Cigarette smoking during pregnancy lowers aromatase cytochrome P-450 in the human placenta.

To clarify whether cigarette smoking during pregnancy causes an organic alteration in placental estrogen producing ability, we determined the catalytic activity of aromatase by the tritiated water assay, and tissue level of aromatase cytochrome P-450 (P-450arom) by the specific enzyme-linked immunosorbent assay, in placental samples from nonsmokers and smokers. As pregnancy progressed, both aromatase activity and P-450arom concentration increased in placentas from nonsmokers and smokers. However, the gradient of the increase was significantly less in heavy smokers (> or = 20 cigarettes a day) than in normal and moderate smokers (< 20 cigarettes a day).

Catalytic efficiency of expressed aromatase following site-directed mutagenesis.

Mutant aromatase cytochrome P-450s, expressed in CHO cells after transfection with cDNAs, have been characterized in terms of their catalytic efficiencies. After solubilization from microsomes, specific aromatase P-450 content of wild-type and mutants Pro308Phe, Asp309Asn, Asp309Ala and Phe406Arg was quantitated by a sandwich enzyme-linked immunosorbent assay (ELISA). Microsomal aromatase activity was determined by the 3H-water method using [1 beta-3H]androstenedione as substrate.

Competitive product inhibition of aromatase by natural estrogens.

In order to better understand the function of aromatase, we carried out kinetic analyses to assess the ability of natural estrogens, estrone (E1), estradiol (E2), 16 alpha-OHE1, and estriol (E3), to inhibit aromatization. Human placental microsomes (50 micrograms protein) were incubated for 5 min at 37 degrees C with [1 beta-3H]testosterone (1.24 x 10(3) dpm 3H/ng, 35-150 nM) or [1 beta-3H,4-14C]androstenedione (3.

Multiple functions of aromatase and the active site structure; aromatase is the placental estrogen 2-hydroxylase.

Androgen aromatase was found to also be estrogen 2-hydroxylase. The substrate specificity among androgens and estrogens and multiplicity of aromatase reactions were further studied. Through purification of human placental microsomal cytochrome P-450 by monoclonal antibody-based immunoaffinity chromatography and gradient elution on hydroxyapatite, aromatase and estradiol 2-hydroxylase activities were co-purified into a single band cytochrome P-450 with approx.

Studies on the mechanism of aromatase and other cytochrome P450 mediated deformylation reactions.

Aromatase is a microsomal cytochrome P450 that converts androgens to estrogens by three sequential oxidations. The isolation of the 19-hydroxy and 19-oxo androgens suggests that the first two oxidations occur at the C19 carbon. However, the mechanism of the third oxidation, which results in C10--C19 bond cleavage, has not been determined.

Structure-function studies of human aromatase.

Site-directed mutagenesis experiments have been carried out to determine the structure-function relationship of human aromatase. By sequence comparison, the region in aromatase that corresponds to the distal helix of cytochrome P-450cam has been identified to be Gln-298 to Val-313. Eight aromatase mutants with changes in this region, i.

Germ cells of the mouse testis express P450 aromatase.

Estrogen production within the testis has been a subject of considerable controversy for many years. Several studies have shown that both Sertoli and Leydig cells produce estrogen during different stages of development. Therefore, we have conducted experiments to localize aromatase, a cytochrome P450 enzyme that converts androgen to estrogen, within the testis.

An aromatase-associated cytoplasmic inclusion, the "stigmoid body," in the rat brain: II. Ultrastructure (with a review of its history and nomenclature).

The ultrastructure of aromatase-associated "stigmoid (dot-like) structures," which were detected in a previous study using light-microscopic immunohistochemistry (Shinoda et al.: J. Comp.

Metabolism-based transformation of myoglobin to an oxidase by BrCCl3 and molecular modeling of the oxidase form.

The stoichiometric reductive debromination of BrCCl3 to a trichloromethyl radical by myoglobin caused the prosthetic heme to become covalently cross-linked to the protein moiety and transformed myoglobin from an oxygen storage protein to an oxidase. This was shown in experiments in which oxygen consumption was measured during redox cycling of the altered myoglobin in the presence of ascorbate or an enzymatic reducing system containing diaphorase and NADH. Redox cycling eventually led to loss of the protein-bound heme adduct and oxidase activity of myoglobin.

Inhibition of cytochromes P450 by nitric oxide and a nitric oxide-releasing agent.

The effect of nitric oxide (NO) on cytochrome P450-mediated benzyloxyresorufin and ethoxyresorufin O-dealkylase activity of rat hepatic postmitochondrial (S-9) or microsomal subfractions, or purified rat liver CYP2B1, was examined. Two distinct inhibitory phases were observed regardless of whether the NO was added prior to initiation of the reactions with NADPH or during the course of substrate turnover. The first was a reversible inhibition characterized by complete cessation of catalytic activity, the duration of which was NO concentration-dependent with an IC50 in the range of 8-60 microM.

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.

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.

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 use of stable isotopes to identify reactive metabolites and target macromolecules associated with toxicities of halogenated hydrocarbon compounds.

1. Halogenated compounds, such as the inhalation anaesthetics, halothane and enflurane, and the chemicals chloroform, carbon tetrachloride, and bromotrichloromethane can cause hepatotoxicity, nephrotoxicity, and inactivation of cytochromes P-450. Each of these toxicities is mediated by reactive metabolites.

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.

[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.

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.

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.

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.

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.

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.

[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.

Multiple steroidogenic cell populations in the thecal layer of preovulatory follicles of the chicken ovary.

The thecal layer of the preovulatory follicle of the chicken ovary produces primarily androgens and estrogens. However, the precise cellular sites of androgen and estrogen synthesis in the thecal layer have not been identified. Therefore, our aims were 1) to identify steroidogenic cells in the thecal layer of the preovulatory follicles by localizing specific steroidogenic enzymes in these cells by immunocytochemistry, and 2) to confirm that these cells which contained the specific steroidogenic enzymes secreted the expected steroid in a short term cell incubation.