Biomedical subjects

Y Osawa

Publications and source records attributed to Y Osawa.

At least 145 records · Page 8Linked to original sources

PubMed1990

[A study of non-aromatizing androgen C10-19lyase in adrenal tissue].

The conversion of androgen to estrogen is mediated by aromatase activity, and 19-hydroxylase and C10-19lyase are seen to be involved in C19-demethylation. The present study is to demonstrate C10-19lyase activity in adrenal tissue. Fetal bovine and pig adrenal tissue homogenates were incubated with 14C-androstenedione(A) and NADPH under air. The products 14C-19-OHA, 14C-19-norA, with added carrier standard 3H-19-OHA, 3H-19-norA were separated and purified by T.L.C.. The identification of 19-norA was established by recrystallization and that of 19-OHA by C.C.D.. The 3H/14C ratios of the products 19-norA were found to be constant in repeated crystallization. The amount of 19-OHA from A in fetal adrenal tissue was 30.0nmol/h/g protein, and that of 19-norA from A was 12.59pmol/h/g protein. C10-19lyase activity from 19-OHA, calculated by the amounts of 19-norA in fetal, bovine and pig adrenal tissue, was 49.7, 3.50, 10.3pmol/h/g protein, respectively. This is the first report to demonstrate androgen C10-19lyase in human fetal adrenal tissue, and it is assumed that fetal adrenal tissue forms 19-norA via 19-OHA.

Adrenal Glands↗

PubMed1990

Metabolism-based covalent bonding of the heme prosthetic group to its apoprotein during the reductive debromination of BrCCl3 by myoglobin.

The reductive metabolism of BrCCl3 by ferrous myoglobin leads to the alteration of the prosthetic heme to form products that can be dissociated from the protein and to those that are irreversibly bound to the protein. The major dissociable or soluble heme metabolites have recently been characterized. In this study, the irreversibly bound heme product was characterized by Edman degradation, amino acid analysis, and electronic absorption and mass spectrometry of peptides derived from the altered protein. It was found that the prosthetic heme was modified by a CCl2 moiety derived from BrCCl3 and was covalently bound to histidine residue 93, the normal proximal ligand to the heme-iron. The data are consistent with a mechanism by which the trichloromethyl radical reacts with the heme to form an intermediate that either can alkylate the proximal histidine residue or form soluble metabolites. The covalent bonding of the heme prosthetic moiety to the apoprotein likely leads to a change in the tertiary structure of the protein that may be responsible for its altered catalytic activity as well as its enhanced susceptibility to proteolysis. Similar processes may account, at least in part, for the covalent alteration of the heme prosthetic group of other hemoproteins caused by xenobiotics and endogenous substrates.

Amino Acid Sequence↗

PubMed1990

Involvement of specific placental antigen X-P2 in rat olfaction: an immunohistochemical study in the olfactory bulb.

Human placental antigen X-P2 (hPAX-P2), an antigen complex associated with cytochrome P-450 of aromatase within estrogen synthesizing tissues, has been reported to be present in a distinct group of rat primary olfactory receptors involved in suckling behavior. In this study, most of the mitral and tufted cells in the rat olfactory bulb were found to possess hPAX-P2 immunoreactivity. This suggests that the activity of these cells can be hormonally modulated and that hPAX-P2 is involved in rat olfaction not only at the receptor level but also at integrative brain levels via the secondary projecting neurons of the olfactory pathway.

Animals↗

PubMed1990

Immunocytochemical localization of aromatase in immature rat ovaries treated with PMSG and hCG, and in pregnant rat ovaries.

Immunocytochemical localization of aromatase cytochrome P-450 was examined in immature rat ovaries treated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG), and in pregnant rat ovaries. It is well known that PMSG and hCG treatments induce ovulation about 12 h after hCG injection. At 24 h after hCG injection, many antral follicles were recognized in immature rat ovaries and only the granulosa cells in the antral follicles were stained weakly with the anti-aromatase antibody. At 0 to 9 h after hCG injection, in addition to the antral follicles, some large Graafian follicles could be observed in the rat ovaries, and the granulosa cells of these follicles were positively stained for aromatase. Each follicle was surrounded by the basal lamina which shows lineally distinct positive reaction against anti-laminin antibody. At 12 h after hCG injection, some large Graafian follicles without oocyte were weakly positive to the anti-aromatase antisera, and the outline of their basal lamina stained with anti-laminin antibody became irregular in shape and fragmentous. At 15 to 18 h after hCG injection, the luteinized cysts could be seen, and the granulosa-lutein cells of these cysts were almost negative for aromatase. Fragmentous reaction to the anti-laminin antibody was observed around the luteinized cysts. In the ovaries of day 4 in pregnancy, only the granulosa cells of the large antral follicles were weakly stained, but corpora lutea negatively reacted to the anti-aromatase antibody. At 7 to 19 days in gestation, both the granulosa cells of antral follicles and pregnant luteal cells were positively stained against aromatase antisera.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Synthesis of 19-hydroxy-[7-2H2]androstenedione for human metabolism studies.

19-Hydroxyandrostenedione (19-OHA), highly labeled with deuterium at position 7, was synthesized from unlabeled androstenediol diacetate. The deuterium labels were introduced into the 7-keto compound with dichloroaluminum deuteride to obtain [7-2H2]androstenediol. The labeled androstenediol diacetate was converted to the labeled 19-OHA by a five-step sequence without appreciable loss of deuterium. The labeled 19-OHA is useful as an internal standard for gas chromatography-mass spectroscopy analysis of the endogenous levels and as a tracer for in vivo metabolic studies.

Androstenediol↗

PubMed1990

Serum level of 19-hydroxyandrostenedione during pregnancy and at delivery determined by gas chromatography/mass spectrometry.

19-Hydroxyandrostenedione (19-OHA) is secreted from the adrenal glands in men and women and also from the placenta during pregnancy. It has been found to cause hypertension in animal models. We have synthesized [7,7-2H2]-19-OHA with high deuterium content and, together with [7,7-2H2]A and [9,11-2H2]estrone (E1), have developed a quantitative assay of serum level 19-OHA, A, and E1 using the gas chromatography/mass spectrometry-mass fragmentography method to monitor individual subjects throughout pregnancy. The labeled 19-OHA, used as internal standard, showed only 6.73% of unlabeled compound. Recovery of standard 19-OHA, A, and E1 (5,000 pg each) added to male plasma was 97.4 +/- 2.3%, 96.3 +/- 2.1%, and 100.1 +/- 4.1% (mean +/- SD), respectively; the intraassay coefficient of variation was 2.1%, 3.5%, and 3.8%, respectively. Ten pregnant subjects without complications and 10 pregnant subjects near term with hypertension were selected (with informed consent). The 19-OHA and E1 serum concentrations of maternal venous blood from uncomplicated pregnancies increased significantly as gestation progressed (19-OHA: first trimester, 225 +/- 72; second trimester, 656 +/- 325; third trimester, 1,518 +/- 544 pg/ml), reaching the highest level at delivery (19-OHA: 1,735 +/- 684 pg/ml). Whereas a positive correlation was found between the level of 19-OHA and E1, no apparent change of the A level was observed during pregnancy. Levels of the three steroid hormones in pregnancy complicated by hypertension in the second and third trimester were not found to be significantly different from those of normal pregnancy (19-OHA of hypertensive subjects: second trimester, 762 +/- 349; third trimester, 1,473 +/- 491 pg/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Androstenedione↗

PubMed1990

Reversible inhibition of human placental microsomal aromatase by CGS 18320B and other non-steroidal compounds.

The effect of bis-(p-cyanophenyl)imidazo-1-yl-methane hemisuccinate (CGS 18320B) and other non-steroidal compounds on the aromatization of androstenedione by human placental microsomal aromatase was studied. CGS 18320B exhibited competitive inhibition with an apparent Ki of 0.16 nM, a 90 and 3800-fold increase in affinity compared to 4-hydroxyandrostenedione and amino-glutethimide, respectively. The inhibition is not time-dependent, indicating that the active site interaction is reversible. CGS 18230B showed a two-fold increased affinity as compared to 4-(5,6,7,8-tetrahydroimidazo[1,5a]pyridin-5-yl)benzonitrile (CGS 16949A) and cis-1-[(4-[(1-imidazoyl)methyl]cyclohexyl)methyl]-imidazole succinate (CGS 14796C) which showed Ki values of 0.35 and 0.39 4M, respectively. 1-[2-[1-(4-carboxyphenyl)-3-ureido]ethyl]-2-(4-pyridyl)-2-imidazoline monohydrochloride (CGP 15720A) showed negligible inhibition.

Androstenedione↗

PubMed1990

Two-stage model of visual pattern discrimination learning in macaque monkeys (Macaca mulatta and Macaca fuscata).

To investigate the nature underlying the process of pattern discrimination learning, a series of seven experiments on seven working assumptions were undertaken. The main findings are as follows. The pattern discrimination learning consists of two time-dependent stages: The initial or first learning stage is the period of performance at chance, and the succeeding or second stage is the period of performance from just above the chance to a criterion level. The duration of the first stage is dependent on the degrees of cue-response separations, whereas that of the second stage is independent of. During the first stage, monkeys do not attend to the discriminative cue even at small cue-response separations, whereas during the second stage, they achieve pattern discrimination, or pattern perception and cognition, regardless of cue-response separations. After having learned the first pattern task with a cue-response separation, they learned new pattern tasks by means of the second stage, showing marked saving of the duration of the first stage. The findings in the present studies indicate that the first stage of learning is predominantly involved in the process of attending to the discriminative cues remote from the response site (a selective attention to the cue), whereas the second stage is concerned with the process of perception and cognition of the discriminative cue.

Animals↗

PubMed1990

Aromatase inhibitors in cigarette smoke, tobacco leaves and other plants.

A chance observation that cigarette smoke interferes with the aromatase assay led us to investigate tobacco leaf and smoke extracts for the presence of aromatase inhibitors. The highest inhibitory activity was found in the basic fraction of cigarette smoke. Further purification of this fraction led to the identification of N-n-octanoylnornicotine. Synthesis and testing of a series of acylated nornicotines and anabasines for their ability to inhibit aromatase showed an interesting correlation of activity with the length of the acyl carbon chain, with maximum activity at C-11. The acylated derivatives showed activity which was significantly greater than that of nicotine and anabasine. In vivo studies in rats indicated that administration of this inhibitor delayed the onset of NMU-induced breast carcinoma and altered the estrus cycle. These in vivo studies suggest that tobacco alkaloid derivatives exert their effects by suppression of the aromatase enzyme system. Toxicity studies indicated relatively low toxicity with LD50 for N-n-octanoylnornicotine = 367 mg/kg body weight. When extracts from thirty five varieties of vegetables, plant leaves, and fruits were analyzed, seventeen showed quantitatively significant aromatase inhibition which was comparable to that of green tobacco leaf, suggesting that naturally occurring substances may affect endocrine function through aromatase inhibition.

Anabasine↗

PubMed1989

Screening of aromatase-containing neurons in rat forebrain: an immunohistochemical study with antibody against human placental antigen X-P2 (hPAX-P2).

Aromatase-containing neurons were immunohistochemically examined in rat brains by using a polyclonal antibody against human placental antigen. The antibody recognizes cytochrome P-450 portion of aromatase, an enzyme converting androgen to estrogen. A large group of strongly immunoreactive cells was identified in the ventral pallidum, which extends caudally from the area surrounding the islands of Calleja. Other strongly or moderately stained cell groups were observed in the cerebral cortex, the amygdaloid area, the nucleus of the diagonal band, and the area anterior to the posterior commissure. Only a few stained cells were present in the medial preoptic region. These findings cast doubt upon the previous assumption, based on biochemical analysis of tissue samples, that the center of the aromatizing system is in the medial preoptic region. They indicate instead that most aromatase-containing neurons of rats lie within the ventral pallidum ventromedially adjacent to the preoptic area.

Animals↗

PubMed1989

[The newly developed method of 19-OH-A as intermediate of estrone biosynthesis by GC-MS].

To study the serum levels of 19-hydroxyandrostenedione (19-OH-A), known as an obligatory intermediate of estrogen biosynthesis and considered to be one of the hypertensinogens, a method using GC-MS with application of synthesized [7,7-d2]androstenedione (A), [7,7-d2] 19-OH-A and [9,11-d2]estrone(E1) as internal standards was newly developed. Normal pregnant women and pregnant women complicated with hypertension near term were selected for the study. The levels of 19-OH-A and E1 increased significantly as gestation progressed [19-OH-A; 224.7 +/- 72.1 (1st trimester), 655.5 +/- 325.4 (2nd trimester). 1517.8 +/- 543.6 (3rd trimester)pg/ml], and a positive correlation was found between the levels of the two steroids. No apparent change was observed in A levels during the course of pregnancy. The mean levels of 19-OH-A in pregnancy complicated with hypertension at 2nd and 3rd trimester were 761.7 +/- 348.9 and 1473.0 +/- 491.4 pg/ml, which were compatible with those in normal pregnancy. The levels of 19-OH-A at delivery in maternal vein (MV) were 1735.1 +/- 683.9 pg/ml. Significantly higher levels of 19-OH-A were found in umbilical vein (UV) (1977.2 +/- 564.9 pg/ml) than those in umbilical artery (109.7 +/- 49.1 pg/ml). 19-OH-A concentration in term placental tissue was 16.3 ng/g.w.w. tissue. This is the first report to demonstrate the serum 19-OH-A levels measured by GC-MS and also to demonstrate the levels in the cord blood. The results indicate that 19-OH-A may be the product of pregnancy and may be derived from the feto-placental compartment.

Androstenedione↗

PubMed1989

[Non-aromatizing androgen 19-hydroxylase in human fetal organs].

19-hydroxyandrostenedione (19-OH A), known for many years as an obligatory intermediate of estrogen biosynthesis, has recently been found to be an amplifier of aldosterone action and in itself a hypertensinogenic steroid (J. Steroid Biochem., 16; 329, 1982). To search the presence of a non-aromatizing andorgen 19-hydroxylase in fetal organs, metabolism of labeled androstenedione(A) in fetal adrenal, liver, lung, brain and kidney was studied. 19-hydroxylase activity was calculated by the amounts of 19-OH A formed from A. The tissue homogenate was incubated with [4-14C]A and NADPH for different periods of time under air. The product, [4-14C] 19-OH A with added carrier standard [6,7-3H] 19-OH A was separated and purified by TLC, acetylation and TLC to constant 3H/14C ratio. The identity of the product was established by C.C.D. and by GC-MS analysis of purified product 19-AcOA in a larger scale adrenal incubation of non-labeled A. 19-hydroxylase activity was 0.9 pmol/min/mg protein for adrenal, but there was a negligible quantity of 19-OH A in lung, brain and kidney. Estrogen production was also assayed by 3H-water method using [1 beta-3H, 4-14C] A (3H/14C = 69.9). No detectable amount of estrogen was found in incubation of any homogenate except for liver. These results indicate that the non-aromatizing 19-hydroxylase activity in fetal adrenal gland is much higher than that in other organs. This is the first report to demonstrate the presence of 19-hydroxylase in human fetal adrenal tissue. It is suggested that 19-OH A secreted from fetal adrenal may also be associated with serum concentration of fetal circulation.

Adrenal Glands↗

PubMed1989

"Necklace olfactory glomeruli" form unique components of the rat primary olfactory system.

A distinct subset of rat primary olfactory neurons was identified immunohistochemically by means of a polyclonal antibody against human placental antigen X-P2 (hPAX-P2), an incompletely characterized substance found in all estrogen-biosynthetic organs. The subset of olfactory receptor cells was distributed widely over the olfactory epithelium with some degree of concentration on the dorsocaudal walls of nasal subcavities. The subset formed unique "necklace olfactory glomeruli," which were composed of seven to nine solitary glomeruli located in the caudal end of the olfactory bulb. One of them was located in the "modified glomerular complex" reported to be involved in rat suckling behavior. The projectional patterns of the necklace olfactory system, albeit diffuse, indicated some degree of spatial correspondence between zones of olfactory epithelium and specific glomeruli. Axons emanating from neighboring cells can project to several glomerular loci. From the necklace olfactory system, an average of 150-200 receptor cells were estimated to converge onto a single necklace glomerulus.

Animals↗

PubMed1989

Further immunocytochemical study on the localization of aromatase in the ovary of rats and mice.

The precise localization of estrogen biosynthesis in the ovary of rats and mice were immunocytochemically studied using new antisera against aromatase cytochrome P-450. The positive reaction for aromatase was detected mainly on the granulosa cells of large, apparently preovulatory follicles. In addition, the cells of some corpora lutea showed very weak positive reaction but most corpora lutea were negative to the staining. Those cells such as the granulosa cells of smaller follicles, the theca interna cells, the interstitial gland cells, oocytes, peritoneal epithelial cells were entirely negative. These results indicate that in the ovary of rats and mice, the granulosa cells of preovulatory follicles are the main site for synthesis of estrogen from androgen which is provided by the theca interna cell and the interstitial gland cell.

Animals↗

PubMed1989

Immunologic identification of the aromatase enzyme system in human endometrium.

Confluent human endometrial stromal cells were cultured in medium with no hormone or supplemented with medroxyprogesterone acetate (MPA), estradiol (E2), and porcine relaxin (RLX) for 5 days. These stromal cells were then labeled with [35S]methionine for 3 h. The radioactive proteins in the particulate fraction of cell homogenate were extracted by detergent and incubated with antisera to purified placental aromatase cytochrome P-450 (P-450arom) and NADPH-cytochrome P-450 reductase to isolate the radio-labeled aromatase enzyme components. Analysis of the radio-labeled protein, isolated by antibody to the cytochrome P-450arom from different preparations (P45FBIII or R-8-2) showed a major band at molecular weight 54k on SDS polyacrylamide gel electrophoresis (SDS-PAGE). The intensity of 54k band was stronger in hormone treated stromal cells than that of control in parallel with the increase of aromatase activity. The radio-labeled protein isolated by anti-NADPH cytochrome P-450 reductase, REDFBIV, showed a major band at the molecular weight 73k on SDS-PAGE with comparable intensity in control and hormone treated samples. Thus, the apparent molecular weights of endometrial cytochrome P-450arom and cytochrome P-450 reductase were identical to placental aromatase enzyme system. When a secretory endometrium and a decidua were labeled with [35S]methionine, the cytochrome P-450arom was detected only in the decidua. NADPH cytochrome P-450 reductase was detected both in the endometrium and the decidua. These results show that antisera to placental aromatase enzyme system cross reacts with the endometrial aromatase enzyme components. The synthesis of cytochrome P-450arom was stimulated by MPA, E2 and RLX while the synthesis of the NADPH-cytochrome P-450 reductase aromatase component was not affected by the hormone.

Aromatase↗

PubMed1989

An enzyme-linked immunosorbent assay for quantitation of aromatase cytochrome P-450.

Traditionally, aromatase has been quantified as aromatase activity according to its ability to produce estrogen from androgen. We have developed a quantitative assay based on the protein mass of catalytically active aromatase cytochrome P-450. A solid phase sandwich enzyme-linked immunosorbent assay for aromatase cytochrome P-450 has been devised using mouse monoclonal antibody (MAb3-2C2) and rabbit polyclonal antiserum (PAb R-8-2). Two rabbit antisera (PAb R-8-1 and R-8-2) were raised by immunization against human placental aromatase cytochrome P-450 which had been isolated by immunoaffinity chromatography of MAb3-2C2-coupled to Sepharose 4B resin. Both antisera were capable of suppressing human placental aromatase activity with IC50 values of 0.6 and 0.8 microliter/ml incubate, respectively, and showed monospecific to aromatase cytochrome P-450 in the Western blot analyses. Solubilized human placental microsomal samples were incubated in microtiter wells precoated with MAb3-2C2. The unbound proteins were washed out, and the aromatase cytochrome P-450 bound with the MAb3-2C2 in the wells was then reacted with PAb R-8-2, the binding of which was subsequently probed with goat antirabbit immunoglobulin G antibody alkaline phosphatase conjugate. Immunoaffinity-purified aromatase cytochrome P-450 of human placental microsomes was used for the standard, with the current assay detection limit at 1 ng/ml. There was a positive correlation between aromatase activity and the immunoreactive aromatase cytochrome P-450 level in solubilized microsomal samples after preincubation at 22 and 37 C, indicating that the enzyme-linked immunosorbent assay measures the level of aromatase cytochrome P-450 that has catalytic activity. The mean level of aromatase cytochrome P-450 in solubilized human term placental microsomes was 16.4 +/- 10.3 (+/- SD) micrograms/ml, corresponding to 0.38 +/- 0.19% of the original microsomes. The mean specific activity of aromatization of the solubilized samples was 0.650 +/- 0.163 nmol estrogen formed/min.mg protein. These results indicate that aromatase in the solubilized placental microsomal fraction has catalytic ability of 5.3 +/- 1.6 min-1 based on the immunoassayable cytochrome P-450.

Antibodies, Monoclonal↗

PubMed1988

[A study of non-aromatizing androgen 19-hydroxylase in sheep adrenal].

We reported on the unusually high isotope effect of non-aromatizing androgen 19-hydroxylase in sheep and dog adrenals and the validity of the [3H] water method using [19-3H3] androgen. We have extended the study to examine whether this 19-hydroxylation is catalyzed by a cytochrome P-450 dependent enzyme. Sheep adrenal homogenate (1.65 mg prot.) was incubated in the presence of NADPH (5.6mM) with [19-3H3, 4-14C]-androstenedione (A) (3.2 microM, 8.24 x 10(4) dpm 3H/micrograms, 3H/14C = 17.2) in a total of 1.2 ml PO4 buffer under air at pH 7.4 for 2, 5 and 10 min. [19-3H2, 4-14C]-19-hydroxy-A (19-OHA) with added carrier was purified through extraction, TLC, acetylation to form 19-AcOA, and further TLC to give 19-hydroxylase activity as assessed by the product isolation method. Simultaneously, the [3H] water was measured by distillation, and with correction by the apparent kinetic isotope effect (KH/KT = 11.8), used for assessment of 19-hydroxylase activity. The effects on the hydroxylation by cofactor (NADPH, NADH), incubation atmosphere (N2, CO/O2), cytochrome P-450 inhibitors (metyrapone, clotrimazole) and heating were measured by both methods. Compared to the complete system (89.6pmol/min/mg as 100%), carbon monoxide suppressed 15.8, 59.3 and 86.4% of the 19-hydroxylation when a CO/O2 ratio of 0.1, 1 and 9 was used, respectively. Replacement to nitrogen atmosphere decreased the activity by 93.8%. Replacement of NADPH with NADH (7.5mM) caused more than a 92.1% decrease in activity. Metyrapone at 50 and 200 microM and and clotrimazole at 2.5 and 10 microM suppressed the activity by 82.8, 90.4, 85.4 and 94.9%, respectively. A larger scale sheep adrenal incubation of A (250 microM) under 18O2 atmosphere and isolation of 19-AcOA were carried out in a similar manner. The gas chromatography-mass spectrometry analysis of the purified product showed 48.5% of the product to be 18O-labeled as [M+ + 2], m/e 346. Thus, the non-aromatizing androgen 19-hydroxylase requires NADPH and molecular oxygen. It is strongly inhibited by carbon monoxide and cytochrome P-450 inhibitors. These results indicate that the enzyme system responsible for non-aromatizing androgen 19-hydroxylase in adrenal is a cytochrome P-450 dependent monooxygenase.

Adrenal Glands↗

PubMed1988

Inhibition of human placental microsomal aromatase by thyroid hormone and iodothyronine derivatives.

The initial velocity kinetics of the effect of thyroid hormone and analogs on human placental microsomal aromatase were studied. Thyroxine and its propionic analog 3,5,3',5'-tetraiodothyropropionic acid show a competitive inhibition with an apparent Ki of 1.4 microM and 8.5 microM, respectively, towards androstenedione aromatization. 3,5,3'-Triiodothyronine with a Ki of 1.8 microM shows a decreased affinity compared to thyroxine. Two analogs, 3,5,3'-triiodothyropropionic acid and 3,5,3'-triiodothyroacetic acid appear to have negligible competition.

Aromatase Inhibitors↗