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T Higashiyama

Publications and source records attributed to T Higashiyama.

48 records · Page 3Linked to original sources
PubMed1993

Cigarette smoking during pregnancy lowers aromatase cytochrome P-450 in the human placenta.

To clarify whether cigarette smoking during pregnancy causes an organic alteration in placental estrogen producing ability, we determined the catalytic activity of aromatase by the tritiated water assay, and tissue level of aromatase cytochrome P-450 (P-450arom) by the specific enzyme-linked immunosorbent assay, in placental samples from nonsmokers and smokers. As pregnancy progressed, both aromatase activity and P-450arom concentration increased in placentas from nonsmokers and smokers. However, the gradient of the increase was significantly less in heavy smokers (> or = 20 cigarettes a day) than in normal and moderate smokers (< 20 cigarettes a day). At term, the mean aromatase activity and P-450arom concentration in placentas from heavy smokers were significantly lower than in nonsmokers and moderate smokers, while aromatase activity per P-450arom (turnover rate) and the mean placental weight were comparable among the three groups. In contrast, the ratio of aryl hydrocarbon hydroxylase activity to aromatase activity was higher in placentas from heavy smokers. Immunohistochemical studies showed that P-450arom was localized in the cytoplasm of syncytiotrophoblasts of chorionic villi in placentas from both nonsmokers and smokers. These results suggest that the induction of placental P-450arom during gestation is suppressed by maternal smoking, resulting in a reduction in estrogen producing ability, while placental xenobiotic P-450 is induced.

Aromatase↗
PubMed1993

Multiple functions of aromatase and the active site structure; aromatase is the placental estrogen 2-hydroxylase.

Androgen aromatase was found to also be estrogen 2-hydroxylase. The substrate specificity among androgens and estrogens and multiplicity of aromatase reactions were further studied. Through purification of human placental microsomal cytochrome P-450 by monoclonal antibody-based immunoaffinity chromatography and gradient elution on hydroxyapatite, aromatase and estradiol 2-hydroxylase activities were co-purified into a single band cytochrome P-450 with approx. 600-fold increase of both specific activities, while other cytochrome P-450 enzyme activities found in the microsomes were completely eliminated. The purified P-450 showed M(r) of 55 kDa, specific heme content of 12.9 +/- 2.6 nmol.mg-1 (+/- SD, n = 4), reconstituted aromatase activity of 111 +/- 19 nmol.min-1.mg-1 and estradiol 2-hydroxylase activity of 5.85 +/- 1.23 nmol.min-1.mg-1. We found no evidence for the existence of catechol estrogen synthetase without concomitant aromatase activity. The identity of the P-450 for the two different hormone synthetases was further confirmed by analysis of the two activities in the stable expression system in Chinese hamster ovarian cells transfected with human placental aromatase cDNA, pH beta-Aro. Kinetic analysis of estradiol 2-hydroxylation by the purified and reconstituted aromatase P-450 in 0.1 M phosphate buffer (pH 7.6) showed Km of 1.58 microM and Vmax of 8.9 nmol.min-1.mg-1. A significant shift of the optimum pH and Vmax, but not the Km, for placental estrogen 2-hydroxylase was observed between microsomal and purified preparations. Testosterone and androstenedione competitively inhibited estradiol 2-hydroxylation, and estrone and estradiol competitively inhibited aromatization of both testosterone and androstenedione. Estrone and estradiol showed Ki of 4.8 and 7.3 microM, respectively, for testosterone aromatization, and 5.0 and 8.1 microM, respectively, for androstenedione aromatization. Androstenedione and testosterone showed Ki of 0.32 and 0.61 microM, respectively, for estradiol 2-hydroxylation. Our studies showed that aromatase P-450 functions as estrogen 2-hydroxylase as well as androgen 19-, 1 beta-, and 2 beta-hydroxylase and aromatase. The results indicate that placental aromatase is responsible for the highly elevated levels of the catechol estrogen and 19-hydroxyandrogen during pregnancy. These results also indicate that the active site structure holds the steroid substrates to face their beta-side of the A-ring to the heme, tilted in such a way as to make the 2-position of estrogens and 19-, 1-, and 2-positions of androgens available for monooxygenation.

Animals↗
PubMed1991

Electrophoretic karyotyping and chromosomal gene mapping of Chlorella.

Molecular karyotypes for six strains of four Chlorella species were obtained by using an alternating-field gel electrophoresis system which employs contour-clamped homogenous electric fields (CHEF). The number and migration pattern of the chromosomal DNA molecules varied greatly from strain to strain: for example, nine separated chromosomes of C. ellipsoidea C87 ranged from 2.5 to 6.5 megabase pairs (mbp) in size, whereas 16 chromosomes of C. vulgaris C169 were from 980 kilobase pairs (kbp) to 4.0 mbp. Depending on the chromosome migration patterns, the six strains were classified into two major chromosome-length polymorphism groups. Using hybridization techniques, the genes for alpha-tublin, chlorophyll-a, b-binding proteins, ribosomal RNAs, and the small subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO) were mapped on the separated chromosomes of C. vulgaris C169. Since Chlorella chromosomes are small enough to separate and isolate individually by CHEF gel electrophoresis under ordinary conditions, they should serve as excellent materials to study the fundamental molecular structure of plant-type chromosomes.

Chlorella↗
PubMed1991

Screening of natural waters for viruses which infect chlorella cells.

By using a plaque assay with the unicellular green alga Chlorella sp. strain NC64A as a host, viruses were screened from natural pond waters collected in Kyoto and Higashi-Hiroshima, Japan. From some samples tested, two kinds of plaques, large (phi = 6 to 10 mm) and small (phi = 2 to 3 mm), were detected with various frequencies. The frequency of plaques in each of the water sources was seasonal; generally, it reached a peak value (8,000 PFU/ml) in May and gradually decreased to the limit of detection (<1) in November before increasing again in early spring. Electron microscopy revealed that the purified and negatively stained viruses were very large (125 to 200 nm) icosahedral particles. The genome isolated from these particles was always a linear double-stranded DNA of 340 to 370 kbp. Electrophoresis patterns of the DNA fragments produced by digestion with restriction enzymes differed considerably from plaque to plaque, even for plaques from the same water source. However, Southern hybridization showed strong homology among all of the virus DNAs tested, indicating relatedness of those viruses. A possible use of the Chlorella virus assay system to monitor the natural population of algal cells and water quality is discussed.

Journal Article↗
PubMed1987

Aromatase.

Aromatase catalyzes the conversion of androgens to estrogens through a series of monooxygenations to achieve the 19-desmolation and aromatization of the neutral steroid ring-A structure. We have separated two forms of aromatase, a major (P2a) and a minor (P3) form, from human term placenta through solubilization and chromatography. Partially purified aromatase in each form was immunoaffinity chromatographed to give a single band (SDS-PAGE) cytochrome P-450 of 55 kDa, utilizing a mouse monoclonal anti-human placental aromatase cytochrome P-450 IgGi (MAb3-2C2) which is capable of suppressing placental aromatase activity. The purified cytochrome P-450 showed specific aromatase activity of 25-30 nmol/min per mg with Km of 20-30 nM for androstenedione on reconstitution with NADPH-cyt P-450 reductase and dilauroyl L-alpha-phosphatidylcholine. This one step represents a higher than 100-fold purification with maintenance of the same Km. The stability analysis showed a half-life of more than 5 yr for solubilized aromatase and 2 months for the aromatase cytochrome P-450 on storage at -90 degrees C. Contrary to the recent claim that estrogen biosynthesis by reconstituted human placental cytochrome P-450 is by trans-diaxial 1 alpha,2 beta-hydrogen elimination, all of our partially purified forms and reconstituted aromatase synthesized estrogens by cis-1 beta, 2 beta-hydrogen elimination. Use of purified aromatase and [19-3H3, 4-14C]androstenedione led us to discover a metabolic switching by aromatase to 2 beta-hydroxylation of androgen. Results of the MAb3-2C2 suppression of aromatase activity in different species and tissues including human, baboons, horses, cows, pigs and rats indicated the presence of various isozymes of aromatase.

Androgens↗
PubMed1982

Multiple forms of aromatase and response of breast cancer aromatase to antiplacental aromatase II antibodies.

Two distinct aromatase-active protein complexes are solubilized by use of deoxycholate and separated by diethylamino-ethyl-cellulose chromatography from lyophilized powder of 900 X g precipitate fraction of human term placenta. Aromatase activity to produce estriol, the major estrogen of human pregnancy, was designated to be aromatase I activity and measured by estriol formation from 16 alpha-hydroxytestosterone. Aromatases II activity was the designation for that which produces estrone plus estradiol and was measured by androstenedione aromatization. Aromatases II and I are eluted with 0.25 M and 0.5 M Tris buffer, respectively, from diethylaminoethyl-cellulose column in an Mr 2 million soluble complex. Each has a minimum active Mr 135,000 subunit, which is isolated by Bio-Gel filtration in the presence of detergents, and consists of a reduced nicotinamide adenine dinucleotide phosphate:cytochrome P-450 reductase (Mr 83,000) and a cytochrome P-450 (Mr 52,000). Aromatase II was found to be the major aromatase, containing approximately five times more aromatase activity, reduced nicotinamide adenine dinucleotide phosphate:cytochrome c reductase activity, cytochrome P-450, and protein than did aromatase I. Antibodies raised in rabbits against aromatase II and its reductase suppressed aromatase II activity of breast cancer tissues, as well as of adult male lung tissue, placental microsomes, and solubilized aromatase. The breast carcinoma specimens responded to the antibodies in different degrees, but there was no response to antibodies against rat liver cytochrome P-450. The results indicate similar antigenic structures for breast cancer and placental aromatase but not for rat liver cytochrome P-450.

Adult↗
PubMed1978

Nonenzymatic hydrolysis reactions of adenosine 5'-triphosphate and its related compounds. III: Catalytic aspects of some cobalt(III) complexes in ATP-hydrolysis.

Trichlorodiethylenetriaminecobalt (III), [CoCl3dien], which is provided with three good leaving ligands and, hence, capable of binding ATP in a characteristic mode, accelerated effectively and specifically hydrolysis of ATP to ADP and Pi. A kinetic study of the reaction indicated that the rate of hydrolysis was first order with respect to the concentration of ATP in the presence of an excess of [CoCl3dien]. The rate constant was calculated to be 1.05 X 10(-2) min-1 at pH 4.0 (50 degrees C), corresponding to a catalysis of the hydrolysis of ATP by a factor of 150. The complex possessing one good leaving ligand, chlorotetraethylenepentaminecobalt(III), and that having two of them in trans-position, dichlorobis(dimethylglyoximato)cobalt(III) only slightly enhanced the hydrolysis of ATP. Dichloro-cis-alpha- and dichloro-cis-beta-triethylenetetraminecobalt(III) complexes, which have two good leaving ligands and allow chelation of ATP in their coordination sphere, exhibited fairly good activities, although the hydrolysis reactions of ATP occurred in two modes as ATP leads to ADP + Pi and ATP leads to AMP + PPi. The mechanism of ATP-hydrolysis reaction with [CoCl3dien] was also discussed on the basis of the kinetic data.

Adenosine Diphosphate↗