Nepmucin/CLM-9, an Ig domain-containing sialomucin in vascular endothelial cells, promotes lymphocyte transendothelial migration in vitro

Nepmucin/CLM-9 is an Ig domain-containing sialomucin expressed in vascular endothelial cells. Here we show that, like CD31, nepmucin was localized to interendothelial contacts and to vesicle-like structures along the cell border and underwent intracellular recycling. Functional analyses showed that nepmucin mediated homotypic and heterotypic cell adhesion via its Ig domain. Nepmucin-expressing endothelial cells showed enhanced lymphocyte transendothelial migration (TEM), which was abrogated by anti-nepmucin mAbs that block either homophilic or heterophilic binding. Notably, the mAbs that inhibited homophilic binding blocked TEM without affecting lymphocyte adhesion. These results suggest that endothelial nepmucin promotes lymphocyte TEM using multiple adhesion pathways.     

Bioluminescence of the arm light organs of the luminous squid Watasenia scintillans

The squid Watasenia scintillans emits blue light from numerous photophores. According to Tsuji [F.I. Tsuji, Bioluminescence reaction catalyzed by membrane-bound luciferase in the "firefly squid", Watasenia scintillans, Biochim. Biophys. Acta 1564 (2002) 189-197.], the luminescence from arm light organs is caused by an ATP-dependent reaction involving Mg2+, coelenterazine disulfate (luciferin), and an unstable membrane-bound luciferase. We stabilized and partially purified the luciferase in the presence of high concentrations of sucrose, and obtained it as particulates (average size 0.6-2 microm). The ATP-dependent luminescence reaction of coelenterazine disulfate catalyzed by the particulate luciferase was investigated in detail. Optimum temperature of the luminescence reaction is about 5 degrees C. Coelenterazine disulfate is a strictly specific substrate in this luminescence system; any modification of its structure resulted in a very heavy loss in its light emission capability. The light emitter is the excited state of the amide anion form of coelenteramide disulfate. The quantum yield of coelenterazine disulfate is calculated at 0.36. ATP could be replaced by ATP-gamma-S, but not by any other analogues tested. The amount of AMP produced in the luminescence reaction was much smaller than that of coelenteramide disulfate, suggesting that the reaction mechanism of the Watasenia bioluminescence does not involve the formation of adenyl luciferin as an intermediate.     

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.     

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.