Yusuke Nagae

Characterization of mouse homolog of brain acyl-CoA hydrolase: molecular cloning and neuronal localization

Acyl-CoA hydrolase could provide a mechanism via its potency to modulate cellular concentrations of acyl-CoAs for the regulation of various cellular events including fatty acid metabolism and gene expression. However, only limited evidence of this is available. To better understand the physiological role of this enzyme, we characterized a mouse brain acyl-CoA hydrolase, mBACH. The cloned cDNA for mBACH encoded a 338-amino-acid polypeptide with >95% identity to the human and rat homologs, indicating that the BACH gene is highly conserved among species. This was supported by the similarity in genomic organization of the BACH gene between humans and mice. Bacterially expressed mBACH was highly active against long-chain acyl-CoAs with a relatively broad specificity for chain length. While palmitoyl-CoA hydrolase activity was widely distributed in mouse tissues, it was marked in the brain, consistent with mBACH being almost exclusively distributed in this tissue, where >80% of the enzyme activity was explained by mBACH present in the cytosol. Immunohistochemistry demonstrated a neuronal localization of mBACH in both the central and peripheral nervous systems. In neurons, mBACH was distributed throughout the cell body and neurites. Although four isoforms except mBACH itself, that may be generated by the alternative use of exons of a single mBACH gene, were cloned, their mRNA levels in the brain were estimated to be negligible. However, a 50-kDa polypeptide besides the major one of 43-kDa seemed to be translated from the mBACH mRNA with differential in-frame ATG triplets used as the initiation codon. These findings will contribute to the functional analysis of the BACH gene using mice including genetic studies.

Identification of cytochrome P450 forms involved in the 4-hydroxylation of valsartan, a potent and specific angiotensin II receptor antagonist, in human liver microsomes

Valsartan is known to be excreted largely as unchanged compound and is minimally metabolized in man. Although the only notable metabolite is 4-hydroxyvaleryl metabolite (4-OH valsartan), the responsible enzyme has not been clarified at present. The current in vitro studies were conducted to identify the cytochrome P450 (CYP) enzymes involved in the formation of 4-OH valsartan. Valsartan was metabolized to 4-OH valsartan by human liver microsomes and the Eadie–Hofstee plots were linear. The apparent Km and Vmax values for the formation of 4-OH valsartan were 41.9–55.8 µM and 27.2–216.9 pmol min−1 mg−1 protein, respectively. There was good correlation between the formation rates of 4-OH valsartan and diclofenac 4′-hydroxylase activities (representative CYP2C9 activity) of 11 individual microsomes (r = 0.889). No good correlation was observed between any of the other CYP enzyme marker activities (CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP4A). Among the recombinant CYP enzymes examined (CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5 and 4A11), CYP2C9 notably catalysed 4-hydroxylation of valsartan. For the specific CYP inhibitors or substrates examined (furafylline, diclofenac, S(+)-mephenytoin, quinidine and troleandomycin), only diclofenac inhibited the formation of 4-OH valsartan. These results showed that CYP2C9 is the only form responsible for 4-hydroxylation of valsartan in human liver microsomes. Although CYP2C9 is involved in valsartan metabolism, CYP-mediated drug–drug interaction between valsartan and other co-administered drugs would be negligible.

Effect of estrogen on induction of micronuclei by mutagens in male mice.

The effect of estrogen on the induction of micronucleated polychromatic erythrocytes (MPCE) by mutagens was examined in male mice. In the dose-response study, a dose-related inhibition of the mitomycin C (MMC)-induced MPCE frequency by estradiol (E2) treatment was observed. In the time study, inhibitory effects of E2 on MPCE frequency by MMC were observed when MMC was administered at 0 or 1 day after injection of E2. The most effective protocol for inhibition was when E2 and MMC were used on the same day. Of mutagens other than MMC, only vincristine (VCR) showed a significant decrease in MPCE frequency when used together with E2. Benzo[a]pyrene (BaP) and 5-fluorouracil (5-FU) showed no significant decrease in MPCE frequency. The data suggest that the induction of micronuclei by mutagens is inhibited by treatment with estrogen, and this could result in a sex difference in the sensitivity of mice employed in the micronucleus test. Mechanisms of the inhibitory effects of estrogen are discussed; these might include a suppression of erythropoiesis and a possible effect on the cell membrane permeability of erythroblasts.

Immunohistochemical localization of acyl-CoA hydrolase/thioesterase multigene family members to rat epithelia

Abstract. Acyl-CoA hydrolases cleave acyl-CoA thioesters to free fatty acids and coenzyme A. The potency of these enzymes may serve to modulate cellular levels of acyl-CoAs to affect various cellular functions, including lipid metabolism. In this study, we investigated the tissue distribution of this multigene family of enzymes, focusing on cytosolic (CTE-I) and mitochondrial acyl-CoA thioesterases (MTE-I) in adult rats, using an anti-CTE-I antibody which recognizes both the isoforms. Western blotting detected them mainly in organs closely related to fatty acid oxidation, of which kidney contained the highest levels of both enzymes. Immunohistochemistry localized the enzymes primarily in the proximal tubules, where a large energy demand is expected and fatty acids represent a major fuel, correlating well with the intrarenal distribution of peroxisomal beta-oxidation. In situ hybridization suggested colocalization of CTE-I and MTE-I in the kidney. The immunoreactivity was also found in various epithelial tissues in the body, including Harderian gland and sebaceous gland. These results demonstrated the distribution of CTE-I and MTE-I in a wide variety of rat tissues, primarily characterized by an epithelial localization, being consistent with their involvement in fatty acid metabolism.