PubMed Health⌕ Search

Biomedical subjects

E R Simpson

Publications and source records attributed to E R Simpson.

At least 163 records · Page 9Linked to original sources
PubMed1989

Structural characterization of the bovine CYP17 (17 alpha-hydroxylase) gene.

The complete exonic and partial intronic sequence of the bovine CYP17 (P45017 alpha) gene has been determined. The gene contains eight exons with exon/intron boundaries which are identical to those determined previously for the human CYP17 gene. The site of initiation of transcription of this gene is located within a 6-base sequence 52 bp from the initiation of translation. Considerable sequence homology (58.7%) is found when approximately 500 bp of the 5'-flanking sequences of the bovine and human CYP17 genes are compared. A computer-based search of this region of bovine CYP17 for consensus sequences associated with binding of transcription factors (i.e., GR, PR, CREB/ATF, AP1, AP2, AP3, AP4, AP5, OTF, CTF/NF1, SP1) shows only the consensus CREB/ATF sequence TGACGT which is also found to be at approximately the same position in the human CYP17 gene. In bovine adrenal cortex, transcription of the CYP17 gene is regulated by the peptide hormone adrenocorticotropin via cAMP. Whether the consensus CREB/ATF sequence is associated with the cAMP-mediated transcription of the CYP17 gene remains to be elucidated.

Adrenal Cortex↗
PubMed1989

Immunohistochemical studies of steroidogenic enzymes (aromatase, 17 alpha-hydroxylase and cholesterol side-chain cleavage cytochromes P-450) in sex cord-stromal tumors of the ovary.

Aromatase, 17 alpha-hydroxylase, and cholesterol side-chain cleavage P-450 cytochromes (P-450AROM, P-450(17 alpha,) and P-450SCC, respectively) were immunohistochemically localized in nine granulosa cell tumors, 15 thecomas, ten Sertoli-Leydig cell tumors, two steroid cell tumors, five fibromas, and five sclerosing stromal tumors. In the thecomas, P-450SCC and P-450(17 alpha) were positive in luteinized theca cells and in cells with vacuolated cytoplasm, while P-450AROM was not observed. In the steroid cell tumors, all the P-450 cytochromes were intensely stained. P-450SCC and P-450(17 alpha) were present in cells with vacuolated cytoplasm in two cases of sclerosing stromal tumor. P-450AROM was weakly demonstrated in one of the granulosa cell tumors. P-450(17 alpha,) P-450SCC, and P-450AROM were all faintly stained in the Sertoli-Leydig cell tumors. No P-450 cytochrome immunoreactivity was observed in any fibroma.

Aromatase↗
PubMed1989

Brenner tumor of the ovary: immunoanalysis of steroidogenic enzymes in 23 cases.

Immunohistochemical analysis of cytochromes P-450 aromatase and P-450 17 alpha-hydroxylase, which catalyze the production of estrogens and androgens, respectively, was performed for 23 cases of ovarian Brenner tumor. Immunoreactivity for P-450 aromatase was observed in the epithelial cells of the tumor distinctly in two cases and faintly in four cases, while immunoreactivity for P-450 17 alpha-hydroxylase was seen in two cases. No immunoreactivity was observed in the stromal cells of the tumors, nor were luteinized cells observed in the cases examined. No correlation was observed between the immunoreactivity of cytochromes P-450 in the epithelial cells of the tumor and endometrial abnormalities. These findings, together with a review of the literature, suggest that the usual Brenner tumor is not associated with specific steroidogenesis. Rare massive Brenner tumors may, however, be capable of androgenic or estrogenic activity.

Aromatase↗
PubMed1989

Regulation of steroid hydroxylase gene expression is multifactorial in nature.

In summary, regulation of steroid hydroxylase gene expression is complex and multifactorial, involving cAMP-dependent and -independent mechanisms required for maintenance of optimal steroidogenic capacity, tissue-specific mechanisms which lead to different steroidogenic pathways in different tissues, and developmental mechanisms which lead to fetal imprinting of steroid hydroxylase expression and which probably overlap with both maintenance and tissue-specific mechanisms. Future studies will involve identification of the trans-acting factors associated with each of these aspects of the multifactorial regulation and characterization of the cis-regulatory elements to which they bind. Such studies will inevitably lead to the identification of genes encoding these trans-acting factors and investigation of their regulation. In this way, it will be possible to work outward from the steroid hydroxylase genes toward the cell surface receptors in order to elucidate the series of events which lead to cAMP-dependent and -independent regulation of steroid hydroxylase gene expression.

Adrenal Cortex↗
PubMed1989

Ultrasound determination of the relationship of radioactive plaques to the base of choroidal melanomas.

There is an assumption that radioactive plaques placed at surgery are, and will remain, in proper relationship to the base of the tumor. The plaque dose is calculated based on this assumption. In fact, factors such as loose sutures, improper diameter estimations, pressure from adjacent rectus muscles, and intervening tissue (oblique muscles) can compromise this relationship. Ultrasound provides a practical method of imaging the tumor and plaque simultaneously. The authors have used postoperative ultrasound to monitor the accuracy of iodine-125 plaque placement in nine cases. Detection of eccentrically placed and malpositioned plaques provides valuable insight which can be used to refine surgical technique. Detection of plaque tilting by oblique muscles can serve as a basis for recalculating dosage. The relationship of plaque margins to vital ocular structures such as the optic nerve can also be determined by ultrasound.

Brachytherapy↗
PubMed1989

Developmental regulation of P-45017 alpha gene expression in fetal bovine adrenal.

Among the multifactorial aspects of regulation of steroid hydroxylase gene expression, it is the developmental process which leads to imprinting of expression of particular steroid hydroxylases in specific cell types. We have begun to investigate the ontogeny of steroidogenesis in fetal bovine tissues. Expression of most steroid hydroxylases and related enzymes is detectable in adrenals of the smallest fetuses studied and continues throughout fetal life. Like the other steroid hydroxylases, P-450(17)alpha is detectable in the earliest fetal adrenals studied. However, following an increase in expression, P-450(17)alpha disappears from the fetal adrenal by 100 days gestational age and remains absent until about 230 days gestational age. The absence of P-450(17)alpha is correlated with the absence of cortisol in the fetal adrenal and the absence of ACTH in fetal plasma. Thus expression of P-450(17)alpha in bovine fetal adrenal appears to be strictly dependent on cAMP while expression of other steroid hydroxylases appears to involve both cAMP-dependent and cAMP-independent mechanisms. Furthermore, P-450(17)alpha is expressed in fetal testis at gestational times when it is absent in fetal adrenal. We have begun to examine binding of nuclear proteins from adrenals of various gestational ages to the 5'-flanking region of the bovine P-450(17)alpha gene. Preliminary evidence indicates the presence of a protein in nuclei of fetal adrenals not expressing P-450(17)alpha that is not present in fetal adrenals of other gestational ages.

Adrenal Glands↗
PubMed1989

Regulation of cholesterol side-chain cleavage and 17 alpha-hydroxylase/lyase activities in proliferating human theca interna cells in long term monolayer culture.

In this report we describe the development and characterization of a long term culture system to study regulation of the expression of 17 alpha-hydroxylase, cholesterol side-chain cleavage, and 3 beta-hydroxysteroid dehydrogenase in human theca interna cells. Conditions have been established for the dispersal, growth, freezing, and storage of functional human theca interna cells isolated from preovulatory follicles of women undergoing laparoscopy for gamete intrafallopian tube transfer and in vitro fertilization procedures. Theca interna cells grown under these conditions have a doubling rate of 28-32 h and are morphologically distinct from human granulosa cells grown under the same conditions. Theca interna cells were grown, passed for successive passages, and transferred into serum-free medium containing forskolin, hCG, LH, or cAMP analogs. There was a time- and dose-dependent increase in 17 alpha-hydroxylase activity and progesterone synthesis from endogenous precursors. Added pregnenolone was converted to 17 alpha-hydroxypregnenolone, which was further converted primarily to dehydroepiandrosterone and, to a much lesser extent, androstenedione. Progesterone was converted to 17 alpha-hydroxyprogesterone and 16 alpha-hydroxyprogesterone. In studies using 17 alpha-hydroxyprogesterone as substrate, no metabolism to androstenedione or any other product was detectable. Similarly, 4-pregnen-20 alpha-ol-one (20 alpha-dihydroprogesterone) was not metabolized to any detectable products. Northern analysis performed on total RNA obtained from forskolin-stimulated theca interna cultures verified that the increase in 17 alpha-hydroxylase activity was associated with a corresponding increase in levels of mRNA specific for 17 alpha-hydroxylase cytochrome P-450. Message levels for cholesterol side-chain cleavage P-450 were similarly increased in cells treated with forskolin. No detectable mRNA encoding aromatase cytochrome P-450 was discerned. This procedure for the preparation and study of proliferating human theca internal cells provides an opportunity to study regulation of the expression of steroidogenic enzymes and other cellular processes unique to human ovarian cells.

Aldehyde-Lyases↗
PubMed1989

Immunolocalization of aromatase, 17 alpha-hydroxylase and side-chain-cleavage cytochromes P-450 in the human ovary.

Immunohistochemical localization of cholesterol side-chain-cleavage, 17 alpha-hydroxylase and aromtase cytochromes P-450 was performed in 35 morphologically normal human premenopausal ovaries by using specific antibodies against the enzymes. In well-developed ovarian follicles in the late stages of follicular growth, immunoreactivity of P-450AROM was only seen in granulosa cells while P-450(17 alpha) and P-450SCC activity was confined to theca interna cells, confirming that follicular oestrogen is produced in granulosa cells by the aromatization of androgens derived from the theca interna cells. In the corpus luteum, this functional differentiation is maintained, since immunoreactivity of P-450AROM was exclusively present in luteinized granulosa cells while that of P-450(17 alpha) was present in luteinized theca calls. Immunoreactivity of P-450SCC was present in both types of cells in the corpus luteum.

Animals↗
PubMed1988

The expression of a functional cDNA encoding the chicken cytochrome P-450arom (aromatase) that catalyzes the formation of estrogen from androgen.

A complementary DNA (cDNA) copy of the aromatase P-450 has been isolated from a chicken ovary library using as probe a partial cDNA believed to encode the human placental aromatase. The predicted amino acid sequence of the chicken aromatase cDNA possesses regions of homology to that of its human counterpart, but only limited homology to other cytochrome P-450 enzymes. The introduction of the cDNA clone into COS-1 cells results in the production of high levels of aromatase activity. The chicken enzyme is targeted to the appropriate subcellular fraction in the transfected COS cells, and the apparent Km of the chicken aromatase activity, measured in microsomes prepared from the transfected cells, is similar to that of the enzyme prepared from chicken ovary microsomes. These findings establish that the cDNA clone encodes chicken ovarian aromatase and demonstrate that this protein can catalyze the three successive oxidation reactions necessary to form estrogen from androgen.

Amino Acid Sequence↗
PubMed1988

Transcription of the bovine adrenodoxin gene produces two species of mRNA of which only one is translated into adrenodoxin.

The 5'-end of the bovine adrenodoxin gene contains unique structural characteristics. Exons 1 and 2 appear to encode different presequences for this mitochondrial precursor protein. However, exon 1 contains a stop codon (TAA) in place of amino acid 15. Thus, while two species of bovine adrenodoxin mRNA arise from a single gene, only the abundant mRNA species (90%) is translated into the adrenodoxin precursor which contains the presequence encoded by exon 2. The minor mRNA species (10%) contains the sequence encoded by exon 1 and cannot be translated into an adrenodoxin precursor. Presumably the sequence within exon 2 is removed from this minor mRNA species by alternative splicing. Furthermore, the initiation of transcription of the major adrenodoxin mRNA (exon 2) lies within intron 1 of this unusual gene, while that for the other mRNA (exon 1) lies in the 5'-flanking region. Thus, the adrenodoxin gene is the first example of a gene encoding a mitochondrial protein which falls into the category of genes having alternate promoters utilizing a pattern of alternative splicing.

Adrenodoxin↗
PubMed1988

Combined 17 alpha-hydroxylase/17,20-lyase deficiency due to a stop codon in the N-terminal region of 17 alpha-hydroxylase cytochrome P-450.

Steroid 17 alpha-hydroxylase (cytochrome P-450(17)alpha) mediates both 17 alpha-hydroxylase and 17,20-lyase activities. A relatively rare disease, 17 alpha-hydroxylase deficiency is characterized by defects in either or both of these activities. The molecular basis for variability of the defect is not well understood. We have determined the exonic sequence of the mutant P-450(17)alpha gene from one Japanese patient with combined 17 alpha-hydroxylase/17,20-lyase deficiencies. A stop codon (TGA) due to a single point mutation was found at the position of amino acid 17 in exon 1 of the P-450(17)alpha gene. The presence of a stop codon in the N-terminal region of this gene leads to the absence of a functional P-450(17)alpha protein in adrenal cortex and ovary, and consequently hypertension, primary amenorrhea and osteoporosis in this patient.

Adrenal Cortex↗
PubMed1988

Effects of epidermal growth factor and insulin-like growth factor I on the levels of mRNA encoding aromatase cytochrome P-450 of human ovarian granulosa cells.

The effects of growth factors to regulate the activity of aromatase, as well as the synthesis of aromatase cytochrome P-450 (P-450AROM) have been studied in human ovarian granulosa cells obtained from women undergoing oocyte retrieval. Insulin-like growth factor I (IGF-I) increased aromatase activity as well as the synthesis of P-450AROM, in a concentration-dependent fashion. The levels of hybridizable mRNA species encoding cytochrome P-450AROM were also increased with IGF-I treatment. By contrast, epidermal growth factor (EGF) had no effect on these parameters when added alone, but markedly inhibited the action of follicle-stimulating hormone (FSH) to stimulate aromatase activity, and the synthesis of cytochrome P-450AROM, as well as its ability to increase the levels of mRNA encoding the enzyme. It is concluded that these growth factors have opposite effects on aromatase activity, and that these actions reflect, in part, changes in the synthesis of cytochrome P-450AROM, which in turn are the consequence of changes in the levels of mRNA encoding this enzyme.

Aromatase↗
PubMed1988

Isolation of a full-length cDNA insert encoding human aromatase system cytochrome P-450 and its expression in nonsteroidogenic cells.

The isolation and cloning of a full-length cDNA insert complementary to mRNA encoding human aromatase system cytochrome P-450 is reported. The insert contains an open reading frame encoding a protein of 503 amino acids. This gene is clearly a member of the cytochrome P-450 gene superfamily, because the sequence contains regions of marked homology to those of other members, notably a putative membrane-spanning region, I helix, Ozols, and heme-binding regions. The cDNA was inserted into a modified pCMV vector and expressed in COS-1 monkey kidney tumor cells. The expressed protein was similar in size to human placental aromatase system cytochrome P-450, as detected by immunoblot analysis, and catalyzed the aromatization of androstenedione, testosterone, and 16 alpha-hydroxyandrostenedione. This activity was inhibited by the known aromatase inhibitors, 4-hydroxyandrostenedione and econazole. Thus the several steps involved in the aromatization reaction appear to be catalyzed by a single polypeptide chain, which can metabolize the three major physiological substrates.

Amino Acid Sequence↗
PubMed1988

Simultaneous transfection of COS-1 cells with mitochondrial and microsomal steroid hydroxylases: incorporation of a steroidogenic pathway into nonsteroidogenic cells.

Transfected, nonsteroidogenic COS-1 cells derived from monkey kidney are found to be capable of supporting the initial and rate-limiting step common to all steroidogenic pathways, the side-chain cleavage of cholesterol to produce pregnenolone. Endogenous COS-1 kidney cell renodoxin reductase and renodoxin are able to sustain low levels of this activity catalyzed by bovine cholesterol side-chain cleavage cytochrome P450 (P450scc) whose synthesis is directed by a transfected plasmid containing P450scc cDNA. Double transfection with both P450scc and adrenodoxin plasmids leads to greater pregnenolone production and indicates that adrenodoxin plays a role as a substrate for this reaction or that bovine adrenodoxin serves as a better electron donor than the endogenous iron-sulfur protein renodoxin. Also it is found that both the bovine adrenodoxin and P450scc precursor proteins are proteolytically processed upon their uptake by COS-1 cell mitochondria to forms having the same electrophoretic mobility as mature bovine adrenodoxin and P450scc. Following triple transfection of COS-1 cells with P450scc, adrenodoxin, and 17 alpha-hydroxylase cytochrome P450 plasmids, pregnenolone produced in mitochondria by the side-chain cleavage reaction can be further metabolized in the endoplasmic reticulum to 17 alpha-hydroxypregnenolone and dehydroepiandrosterone. Although this functional steroidogenic pathway can be incorporated into this nonsteroidogenic cell type, it is found to be nonresponsive to cAMP, a potent activator of steroid hormone biosynthesis in adrenal cortex, testis, and ovary. Thus the cellular mechanisms necessary to support both microsomal and mitochondrial steroid hydroxylase activities appear not to be tissue specific, whereas the acute cAMP-dependent regulation of steroidogenesis is not present in transformed kidney (COS-1) cells.

Adrenodoxin↗