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Biomedical subjects

Y Osawa

Publications and source records attributed to Y Osawa.

At least 163 records · Page 9Linked to original sources
PubMed1987

Radiometric assay for cytochrome P-450-catalyzed progesterone 16 alpha-hydroxylation and determination of an apparent isotope effect.

In the course of studies on the oxygenation of steroids by purified P-450 cytochromes, particularly rabbit liver microsomal cytochrome P-450 form 3b, a rapid and reliable radiometric assay has been devised for progesterone 16 alpha-hydroxylation. In view of the lack of a commercially available, suitably tritiated substrate, [1,2,6,7,16,17-3H]progesterone was treated with alkali to remove the label from potential hydroxylation sites other than the 16 alpha position. The resulting [1,7,16-3H]progesterone was added to a reconstituted enzyme system containing cytochrome P-450 form 3b, NADPH-cytochrome P-450 reductase, and NADPH, and the rate of 16 alpha-hydroxylation was measured by the formation of 3H2O. This reaction was shown to be linear with respect to time and to the cytochrome P-450 concentration. An apparent tritium isotope effect of 2.1 was observed by comparison of the rates of formation of tritium oxide and 16 alpha-hydroxyprogesterone, and the magnitude of the isotope effect was confirmed by an isotope competition assay in which a mixture of [1,7,16-3H]progesterone and [4-14C]progesterone was employed.

Animals↗
PubMed1987

Equilin and equilenin biosynthesis. Stereochemistry of aromatization of 3-hydroxy-3,5,7-androstatrien-17-one by horse placenta.

The metabolic pathway leading to equilin and equilenin biosynthesis in the pregnant mare is different from that of estrone and estradiol and it is apparently cholesterol-independent. The precise precursors and intermediates and the stereomechanism of equine placental aromatization have not been established. [1,2-3H, 4-14C]3-Hydroxy-3,5,7-androstatrien-17-one was synthesized as a potential substrate and the 3H-distribution was analyzed by biochemical and chemical derivatization methods. The substrate was converted to equilin, equilenin and Heard's ketone by horse placental microsomes with a sp. act. of 74, 18 and 2.8 pmol/h/mg, respectively, and only to equilin by human placental microsomes with a rate of 26 pmol/h/mg. Analysis of the loss of 3H-labeling during aromatization showed the stereospecific 17 beta,2 beta-cis hydrogen elimination for equine estrogen biosynthesis both by horse and human placental microsomes. This is the same as for estrone and estradiol biosynthesis by both placentas. The biosynthesis of Heard's ketone, a non-phenolic ring-B aromatic C18 steroid, by horse placental microsomes was found to involve none of the four hydrogens at C-1 and C-2. This refutes the previous postulate that Heard's ketone arises from equilenin by reduction of the ring-A.

17-Ketosteroids↗
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↗
PubMed1987

Stereochemistry of the functional group determines the mechanism of aromatase inhibition by 6-bromoandrostenedione.

A selective inhibitor of aromatase (estrogen synthetase) would be a useful pharmacological tool with potential therapeutic application. We have found that 6 alpha-bromoandrostenedione (6 alpha-BrA) is a competitive inhibitor of human placental aromatase with respect to androstenedione, with an apparent Ki of 3.4 nM, while 6 beta-BrA is a mechanism-based irreversible inhibitor with an apparent Ki of 0.8 microM and a kinact of 0.025 min-1. Aromatase activity was measured by tritium release into water from the 1 beta position of [1(-3)H,4(-14)C]androstenedione in reaction mixtures containing NADPH and the aromatase. Time-dependent inhibition was assessed by preincubation of inhibitors with either the 900 X g placental pellet or placental microsomes in the presence of NADPH. Aliquots were taken at intervals, diluted, and assayed for aromatase activity with androstenedione and additional NADPH. The time-dependent inhibition by 6 beta-BrA was dependent on the concentration of this compound and the presence of NADPH, while the addition of excess substrate in the preincubation mixture hindered the inactivation. Both epimers were ineffective in inhibiting rabbit liver microsomal drug-metabolizing activities in a competitive or time-dependent manner. This indicates a high selectivity of 6-BrA inhibition among P-450 cytochromes. These and other 6-substituted androgens may be useful probes into the nature of the active site and mechanism of action of aromatase.

Androstenedione↗
PubMed1986

The apparent shift in distribution of aromatase activity in the subcellular fraction of human term placenta stored at -96 degrees C.

The effects of storage at -96 degrees C on the distribution of the total aromatase activity in the homogenate, 900 xg pellet and 900 xg supernatant of two human term placentas were studied. Both specific and total aromatase activities in the 900 xg pellet increased through storage at -96 degrees C, while that in the 900 xg supernatant decreased. The ratio of total aromatase activity between the 900 xg pellet and 900 xg supernatant was about 2:1 in fresh placenta, but the ratio was about 19:1 in placenta stored at -96 degrees C for 3.5 months. Total aromatase activity in the tissue homogenate remained fairly constant throughout five months of storage at -96 degrees C. These data indicate that although active microsomal aromatase is thought to be available only from fresh placenta and the activity is lost quickly during storage of the tissue, aromatase itself is stable for months at -96 degrees C and the 900 xg pellet prepared from the frozen tissue contains almost the total original aromatase activity in the fresh placental tissue.

Aromatase↗
PubMed1985

Immunocytochemical localization of aromatase in rat testis.

The immunocytochemical localization of aromatase in the testes of young and adult rats was investigated by an indirect-immunofluorescent method using antihuman placental aromatase-II cytochrome P-450 antibody. In both young (1 and 2 weeks old) and adult rats, only the Leydig cells in the interstitial tissue showed a positive immunoreaction for aromatase, while the germ cells and Sertoli cells in the seminiferous tubule were entirely negative. In addition, electron microscopy revealed that the Leydig cells in the testes of young as well as adult rats have a well-developed smooth endoplasmic reticulum, mitochondria with tubulovesicular cristae, and a few lipid droplets, these structures being characteristic of steroid secretory cells. On the basis of these results, we suggest that estrogens are mainly synthesized in Leydig cells of the testes.

Animals↗
PubMed1985

High-performance liquid chromatographic determination of aromatization of 16 alpha-hydroxylated androgens with human placental microsomes.

A sensitive nonradiometric assay of aromatization of 16 alpha-hydroxylated androgens, 16 alpha-hydroxy-4-androstene-3,17-dione (16 alpha-OHA), and 16 alpha-hydroxytestosterone (16 alpha-OHT), has been developed using reverse-phase high-performance liquid chromatography with voltametric detector. The estrogens produced by human placental microsomes, estriol (E3) and 16 alpha-hydroxyestrone (16 alpha-OHE1), were simultaneously detected in quantities as low as 1-2 ng using 3-methoxy-1,3,5(10)-estratriene-2, 16 alpha,17 beta-triol as an internal standard. E3 was the only estrogen detected from the incubate of 16 alpha-OHT with the microsomes and NADPH, while 16 alpha-OHA gave 16 alpha-OHE1 and E3 under the same conditions. Apparent Km and Vmax of the microsomal aromatase for 16 alpha-OHA and 16 alpha-OHT were 2.56 microM and 71.4 pmol/min/mg and 13.33 microM and 15.4 pmol/min/mg, respectively.

Androgens↗
PubMed1985

Erythrocyte membrane abnormalities in patients with amyotrophic chorea with acanthocytosis. Part 1. Spin labeling studies and lipid analyses.

Low fluidity was observed in the erythrocyte membranes of familial chorea-acanthocytosis by spin labeling with 12NS. The high content of the saturated fatty acids support the finding of low fluidity. Other lipid contents (cholesterol and phospholipids) and the cholesterol/phospholipid ratio were within normal ranges. The sharp decrease in the parameter, (h0/h-1)1/2, suggests phase separation deep inside the patients' erythrocyte membrane matrix. This distortion of the membrane matrix may be caused by membrane components other than the lipid components.

Acanthocytes↗
PubMed1985

Erythrocyte membrane abnormalities in patients with amyotrophic chorea with acanthocythosis. Part 2. Abnormal degradation of membrane proteins.

Bands 2.1, 3 and 4.2 of the erythrocyte membrane proteins in chorea-acanthocytosis are self-digested faster than those in normal controls. The junction between spectrin and band 3 protein at the cytoplasmic side of the patients' erythrocyte membranes may have some conformational defects. The conformational defects of the proteins may be responsible for the low fluidity in the interior of the patients' erythrocyte membranes. These basic molecular defects may be widespread in other extraneural cell membranes as well as in neuronal cell membranes.

Blood Proteins↗
PubMed1985

Angiotensin II induces the release of 19-hydroxyandrostenedione in man.

Plasma concentrations of 19-hydroxyandrostenedione [19-hydroxy-4-androstene-3,17-dione (19-OH-A-dione)], which we reported to be an amplifier of the action of aldosterone on the basis of results obtained in bioassays using adrenalectomized rats, were measured in man using a specific and sensitive RIA. Plasma 19-OH-A-dione concentrations (mean +/- SE) in normal subjects were 56 +/- 4 pg/ml in men and 51 +/- 4 pg/ml in women. Plasma 19-OH-A-dione rose significantly during ACTH stimulation and declined significantly during dexamethasone suppression. Plasma 19-OH-A-dione levels in the adrenal vein were an order of magnitude higher than those in the inferior vena cava. These results demonstrate that 19-OH-A-dione is directly secreted by the adrenal cortex and that its secretion is under the control of ACTH. As the action of 19-OH-A-dione may be closely related to that of aldosterone, the response of 19-OH-A-dione to angiotensin II infusion was evaluated and compared with that of aldosterone. During the infusion of graded doses (0.5-4.0 ng/kg . min) of angiotensin II, plasma 19-OH-A-dione increased significantly after the infusion of angiotension II at rates of 1.0 and 2.0 ng/kg . min. It then declined after the infusion of angiotensin II at a rate of 4.0 ng/kg . min. In contrast, plasma aldosterone did not increase significantly until the infusion rate reached 4.0 ng/kg . min. These results indicate that the secretion of 19-OH-A-dione is under the control of angiotensin II. Similar changes in 19-OH-A-dione and aldosterone were found during postural changes. In those subjects who had a small increase in plasma aldosterone when they were upright, 19-OH-A-dione significantly increased. In contrast, in those subjects who had a large increase in plasma aldosterone when upright, 19-OH-A-dione significantly declined. These results suggest that a small elevation of endogenous angiotensin II induces an elevation of plasma 19-OH-A-dione, and a sharp increase in endogenous angiotensin II induces an increase in plasma aldosterone and a decline in plasma 19-OH-A-dione. As angiotensin II stimulated the release of 19-OH-A-dione when the secretion of ACTH was suppressed, angiotensin II acted directly on the adrenal cortex. 19-OH-A-dione is a newly recognized biologically active adrenal C19 steroid which is regulated by both ACTH and the renin-angiotensin system in man.

Adrenocorticotropic Hormone↗
PubMed1984

Additive effects of aminoglutethimide, testololactone, and 4-hydroxyandrostenedione as inhibitors of aromatase.

In vitro p450 spectral data suggested that combinations of aromatase inhibitors might produce enhanced biologic effects. If correct, two clinically available aromatase inhibitors, aminoglutethimide (AG) and testololactone (TL) could potentially be given together at lower than usual dosage with reduction of patient side effects and preservation of aromatase inhibition. Using a [3H]water aromatase assay and a placental microsomal system, AG and TL were tested individually and in combination over their respective dose response ranges. Additive effects of these two compounds were observed. Another inhibitor, 4-hydroxyandrostenedione, given with AG produced similar additive inhibition. These data provide a basis for a future trial of AG and TL in combination in patients with breast carcinoma.

Aminoglutethimide↗
PubMed1983

Binding of thyroid hormone by heme proteins may signal protein age.

Cellular aging is thought to include the accumulation of proteins with post-translational modifications that alter degradation rates or function or both. Protein catabolism has been regarded as a randomly occurring process, but recent observations have suggested mechanisms by which old and young cells may distinguish recently-synthesized protein molecules from "old" proteins. Hemoglobin, myoglobin and other heme proteins bind thyroid hormone progressively with time and in a poorly-dissociable manner. This cumulative acquisition of thyroid hormone is postulated to be an index of molecular aging.

Erythrocytes↗
PubMed1983

Outer and inner membrane preparations of extreme thermophile and their physico-chemical properties.

Outer and inner (cytoplasmic) membranes were partially purified from the gram negative extremely thermophilic bacteria, Thermus thermophilus HB-8 by sucrose density gradient centrifugation. In spite of our efforts to separate them, the inner membrane fraction contained some outer membrane components as determined by enzyme assay and electrophoresis. When studied by 5DS spin labeling, the outer membranes showed a larger 2T11 value (lower fluidity) than the inner membranes, although the fatty acid compositions were similar. The inner membranes of the cells cultured at higher temperature showed a larger 2T11 value than the cells cultured at lower temperature. A similar phenomenon was observed with the TEMPO parameter of liposomal membranes. The upper break point (Th) of the inner membranes observed by spin labeling was slightly lower than the culture temperature of the cells, and the lower break point (T1) corresponded well to the lowest temperature limit of growth. The calorimetric heating curve of the inner membranes had a broader temperature range of transition than that of the liposomal membranes. The transition temperature observed by calorimetry seems to reflect the melting properties of the membrane lipids, while fatty acid spin probe probably reports the local environment of the membrane, which is more directly related to its biological function.

Adaptation, Physiological↗
PubMed1982

Norethisterone, a major ingredient of contraceptive pills, is a suicide inhibitor of estrogen biosynthesis.

Norethisterone (17 alpha-ethynyl-19-nortestosterone) is an effective irreversible inhibitor of estrogen synthetase (aromatase), the enzyme responsible for the conversion of androgens to estrogens, even at a 2 X 10(-6) molar concentration. This irreversible inactivation, which is directed toward the active site of aromatase and requires the cofactor-reduced nicotinamide adenine dinucleotide phosphate, is both time- and concentration-dependent. Ethisterone (17 alpha-ethynyltestosterone), in contrast, is not a suicide inhibitor of aromatase even at concentrations of 10(-4) molar.

Aromatase Inhibitors↗
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↗