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Biomedical subjects

Y Osawa

Publications and source records attributed to Y Osawa.

At least 19 recordsLinked to original sources
PubMed1998

Metabolism-based inactivation of penile nitric oxide synthase activity by guanabenz.

Guanabenz (Wytensin) was shown to inactivate nitric oxide synthase (NOS) activity in vitro and in vivo. In in vitro studies with the use of a cytosolic fraction from penile tissue, the inactivation was found to depend on NADPH, time, and the concentration of guanabenz. The L-, but not the D-, isomer of arginine could protect from the inactivation, suggesting an active site-directed event. The kinetics of inactivation could be described by an apparent dissociation constant for the initial reversible complex (Ki) and a pseudo first-order inactivation constant (kinact) of 38.5 microM and 0.179 min-1, respectively. In in vivo studies, guanabenz was shown to inhibit penile cytosolic NOS activity in a dose- and time-dependent manner. Treatment of rats with guanabenz (5 mg/kg/day) for 4 days caused a decrease of approximately one-half in the NOS activity of the penile cytosolic fraction with a concomitant loss in the amount of immunodetectable NOS protein. Treatment for 4 days at a dose of 0. 5 mg/kg/day showed a similar decrease in activity, whereas a dose of 0.05 mg/kg/day showed no effects. Due to the multitude of processes that are regulated by NO, the inactivation of NOS is a potential mechanism to be considered in a variety of biological effects associated with drugs.

Animals↗
PubMed1997

Endogenous production of tumour necrosis factor is required for manganese superoxide dismutase expression by irradiation in the human monocytic cell line THP-1.

Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme that scavenges superoxide (O2-) ions. We studied the regulation of MnSOD gene expression by irradiation and the mechanisms in human monocytic cell line THP-1. We found that irradiation induced expression of the MnSOD gene through the autocrine mechanism, involving the production of tumour necrosis factor (TNF). Irradiation increased TNF production in THP-1 cells, and TNF increased the levels of MnSOD transcripts. Supernatant from irradiated THP-1 cells induced the expression of MnSOD mRNA, and anti-TNF antibody blocked the induction of MnSOD mRNA. Irradiation also increased the levels of MnSOD mRNA in other myelocytic cell lines, HL60 and KG-1, and the ovarian cancer cell line SK-OV-3. Moreover, increased levels of MnSOD mRNA were observed in mature myeloid cells, including macrophages and granulocytes, as well as in immature cells. However, irradiation did not increase the level of MnSOD mRNA in THP-1 cells with prolonged exposure to PMA. We also found that irradiation increased the rate of MnSOD transcription, and irradiation stabilized MnSOD mRNA in THP-1 cells. Our results indicate that the endogenous production of TNF is required, at least in part, for the induction of MnSOD mRNA expression by irradiation in THP-1 cells, and the increased levels of MnSOD transcripts on irradiation occur through a pathway involving protein kinase C activation. Our results also indicate that the increase in MnSOD mRNA caused by irradiation is regulated by both transcriptional and post-transcriptional mechanisms.

Autocrine Communication↗
PubMed1997

Hydrogen peroxide-mediated alteration of the heme prosthetic group of metmyoglobin to an iron chlorin product: evidence for a novel oxidative pathway.

Treatment of metmyoglobin with H2O2 is known to lead to the crosslinking of an active site tyrosine residue to the heme [Catalano, C. E., Y. S. Choe, and P. R. Ortiz de Montellano (1989) J. Biol. Chem. 264, 10534-10541]. We have found in this study that this reaction also leads to an altered heme product not covalently bound to the protein. This product was characterized by visible absorption, infrared absorption, and mass and NMR spectrometry as an iron chlorin product formed from the saturation of the double bond between carbon atoms at positions 17 and 18 of pyrrole ring D with concomitant addition of a hydroxyl group on the carbon atom at position 18 and lactonization of the propionic acid to the carbon atom at position 17. Studies with the use of (18)O-labeled H2O2, O2, and H2O clearly indicate that the source of the added oxygen on the heme is water. Evidently, water adds regiospecifically to a cationic site formed on a carbon atom at position 18 after oxidation of the ferric heme prosthetic group with peroxide. Prolonged incubation of the reaction mixture containing the iron hydroxychlorin product led to the formation of an iron dihydroxychlorin product, presumably from a slow addition of water to the initial iron hydroxychlorin. The iron chlorin products characterized in this study are distinct from the meso-oxyheme species, which is thought to be formed during peroxide-mediated degradation of metmyoglobin, cytochrome P450, ferric heme, and model ferric hemes, and give further insight into the mechanism of H2O2-induced heme alterations.

Animals↗
PubMed1997

Preparation and characterization of the F (ab)2 fragments of an aromatase activity-suppressing monoclonal antibody.

The preparation and characterization of the Fab and F(ab')2 fragments of a murine monoclonal antibody specific for aromatase cytochrome P-450 and which is suppressive of estrogen biosynthesis are described. This monoclonal antibody, MAb3-2C2, was purified from murine ascites using protein A affinity chromatography and digested with immobilized papain to produce antibody fragments. The Fab and F(ab')2 fragments were then purified using protein A affinity chromatography and S-200 HR size exclusion chromatography. The Fab fragment was further purified using S-100 HR size exclusion chromatography. Both the Fab and F(ab')2 fragments of the MAb3-2C2 suppressed aromatase activity in a dose-dependent manner. While the F(ab')2 fragment (110 kDa) maintained potent suppressive activity, the Fab fragment (42 kDa) required a higher concentration to suppress aromatase activity as compared to the IgG.

Antibodies, Monoclonal↗
PubMed1997

Expression of aromatase cytochrome P450 protein and messenger ribonucleic acid in human endometriotic and adenomyotic tissues but not in normal endometrium.

To determine whether local estrogen production takes place in endometriotic or adenomyotic tissues, in eutopic endometrium from patients with endometriosis or adenomyosis, and in normal endometrium, tissue specimens were examined by immunohistochemistry, catalytic activity, and mRNA expression for aromatase cytochrome P450 (P450arom). P450arom was immunohistochemically localized in the cytoplasm of glandular cells of endometriotic and adenomyotic tissues, and of eutopic endometrium from patients with the respective diseases, whereas estrogen receptors and progesterone receptors were localized in the nuclei of the glandular cells and stroma. Aromatase activity in the microsomal fraction of adenomyotic tissues was inhibited by the addition of danazol, aromatase inhibitors, and anti-human placental P450arom monoclonal antibody (mAb3-2C2) in a manner similar to such inhibition in other human tissues. Reverse transcription polymerase chain reaction and Southern blot analysis also revealed P450arom mRNA in these tissues. However, neither P450arom protein activity nor mRNA was detected in endometrial specimens obtained from normal menstruating women with cervical carcinoma in situ but without any other gynecological disease. These results suggest that at a local level, endometriotic and adenomyotic tissues produce estrogens, which may be involved in the tissue growth through interacting with the estrogen receptor.

Adult↗
PubMed1997

Seasonal changes in spermatogenesis and testicular steroidogenesis in the male black bear Ursus americanus.

American black bears, Ursus americanus, are seasonal breeders with a mating season in late spring to early summer. The objectives of this study were to determine whether there are seasonal changes in spermatogenesis and immunolocalization of testicular steroidogenic enzymes, and to correlate these changes with peripheral steroid concentrations. Three captive mature bears were maintained in open cages during the summer season and provided with chambers for denning during the winter. Testicular biopsies and blood samples were obtained from anaesthetized bears on 12 March, 15 June, 12 October and 15 January. Steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3 beta-hydroxysteroid dehydrogenase (3 beta HSD), porcine testicular 17 alpha-hydroxylase cytochrome P450 (P450c17) and human placental aromatase cytochrome P450 (P450arom). Spermatogenesis changed seasonally: spermatogonia and degenerating spermatocytes were observed in October; spermatogonia and primary spermatocytes were present in January; spermatogonia, spermatocytes and round spermatids were present in March; and spermatogonia through spermatozoa were present in June. P450scc and P450c17 were immunolocalized in spermatids and Leydig cells in June, whereas in October these enzymes were present only in Leydig cells. 3 beta HSD was localized in Leydig cells in June and October with more intense staining in June. Localization of P450arom changed seasonally: no immunostaining in October; positive immunostaining in Sertoli cells in January; more extensive immunostaining in Sertoli cells, peritubular-myoid cells and round spermatids in March; and strong immunostaining in Sertoli cells and round and elongating spermatids in June. Serum testosterone and oestradiol concentrations changed seasonally: testosterone and oestrogen were low in October and January, slightly higher in March, and high in June. The present study demonstrates that in the black bear seasonal changes in spermatogenesis are accompanied by changes in the immunolocalization of testicular steroidogenic enzymes that are correlated with changes in serum testosterone and oestradiol concentrations. The presence of P450arom in Sertoli cells at the beginning of testicular recrudescence suggests that aromatase and oestrogen may play a role in re-initiating spermatogenesis.

3-Hydroxysteroid Dehydrogenases↗
PubMed1997

Antibody responses against Em18 and Em16 serodiagnostic markers in alveolar and cystic echinococcosis patients from northwest China.

Western blot analysis was carried out in order to evaluate new serodiagnostic markers, Em18 and Em16, for differentiation of alveolar echinococcosis (AE) from cystic echinococcosis (CE) using 36 serum samples from hydatid patients from Xinjiang, China, where AE and CE are both endemic and one double infection case has been reported. All AE cases except one (5/6) who exhibited a calcified lesion and a single case of double infection showed antibody responses against Em18 and Em16. Some of CE patient sera (6/22) showed antibody response against Em16 except one who showed that against Em18. Analyses of IgG subclass responses against Em18 and Em16 were carried out using all serum samples showing antibody responses against Em18 and/or Em16 (seven CE, five AE, and one AE + CE) and additional samples of three CE and 22 AE from Sichuan, China. IgG4 was the most predominant antibody subclass. Em18 and Em16 were recognized by both IgG4 and IgG1 (in most cases) or by either IgG4 or IgG1 (in minor cases) or by IgG3 (in very rare cases). Neither Em18 nor Em16 was recognized by IgG2 antibodies. The usefulness of Em18 and Em16 as potential new markers for serological differentiation of human AE and CE, respectively, is discussed.

Antibody Formation↗
PubMed1997

Diverse function of aromatase and the N-terminal sequence deleted form.

The diverse function of human placental aromatase including estradiol 6alpha-hydroxylase and cocaine N-demethylase activity are described, and the mechanism for the simultaneous metabolism of estradiol to 2-hydroxy- and 6alpha-hydroxyestradiol at the same active site of aromatase is postulated. Comparison of aromatase activity is also made among the wild type and N-terminal sequence deleted forms of human aromatase which are recombinantly expressed in Escherichia coli. Aromatase cytochrome P450 was reconstituted and incubated with [6alpha,7alpha-(3)H2,4-(14)C]estradiol, 7-ethoxycoumarin, and [N-methyl-(3)H3]cocaine. 6Alpha-hydroxy[7alpha-(3)H,4-(14)C]estradiol was isolated as the metabolite of estradiol and the 3H-water release method based on the 6alpha-3H label was established. The initial rate kinetics of the 6alpha-hydroxylation gave Km of 4.3 microM, Vmax of 4.02 nmol min(-1) mg(-1), and turnover rate of 0.27 min(-1). Testosterone competed dose-dependently with the 6alpha-hydroxylation and showed the Ki of 0.15 microM, suggesting that they occupy the same binding site of aromatase. The deethylation of 7-ethoxycoumarin showed Km of 200 microM, Vmax of 12.5 nmol min(-1) mg(-1) and turnover rate of 1.06 min(-1). The N-demethylation of cocaine was analysed by the 3H-release method, giving Km of 670 microM, Vmax of 4.76 nmol min(-1) mg(-1), and turnover rate of 0.49 min(-1). All activity was dose-responsively suppressed by anti-aromatase P450 monoclonal antibody MAb3-2C2. The N-terminal 38 amino acid residue deleted form of aromatase P450 was expressed in particularly high yield giving a specific activity of 397 +/- 83 pmol min(-1) mg(-1) (n = 12) of crude membrane-bound particulates with a turnover rate of 2.6 min(-1).

Aromatase↗
PubMed1996

Identification and characterization of three isotypes of protein phosphatase inhibitor-2 and their expression profiles during testis maturation in rats.

cDNAs for three isotypes of inhibitor-2(I-2), I-2 alpha 1, I-2 alpha 2 and I-2 beta were isolated from a rat testis library. I-2 alpha 2 and I-2 beta are new forms. The former is an alternatively spliced form of I-2 alpha 1, encoding a protein with substitution of three amino acids for 14 amino acids of the I-2 alpha 1 protein at the C-terminus. The latter is derived from a different gene and encodes a 126-amino-acid protein having highly conserved regions with the I-2 alpha 1 protein from amino acid positions 22-47 and 111-126. I-2 alpha 2 and I-2 beta are expressed exclusively in the testis, and the expressions of all three forms of I-2 coincide with sperm cell maturation. The half-maximal inhibitory concentration of the GST-I-2 alpha 2 fusion protein on the PP1 gamma 2 catalytic subunit is the same as that of GST-I-2 alpha 1, being 10 nM. However, the half-maximal inhibitory concentration of GST-1-2 beta is 100-fold higher, being 1 microM. GST-I-2 beta showed no competition with GST-I-2 alpha 1. and its biological significance is unknown.

Amino Acid Sequence↗
PubMed1996

Immunohistochemical localization of estrogen receptors within aromatase-immunoreactive neurons in the fetal and neonatal rat brain.

We elucidated the anatomical relationship between estrogen receptors and aromatase, the enzyme converting androgens to estrogens, in the fetal and neonatal rat brain by means of double immunohistochemical labeling, using antibodies against rat estrogen receptors and human placental aromatase cytochrome P450. Numerous aromatase-immunoreactive neurons were found in the medial preoptic area, the bed nucleus of the stria terminalis, the medial amygdaloid nucleus and the ventromedial nucleus. Estrogen receptors were also abundant in these areas. Most of the aromatase-immunoreactive neurons showed immunoreactivity for estrogen receptors in the medial subdivision of the bed nucleus of the stria terminalis and in the posterodorsal, division of the medial amygdaloid nucleus. There were also many double-labeled cells in the ventromedial nucleus. However, in the medial preoptic area the localization of aromatase-immunoreactive neurons was distinct from that of neurons containing estrogen receptors. These results suggested that estrogens, which are converted from androgens in aromatase-containing neurons, are involved in the sexual differentiation of the brain through estrogen receptors within aromatase-immunoreactive neurons in the bed nucleus of the stria terminalis, the medial amygdaloid nucleus and the ventromedial nucleus, but through estrogen receptors in aromatase-immunonegative neurons in the medial preoptic area.

Animals↗
PubMed1996

Preparation of an activity-inhibiting monoclonal antibody against human placental aromatase cytochrome P450.

We produced a murine monoclonal antibody (MAb) to human placental aromatase cytochrome P450. This MAb, designated MAb3-2C2, was selected on its ability to suppress aromatase activity. The specificity of this MAb was assessed by selective immunoprecipitation of 125I-labeled aromatase cytochrome P450 as well as by the identification of a 55-kDa protein, which was enriched and purified by immunoaffinity chromatography on a MAb-coupled Sepharose 4B column. The MAb was able to suppress both human placental and ovarian microsomal aromatase. Species differences of aromatase were recognized by MAb3-2C2 on the basis of differential immunosuppression of aromatase activity. The antibody had no effect on non-aromatase cytochrome P450s. MAb3-2C2 gave negative results with human placental aromatase P450 in the Western blot analysis. The data presented indicate that MAb3-2C2 is specific for aromatase cytochrome P450 and that its epitope is located in a fragile tertiary conformation of the enzyme, thus making it capable of sensitively affecting catalysis.

Animals↗
PubMed1996

Covalent crosslinking of the heme prosthetic group to myoglobin by H2O2: toxicological implications.

It is known that treatment of myoglobin with H2O2 leads to covalent alteration of the heme prosthetic group with concomitant formation of a protein bound heme adduct and transforms myoglobin from an oxygen storage protein to an oxidase. In the current study it was shown, with the use of 14C-labeled heme reconstituted into apomyoglobin, that up to 88% of the oxidatively altered heme can be accounted for by the protein bound product. Furthermore, a partially purified preparation of the protein bound heme adduct was introduced into human fibroblasts using the method of osmotic lysis of pinosomes and found to cause cell death (40%) within 1 h, as evidenced by trypan blue exclusion. Native myoglobin introduced into cells in the same manner or extracellular treatment by the protein bound heme adduct had no effect on cell viability. The extent of cell death could be decreased (50%) by N-acetyl-L-cysteine, indicating a potential role for reactive oxygen intermediates in this process. These results show that the covalently altered myoglobin can elicit cellular damage and suggests that similar processes may occur in vivo in pathologic conditions such as that involving cardiac ischemia and reperfusion injury, where covalently altered myoglobin may form.

Acetylcysteine↗
PubMed1996

Effector mechanisms responsible for gamma interferon-mediated host resistance to Legionella pneumophila lung infection: the role of endogenous nitric oxide differs in susceptible and resistant murine hosts.

To facilitate identification of the effector mechanism(s) responsible for gamma interferon (IFN-gamma)-mediated host resistance to Legionella pneumophila, a murine model of legionellosis in BALB/c mice with a targeted disruption in the IFN-gamma gene (gamma knockout [GKO] mice) was developed. Immunocompetent BALB/c mice and GKO mice were inoculated intratracheally with virulent L. pneumophila (10(6) bacteria per mouse), and bacterial clearance and the pulmonary inflammatory response were assessed. L. pneumophila did not replicate in, and was rapidly cleared from, the lungs of immunocompetent BALB/c mice, demonstrating that immunocompetent BALB/c mice are resistant to replicative L. pneumophila pulmonary infections. In contrast, similarly infected GKO mice developed persistent, replicative intrapulmonary L. pneumophila infections with extrapulmonary dissemination of the bacteria to the spleen. Histopathologic and flow cytometric analysis of L. pneumophila-infected lung tissue demonstrated that while immunocompetent BALB/c mice develop multifocal pneumonitis which resolves, similarly infected GKO mice develop diffuse pneumonitis with persistent neutrophil recruitment into the lung. Intratracheal administration of exogenous IFN-gamma to L. pneumophila-infected GKO mice facilitated intrapulmonary clearance of the bacteria, confirming the pivotal role of IFN-gamma in innate host defenses to L. pneumophila lung infection in this murine host. The potential role of endogenous reactive nitrogen intermediates, including nitric oxide (NO), in IFN-gamma-mediated resistance to L. pneumophila pulmonary infections in immunocompetent BALB/c mice was subsequently assessed. Macrophage inducible nitric oxide synthetase (an enzyme responsible for the production of NO) was induced in alveolar cells from L. pneumophila-infected immunocompetent BALB/c mice (with maximal expression at 48 h postinfection) but was not induced in similarly infected GKO mice. However, administration of the NO synthetase inhibitor N-monomethyl-L-arginine did not significantly inhibit clearance of L. pneumophila from the lung of immunocompetent BALB/c mice (compared with that in similarly infected mice not administered N-monomethyl-L-arginine). In contrast, we have previously demonstrated that IFN-gamma-induced host resistance to replicative L. pneumophila lung infections in a susceptible murine host (A/J mice) is mediated, in part, by endogenous NO. Taken together, these studies identify a differing role of endogenous NO in IFN-gamma-mediated resistance to L. pneumophila pulmonary infection in susceptible and resistant murine hosts.

Animals↗
PubMed1996

Diverse functions of aromatase: O-deethylation of 7-ethoxycoumarin.

In studying the diverse functions of aromatase we found that purified and reconstituted aromatase also catalyzes O-deethylation of 7-ethoxycoumarin. Aromatase cytochrome P450 was purified from human term placentas by monoclonal antiaromatase P450 antibody-Sepharose 4B column chromatography. Kinetic analysis of the O-deethylation of 7-ethoxycoumarin by reconstituted aromatase showed Km of 200 microM, Vmax of 12.5 nmol.min-1.mg-1, and turnover rate of 1.06 min-1. 7-Ethoxycoumarin competitively inhibited androstenedione aromatization, the Ki was 180 microM. Fadrozole (CGS16949A), a specific competitive aromatase inhibitor, and MAb3-2C2, an antiaromatase P450 monoclonal antibody, inhibited both aromatase and 7-ethoxycoumarin O-deethylase activities dose responsively. The IC50 of Fadrozole was 33 nM for aromatase and 67 nM for 7-ethoxycoumarin O-deethylase. The IC50 of MAb3-2C2 was 1.1 micrograms IgG for aromatase and 4.0 micrograms IgG for 7-ethoxycoumarin O-deethylase. These results indicate that the two enzyme activities are catalyzed by the same active site of the cytochrome P450. Contrary to the previous postulate on the mechanism-based inactivation of microsomal aromatase by 4-androstene-3,6,17-trione, we found that with purified aromatase, both the initial 19-hydroxylase and the after lyase reactions are simultaneously inactivated by the steroid suicide inhibitor.

Androstenes↗
PubMed1996

Granulosa-cumulus-corona expansion and aromatase localization in preovulatory follicles in superovulated heifers.

Granulosa-cumulus and cumulus-corona expansion as well as aromatase localization within ovarian follicles were monitored during the preovulatory period in superovulated cattle that were blood sampled every 2'nd h for LH analyses. Granulosa-cumulus as well as cumulus-corona expansion were studied by means of transmission electron microscopy and computerized image analysis. Localization of aromatase, an enzyme involved in estrogen synthesis, was determined immunocytochemically using anti-human placental aromatase cytochrome P-450 antisera. Nuclear oocyte maturation was determined by aceto:orcein staining. Significant cell dissociation within the granulosa-cumulus stalk occurred before the breakdown of the germinal vesicle, i.e. the oocyte nucleus, during the period up to 5-7 h after the LH peak, i.e. the highest LH concentration during the surge. Significant increase in intercellular spacing between the cumulus-corona cells occurred at 13-15 and 19-21 h after the LH peak. Before the LH peak all layers of granulosa cells were immunocytochemically stained for aromatase. At 5-7 h after the LH peak, however, only the granulosa cell layers located near the basal lamina were stained, and at all later intervals staining was absent. The granulosa cells of primary and secondary follicles, the interstitial gland cells, the theca interna cells and the oocytes in all follicles were immunocytochemically unstained.

Animals↗
PubMed1996

[In vitro chemosensitivity test for seven undifferentiated thyroid carcinoma cell lines using MTT assay].

In the current study, we ran a chemosensity test on 7 human undifferentiated carcinoma cell lines against 10 anticancer compounds using MTT assay. Efficacy was estimated by comparing fifty percent inhibitory concentration (IC50) with the peak plasma concentration. The results showed that the 7 cell lines could be divided into two groups which had different (high or low) chemosensitivity, and that in the high sensitivity group, ACD, VCR, and etoposide were indicated as useful drugs. Our results suggest that it may be impossible to rescue all undifferentiated carcinoma patients by chemotherapy alone because the tumor may consist of 2 clones which have a different chemosensitivity.

Antineoplastic Agents↗