Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q8NEX9 (reductase)
 26,410 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Aromatase, an enzyme complex localized in the endoplasmic reticulum of estrogen-producing cells, is composed of NADPH-cytochrome P-450 reductase, and aromatase cytochrome P-450 (cytochrome P-450AROM). To define the molecular mechanisms involved in the multifactorial regulation of cytochrome P-450AROM in estrogen-producing cells, we have isolated a cDNA specific for human cytochrome P-450AROM and have used this cDNA to isolate the human cytochrome P-450AROM gene. The cDNA sequence encodes a polypeptide of 503 amino acids and contains--near the carboxy-terminus, a region of high homology with the putative heme-binding regions of other P-450 cytochromes. COS1 cells transfected with an expression plasmid containing the cytochrome P-450AROM cDNA had the capacity to aromatize testosterone, androstenedione and 16 alpha-hydroxyandrostenedione, suggesting that a single polypeptide catalyzes all steps of the aromatization reaction using either of the three major C19-substrates. The human cytochrome P-450AROM gene is greater than 52 kb in size and consists of 10 exons and 9 introns. Hormonally induced changes in aromatase activity of human ovarian granulosa and adipose stromal cells are associated with comparable changes in cytochrome P-450AROM gene expression and synthesis, whereas the reductase component is only modestly affected. Studies are in progress to define the molecular mechanisms involved in the regulation of cytochrome P-450AROM gene expression in estrogen-producing cells.
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PMID:Use of molecular probes to study regulation of aromatase cytochrome P-450. 169 30

Using human adipose stromal cells in monolayer culture as a model system for study of the regulation of aromatase activity, as well as polyclonal antibodies raised in this laboratory against aromatase cytochrome P-450 (cytochrome P-450AROM), it was found that the rate of synthesis of cytochrome P-450AROM was stimulated by dibutyryl cyclic AMP. This stimulation was attenuated by epidermal growth factor and was potentiated by phorbol esters. These changes in cytochrome P-450AROM synthesis were associated with comparable changes in the levels of translatable cytochrome P-450AROM mRNA, as well as with changes in the activity of aromatase of these cells. By contrast, there was little change in the synthesis of the reductase component of the aromatase enzyme complex in response to these factors. The increase in mRNA was blocked by cycloheximide, indicative of a requirement for protein synthesis in mediating this inductive response. It is concluded that aromatase activity is regulated primarily by changes in the level of mRNA encoding cytochrome P-450AROM, and that such changes are likely a reflection of changes in the rate of transcription of the gene encoding this enzyme. Increases in the levels of cytochrome P-450AROM mRNA are apparently mediated by a regulatory protein(s), similar to that found for other steroidogenic forms of cytochrome P-450.
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PMID:Regulation of estrogen biosynthesis in human adipose stromal cells. Effects of dibutyryl cyclic AMP, epidermal growth factor, and phorbol esters on the synthesis of aromatase cytochrome P-450. 303 80

The relationship of function to structure of aromatase cytochrome P450 (P450arom; the product of the CYP19 gene) has been examined by means of sequence alignment and site-directed mutagenesis. Comparison has been made between the sequence of P450arom and the two soluble bacterial cytochrome P450 isoforms, whose three-dimensional structure has been determined (P450BM3 and P450cam). From this comparison, it appears that although there is a similarity of overall structure in cytochromes P450, there is enough significant difference in the regions involved in substrate recognition and substrate binding that residues believed to be involved, even in the known structures, must be tested. With this in mind, we have generated a detailed alignment of P450arom, including the definition of putative alpha-helices and beta-sheets based on comparison of the alignments of P450BM3 and P450cam, generated from their three-dimensional structure, and have made mutations in regions we believe to be involved in substrate recognition at the solvent surface and orientation in the heme pocket. We have mutated F116 and F134 to determine if they are present in the heme pocket, and Q225 and L228 to determine if they are a part of the substrate recognition loop. Although F116E is essentially inactive and may be a folding mutant or may inhibit reductase binding, F134E is more active than the wild type and may be located in the heme pocket facilitating the hydrogen abstraction from C2 of androstenedione. Mutations at Q225 and L228 also result in the anticipated changes in the apparent Km and maximum velocity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Functional domains of human aromatase cytochrome P450 characterized by linear alignment and site-directed mutagenesis. 814 67

The cDNA encoding human placental cytochrome P450 aromatase (CYP19) was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression vector system. The recombinant protein product was characterized by Northern and Western blot analyses as well as by direct measurement of aromatase activity. The expressed enzyme proved to be both catalytically active in the presence of P450 reductase and immunologically reactive with polyclonal antibodies raised against human placental aromatase. The activity of aromatase increased 10% after the addition of 0.1 microgram/ml hemin chloride to the culture medium. However, the level of aromatase protein decreased considerably when the concentration of hemin chloride reached 10 micrograms/ml indicating that hemin chloride has toxic effects on the lepidopteran insect cell line. In conclusion, the baculovirus system is suitable for high level expression of functional human placental CYP19.
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PMID:Expression of human placental cytochrome P450 aromatase (CYP19) cDNA in insect cells using a luciferase based baculovirus vector. 828 Jan 69

Using the reverse transcription polymerase chain reaction, mRNAs encoding steroidogenic P450s as well as NADPH-cytochrome P450 reductase (P450 reductase), adrenodoxin and the transcription factor steroidogenic factor 1 (SF-1) were all detected in rodent brain, but their distribution between brain regions varied. Adrenodoxin and P450 reductase were detected in all regions, suggesting the presence of both mitochondrial and microsomal P450s throughout the brain. Messenger RNAs encoding P450scc (CYP11A1) and P45017 alpha (CYP17) were also detected in all brain regions, this being the first report of CYP17 in the brain. P450c21 (CYP21) was detected only in the brain stem. P45011 beta (CYP11B1) and P450aldo (CYP11B2) are expressed in rat brain, but not in mouse brain; CYP11B1 primarily in the cerebrum, whereas CYP11B2 was detected in all brain regions. In both species, highest levels of aromatase P450 (CYP19) mRNA were detected in the cerebrum. SF-1 expression was restricted to the cerebrum minus cortex. Thus, although SF-1 is required for high level expression of the steroidogenic enzymes in adrenals and gonads, other factors may influence the expression of these genes in the brain. If the mRNAs detected by RT-PCR are indeed translated into functional enzymes, these studies suggest that different brain regions have different capacities for local steroid hormone production and metabolism. This raises the technical challenge of locating the specific sites of synthesis as well as the function of such locally produced ligands.
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PMID:Messenger RNAs encoding steroidogenic enzymes are expressed in rodent brain. 875 Aug 63

Implantation is initiated on day 4 in the mouse and on day 13 in the pig. The preimplantation pig blastocyst synthesizes steroid hormones, but whether preimplantation rodent embryos also have this ability has remained unresolved for the last two decades. In this study, the mRNAs encoding NADPH-cytochrome P450 reductase (P450-reductase), adrenodoxin, lanosterol 14-demethylase P450 (CYP51), 17 alpha-hydroxylase P450 (CYP17), cholesterol side-chain cleavage P450 (CYP11A1), sterol 27-hydroxylase P450 (CYP27), and aromatase P450 (CYP19) were examined in day 4 mouse blastocysts (day 1 = vaginal plug) and in day 13 and 16 pig blastocysts using reverse transcription-polymerase chain reaction (RT-PCR). In mouse blastocysts, mRNAs of P450-reductase, adrenodoxin, and CYP51, but not CYP17, CYP11A1, CYP27, and CYP19, were detected. In agreement with this finding, no aromatase protein could be detected by immunohistochemistry. By contrast, all these mRNAs were detected in the pig blastocyst. Furthermore, both the ovarian and placental types of aromatase (CYP19) mRNAs were detected in the pig blastocyst on days 13 and 16 of pregnancy, although the ovarian form was more abundant. Both forms of aromatase were much higher in day 13 than in day 16 pig blastocysts. The results provide definitive evidence that the preimplantation mouse blastocyst, as opposed to the pig blastocyst, has no ability to synthesize estrogen and no steroidogenic capacity. Maternal estrogen synthesis is essential for implantation of the mouse blastocyst.
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PMID:Preimplantation mouse blastocysts fail to express CYP genes required for estrogen biosynthesis. 905 33

The effects of anticonvulsants on the activities of cytochromes P-450(17alpha,lyase) (CYP17), P-450arom (CYP19), P-450C21 (CYP21), P-450SCC (CYP11A1), and P-450(11beta) (CYP11B1) mono-oxygenase systems were studied using rat testicular microsomes, human placental microsomes, bovine adrenocortical microsomes, bovine adrenocortical mitochondria and purified cytochrome P-450(11beta). Phenytoin, clonazepam and carbamazepine inhibited the steroidogenesis catalysed by these cytochrome P-450 mono-oxygenase systems and the Ki values for each anticonvulsant were determined. Neither hydantoin nor sodium valproate inhibited the activities of steroidogenic cytochromes P-450. When the activities of cytochromes P-450arom and P-450C21 were measured in the presence of anticonvulsants, the Ki values (0.15 mM) for phenytoin were close to the plasma concentration of phenytoin under therapeutic conditions. Phenytoin, clonazepam and carbamazepine directly inhibited the monooxygenase activities of cytochromes P-450, because they did not affect the activities of NADPH-cytochrome P-450 reductase, NADPH-adrenoferredoxin reductase and adrenoferredoxin.
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PMID:Direct inhibitions of the activities of steroidogenic cytochrome P-450 mono-oxygenase systems by anticonvulsants. 918 61

Heterocyclic amines (HCAs) that are present in cooked foods require metabolic activation to exert their genotoxicity. They undergo activation via N-hydroxylation by cytochrome P450 1A2 (CYP1A2), followed by O-esterification by O-acetyltransferase (OAT). To develop a Salmonella tester strain that is highly sensitive to mutagenic HCAs, we introduced a coexpression plasmid (p1A2OR) carrying human CYP1A2 and NADPH-CYP reductase cDNAs and an expression plasmid (pOAT) carrying Salmonella OAT to Salmonella typhimurium TA1538 to yield a TA1538/ARO strain. The TA1538/ARO strain was proven to express the enzymes, as indicated by high activities of 7-ethoxyresorufin O-deethylase and isoniazid N-acetylase. The TA1538/ARO strain exhibited very high sensitivity to mutagenic HCAs 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and a somewhat higher sensitivity to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine compared with the parent Ames tester strain TA1538. The minimum concentrations of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, IQ, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine giving positive results were defined by evidence that the number of colonies increased in a dose-dependent manner and reached a number two times higher than that obtained by vehicle alone as a control in the TA1538/ARO strain at concentrations of 0.3, 3, 30, and 1000 pM, respectively. When the membrane and cytosol fractions prepared from TA1538/ARO were added to a mixture containing the parental TA1538, the sensitivity of TA1538 to IQ was much lower than that seen with TA1538/ARO. These results indicate that the intracellular expression of drug-metabolizing enzymes makes the established strain of Salmonella highly sensitive to mutagenic HCAs.
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PMID:Establishment of a Salmonella tester strain highly sensitive to mutagenic heterocyclic amines. 958 21

Sex determination in egg-laying amniotes may be fundamentally different from that of placental mammals. The mammalian ovary differentiates normally in the absence of estrogen, whereas estrogen seems to be crucial for proper ovarian development in birds, reptiles, and lower vertebrates. Estrogens are produced normally by the biosynthetic conversion of androgens by the enzyme aromatase (CYP19), which is the sole mediator of this reaction. Aromatase inhibitors are capable of reversing females to males in turtles and chickens; therefore, a role for aromatase as the female sex determinant has been postulated for species in which sex determination is temperature-dependent. The entire aromatase coding sequence (1,509 base pairs) from adult terrapin ovaries was cloned, and Northern analysis indicates a single transcript (2.4 kb) for adult ovaries, whereas male and female brains express a 2.4-kb as well as a 9.6-kb transcript. Using a sensitive (attomole sensitivity) competitive RT-PCR technique, aromatase transcript abundance was quantified during embryonic development for embryos treated with and without estrogen. Aromatase is transcribed, well before the temperature-sensitive, (stage 12), at both male and female temperatures in the brain. There is a switch to lower aromatase transcript abundance in the female brain concurrent with an exponential rise of aromatase transcript in the putative ovary. Transcripts remain below the detection limits in the putative testes but exhibit female levels of aromatase transcript when treated with estrogen. Aromatase mRNA levels are generally reduced in the brain by estradiol application. On the basis of these findings, we have postulated a model based on the competition between 5 alpha-reductase and P450 aromatase for androgen substrate in both the brain and the undifferentiated gonad to explain the TSD phenomenon in reptiles.
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PMID:Embryonic brain-gonadal axis in temperature-dependent sex determination of reptiles: a role for P450 aromatase (CYP19). 966 30

A co-expression plasmid (p1A2OR) carrying human CYP1A2 and NADPH-P450 reductase cDNAs and an expression plasmid (pOAT) carrying Salmonella TA1538/ARO cells. The CYP and OAT expressed in the Salmonella cells showed catalytic activity. The TA1538/ARO strain exhibited high sensitivity to heterocyclic amines (HCAs) such as 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimenthylimidazo[4,5-f]quinoline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) as compared with the parent Ames tester strain TA1538. It is suggested that the intracellular expression of drug-metabolising enzymes makes the Salmonella cells highly sensitive to the HCAs.
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PMID:Establishment of a Salmonella tester strain highly sensitive to mutagenic heterocylic amines. 1050 88


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