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Biomedical subjects

W L Miller

Publications and source records attributed to W L Miller.

At least 253 records · Page 14Linked to original sources
PubMed1985

Progesterone inhibits basal production of follicle-stimulating hormone in ovine pituitary cell culture.

Progesterone (P4) regulation of FSH production was studied in dispersed ovine pituitary cell culture. FSH secretion into culture media was decreased by 30-70% in the presence of 10(-7) M P4. The ED50 of P4 was 2 X 10(-10) M. Decreased FSH secretion was observed as early as 9-12 h after treatment with P4 (10(-7) M). FSH secretion rebounded to control levels after P4 was removed for 4 days. The intracellular FSH content also decreased after P4 treatment, suggesting that decreased FSH secretion is due to decreased biosynthesis. Two progesterone metabolites, 5 alpha-dihydroprogesterone and 17 alpha-hydroxyprogesterone, showed relative activities of 39% and 28%, respectively, compared to P4. Although testosterone (10(-7) M) inhibited FSH secretion by 10%, it may have done so only via conversion to estradiol. Dihydrotestosterone, androstanedione, and cortisol had no effect on FSH secretion. P4 consistently showed activity in 2-day-old ovine culture, but its effect essentially disappeared by day 10 or 12 of culture. When insulin was included in the culture medium (medium 199 plus 10% charcoal-treated anestrous ewe serum), the effect of P4 was maintained for up to 20 days, and FSH production, in general, was augmented with insulin. These results indicate that P4 can specifically inhibit FSH production in ovine pituitary cell culture, but there is no long term maintenance of the P4 response unless insulin is present. Finally, it is known that 17 beta-estradiol also inhibits FSH production in ovine pituitary cultures. Therefore, our results establish that FSH production is a phenomenon that is regulated similarly by both estradiol and P4.

17-alpha-Hydroxyprogesterone↗
PubMed1984

Molecular cloning of DNA complementary to bovine adrenal P450scc mRNA.

P450scc is the rate-limiting hormonally regulated enzyme that cleaves the cholesterol side chain. Translation of bovine adrenocortical mRNA and immunoprecipitation with rabbit anti-bovine P450scc indicates P450scc mRNA represents 1% of the total. DNA complementary to bovine adrenocortical mRNA was cloned in the PstI site of pBR322 by dC X dG tailing and high-efficiency transformation. A clone containing sequences complementary to P450scc mRNA was identified by hybrid-selected translation only when plasmid DNA was first purified by CsCl gradient centrifugation. As is often the case with hybrid-selected translation, the clone identified contains a small insert.

Adrenal Glands↗
PubMed1984

Interaction of small nuclear ribonucleoproteins with simian virus 40 in CV-1 cells: is U2 snRNA involved in regulating replication?

The small nuclear RNAs (snRNAs) in African Green Monkey kidney cells (CV-1 cells) were examined by polyacrylamide gel electrophoresis. Methodology was developed to improve their extraction from enriched fractions. Cellular fractionation studies and subsequent analysis of these RNAs indicate that they are tightly associated with chromatin. Treatment of cells with alpha-amanitin totally suppressed transcription of U1, U2, U4, U5, and partially suppressed transcription of U6, suggesting that these snRNAs are transcribed by RNA polymerase II. Upon infection of the cells by simian virus 40 (SV40), overall transcription of these and other cellular RNAs was stimulated. Gel filtration and formaldehyde crosslinking studies indicated that the ribonucleoproteins (snRNPs) containing snRNAs are associated with the viral minichromosome. Nucleotide sequence comparisons show extensive sequence complementarity between the 5' end of U2 RNA, the replication origin of SV40, and a prokaryotic RNA (RNA I) that is involved in control of plasmid replication. The clustered homologies between these RNAs and the association of snRNAs with the SV40 chromosome suggest that snRNAs may be evolutionarily related to small RNAs from plasmids and are consistent with an hypothesis that U2 RNA may be involved in DNA replication.

Antigens, Viral, Tumor↗
PubMed1984

Biosynthesis and secretion of follicle-stimulating hormone beta-subunit from ovine pituitary cultures: effect of 17 beta-estradiol treatment.

An assay was developed to detect tritium-labeled ovine FSH beta-subunit [( 3H]oFSH beta) secreted from primary ovine pituitary cultures. This procedure used affinity-enriched antibodies raised against reduced and carbamylmethylated oFSH beta (RCM-oFSH beta) in a two-cycle immunoextraction procedure. A discrete species with an apparent mol wt of 21,000 was detected in sodium dodecyl sulfate electrophoretic patterns of immunoextracts from culture medium. This species was identified as RCM-[3H]oFSH beta by its comigration with highly purified RCM-oFSH beta, its reduction in culture media after cultures were treated with 17 beta-estradiol, which normally decreases radioimmunoassayable oFSH; and its displacement from the extracting antibodies by excess unlabeled RCM-oFSH beta. The assay was used in a pulse-chase study to determine that [3H]oFSH beta is secreted within 1-2 h of its synthesis. Prior treatment of cultures with 17 beta-estradiol did not change this timing of secretion.

Animals↗
PubMed1984

Cortisol augments synthesis of growth hormone, but does not alter synthesis of prolactin and proopiomelanocortin, in the 120- to 125-day fetal ovine pituitary.

In adult animal pituitaries or in cultured pituitary tumor cells, glucocorticoids are regulators of GH, PRL, and proopiomelancortin (POMC) synthesis. However, ovine fetal plasma cortisol concentrations are low until shortly before parturition, suggesting that cortisol may not normally regulate hormone synthesis in the fetal pituitary. To investigate whether cortisol could affect fetal synthesis of GH, PRL, and POMC, we obtained fetal pituitary tissue from normal fetuses and from fetuses which had received cortisol infusion for 48 h. Tissues were labeled in short term organ culture and the newly synthesized proteins were displayed by two-dimensional gel electrophoresis and autoradiography. Results were quantified by computerized integration of the area and density of the autoradiographic spots after high resolution television scanning. Cortisol infusion augmented synthesis of GH in comparison to controls (P = 0.01), but did not alter PRL synthesis. Cortisol also did not inhibit POMC synthesis in either the anterior pituitary or the neurointermediate lobe. These data suggest that the pituitary-adrenocortical slow feedback inhibition of POMC synthesis is not functional in the ovine fetus at 120 to 125-days gestation, but that pituitary somatotropes are responsive to glucocorticoids at this stage of fetal development.

Animals↗
PubMed1984

Porcine ovarian inhibin preparations sensitize cultured ovine gonadotrophs to luteinizing hormone-releasing hormone.

Treatment of ovine pituitary cell cultures with an acetone powder of porcine follicular fluid (APPFF; 50 micrograms/ml) decreased FSH secretion 60%, did not alter basal LH secretion, but increased by 2- to 3-fold the effectiveness of LHRH or D-Lys6-LHRH (10(-8) M) in releasing LH. Chromatography of APPFF on Matrex Gel Red A (MGRA) yielded a protein fraction (MGRA-IV) in which both FSH-inhibiting and LHRH-enhancing activities were enriched 8-fold. Both activities were destroyed by trypsin, but both were highly resistant to heat. The apparent mol wt of the active substance(s) was greater than 10,000. The LHRH-enhancing effect of MGRA-IV was reversible and declined, with an apparent half-life of 7 h, when MGRA-IV treatment was discontinued. There was too little estrogen in either APPFF or MGRA-IV to account for any of the activities. These results demonstrate that porcine follicular fluid contains LHRH-enhancing activity along with classical inhibin activity and that both activities may be linked in one molecule. These dual activities may be important in a number of species.

Animals↗
PubMed1983

Regulation of beta-chain mRNA of ovine follicle-stimulating hormone by 17 beta-estradiol.

17 beta-Estradiol (E2) rapidly and reversibly decreases the synthesis of follicle-stimulating hormone (FSH) in primary dispersed cell cultures of ovine pituitaries. Similarly, E2 also causes a decrease in the messenger RNA for the beta subunit of ovine FSH (FSH beta-mRNA) as measured in an in vitro translation assay. These results are consistent with the concept that E2 directly regulates either transcription of the FSH beta gene or processing of FSH beta-mRNA in sheep. Of additional interest is the observation that pituitary cultures from various species respond differently to E2 in terms of FSH synthesis. For example, E2 stimulates FSH synthesis in rat pituitary cultures, has no effect in similar rabbit cultures, and inhibits FSH synthesis in ovine cultures. Thus, a set of eukaryotic 'mutants' may exist to aid studies of the effect of E2 on FSH synthesis and secretion.

Animals↗
PubMed1983

Clinical and laboratory findings in childhood mixed connective tissue disease: presence of antibody to ribonucleoprotein containing the small nuclear ribonucleic acid U1.

Seven children and adolescents are described with mixed connective tissue disease. The patients had varying clinical features, commonly characterized by Raynaud phenomenon, arthritis, abnormal pulmonary function, and esophageal dysmotility. All patients had speckled antinuclear antibodies and high titers (greater than 1:100,000) of antibodies to ribonuclease-sensitive extractable nuclear antigen. We prepared extractable nuclear material from radioactively labeled HeLa cells, analogous to classic extractable nuclear antigen. Sera from all seven patients precipitated ribonucleoprotein containing the small nuclear ribonucleic acid species U1 from the HeLa cell extract. Antibody to U1 ribonucleoprotein was not found in sera from 51 of 53 children and adults having a variety of autoimmune and other diseases, nor in sera from nine normal individuals. The U1 ribonucleoprotein appears to be the component of extractable nuclear antigen characteristically reacting with sera from patients with mixed connective tissue disease. The finding of a distinct molecular marker in all children studied with mixed connective tissue disease indicates that this is a distinct disease entity and not a heterogeneous population of immune disorders.

Adolescent↗
PubMed1983

Synthesis and platelet aggregation inhibiting activity of prostaglandin D analogues.

Several prostaglandin D (PGD) analogues have been synthesized, incorporating the following variations: (a) varying degrees of side-chain unsaturation, (b) C-9 hydroxy removed or in the unnatural 9 beta configuration, (c) metabolically stabilized analogues (e.g., 15-methyl, 16,16-dimethyl, 17-phenyl, etc.), and (d) delta 12 isomers resulting from decomposition of PGD2. With regard to their ability to inhibit adenosine diphosphate (ADP) induced human platelet aggregation: (a) PGD3 greater than or equal to PGD2 greater than PGD1 greater than 13,14-dihydro-PGD1, (b) the 9 beta- and 9-deoxy-PGD2 analogues are more potent than PGD2, (c) metabolically stabilized analogues with bulky substituents at or near C-15 have substantially reduced antiaggregatory activity relative to PGD2 and (d) the delta 12 isomers of PGD2 are much less active than PGD2.

Animals↗
PubMed1983

Synthesis and biological activity of prostaglandin lactones.

Most of the primary prostaglandins and several biologically important prostaglandin analogues were converted to 1,9-, 1,11- or 1,15-lactones, in order to investigate the biological profiles of these internal esters and to assess their potential as prodrugs for the corresponding open-chain hydroxy acids. In each case, the key lactonization step was done using Corey's "double activation" procedure (cyclization of omega-hydroxy-2-pyridinethiol esters). In general, the 1,9-lactones exhibited less than 1% of the biological activity of the parent hydroxy acids in the standard prostaglandin test systems. The 1,11- and 1,15-lactones, on the other hand, were essentially equal to the parent hydroxy acids as antifertility agents (a 4-day assay which would allow time for in vivo enzymatic lactone hydrolysis). The 1,11- and 1,15-lactones exhibited very low activity in acute or in vitro screens (e.g., rat blood pressure and gerbil colon stimulation), assays which more closely reflect the intrinsic activity of the lactones themselves. These results are consistent with the observed relative ease of enzymatic hydrolysis of the prostaglandin lactones (1,15 greater than or equal to 1,11 much greater than 1,9). Several of the lactones whose parent hydroxy acids are resistant to metabolic inactivation (e.g., 15-methyl, 16-phenoxy, and 17-phenyl) exhibited potent abortifacient activity in the hamster. These lactones, with greatly diminished activity in the blood pressure and smooth muscle assays (indicators of potential side effects), represent a therapeutically useful class of antifertility agents.

Animals↗
PubMed1983

Estrogenic and antiestrogenic effects of enclomiphene and zuclomiphene on gonadotropin secretion by ovine pituitary cells in culture.

17 beta-Estradiol (E2) alters gonadotropin secretions in ovine pituitary cell cultures by 1) augmenting the LH response to LHRH and 2) inhibiting the basal secretion of FSH. These responses were used to study the estrogenic and antiestrogenic effects of enclomiphene, zuclomiphene, and their commercial mixture, Clomid. In terms of the LH response to LHRH, Clomid and enclomiphene (10(-6) M) acted as E2 antagonists because they blocked the action of E2 (10(-10) M). By themselves they did not alter the LHRH response. Previous studies using rat pituitary cultures showed opposite results, since both enclomiphene and Clomid acted as estrogens in rat cultures. Apparently, species differences are involved. One conclusion from these data, therefore, is that it would be unwise to predict how Clomid acts in any species without direct experimentation. Zuclomiphene (10(-7)--10(-5) M) acted as a E2 agonist, in terms of the LH response to LHRH, because it sensitized cultures to LHRH. Surprisingly, all of the clomiphenes, including zuclomiphene, acted primarily as E2 antagonists when FSH secretion was observed. At a concentration of 10(-6) M, they blocked the inhibitory effects of E2 (10(-10 M) on FSH secretion, but enclomiphene and Clomid also exhibited minor estrogenic action when administered alone. The clear but unexpected role of zuclomiphene as both E2 agonist (as measured by LH secretion) and antagonist (as measured by FSH secretion) suggests that zuclomiphene may be an especially useful compound for dissociating the effects of E2 on FSH and LH in vivo, at least in sheep. Of more theoretical interest is the fact that zuclomiphene can affect two E2-responsive systems oppositely. Both systems appear to reside in the same subset of pituitary cells.

Animals↗
PubMed1983

Endocrine, histological, and biochemical studies of adrenocorticotropin-producing islet cell carcinoma of the pancreas in childhood with characterization of proopiomelanocortin.

Two 8-yr-old children, a boy and girl, are described with Cushing's syndrome secondary to ectopic ACTH-secreting pancreatic islet cell carcinomas. The girl, seen 28 yr ago, had strong presumptive evidence of ectopic ACTH production and hypercalcemia. The boy, studied recently, had strikingly elevated concentrations of plasma ACTH (1,500 pg/ml) and beta-lipotropin (beta LPH; 2,500 pg/ml) and showed no suppression of urinary 17-hydroxycorticoids or cortisol with low and high dose dexamethasone. He had increased plasma calcitonin (257 pg/ml), glucagon (442 pg/ml), lactate dehydrogenase (497 IU/liter), and alpha-fetoprotein (5,144 pg/ml). He also had hypokalemic alkalosis with elevated plasma deoxycorticosterone (70 ng/ml) and PRA (6.9 ng/ml.h) but normal plasma aldosterone (8.2 ng/dl) and 18-hydroxycorticosterone (7.6 ng/dl). Preoperative localization of the tumor was accomplished by computed tomographic scan of the abdomen with concurrent barium enema. Cell-free translation of the tumor mRNA produced authentic proopiomelanocortin of 35,000 mol wt, indicating that the ACTH and beta LPH were produced by the tumor from a common precursor. After removal of a large amount of metastatic tissue from the boy, clinical progression of the remaining tumor was monitored by measuring plasma ACTH and beta LPH. Episodic secretion of ACTH and beta LPH was demonstrated by taking frequent plasma samples while suppressing pituitary ACTH with oral dexamethasone. Chemotherapy and radiation proved ineffective in controlling the growth of his tumor.

Adenoma, Islet Cell↗
PubMed1982

Synthesis of growth hormone, prolactin, and proopiomelanocortin by intact adult ovine pituitary tissue in vitro.

Synthesis of GH, PRL, and proopiomelanocortin (POMC) by the adult ovine pituitary was examined by culturing intact tissue explants in vitro and analyzing newly synthesized proteins by two-dimensional gel electrophoresis. Spots on autoradiographs of two-dimensional gels were identified by comigration with known standards, by analysis of tryptic peptides, or both. GH and PRL are the predominant proteins synthesized in the adult ovine pituitary, but their syntheses could not be detected in the neurointermediate lobe of the pituitary, the cerebral cortex, hypothalamus, mammillary body, or placenta. As quantified by cell-free translation in rabbit reticulocyte lysate, 14% of female ovine pituitary mRNA encodes pre-GH and 30% encodes pre-PRL. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, ovine pre-GH and pre-PRL have molecular weights of 24,500 and 26,000, respectively, indicating they contain leader peptides of 26-30 amino acids, as found in bovine, rat, and human pre-GH and pre-PRL. POMC, the precursor to corticotropin, endorphin, and other peptides, was synthesized primarily in the neurointermediate lobe of the pituitary, but synthesis was also detected in the anterior lobe. POMC synthesis was not detected in the hypothalamus, cerebral cortex, mammillary body, or placenta.

Animals↗