Human aquaporin adipose (AQPap) gene. Genomic structure, promoter analysis and functional mutation.

Aquaporin adipose (AQPap), which we identified from human adipose tissue, is a glycerol channel in adipocyte [Kishida et al. (2000) J. Biol. Chem. 275, 20896-20902]. In the current study, we determined the genomic structure of the human AQPap gene, and identified three AQPap-like genes that resembled (approximately 95%) AQPap, with little expression in human tissues. The AQPap promoter contained a putative peroxisome proliferator response element (PPRE) at -46 to -62, and a putative insulin response element (IRE) at -542/-536. Deletion of the PPRE abolished the pioglitazone-mediated induction of AQPap promoter activity in 3T3-L1 adipocytes. Deletion and single base pair substitution analysis of the IRE abolished the insulin-mediated suppression of the human AQPap gene. Analysis of AQPap sequence in human subjects revealed three missense mutations (R12C, V59L and G264V), and two silent mutations (A103A and G250G). The cRNA injection of the missense mutants into Xenopus oocytes revealed the absence of the activity to transport glycerol and water in the AQPap-G264V protein. In the subject homozygous for AQPap-G264V, exercise-induced increase in plasma glycerol was not observed in spite of the increased plasma noradrenaline. We suggest that AQPap is responsible for the increase of plasma glycerol during exercise in humans.     

Production of a Doubly Chiral Compound, (4R,6R)-4-Hydroxy-2,2,6-Trimethylcyclohexanone, by Two-Step Enzymatic Asymmetric Reduction

A practical enzymatic synthesis of a doubly chiral key compound, (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone, starting from the readily available 2,6,6-trimethyl-2-cyclohexen-1,4-dione is described. Chirality is first introduced at the C-6 position by a stereoselective enzymatic hydrogenation of the double bond using old yellow enzyme 2 of Saccharomyces cerevisiae, expressed in Escherichia coli, as a biocatalyst. Thereafter, the carbonyl group at the C-4 position is reduced selectively and stereospecifically by levodione reductase of Corynebacterium aquaticum M-13, expressed in E. coli, to the corresponding alcohol. Commercially available glucose dehydrogenase was also used for cofactor regeneration in both steps. Using this two-step enzymatic asymmetric reduction system, 9.5 mg of (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone/ml was produced almost stoichiometrically, with 94% enantiomeric excess in the presence of glucose, NAD⁺, and glucose dehydrogenase. To our knowledge, this is the first report of the application of S. cerevisiae old yellow enzyme for the production of a useful compound.     

Preparation and handling of aequorin solutions for the measurement of cellular Ca2+

Main characteristics of the various types of aequorin presently available for measuring cellular Ca2+, i.e. heterogeneous aequorin, isoaequorins, recombinant aequorin, fluorescein-labeled aequorin and semi-synthetic aequorins, are summarized. Basic techniques of preparing and handling the solutions of those aequorins for measuring Ca2+, including such techniques as concentrating aequorin solutions, freeze-drying, changing buffer composition, and the regeneration of active aequorin, are described.     

Branched-chain amino acids regulate type I tropocollagen and type III tropocollagen syntheses via modulation of mTOR in the skin

Abstract

Branched-chain amino acids (BCAAs) exhibit many physiological functions. However, the potential link and mechanism between BCAA and skin function are unknown. We examined the effects of deletion of branched-chain α-keto acid dehydrogenase kinase (BDK), a key enzyme in BCAA catabolism, on type I and III tropocollagen syntheses in mice. Leucine and isoleucine levels were significantly lower in the skin of BDK-KO mice compared with wild-type mice. No changes in valine concentrations were observed. The levels of type I and III tropocollagen proteins and mRNAs (COL1A1 and COL3A1) were significantly lower in the skin of BDK-KO mice compared with wild-type mice. The phosphorylation of p70 S6 kinase, which indicates mammalian target of rapamycin (mTOR) activation, was reduced in the skin of BDK-KO mice compared with wild-type mice. These findings suggest that deficiencies of leucine and isoleucine reduce type I and III tropocollagen syntheses in skin by suppressing the action of mTOR.