference CO-reduced spectra.

Alleles↗
PubMed2002

Current progress on esterases: from molecular structure to function.

This article reports on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the April 2001 Experimental Biology meeting. Current developments in molecular-based studies into the structure and function of cholinesterases, carboxylesterases, and paraoxonases are described. This article covers mechanisms of regulation of gene expression of the various esterases by developmental factors and xenobiotics, as well as the interplay between physiological and chemical regulation of enzyme activity.

Animals↗