Mikiko Ikeda

THE POLYMORPHISM IN THE CAUDAL-RELATED HOMEODOMAIN PROTEIN Cdx-2 BINDING ELEMENT IN THE HUMAN VITAMIN D RECEPTOR GENE

The major physiological activity of 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3] is the regulation of calcium absorption in the small intestine, and the level of vitamin D receptor (VDR) is an important factor in this regulation. In a previous study, we indicated that the caudal‐related homeodomain Cdx‐2 played an important role in the intestine‐specific transcription of the human VDR gene. In this study, the polymorphism was identified in the core sequence 5′‐ATAAAAACTTAT‐3′ in the Cdx‐2 binding site in the VDR gene promoter. In 261 Japanese women with genotyped VDR polymorphisms, 48 were genotype Cdx‐A (adenine at −3731 nucleotides [nt] relative to the transcription start site of human VDR gene 5‐ATAAAAACTTAT), 82 were genotype Cdx‐G (guanine at −3731 nt, 5′‐GTAAAAACTTAT‐3′), and 131 were genotype Cdx‐A/G (heterozygote). In postmenopausal Japanese women, the bone mineral density (BMD) in the lumbar spine (L2‐L4) with the Cdx‐G homozygote was 12% lower than that with the Cdx‐A homozygote (p < 0.05). In electrophoretic gel mobility shift assay (EMSA), the oligonucleotide with Cdx‐G allele markedly decreased the binding to Cdx‐2 compared with that in the Cdx‐A allele. The transcriptional activity of the VDR promoter with Cdx‐G allele was decreased to 70% of the Cdx‐A allele. In addition, in the herpes simplex virus thymidine kinase promoter, the Cdx‐2 binding element with the G allele showed significantly lower transcriptional activity than that of the A allele. Thus, the polymorphism in the Cdx‐2 binding site of the VDR gene (Cdx‐polymorphism) would affect the expression of VDR in the small intestine. In addition, this polymorphism may modulate BMD in postmenopausal Japanese women.

Metabolism of trichloroethylene

Abstract Trichloroethylene was metabolized to chloral hydrate, trichloroethanol and trichloroacetic acid in vitro . The three metabolites in the incubation mixture were determined by gas-liquid chromatography using an electron capture detector. The kinetics of the individual steps of the metabolism of trichloroethylene were investigated in rat liver subcellular fractions or recombined fractions. The general features of trichloroethylene metabolism in vitro were demonstrated by the conversion of trichloroethyleme to the three metabolites (6 per cent total yield) by the 700 g supernatant fraction of rat liver in 2 hr. Oxidation of trichloroethylene to chloral hydrate occurred only in the microsomal fraction of rat liver, as previously reported by Byington and Leibman [5]. (This step was rate-limiting and was stimulated by both phenobarbital and 3-methylcholanthrene pretreatment.) Reduction of chloral hydrate to trichloroethanol occurred in the cytosol of rat liver. This activity was separated into at least three fractions by a DEAE cellulose column—one of them was NADH-dependent and the others were NADPH-dependent.) The formation of trichloroacetic acid from chloral hydrate required cytosol or mitochondria with NAD.

Molecular cloning and sequencing of the cDNA for vacuolar H+-pyrophosphatase from Chara corallina1.

We have cloned a cDNA for vacuolar proton-translocating pyrophosphatase of Chara corallina that is one of the closest green algae to the land plants. The deduced protein consists of 793 amino acid residues. Its sequence is 71% identical to the H+-pyrophosphatases of land plants, and is less than 46% identical to those of marine alga and phototrophic bacterium.

SACCHAROMYCES CEREVISIAE CULTURED UNDER AEROBIC AND ANAEROBIC CONDITIONS :AIR-LEVEL OXYGEN STRESS AND PROTECTION AGAINST STRESS

Cells of Saccharomyces cerevisiae were grown aerobically and anaerobically, and levels of the protective compounds, cysteine and glutathione, and activities of defensive enzymes, catalase and superoxide dismutase, against an oxygen stress were determined and compared in both cells. Aerobiosis increased both the compounds and enzyme activities. The elevated synthesis of glutathione could be associated with the increased levels of cysteine which in its turn was found to be controlled by the oxygen-dependent activation of cystathionine beta-synthase.

Preparative high-yield electroelution of proteins after separation by sodium dodecyl sulphate—polyacrylamide gel electrophoresis and its application to analysis of amino acid sequences and to raise antibodies

A method for the preparative high-yield electroelution of proteins from sodium dodecyl sulphate (SDS) polyacrylamide gel strips was established. The method consisted of SDS-polyacrylamide gel electrophoresis, detection of proteins with sodium acetate and electrophoretic elution at 200 V for 3 h by utilizing a horizontal flat-bed gel electrophoresis apparatus. Standard proteins with molecular masses of 14-66 kilodalton (cytochrome c, aldolase, ovalbumin and bovine serum albumin) were recovered with an average yield of 73.6 +/- 2.3%. A membrane-bound protein, rat skeletal muscle Ca(2+)-ATPase (100 kilodalton) was also well recovered (over 60%). This method was applicable to the purification of proteins required for N-terminal amino acid sequencing and to raise antibodies.

A vacuolar ATPase and pyrophosphatase in Acetabularia acetabulum

Vacuole-rich fractions were isolated from Acetabularia acetabulum by Ficoll step gradient centrifugation. The tonoplast-rich vesicles showed ATP-dependent and pyrophosphate-dependent H(+)-transport activities. ATP-dependent H(+)-transport and ATPase activity were both inhibited by the addition of a specific inhibitor of vacuolar ATPase, bafilomycin B1. A 66 kDa polypeptide present in the preparation cross-reacted with the anti-IgG fractions against the alpha and beta subunits of Halobacterium halobium ATPase and with the antibody against the A subunit (68 kDa subunit) of mung bean vacuolar ATPase. A 56 kDa polypeptide present in the vacuole preparation showed cross-reactivity with the antibody against the B subunit (57 kDa) of mung bean vacuolar ATPase but not with the anti-beta subunit of H. halobium ATPase. A 73 kDa polypeptide cross-reacted with the antibody against inorganic pyrophosphatase of mung bean vacuoles. These results suggest that vacuolar membrane of A. acetabulum equipped energy transducing systems similar to those found in other plant vacuoles.

Mercury uptake by acatalasemia mice and their erythrocytes, lung and liver homogenates

SummaryErythrocytes, lung and liver homogenates prepared from actalasemia mice showed a decrease in their in vitro ability to take up mercury from air saturated with mercury vapor when compared with those of normal mice. Acatalasemia mice had decreased levels of mercury in the lung and blood, and increased levels in the liver following exposure in vivo to metallic mercury vapor (10 mg/m3) for 30 min. Pretreatment of normal and acatalasemia mice with aminotriazole resulted in decreases in the mercury content of lung and blood, and an increase in the liver content, in comparison with non-treated mice.

A simple and specific determination of glycine in biological samples

Abstract A simple specific assay was developed for the determination of glycine in a solution containing other amino acids. Hippuric acid was obtained after reacting glycine with benzoyl chloride and was extracted with ethyl acetate. It was then reacted with acetic anhydride, p -dimethylaminobenzaldehyde, and pyridine for color development. The amount of glycine (1 to 100 μg) in the original solution could be determined by measuring the absorbance (458 nm) of this chromogen. This procedure was applied on an amino acid mixture, urine, serum, blood, and liver homogenate.

Studies on NADPH-dependent chloral hydrate reducing enzymes in rat liver cytosol

Abstract Chloral hydrate, a sedative hypnotic and also a major metabolite of trichloroethylene in higher animals, is reduced to trichloroethanol by liver extracts. The reducing activity in rat liver cytosol could be separated into four fractions [one NADH- (F 1 ) and three NADPH-dependent (F 2 , F 3 and F 4 )] by DEAE-cellulose column chromatography. By several procedures, F 2 was purified over 1000-fold and F 4 was purified over 600-fold from liver cytosol. As judged from polyacrylamide gel electrophoresis performed with and without the addition of sodium dodecylsulfate, the final preparations were essentially homogeneous. They differed in molecular weight, mobility on polyacrylamide gel electrophoresis, pH optimum, substrate specificity, and sensitivity to inhibitors. The molecular weights were estimated to be 36,000 and 32,500 for F 2 and F 4 , respectively, by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. The estimation of molecular weights by thin-layer gel chromatography indicated that the enzymes were monomers. An examination of over thirty substrates revealed that both enzymes catalyzed the reduction of long-chain aliphatic, alicyclic and aromatic aldehydes as well as halogenated acetaldehyde. The F 2 enzyme acted on d -glucuronate, indicating that it was identical to the aldehyde reductase recently reported by other workers ( l -gulonate: NADP + 1-oxidoreductase EC 1.1.1.19). The F 4 enzyme, on the other hand, preferentially acted on C 24 3-ketosteroids.

Improved gas chromatographic method of determining diclofenac in plasma

Diclofenac was converted into either its methyl or ethyl ester in methanol or ethanol containing 0.1% or 0.5% sulfuric acid, respectively. The ester was extracted and subjected to gas--liquid chromatography with electron-capture detection. The esterification resulted in an increased sensitivity of the gas chromatographic detection, three times better than that previously reported for the formation of indolone ring in trifluoroethanol containing 0.5% sulfuric acid.