Diffuse hemorrhage of the breast caused by underlying carcinoma of the breast: a case report.
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Adenocarcinoma↗
Biomedical subjects
W L Miller
Publications and source records attributed to W L Miller.
Congenital adrenal hyperplasia.
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Adrenal Glands↗
Structure of a bovine gene for P-450c21 (steroid 21-hydroxylase) defines a novel cytochrome P-450 gene family.
P-450c21, a cytochrome P-450 enzyme [steroid 21-monooxygenase (steroid 21-hydroxylase), EC 1.14.99.10], mediates the 21-hydroxylation of glucocorticoid and mineralocorticoid hormones in the adrenal gland. The complete sequence of a bovine P-450c21 gene shows it is 3447 base pairs long and contains 10 exons. The intron/exon organization and encoded amino acid sequence indicate that P-450c21 represents a unique family of genes in the P-450 gene superfamily. Primer extension and S1 nuclease protection experiments identified several cap sites for initiation of transcription; the principal cap site produces mRNA with a 5' untranslated region only 11 bases long. S1 nuclease protection experiments confirm that P-450c21 is actively expressed in the adrenal and the testis, an organ not known to secrete 21-hydroxylated steroids.
Amino Acid Sequence↗
Human cholesterol side-chain cleavage enzyme, P450scc: cDNA cloning, assignment of the gene to chromosome 15, and expression in the placenta.
Conversion of cholesterol to pregnenolone is mediated by P450scc [cholesterol, reduced-adrenal-ferrodoxin: oxygen oxidoreductase (side-chain-cleaving), EC 1.14.15.67]. RNA from several human adrenal samples was translated in vitro and immunoprecipitated with anti-bovine P450scc, indicating that P450scc mRNA represents about 0.5% of human adrenal mRNA in normal, hypertrophied, and malignant adrenals. A 1626-base-pair human adrenal P450scc cDNA was cloned in bacteriophage lambda gt10. Primer extension data indicated P450scc mRNA is about 1850 bases long and that all adrenal P450scc mRNA has the same 5' end. A full-length clone containing 1821 bases was obtained from a human testis cDNA library to yield the complete sequence. The encoded human preP450scc contains 521 amino acids with a molecular weight of 60189.65. The testis and adrenal sequences were identical; the human cDNA and amino acid sequences are 82% and 72% homologous, respectively, with the bovine sequences. P450scc cDNA was used to probe DNA from a panel of mouse-human somatic cell hybrids, showing that the single human P450scc gene lies on chromosome 15. The human P450scc gene is expressed in the placenta in early and midgestation; primary cultures of placental tissue indicate P450scc mRNA accumulates in response to cyclic AMP.
Amino Acid Sequence↗
Regulation of ovine pituitary glycoprotein hormone alpha subunit mRNA by 17 beta-estradiol in cell culture.
Messenger RNA (mRNA) activity for the alpha glycoprotein subunit in ovine pituitary cell cultures was quantified using a wheat germ translation assay. [35S] Cysteine alpha translation product normally accounted for 0.3-0.9% of total 35S-product in translations of total culture RNA. Treatment of cultures with 1 nM 17 beta-estradiol (E2) decreased alpha mRNA activity by 50% and 92% after 1.8 and 8 days, respectively. Alpha mRNA activity decreased by 1/2 in 51 h, which is about three times slower than the rate of decrease reported for follicle-stimulating hormone (FSH) beta mRNA activity in similarly treated cultures (Alexander and Miller, 1982). The effective dose (ED50) for E2 in vitro was 30 pM (8 pg/ml), which is within the physiological range for ewes. These results indicate that E2 can act directly on the ovine pituitary to decrease alpha mRNA activity coordinately with mRNA activity for FSH beta.
Animals↗
Vascular smooth muscle responsiveness in a hibernator: effects of season and temperature.
To determine the effects of season, acclimation state, and hibernation on the reactivity of vascular smooth muscle from a hibernant species, strips of thoracic aorta, renal and femoral arteries, and portal vein obtained from adult woodchucks, Marmota monax, were suspended for isometric tension measurements in physiological salt solution. These blood vessels exhibited no seasonal variation in resting tension, connective tissue content, or maximum tension developed to norepinephrine. However, the concentration-response curves to norepinephrine in both aortic and portal vein strips from animals tested in May and June were shifted to the left of those from animals tested in either August or November through February. This increased sensitivity to the catecholamine was seen also in renal vessels from hibernating compared with nonhibernating animals. Decreasing organ bath temperature from 37 to 28 degrees C increased tension developed in response to norepinephrine in aortic and renal strips, whereas that of the femoral artery was unchanged. With further cooling to 17 degrees C, the responses to norepinephrine in aortic and renal strips were similar to the responses at 37 degrees C. The contraction developed to 40 mM KCl was diminished in all tissues at 28 degrees C. Blockade of beta-adrenergic receptors did not augment the response to norepinephrine at 37 degrees C. Contractions of the woodchuck aorta in calcium-free medium were sustained longer than comparable tissue obtained from a rabbit. These data suggest that receptor-mediated processes are modulated in hibernating animals. This modulation varies among vascular beds and may act to maintain or divert perfusion of the tissue through entry, during, and arousal from hibernation.
Acclimatization↗
Study of cholesterol side-chain cleavage (20,22 desmolase) deficiency causing congenital lipoid adrenal hyperplasia using bovine-sequence P450scc oligodeoxyribonucleotide probes.
Conversion of cholesterol to pregnenolone is mediated by the cholesterol side-chain cleavage (SCC) enzyme, P450scc. Deficient SCC activity causes congenital lipoid adrenal hyperplasia (also known as 20,22 desmolase deficiency), a potentially lethal defect in the synthesis of all steroid hormones. To probe for possible genetic defects causing this disease we synthesized four oligodeoxyribonucleotides containing 63 to 72 bases corresponding to portions of the bovine complementary DNA (cDNA) sequence for P450scc. The bovine oligonucleotides were labeled and used directly to probe Southern blots of normal human genomic DNA, revealing a pattern indicating there is a single P450scc gene in the human genome. Hybridization to Northern blots of normal human and bovine adrenal messenger RNA indicates that P450scc messenger RNA is about 2.0 kilobases long in both species. Hybridizations of the oligonucleotides to genomic DNA from three unrelated patients with SCC deficiency did not detect a deletion in the human P450scc gene. The bovine sequence oligonucleotides were then used to isolate a human P450scc cDNA clone. The isolated P450scc cDNA fragment contains 818 bases encoding 239 amino acids of the protein, the translation termination signal, and 98 bases of the 3' untranslated region. The sequence of this carboxy-terminal half of the human P450scc protein is 72% homologous with the bovine sequence and contains an additional amino acid not found in bovine P450scc; the human and bovine nucleotide sequences are 81% homologous. Repetition of the genomic DNA blotting studies with the cDNA probe gave the same results obtained with the bovine-sequence oligonucleotide probes, confirming that SCC deficiency is not due to a deletion in the regions of the P450scc hybridizing with the probes. Long, chemically synthesized heterologous sequence oligonucleotides containing unknown numbers of base mismatches with human sequences may thus be used to study human genes so that access to a cDNA is not necessary for such studies.
Adrenal Hyperplasia, Congenital↗
A direct pituitary action of progesterone on basal secretion of follicle-stimulating hormone in ovine cell culture: dependence on ovaries in vivo.
Progesterone (P4) inhibits FSH production by 60-70% in cell cultures of pituitaries from normal ewes. This communication reports that P4 is much less effective in inhibiting FSH production in cultures prepared from pituitaries of ovariectomized ewes. P4 (10(-7) M) inhibited 61 +/- 5% of basal FSH secretion in cultures from intact anestrous ewes. When cultures were prepared from pituitaries collected on day 9, 21, or 42 after ovariectomy, P4 inhibited FSH secretion by only 46 +/- 3%, 16 +/- 2%, and 10 +/- 2%, respectively. One group of ewes received implants of 17beta-estradiol (E2) at the time of ovariectomy. When cultures were prepared from these ewes 35 days after ovariectomy, P4 inhibited FSH secretion 56 +/- 5%, not significantly different (P greater than 0.05) from inhibition in normal cultures. Furthermore, under similar culture conditions, E2 and a porcine ovarian inhibin preparation inhibited FSH secretion regardless of the length of time after ovariectomy. These in vitro results suggest that ovariectomy causes a time-dependent decrease in pituitary responsiveness to P4 in vivo. Since E2 can maintain P4 sensitivity in pituitaries from ovariectomized ewes, E2 may be the only ovarian factor required to maintain pituitary responsiveness to P4 in vivo.
Animals↗
Progesterone antagonizes the ability of porcine ovarian inhibin to sensitize ovine pituitary cell culture to luteinizing hormone-releasing hormone: dependence on ovaries in vivo.
Progesterone (P4) and a porcine follicular preparation of inhibin (MGRA-IV) have opposite actions on regulation of the ability of LHRH to release LH in ovine pituitary cell culture. Both P4 and inhibin change the response to LHRH. The ability of inhibin to sensitize cultures to LHRH (126-273%) was greatly inhibited (up to 100%) in the presence of P4 (10(-7) M). The inhibitory action of P4 on LHRH-stimulated and inhibin-sensitized LHRH-stimulated LH secretion in ovine pituitary cell culture was dependent on the presence of ovaries in vivo. P4 inhibited 68% of LHRH-stimulated LH secretion in pituitary cultures from intact ewes. However, when cultures were prepared from pituitaries collected on days 9, 21, and 42 after ovariectomy, P4 inhibited LHRH-stimulated LH secretion by only 36%, 13%, and 0%, respectively. Ovariectomy had no effect (P greater than 0.05) on the sensitizing action of inhibin on LHRH-stimulated LH secretion, but ovariectomy did cause a time-dependent decline in the inhibitory action of P4 on inhibin-sensitized LHRH-stimulated LH secretion. Furthermore, when cultures were prepared from pituitaries collected from ewes ovariectomized for 35 days but treated with estradiol implants, both LHRH-stimulated and inhibin-sensitized LHRH-stimulated LH secretion were inhibited as well by P4 as in pituitary cultures from intact ewes. These results suggest that although P4 can completely inhibit the sensitizing action of inhibin on LHRH-stimulated LH secretion, its inhibitory action is dependent on the presence of ovaries or estradiol in vivo.
Animals↗
Hormonal regulation of P450scc (20,22-desmolase) and P450c17 (17 alpha-hydroxylase/17,20-lyase) in cultured human granulosa cells.
Conversion of cholesterol to pregnenolone in man is mediated by a single enzyme, P450scc. To study possible regulation of the single P450scc gene in ovarian steroid synthesis, we incubated human granulosa cells with potential hormonal stimulators, measured P450scc mRNA accumulation by hybridization to 32P-labeled human P450scc cDNA, and compared the results to secretion of progesterone into the culture medium. Primary cultures of human granulosa cells were optimally responsive after 8-14 days of culture. Incubation with hCG (1.0-100 ng/ml), FSH (1.0-50 ng/ml), and (Bu)2cAMP (0.02-2.0 mM) increased P450scc mRNA accumulation and progesterone secretion in dose-dependent fashions. Maximal stimulation increased P450scc mRNA accumulation and progesterone secretion to 490% and 240% of control values, respectively, with hCG, to 166% and 168% with FSH, and to 495% and 380% with (Bu)2cAMP. PRL (to 100 ng/ml), ACTH (10(-6) M), and butyric acid (2 mM) had no significant effect on progesterone secretion or P450scc mRNA accumulation. These data indicate gonadotropin-specific stimulation of cAMP-mediated regulation of P450scc mRNA accumulation in human granulosa cells, presumably mediated by increased P450scc gene transcription. Ovarian estrogen synthesis may require both thecal and granulosa cells, although this two-cell theory of estrogen synthesis is unproven in man. To examine this theory, we probed the same blots used in the experiments described above with 32P-labeled human P450c17 cDNA (P450c17 is the single enzyme mediating both 17 alpha-hydroxylase and 17,20-lyase activities). Only miniscule amounts of P450c17 mRNA were found in the human granulosa cells, and the amounts did not increase in response to any of the above stimuli. These data strongly support the two-cell theory of human ovarian estrogen synthesis.
Aldehyde-Lyases↗
Assignment of the gene for adrenal P450c17 (steroid 17 alpha-hydroxylase/17,20 lyase) to human chromosome 10.
P450c17 is the single enzyme mediating both 17 alpha-hydroxylase and 17,20 lyase activities. We identified several human P450c17 cDNA clones in a human adrenal cDNA library we constructed in lambda gt10. A short clone containing the 3'-terminal 650 bases of the full-length sequence was used to examine Southern blots of DNA from normal persons and from a panel of mouse/human somatic cell hybrid lines. The pattern of hybridization of this cDNA to normal human DNA cut with 8 restriction endonucleases suggests the human genome has two (or more) P450c17 genes. The pattern of hybridization to the somatic cell hybrid cell lines, each containing a limited, known number of human chromosomes, indicates the human adrenal P450c17 gene lies on chromosome 10. The chromosomal locations of the other P450c17 genes could not be determined.
Adrenal Glands↗
Developmental expression of genes for the stereoidogenic enzymes P450scc (20,22-desmolase), P450c17 (17 alpha-hydroxylase/17,20-lyase), and P450c21 (21-hydroxylase) in the human fetus.
Fetal adrenal steroidogenesis is required for the production of placental estrogen, and fetal testicular steroidogenesis is required for the development of male external genitalia. We studied the ontogeny and tissue specificity of expression of the genes for three steroidogenic enzymes: P450scc (the cholesterol side-chain cleavage enzyme), P450c17 (17 alpha-hydroxylase/17,20-lyase), and P450c21 (21-hydroxylase) in the human fetus. RNA from fetal tissues was probed with homologous human P450scc, P450c17, and P450c21 cDNAs cloned in our laboratory. At 20-21 weeks gestation, P450scc mRNA was most abundant in the adrenal, followed by testis, placenta, and ovary. P450c17 mRNA was also most abundant in the adrenal, followed by testis and ovary, but was undetectable in the placenta. P450c21 mRNA was detected only in the adrenal. None of these mRNAs was detected in kidney, liver, spleen, intestine, or muscle. Twenty-two fetal testis samples (13-25.8 weeks gestation) were studied. P450scc and P450c17 mRNAs were most abundant at 14-16 weeks and diminished to 35 and 19% of their peak values, respectively, by 20-25.8 weeks. Ovarian P450scc and P450c17 mRNAs were present, respectively, in only 6.2% and 1.8% of the maximum amount in the testis and did not vary detectably from 14.9 to 21.5 weeks gestation. The testicular and ovarian steroidogenic enzyme mRNA data correlate well with previously reported changes in gonadal steroidogenesis with gestational age. The presence of P450scc mRNA, but not P450c17 mRNA, in the placenta indicates that the placenta is able to initiate the synthesis of some steroid hormones, but is not able to synthesize estrogen de novo. Since P450c21 was found only in the adrenal, the extraadrenal 21-hydroxylation of progesterone to deoxycorticosterone, a common event in the fetus, is probably mediated by an enzyme(s) other than P450c21.
Aldehyde-Lyases↗
Cytoprotective and antisecretory properties of a non-diarrheogenic and non-uterotonic prostacyclin analog: U-68,215.
U-68,215 [15-Cyclohexyl-9-deoxo-13,14-dihydro-2',9 alpha-methano-4,5,6,16,17,18,19,20-octanor-3-oxa-3,7-(1', 3'-interphenylene)-PGE1] is a stable prostacyclin analog. When given orally to rats, it is cytoprotective for the stomach (ED50: 0.8 micrograms/kg) and the intestine (ED50: 22 micrograms/kg), is gastric antisecretory (ED50: 35 micrograms/kg) and antiulcer (aspirin) (ED50: 5 micrograms/kg). The oral antisecretory ED50 in dogs is 50 micrograms/kg. It has a long duration of gastric cytoprotection: 8-10 hours compared to 3 hours for 16,16-dimethyl PGE2. Unlike most prostaglandins of the E type, it is not diarrheogenic (not enteropooling), it does not induce cellular proliferation of the gastrointestinal mucosa, when given twice a day for eight days, it is not uterotonic (in monkeys), and it does not prevent embryo implantation in hamsters. It inhibits ex vivo platelet aggregation (ED50: 300 micrograms/kg), but does not promote bleeding from cut vessels nor from gastric ulcers. U-68,215 lowers blood pressure at an oral dose corresponding to 1-5 times the antisecretory ED50 in rats and dogs, and to 150 times the cytoprotective ED50 in rats. It may be of therapeutic value in the treatment of conditions where inhibition of gastric acid secretion is desirable, e.g., gastric and duodenal ulcer, and in conditions responding to cytoprotection, e.g., stress ulcers, hemorrhagic gastritis and gastric erosions associated with nonsteroidal antiinflammatory drugs.
Alprostadil↗
Cloning and characterization of the bovine gene for steroid 21-hydroxylase (P-450c21).
Steroid 21-hydroxylase activity is required for the synthesis of both cortisol and aldosterone. Using two manually synthesized oligonucleotide probes, we screened a bovine genomic DNA library and identified a phage, lambda E11, carrying the gene for the steroid 21-hydroxylase, P-450c21. The identity of lambda E11 was initially proven by initiating dideoxy sequencing from the two oligonucleotides directly on the full-length, uncleaved phage template. Hybridization of total bovine genomic DNA to lambda E11 restriction fragments indicates the presence of repetitive sequences in or near the P-450c21 gene. Northern blots indicate that the mature mRNA exists in two principal forms of about 2.2 and 2.4 kb in length. Southern blots indicate there are two copies of the gene in the bovine genome. Sequence analysis of a 1141-bp Eco RI fragment of the gene shows three complete exons and a portion of a fourth exon, correctly determining the amino acid sequence of 148 amino acids of this important enzyme. This cloned 1141-bp fragment cross-hybridizes to human genomic DNA, indicating it should be a useful probe for studying the human P-450c21 gene in patients having impaired 21-hydroxylase activity.
Amino Acid Sequence↗
Congenital adrenal hyperplasia due to deficient cholesterol side-chain cleavage activity (20, 22-desmolase) in a patient treated for 18 years.
Two siblings, a 9-week-old female and an 18-year-old male pseudohermaphrodite are described with deficient cholesterol side-chain cleavage activity. The female died untreated in 1954; the second sibling, a phenotypically female infant with 46 XY karyotype, was diagnosed at age 5 weeks. Massive adrenal hyperplasia was revealed by intravenous pyelography showing downward displacement of the kidneys. Secretion rates of cortisol, aldosterone, deoxycorticosterone and corticosterone were unmeasurable. Urinary 17-hydroxycorticosteroids (17-OHCS), tetrahydrocortisol, 17-ketosteroids (17-KS), pregnanetriol, pregnanediol, and delta 5-3 beta-ol steroids were not detected during prolonged administration of ACTH. Plasma concentrations and urinary excretion of gonadotrophins were increased. Gonadal mitochondria did not convert radiolabelled cholesterol to pregnenolone. The gluccocorticoid and mineralocorticoid deficiencies have been controlled well by steroid replacement therapy. Plasma ACTH concentrations and plasma renin activity remained strikingly elevated even when supraphysiologic doses of glucocorticoids and mineralocorticoids were given. Oestrogen replacement alone induced a pubertal growth spurt. The differential diagnosis, the effects of long-term steroid replacement therapy, and comparison with previously reported findings are discussed.
Adolescent↗
An approach to the molecular biology of congenital adrenal hyperplasia.
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Adrenal Hyperplasia, Congenital↗
Secretion of ovine luteinizing hormone in vitro: differential positive control by 17 beta-estradiol and a preparation of porcine ovarian inhibin.
17 beta-Estradiol (E2) and an enriched preparation of porcine ovarian inhibin can positively regulate LH secretion in two different ways in ovine pituitary cell cultures. One major effect of these agents is to increase the sensitivity of cultures to low levels of LHRH or LHRH analogs. The second effect is to increase culture responsiveness to high levels of LHRH or its analogs. Prolonged treatment with 1 nM E2, for instance, increased pituitary sensitivity to D-Lys6-LHRH by 10-fold, since it lowered the ED50 of D-Lys6-LHRH from approximately 400 pM to about 40 pM; the maximum LH secretory response to D-Lys6-LHRH (1-100 nM) was not changed, however. In contrast, short term treatment with an E2-free preparation of porcine ovarian inhibin could increase, by 350%, normal LH responsiveness to high levels of D-Lys6-LHRH (1-100 nM), but had no effect on sensitivity; the ED50 remained unchanged. Chronic treatment with inhibin eventually increased sensitivity, and interestingly, a short treatment with E2 (6 h) caused a major increase in both sensitivity and responsiveness, but the responsiveness component usually disappeared between 6 and 48 h of chronic E2 treatment. Treatment with both E2 and the inhibin preparation caused the greatest increase in pituitary sensitivity to D-Lys6-LHRH (ED50 decreased to approximately 30 pM), but the increased responsiveness to D-Lys6-LHRH (1-100 nM) declined with time as with E2-only treatment. The different actions of E2 and the ovarian inhibin preparation seen to perturb different aspects of LH secretion. Therefore, study of these phenomena may uncover novel molecular details governing LHRH-stimulated LH release. Furthermore, these two types of LH secretory control may be physiologically important, at least in sheep, and perhaps in other animals as well.
Animals↗
Progesterone decreases the responsiveness of ovine pituitary cultures to luteinizing hormone-releasing hormone.
Progesterone (P4) regulation of LH secretion in sheep was studied in vitro using dispersed pituitary cell cultures. Neither cell content nor basal secretion of LH were normally altered by P4, but maximal stimulation of LH secretion by LHRH was suppressed up to 70% with 10(-7) M P4, without changing the ED50 of LHRH. The inhibitory effect of P4 appeared within 6 h and became maximal by 24 h but was partially reversed during continued treatment with P4. The inhibitory effect was completely reversed by 24 h after P4 withdrawal. The ED50 of P4 was 10(-9) M. P4 also decreased the sensitizing effect of 17 beta-estradiol (E2) on LHRH-induced LH secretion in ovine pituitary culture. It partially inhibited the E2-induced increase in maximal response to LHRH and decreased the ability of E2 to lower the ED50 of LHRH. Our results clearly show that P4 can act directly on the ovine pituitary to inhibit both normal and E2-sensitized LH responsiveness to LHRH.
Animals↗