Thioesterase activity and subcellular localization of acylprotein thioesterase 1/lysophospholipase 1

Acylprotein thioesterase 1 (APT1), also known as lysophospholipase 1, is an important enzyme responsible for depalmitoylation of palmitoyl proteins. To clarify the substrate selectivity and the intracellular function of APT1, we performed kinetic analyses and competition assays using a recombinant human APT1 (hAPT1) and investigated the subcellular localization. For this purpose, an assay for thioesterase activity against a synthetic palmitoyl peptide using liquid chromatography/mass spectrometry was established. The thioesterase activity of hAPT1 was most active at neutral pH, and did not require Ca²⁺ for its maximum activity. The K_M values for thioesterase and lysophospholipase (against lysophosphatidylcholine) activities were 3.49 and 27.3 μM, and the V_max values were 27.3 and 1.62 μmol/min/mg, respectively. Thus, hAPT1 revealed much higher thioesterase activity than lysophospholipase activity. One activity was competitively inhibited by another substrate in the presence of both substrates. Immunocytochemical and Western blot analyses revealed that endogenous and overexpressed hAPT1 were mainly localized in the cytosol, while some signals were detected in the plasma membrane, the nuclear membrane and ER in HEK293 cells. These results suggest that eliminating palmitoylated proteins and lysophospholipids from cytosol is one of the functions of hAPT1.     

pFJ265, a new cloning vehicle for Streptomyces

A 9.3-kb plasmid, pNM100, was isolated from Streptomyces virginiae (NRRL 15156) and characterized. Streptomyces genes for thiostrepton and neomycin resistance were cloned into pNM100 to yield a small plasmid derivative, pFJ265, that is suitable for Streptomyces gene cloning. pFJ265 is a 9.2-kb nonconjugative plasmid and has a copy number of several hundred per chromosome.     

The 1975 type Japanese diet improves the gut microbial flora and inhibits visceral fat accumulation in mice

ABSTRACT

In this study, the 1975 type Japanese diet was prepared and its effects and related mechanism were examined in mice. Mice were assigned to three experimental groups, the CD group fed a control diet, the MD group fed a modern Japanese diet (MD), and the JD group fed the 1975 type Japanese diet (JD) for 4 weeks. MD and JD were low protein, high fat, and high carbohydrate diets compared to the CD. Total white adipose tissue weights were significantly increased in the MD group compared to those in the CD group and were decreased in the JD group compared to those in the MD group. In the JD group, adipocyte hypertrophy was inhibited and Hsl mRNA expression was enhanced in epididymal adipose tissue and the number of bacteria associated with the production of short chain fatty acids was increased. Therefore, the JD inhibits lipid accumulation in white adipose tissue.     

Detection of herpes simplex and varicella-zoster virus DNA by field-inversion gel electrophoresis from clinical materials.

A simple method using field-inversion gel electrophoresis (FIGE) was applied to detect herpes simplex virus (HSV) and varicella-zoster virus (VZV) genomes in clinical specimens. The whole genomes of these viruses could be detected in small vesicle tissues by the FIGE method regardless of their clinical stages of skin lesions. And the sensitivity of the FIGE method was equivalent to that of an immunofluorescent (IF) method. These data indicated usefulness of the FIGE method to detect the whole genomes of HSV and VZV in clinical specimens.     

Detection of the Leghemoglobin Gene on Two Chromosomes of Phaseolus vulgaris by in situ PCR Linked-Fluorescent in situ Hybridization (FISH)

The leghemoglobin (Lb) gene on the metaphase chromosomes ofPhaseolus vulgaris was amplified by in situ PCR. The amplifiedLb gene could be detected on two chromosomes by fluorescentin situ hybridization (FISH) using the short Lb gene probe. (Received January 9, 1998; Accepted April 30, 1998)