PubMed HealthSearch

Biomedical subjects

Y Osawa

Publications and source records attributed to Y Osawa.

At least 19 recordsLinked to original sources

An aromatase-associated cytoplasmic inclusion, the "stigmoid body," in the rat brain: I. Distribution in the forebrain.

An aromatase-containing neural system was examined in the rat forebrain, using a polyclonal antibody against aromatase-associated human placental antigen X-P2 (hPAX-P2). Numerous dot-like structures, which we have called stigmoid bodies, were immunostained in the preoptico-hypothalamic region, the bed nucleus of the stria terminalis, the medial amygdala, the arcuate nucleus, the subfornical organ, and the area extending from the hypothalamic area to the central gray through the medial forebrain bundle and the periventricular fiber system of the posterior diencephalon. The stigmoid bodies were always found as inclusions in the neuronal cytoplasm. Their diameter was usually 1-3 microns, but exceptionally large forms, over 3 microns, were found in some brain regions, including the area extending from the median preoptic nucleus surrounding the organosum vasculosum laminae terminalis to the anterior medial preoptic nucleus, the periventricular nucleus of the preoptic area, and some parts of the medial preoptic nucleus. Most of these nuclei show sexual dimorphism. The distribution pattern of the hPAX-P2 immunoreactive stigmoid bodies agreed well with that of aromatase activity previously reported in many biochemical studies. Brain regions where the stigmoid bodies were prominent largely coincide with steroid binding locations common to both androgen and estrogen, or regions where both sex steroid receptors are present. Although it still remains to be determined whether aromatase is localized within these stigmoid bodies, it appears likely that they are closely associated with the function of sex steroids at their target sites in the brain.

Animals

Covalent alteration of the prosthetic heme of human hemoglobin by BrCCl3. Cross-linking of heme to cysteine residue 93.

Recent studies have shown that a protein-bound heme adduct formed from the reaction of BrCCl3 with myoglobin was due to bonding of the proximal histidine residue through the ring I vinyl of a heme-CCl2 moiety. The present study reveals that BrCCl3 also reacts with the heme of reduced human hemoglobin to form two protein-bound heme adducts. Edman degradation and mass spectrometry provided evidence that these protein-bound heme adducts were addition products in which heme-CCL2 or heme-CCl3 were bound to cysteine residue 93 of the beta-chain of hemoglobin. It appeared that the cysteine residue was bonded regiospecifically to the ring I vinyl group of the altered heme moiety, because the nonprotein-bound products of the reaction included the beta-carboxyvinyl and alpha-hydroxy-beta-trichloromethylethyl derivatives of the ring I vinyl moiety of heme. The absorption spectra of the protein-bound adducts in both the oxidized and reduced states were highly similar to those described for hemichromes, which are thought to be involved in the formation of Heinz bodies and subsequent red cell lysis.

Bromotrichloromethane

Kinetic properties of aromatase mutants Pro308Phe, Asp309Asn, and Asp309Ala and their interactions with aromatase inhibitors.

Mutant forms of aromatase cytochrome P-450 bearing modifications of amino acid residues Pro308 and Asp309 and expressed in transfected Chinese hamster ovary cells were subjected to kinetic analysis and inhibition studies. The Km for androstenedione for expressed wild type (11.0 +/- 0.3 nM SEM, n = 3) increased 4-, 25- and 31-fold for mutants Pro308Phe, Asp309Asn and Asp309Ala, respectively. There were significant differences in sensitivity among wild type and mutants to highly selective inhibitors of estrogen biosynthesis. 4-Hydroxyandrostenedione (4-OHA) a strong inhibitor of wild type aromatase activity (IC50 = 21 nM and Ki = 10 nM), was even more effective against mutant Pro308Phe (IC50 = 13 nM and Ki = 2.8 nM), but inhibition of mutants Asp309Asn and Asp309Ala was considerably less (IC50 = 345 and 330 nM and Ki = 55 and 79 nM, respectively). Expressed wild type aromatase and Pro308Phe aromatase were strongly inhibited by CGS 16949A (IC50 = 4.0 and 4.6 nM, respectively) whereas mutants Asp309Asn and Asp309Ala were markedly less sensitive (IC50 = 140 and 150 nM, respectively). CGS 18320B produced similar inhibition. Kinetic analyses produced Ki = 0.4 nM for CGS 16949A inhibition of wild type versus 1.1, 37 and 58 nM, respectively, against Pro308Phe, Asp309Asn and Asp309Ala. The results demonstrate significant changes in function resulting from single amino acid modifications of the aromatase enzyme. Our data indicate that mutation in Asp309 creates a major distortion in the substrate binding site, rendering the enzyme much less efficient for androstenedione aromatization. The substitution of Pro308 with Phe produces weaker affinity for androstenedione in the substrate pocket, but this alteration favors 4-OHA binding. Similarly, mutant Pro308Phe exhibits a slightly greater sensitivity to inhibition by CGS 18320B than does the wild type. These results indicate that residues Pro308 and Asp309 play critical roles in determining substrate specificity and catalytic capability in aromatase.

Animals

Role of nitric oxide in NMDA-evoked release of [3H]-dopamine from striatal slices.

Evidence that excitatory amino acids act via N-methyl-D-aspartate (NMDA) receptors to evoke the release of catecholamines from axonal terminals and synaptosomes has been used to argue for the presence of pre-synaptic NMDA receptors. NMDA receptor agonists also generate nitric oxide (NO) which rapidly diffuses through neural tissue. We find that exogenously applied NO evokes [3H]-dopamine release from cultured neurons. This release is not blocked by the NMDA antagonist MK-801 nor by tetrodotoxin. Both NG-nitroarginine which inhibits NO synthesis, and hemoglobin which binds extracellular NO, block NMDA-evoked [3H]-dopamine release from striatal slices. A major role of endogenously-synthesized NO may be to evoke neurotransmitter release in local volumes of neural tissue.

Animals

Increasing aromatase cytochrome P-450 level in human placenta during pregnancy: studied by immunohistochemistry and enzyme-linked immunosorbent assay.

We investigated the immunohistochemical localization of aromatase cytochrome P-450 (P-450arom) using a specific polyclonal antiserum (PAb R-8-2). We compared catalytic activity, as detected by the tritiated water assay, and tissue levels of P-450arom, as detected by the specific enzyme-linked immunosorbent assay, in placental samples from early pregnancy to term. Immunostaining and subsequent detection by light and electron microscopy demonstrated that P-450arom is localized in the microvilli and endoplasmic reticulum of the syncytiotrophoblasts of the chorionic villi, but is not present in the mitochondria, nuclei, or cytotrophoblasts at any time during gestation. The P-450arom concentration and aromatase activity were greater in the microsomal fraction than in the mitochondrial fraction or total homogenate at each gestational stage and increased linearly as pregnancy progressed. However, the specific activity of P-450arom was comparable among subcellular fractions in each gestational period. These results suggest that the nature and localization of P-450arom are unchangeable, and the P-450arom concentration increases during pregnancy. It appears that the aromatase detected in the mitochondrial fraction is a contamination of microsomal aromatase, and the increase in aromatase activity can be attributed to an increase in the number of P-450arom molecules rather than an increase in the catalytic ability of each molecule.

Aromatase

Synthesis of deuterium-labeled 16 alpha,19-dihydroxy C19 steroids as internal standards for gas chromatography-mass spectrometry.

[2 beta,7,7,16 beta-2H4]16 alpha,19-Dihydroxyandrost-4-ene-3,17-dione (14) and [7,7,16 beta-2H3]3 beta,16 alpha,19-trihydroxyandrost-5-en-17-one (16), with high isotopic purity, respectively, were synthesized from unlabeled 3 beta-(tert-butyldimethylsiloxy)-androst-5-ene-17 beta-yl acetate (1). The deuterium introduction at C-7 was carried out by reductive deoxygenation of the 7-keto compound 3 with dichloroaluminum deuteride and that at C-2 beta and/or C-16 beta by controlled alkaline hydrolysis of 16-bromo-17-ketone 11 or 12 with NaOD in D2O and pyridine. [7,7-2H2]3 beta-Hydroxyandrost-5-en-17-one (6), obtained from compound 1 by a five-step sequence, was converted to compound 14 or 16 by an eight-step or seven-step sequence, respectively. The labeled steroids 14 and 16 are useful as internal standards for gas chromatography-mass spectrometry analysis of the endogenous levels.

Androstenedione

Immunohistochemical localization of 17 alpha-hydroxylase/C17-20 lyase and aromatase cytochrome P-450 in the human ovary during the menstrual cycle.

Immunohistochemical localization of 17 alpha-hydroxylase/C17-20 lyase (P-450(17 alpha,lyase)) and aromatase cytochrome P-450 (P-450arom) in normal human ovaries during the menstrual cycle was studied using specific polyclonal antibodies which were raised against corresponding enzymes. In the follicular phase of matured follicles, P-450(17 alpha,lyase) was localized in theca interna cells and P-450arom in granulosa cells. P-450(17 alpha,lyase) was expressed in theca interna cells before P-450arom was expressed in granulosa cells. The corpus luteum showed immunoreactivity to both enzymes and, after menstruation, immunoreactivity decreased gradually until it could not be detected in the corpus albicans. In corpus luteum graviditatis the immunoreactivity continued to be expressed strongly. In some atretic follicles, P-450(17 alpha,lyase) and/or P-450arom continued to be expressed. In the stromal layer, P-450(17 alpha,lyase was detected in secondary interstitial cells, which originated from the theca interna of atretic follicles, and P-450arom was detected in hilar cells. Immunoreactivity to both enzymes was also detected in oocytes of developing follicles. These results are consistent with the two cell theory in the human ovary. They also suggest that androgens and oestrogens are produced not only by follicles and corpora lutea but also by stroma and oocytes.

Adult

Immunohistochemical studies on the localization of aromatase and 17 alpha-hydroxylase/C17-20 lyase (17 alpha-lyase) in estrous cycling and pregnant hamster ovaries.

In order to clarify periodic changes in the localization of enzymes engaged in estrogen biosynthesis during the estrous cycle, immunohistochemical and fine structural studies were performed using estrous cycling and pregnant hamster ovaries. Results showed that ovulation takes place at midnight between Day 4 and Day 1 in the regular 4 day-cycle hamster. Immunoreactivity for aromatase is localized in the granulosa cells of the secondary follicle and granulosa lutein cells during the morning (10:00 am) of Day 1 to the evening (5:00 pm) of Day 4; in the night (9:00 pm) of Day 4, only the granulosa cells of the Graafian follicle showed a strong immunoreaction. As for 17 alpha-lyase, theca interna cells of the secondary follicle are immunopositive throughout Day 2 to the morning (10:00 am) of Day 4. Only a few cells in the theca interna of the Graafian follicles are immunopositive in the evening (5:00 pm) of Day 4. No positive cells for this enzyme were detected in the night (9:00 pm) of Day 4 or morning (10:00 am) of Day 1. The rapid decrease of estrogen biosynthesis occurring just before ovulation is considered to be due to the disappearance of 17 alpha-lyase in the theca interna cells of the ovary. On Day 10 of pregnancy, the granulosa cells of the secondary follicles and both the granulosa and theca lutein cells of the corpora lutea are immunostained with the aromatase antibody, while the theca interna cells of the secondary follicles reacted positively to the 17 alpha-lyase antibody. Only the granulosa and theca interna cells from the large preovulatory Graafian follicle of Day 4 (proestrus) which are positively stained for aromatase as well as 17 alpha-lyase show ultrastructural features typical of steroid secretory cells.

Aldehyde-Lyases

Heme oxygenase: expression in human retina and modulation by stress agents in a human retinoblastoma cell model system.

PCR and Southern blot analyses demonstrate that mRNA for heme oxygenase (HO), a well known "stress protein" in a number of tissues, is present in human retina. Western and northern blots show that the protein and mRNA are also expressed in human Y-79 retinoblastoma cells in culture and that the HO enzyme is rapidly induced by its substrate, heme. Moreover, HO is also induced by two chemicals, sodium arsenite and menadione, that act as agents of oxidative stress. HO is the regulatory enzyme in the heme degradative pathway and an increase in its activity could lead to the accumulation of bilirubin, an antioxidant, in the cell at the expense of heme, a prooxidant. The HO pathway may thus be of importance in protecting the retina against oxidative stress in vivo. Moreover, the Y-79 culture system should provide an excellent model for use in examining stress mechanisms in retinal cells at a molecular level.

Arsenic

[Importance of sperm morphology assessment in human in vitro fertilization].

Two hundred eighteen IVF cycles were analyzed in order to clarify the influence of the strictly normal morphology (SNM) of sperm on IVF outcome. SNM was defined according to Kruger et al.'s strict criteria (1988) with our modifications. IVF cycles were divided into 3 groups: %SNM greater than or equal to 40% (69 cycles), 40% less than %SNM greater than or equal to 12% (123 cycles) and %SNM less than 12% (26 cycles). The rates of embryo transfer and pregnancy per cycle and the rate of abortion per pregnancy in the three groups were as follows: 81%, 39%, 19% in %SNM greater than or equal to 40%; 71%, 24%, 30% in 40% greater than %SNM greater than or equal to 12%; and 58%, 7.7%, 50% in %SNM less than 12%. The pregnancy rate per cycle significantly decreased as the %SNM decreased (p less than 0.01), and there was a tendency toward an increase in the abortion rate per pregnancy as the %SNM decreased. The results suggest that %SNM is a useful parameter to use in predicting the IVF outcome.

Abortion, Spontaneous

[Studies on changes in low esophageal sphincter function after esophageal transection or esophago-esophagostomy in children].

An aim of the present study is to clarify the changes of esophageal function after surgical treatments of the esophageal varices or the congenital esophageal stenosis in children. Esophageal manometric studies were performed in sixteen children undergoing the esophageal transection with paraesophageal devascularization or the esophago-esophagostomy with partial esophagectomy before, within 1 month and over 7 months after the operation. The pressure of lower esophageal sphincter (LES), the length of LES, the LES relaxation test and the gastroesophageal reflux (GER) inducing test were measured. 1) Esophageal transection; The pressure dropped within 1 month and returned to the preoperative level over 11 months after the operation. Temporal deterioration of LES function was observed during the early postoperative days. However, return to the preoperative state was gained over 1 year. 2) Esophago-esophagostomy; The pressure and the length of LES were not changed after the operation. The LES function was disturbed preoperatively and moreover long postoperatively, in the case when congenital stenosis closely existed to the esophago-gastric junction. Degree of the postoperative disturbance of the LES function may be influenced by the distance to the part of the transection or the partial esophagectomy from the E-C junction.

Anastomosis, Surgical

Theoretical studies on the mechanism of conversion of androgens to estrogens by aromatase.

Semiempirical molecular orbital calculations (AM1) were used to model several possible reaction mechanisms for the third oxidation of the aromatase-catalyzed conversion of androgens to estrogens. The reaction mechanisms considered are based on the assumption that the third oxidation is initiated by 1 beta-hydrogen atom abstraction. Homolytic cleavage of the C10-C19 bond was modeled for both the 3-keto and 2-en-3-ol forms of the androgen 1-radicals. The addition of a protein nucleophile to the 19-oxo intermediate was also considered, and -OCH3, -SCH3, and -NHCH3 were used to represent the Ser, Cys, and Lys adducts. The transition states were estimated and optimized from the reaction coordinates obtained by constraining and increasing the C10-C19 bond lengths. The enthalpies of activation range from 14 to 21 kcal and are approximately 2 kcal lower for cleavage of the enol form. Given the tendency for AM1 to overestimate activation energies, all reactions may be energetically accessible. Other reactions modeled include a homolytic cleavage reaction from a thioether radical cation and the direct additions of oxygen radical compounds to the carbonyl of the 1-radical-2-en-3-ol-19-oxo androgen. A mechanism is proposed in which the 19-oxo intermediate is subject to initial nucleophilic attack by the protein. Since rotation of the 19-carbonyl can bring the oxygen within 2.1 A of the 2 beta-hydrogen, the formation of a tetrahedral intermediate can occur with concomitant removal of the 2 beta-proton. Enolization activates the C1-position for hydrogen atom abstraction, since the resulting radical is resonance stabilized.(ABSTRACT TRUNCATED AT 250 WORDS)

Androgens

Structure-activity studies of non-steroidal aromatase inhibitors: the crystal and molecular structures of CGS 16949A and CGS 18320B.

The crystal and molecular structures of 4-(5,6,7,8-tetrahydroimidazo[1,5-a]pyridin-5-yl)benzonitrile hydrochloride (CGS 16949A) and bis(p-cyanophenyl)imidazo-1-yl methane hemisuccinate (CGS 18320B) have been determined as part of structure-activity relationship studies of non-steroidal aromatase inhibitors. CGS 18320B crystallizes with two inhibitor molecules in the asymmetric unit that are similar in conformation. The cyanophenyl groups and the imidazole moieties in the CGS 18320B molecules display a propellor-like arrangement. The orientation of the imidazole ring in CGS 16949A, which is constrained by the piperidine ring, differs by about 80 degrees from the orientations in both CGS 18320B molecules. The conformations of both compounds are consistent with the proposed model (Banting et al. (1988) J. Enz. Inhibit., 2, 216) for inhibitor binding by positioning of the cyanophenyl group in the steroid A-ring binding site and interaction of the imidazole nitrogen with the iron of the haem.

Aromatase Inhibitors

Synthesis and some reactions of 6-bromoandrogens: potential affinity ligand and inactivator of estrogen synthetase.

The synthesis of epimeric 6-bromo-4-androstene-3,17-dione (1a and 1b), 6-bromotestosterone (2a and 2b) and its acetate (3a and 3b), and 6-bromo-16 alpha-acetoxy-4-androstene-3,17-dione (5a and 5b), and 6 beta-bromo-16 alpha-hydroxy-4-androstene-3,17-dione (4) is described. The interconversions among compounds 1, 2, and 3 are also studied. The 6 beta-isomer (1b, 2b, and 3b) was epimerized to the 6 alpha-isomer (1a, 2a and 3a) in carbon tetrachloride or chloroform-methanol (9:1) and the 6 alpha-isomer was isolated by fractional crystallization from the epimeric mixture. 6 alpha-Bromo isomer 1a was also epimerized back to 6 beta-bromo isomer 1b in chloroform-methanol (9:1). Two polymorphic forms of 6 beta-bromotestosterone acetate (3b) were isolated (mp. 114--117 degrees and 138--141 degrees). The 6 beta-bromo isomers were found to be unstable in methanol and decomposed to give 5 alpha-androstane-3,6-dione derivative (6). The results of irreversible inactivation of human placental androgen aromatase with some of these 6-bromoandrogens are discussed.

Methods

Elevation of visual pattern discrimination limen in monkeys with total removal of inferotemporal cortex.

Attempts were made to test the validity of the general view that inferotemporal lesions in monkeys do not result in a visual acuity disorder. It was found in the first experiment that monkeys with total removal of the inferotemporal visual area (TIT monkeys) showed a significant elevation of the discrimination limen for visual patterns of reduced sizes even when compared to monkeys with removal of lateral striate cortex (LS monkeys); yet in a food-morsel (raisin) detection test the TIT monkeys performed as well as normal monkeys, although the LS monkeys showed significant deficits. A second experiment was conducted, in which the same subjects were tested with the same patterns as in the first experiment but with the stimulus background changed from the square used earlier to an enlarged disc. While both normal and LS monkeys performed this task easily, the TIT monkeys could not learn the discrimination within the training limit of 1,200 trials. The results suggest that such a marked elevation of the discrimination limen in the TIT monkeys as found in the first experiment is not attributable to a visual acuity disorder, but is explained as due to an impairment of the pattern perception mechanism.

Animals