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Biomedical subjects

Y Osawa

Publications and source records attributed to Y Osawa.

At least 91 records · Page 5Linked to original sources
PubMed1995

Inhibition of aromatase activity in human placental microsomes by 13-retro-antiprogestins.

Mifepristone (RU 486), used clinically for the termination of early pregnancy, and its acetyl and 13-retro (13 alpha) analogs show potent antiproliferative effects against estrogen-dependent human breast tumors and endometriosis. However, there has been no report on direct inhibition of aromatase by antiprogesterones. Aromatase inhibitors have been shown to be effective against estrogen-dependent breast cancer. We evaluated the inhibition of aromatase by various antiprogestins (ZK 112.993, ZK 98.734, ZK 114.043, ZK 98.299, and ZK 114.863). Human placental microsomes were incubated with [1 beta-3H,4-14C] androstenedione (3-114 nM) in the presence of NADPH, with or without putative inhibitors (10-200 microM). Aromatase activity was assessed by tritium release to water from the 1 beta-position of the substrate. ZK 112.993 and ZK 98.734 did not show any inhibitory effect. The statistical analysis of the data using standard errors was obtained from replicate experiments. ZK 114.043 showed slight inhibition with a Ki of 54.8 +/- 6.4 microM (m +/- SE, n = 6) against androstenedione aromatization. The two 13-retro-steroids, ZK 98.299 and ZK 114.863, showed aromatase inhibition with Ki values of 19.0 +/- 1.5 microM (n = 7) and 12.7 +/- 0.94 microM (n = 7), respectively, which is weak with respect to some known potent inhibitors, but significant when compared with the other antiprogestins which were tested. The results suggest that the unnatural 13-retro-antiprogestin conformation may have a better fit to the aromatase active site than the natural 13 beta-antiprogestin conformation. (Steroids 60:234-238, 1995).

Aromatase Inhibitors↗
PubMed1995

Em18 and Em16, new serologic marker epitopes for alveolar echinococcosis in western blot analysis, are the only two epitopes recognized by commercially available weak positive (cut off) sera for Em2plus-ELISA.

The assay system for antibody responses against Em2, the most specific antigen for serodiagnosis of alveolar echinococcosis (AE), has been established by enzyme-linked immunosorbent assay (ELISA) but not by Western blot assay, since Em2 antigen is not protein but carbohydrate in nature. Recently we reported that previously undescribed protein epitopes, designated Em18 and Em16 due to their molecular weights, were good serologic markers for AE by Western blot analysis. It has been shown that Em18 and Em16 are the only two epitopes recognized by commercially available weak positive (cut off) sera for the Em2plus-ELISA.

Antibodies, Helminth↗
PubMed1995

Rooster testicular germ cells and epididymal sperm contain P450 aromatase.

We recently found that cytochrome P450 aromatase (P450arom) is present in germ cells of the mammalian testis and is capable of converting androgens to estrogens in the male reproductive tract. The objective of the present study was to determine whether testicular germ cells and epididymal sperm of an avian species are also capable of synthesizing estrogen. P450arom was localized in the rooster testis and epididymal region by immunocytochemistry, using an antiserum generated against purified human placental cytochrome P450arom. Immunostaining was present in pachytene spermatocytes, round spermatids, elongated spermatids, flagella of late spermatids, and sperm in the epididymal region. A positive reaction was also found in nonciliated cells of the epididymal region. However, the absence of mRNA for P450arom in the epididymal region indicated that the immunoreactive protein present in the epididymal region is not synthesized in this region. The immunoreactive P450arom found in epididymal sperm was shown to be active through use of a 3H2O assay. On the basis of these data, we conclude that rooster testicular germ cells and epididymal sperm are sites for the synthesis of estrogen, a potential regulator or modulator of germinal epithelium in the testis and the epithelium of the epididymal region of the avian species.

Animals↗
PubMed1995

Steroidogenic activity and ultrastructural observation of atretic follicles in the cycling hamster ovary.

In mature hamster ovaries, many follicles undergo atresia and this atretic change occurs in any estrous day. In this study, the localization of enzymes involved in estrogen biosynthesis, Bromodeoxyuridine (BrdU) incorporation, and Proliferating Cell Nuclear Antigen (PCNA) were immunohistochemically examined in the atretic follicles always provided with an atrum variable in size. Moreover, the granulosa cells from the atretic follicle in various but parallel stages of development were examined by scanning and transmission electron microscopes. Very early morphological signs of atresia in these follicles was the pyknotic change of a few granulosa cells lining the antral cavity. In this kind of follicles, the number of BrdU incorporating granulosa cell was decreased and the immunoreactivity of PCNA and aromatase was gradually decreased. Even in the early stage of atresia, some granulosa cells showed a remarkable morphological change characteristic of apoptosis as revealed by scanning and transmission electron microscopy.

Animals↗
PubMed1994

Characterization of the inactivation of nitric oxide synthase by NG-methyl-L-arginine: evidence for heme loss.

The nitric oxide synthases (NOS) are a unique family of P450-type hemoproteins that catalyze the formation of .NO and citrulline from L-arginine, oxygen, and NADPH. NG-Methyl-L-arginine (L-NMA) has been shown to function as a slow, partially uncoupled alternate substrate and mechanism-based inhibitor of the inducible NOS [Olken, N. M., & Marletta, M. A. (1993) Biochemistry 32, 9677-9685]. In this report, the inactivation of NOS by L-NMA has been investigated in detail. Inactivation fails to occur under an argon atmosphere, establishing turnover dependence. The partition ratio, defined as the number of molecules of citrulline formed per NOS monomer inactivated, is 108 +/- 3. By utilizing NG-methyl-L-[2,3-3H2]arginine and NG-[14C]methyl-L-arginine, the stoichiometry of radiolabeling is 0.11 +/- 0.01 equiv of tritium and 0.41 +/- 0.10 equiv of carbon-14 per inactivated NOS monomer. Dialysis under native conditions does not change this stoichiometry. However, dialysis of NOS following denaturation decreases the stoichiometry of radiolabeling to 0.08 +/- 0.04 equiv of tritium and 0.12 +/- 0.04 equiv of carbon-14 per inactivated NOS monomer. Absolute and CO-reduced difference spectroscopy indicates that inactivation of L-NMA is accompanied by a substantial loss of the heme chromophore, which is not prevented by catalase. HPLC analysis of NOS heme following inactivation with L-NMA indicates substantial loss of heme. These findings suggest that multiple mechanisms may contribute to the loss of NOS activity by L-NMA, including heme loss and possibly protein and cofactor modification.

Amino Acid Oxidoreductases↗
PubMed1994

Structure of the novel heme adduct formed during the reaction of human hemoglobin with BrCCl3 in red cell lysates.

It was previously shown that the reductive debromination of BrCCl3 to trichloromethyl radical by human hemoglobin leads to formation of dissociable altered heme products, two of which are identical to those formed from myoglobin and one which is novel. In this study, we have elucidated the structure of this novel adduct with the use of mass spectrometry, as well as 1H and 13C NMR as a substitution product of a -C(Cl) = CCl2 moiety for a beta-hydrogen atom on the prosthetic heme's ring I vinyl group. From studies with the use of 13C-enriched BrCCl3, it was determined that the added carbon atoms were derived from 2 eq of BrCCl3. A mechanism that involves multiple reductive events and a radical cation heme intermediate is proposed. Consistent with this mechanism, cellular reductants were found to selectively enhance the amount of this novel dissociable heme adduct. These studies reveal fine differences between myoglobin and hemoglobin in the accessibility of reactive intermediates to the ring I vinyl group, as well as the potential importance of cellular reductants on the course of heme alteration.

Ascorbic Acid↗
PubMed1994

Selective inhibition of the 12-lipoxygenase pathway of arachidonic acid metabolism by L-arginine or sodium nitroprusside in intact human platelets.

L-arginine (1-100 microM) or sodium nitroprusside (1-100 microM) caused a concentration dependent decrease in the metabolism of exogenously added arachidonic acid via the 12-lipoxygenase pathway in intact human platelets, as determined by the use of an HPLC assay. NG-monomethyl-L-arginine, but not the D-isomer of this compound, diminished the inhibitory effect of L-arginine. The D isomer of arginine had no effect. The cyclooxygenase pathway was much less susceptible to these effects. This study indicated that nitric oxide formed in intact human platelets selectively inhibited 12-lipoxygenase over that of cyclooxygenase and suggests that such inhibition may be an important regulatory mechanism.

Arachidonate 12-Lipoxygenase↗
PubMed1994

Neuronal aromatase expression in preoptic, strial, and amygdaloid regions during late prenatal and early postnatal development in the rat.

Brain aromatase has been considered to be an important clue in elucidating the actions of androgen on brain sexual differentiation. Using highly specific anti-P450arom antiserum, the regional and subcellular distributions were immunohistochemically evaluated in the preoptic, strial, and amygdaloid regions of developing rat brains. Aromatase-immunoreactive (AROM-I) neurons were classified into three groups. The first, in which immunostaining occurs only during certain pre- or neonatal days (E16-P2), included the anterior medial preoptic nucleus, the periventricular preoptic nucleus, neurons associated with the strial part of the preoptic area, and the rostral portion of the medial preoptic nucleus. The second is a striking AROM-I cell group in the "medial preopticoamygdaloid neuronal arc," which extends from the medial preoptic nucleus to the principal nucleus of the bed nucleus of the stria terminalis and the posterodorsal part of the medial amygdaloid nucleus. The AROM-I neurons appeared by E16, reaching a peak in staining intensity between E18 and P2 and diminishing after the perinatal stage. After P14, a third group of AROM-I neurons emerged in the lateral septal nucleus, the oval nucleus of the bed nucleus of the stria terminalis, and the central amygdaloid nucleus. The second group was thought to be the major aromatization center in developing rat brains, while the center might partly shift to the third group of neurons after the late infantile stage. The distribution and developmental patterns were basically similar in males and females, suggesting that the neonatally prominent aromatase is not induced by male-specific androgen surges occurring around birth. On immunoelectron microscopy, subneuronal aromatase was predominantly localized on the nuclear membrane and endoplasmic reticulum, which appeared to be appropriate for the efficient conversion of androgen into estrogen just prior to binding to the nuclear receptors.

Amygdala↗
PubMed1994

6H-pyrazolo[4,5,1-de]acridin-6-ones as a novel class of antitumor agents. Synthesis and biological activity.

The 7-substituted 6H-pyrazolo[4,5,1-de]acridin-6-ones with (aminoalkyl)amino and/or (hydroxyalkyl)amino groups in the side chains were synthesized by bromination using N-bromosuccinimide and the subsequent reaction with amines from the 7-substituted 5-bromo-2-methyl-6H-pyrazolo-[4,5,1-de]acridin-6-one. The substitution reaction of the amines with alkyl bromide (the C2 position) and aryl bromide (the C5 position) was accomplished by choosing the proper reaction conditions. These compounds show DNA intercalating ability in ethidium fluorescence assay and antiproliferative activity against Hela S3 cells. Impressive antitumor activity in vivo against murine P388 leukemia and murine sarcoma 180 solid tumor in mice was demonstrated for the 7-hydroxy analogs. In addition, some of these showed excellent antitumor activity against adriamycin-resistant murine P388 leukemia (P388/ADM) in mice.

Aminoacridines↗
PubMed1994

Steroidogenic activity of atretic follicles in the cycling hamster ovary and relation to ultrastructural observations.

To evaluate the relation between the steroidogenic activity and cell proliferation of individual follicles in mature hamster ovaries during the estrous cycle, the localization of enzymes involved in estrogen biosynthesis and bromodeoxyuridine (BrdU) incorporation were examined immunohistochemically. Moreover, granulosa cells from the early atretic follicle were examined by scanning and transmission electron microscopy. Immunoreactivity for aromatase was localized in the granulosa cells of healthy developing follicles and Graafian follicles, as well as in newly formed granulosa lutein cells. In the healthy follicles of an ovulation cycle, intensity of aromatase immunoreactivity was suddenly decreased on day 3. The theca interna cells of healthy developing follicles were immunopositive for 17 alpha-hydroxylase/C17-C20 lyase (17 alpha-lyase) from day 2 to the morning of day 4, but on the evening of day 4 most theca interna cells were immunonegative except for only a few cells of the large Graafian follicles. BrdU incorporation was observed in the granulosa cells of healthy developing follicles, in the endothelial cells of capillaries around the developing follicles, and of newly formed corpora lutea. Very early morphological signs of atresia was the pyknotic change of a few granulosa cells lining the antral cavity. In that follicle, the number of BrdU-incorporating granulosa cells was suddenly decreased whilst immunoreactivity of aromatase and 17 alpha-lyase were gradually decreased. These data suggest that the mechanism of the loss of aromatase activity from the granulosa cells of atretic follicles appears to differ from that in cycling follicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Immunocytochemical localization of aromatase-containing neurons in the rat brain during pre- and postnatal development.

The present immunohistochemical study demonstrates the ontogenetic appearance of aromatase-immunoreactive neurons in several discrete regions of the hypothalamus and limbic system in the rat brain, using a purified antibody against human placental aromatase cytochrome P450. Immunoreactive cells were first detected in the preoptic area on the 13th day of embryonic life (E13), and additionally in the bed nucleus of the stria terminalis on E15. Labeled cells were also found in the medial amygdaloid nucleus and the ventromedial nucleus on E16, and some were detected in the arcuate nucleus on E19. As gestation progressed, the number and the immunoreactivity of these cells gradually increased and peaked within definite periods of perinatal life and thereafter declined or disappeared. The immunoreactive cells were also found in the central amygdaloid nucleus and the lateral septal nucleus, and in the ventral pallidum, after the 14th day of postnatal life (P14) and 30th day (P30), respectively. The distribution of aromatase-immunoreactive neurons was similar between the sexes, while the immunoreactivity was higher in males than in females after late gestational days. No immunoreaction was detectable in other regions of the telencephalon or midbrain at any time periods studied. The aromatase-immunoreactive neurons in the specific regions may be involved in the sexual differentiation of the brain.

Age Factors↗
PubMed1994

Localization of aromatase in equine Leydig cells.

Stallion testes secrete large amounts of estrogens, but the cellular location of the enzyme that converts androgens to estrogens, cytochrome P450 aromatase, has not been determined. The goal of the present study was to immunocytochemically localize stallion testicular aromatase using a polyclonal antibody generated against human placental cytochrome P450 aromatase. Testes were obtained from 12 stallions from 2 to 23 years of age, during both the breeding and non-breeding seasons. Immunoreactivity was confined to the Leydig cells in all testes examined. No immunostaining was observed in the Sertoli or germ cells. Heterogeneity in the level of immunostaining among individual Leydig cells was observed. The results of this study indicate that in postpubertal, adult, and aged stallions, testicular aromatase is located in Leydig cells.

Animals↗
PubMed1994

Suicide inactivation of aromatase in human placenta and uterine leiomyoma by 5 alpha-dihydronorethindrone, a metabolite of norethindrone, and its effect on steroid-producing enzymes.

Norethindrone (NET; 17 alpha-ethynyl-19-nortestosterone), a progestogen component of the contraceptive pill, irreversibly inhibits aromatase activity in human placental microsomes. However, it is known also to be aromatized in vitro and in vivo to produce a biologically very active estrogen called ethynylestradiol (EE2). It is therefore inappropriate to administer a high dose of NET to estrogen-dependent cancer patients for a prolonged time period. In this study, we focused on 5 alpha-dihydronorethindrone (5 alpha-DHNET), a metabolite of NET that is not aromatizable, and the inhibitory effects of 5 alpha-DHNET on human placental and uterine leiomyoma microsomal aromatase and other steroid synthetases. 5 alpha-Dihydronorethindrone showed weak affinity for both estrogen and progestogen receptors. It inhibited significantly human placental aromatase activity in a dose-dependent manner (Ki = 9.0 mumol/l; Kinact = 0.024/min), as well as that of uterine leiomyoma, but did not influence cholesterol side-chain cleavage or 17 alpha-hydroxylase, 21-hydroxylase or 11 beta-hydroxylase activities. These results suggest that 5 alpha-DHNET may be useful as an aromatase inhibitor, whose use in large doses is expected to reduce the size of estrogen-dependent tumors.

Adult↗
PubMed1994

Immunolocalization of steroidogenic enzymes P450scc, 3 beta HSD, P450c17 and P450arom in the corpus luteum of the Hokkaido brown bear (Ursus arctos yesoensis) in relation to delayed implantation.

The objective was the immunocytochemical localization of steroidogenic enzymes in the corpus luteum of Hokkaido brown bears during the period of delayed implantation. Cholesterol side-chain cleavage cytochrome P450 (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), 17 alpha-hydroxylase cytochrome P450 (P450c17) and aromatase cytochrome P450 (P450arom) were localized as biosynthetic sites of pregnenolone, progesterone, androgens, and oestrogens, respectively. Ovaries containing corpora lutea were obtained from three mature bears during the expected delayed implantation period and ovarian sections were immunostained by the avidin-biotin-peroxidase complex method using polyclonal antibodies generated against steroidogenic enzymes of mammalian origin. P450scc and 3 beta HSD were localized in all luteal cells, whereas P450c17 (0.4-5.1% of 1000 cells) and P450arom (7.1-11.2% of 1000 cells) were localized in only a few luteal cells. These data suggest that luteal cells contain steroidogenic enzymes required for progesterone synthesis but also have a minimum capability for synthesizing androgen and oestrogen during the delayed implantation period in Hokkaido brown bears.

3-Hydroxysteroid Dehydrogenases↗
PubMed1994

Immunocytochemical localization of aromatase in the ovary of superovulated cattle, pigs and sheep.

The localization of aromatase, an enzyme converting androgens to estrogen, in the ovaries of superovulated cattle, pigs and sheep was studied immunocytochemically in the preovulatory and postovulatory period using anti-human placental aromatase cytochrome P-450 antiserum. Immunostaining for aromatase was detected in the granulosa cells of preovulatory follicles of all species studied. Theca interna cells were stained in preovulatory follicles in the pig but not in cattle and sheep. Interstitial gland cells, cumulus cells and oocytes were unstained in all species. In cattle and pig the corpora lutea were unstained whereas they displayed staining in the sheep. Preantral and small antral follicles were unstained during both the preovulatory and postovulatory period in all species. It is concluded that granulosa cells of preovulatory follicles are the main residence for aromatase activity in superovulated cattle, pig and sheep, whereas the activity of theca interna and corpora lutea is species specific.

Animals↗
PubMed1993

Tobacco alkaloid derivatives as inhibitors of breast cancer aromatase.

The inhibition of estrogen biosynthesis by the use of aromatase inhibitors is emerging as a valuable approach to breast cancer therapy. Because smoking has a profound effect on estrogen-related processes we examined the ability of tobacco constituents to suppress estrogen production by breast cancer aromatase. N-n-octanoylnornicotine and N-(4-hydroxyundecanoyl) anabasine suppressed aromatase activity in culture of two human breast cancer cell lines, MDA-MB-231 (IC50 of 310 and 20 microM, respectively) and SK-BR-3 (IC50 of 450 and approximately 2 microM, respectively). MDA-MB-231 cells induced by 250 nM dexamethasone or 1 mM (Bt)2cAMP were slightly more sensitive to both inhibitors. Kinetic analyses showed that inhibition by N-(4-hydroxyundecanoyl)anabasine is competitive with respect to androstenedione as substrate, with apparent Ki values of 0.2 microM against microsomal aromatase activity derived from both (Bt)2cAMP-induced MDA-MB-231 cells and human breast tumor tissue. The corresponding apparent Ki against human placental microsomal aromatase activity was 0.4 microM. These results indicate that acyl derivatives of nornicotine and anabasine block estrogen formation in breast tumor cells and tissue and could contribute to the decreased intra-tissue estrogen levels in women who smoke.

Adenocarcinoma↗
PubMed1993

Differential susceptibilities of the prosthetic heme of hemoglobin-based red cell substitutes. Implications in the design of safer agents.

One approach to the development of an effective red cell substitute has been chemical modification of human hemoglobin to optimize oxygen transport and plasma half-life. Human hemoglobin A0 and two of these modified hemoglobins, one prepared from the cross-linking of the alpha-chains at lysine residue 99 by bis(3,5-dibromosalicyl)fumarate (Hb-DBBF) and the other by acylation of lysine residue 82 of the beta-chain by mono-(3,5-dibromosalicyl)fumarate (Hb-FMDA), were tested by HPLC for their susceptibility to oxidative damage caused by H2O2. Such oxidative insult may occur during ischemia and reperfusion of tissues after transfusion of red cell substitutes to patients with hypovolemic shock and trauma. Hb-DBBF was extremely susceptible to damage of its heme and protein moieties with stoichiometric amounts of H2O2, whereas Hb-FMDA was highly resistant, even at 10-fold molar excess and at an acidic pH of 4.7. Hemoglobin A0 was of intermediate susceptibility, exhibiting alteration of heme and protein moieties at acidic but not neutral pH. Since the degradation of heme can release the potentially toxic agent iron, Hb-FMDA may be a more promising candidate than Hb-DBBF for development as a red cell substitute. A similar approach may be used to assess the susceptibility of other hemoglobin-based red cell substitutes to oxidative damage in order to determine the molecular basis of heme and protein alteration.

Aspirin↗