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Biomedical subjects

W L Miller

Publications and source records attributed to W L Miller.

At least 271 records · Page 15Linked to original sources
PubMed1982

Synthesis and glycosylation of proopiomelanocortin by a Cushing tumor.

ACTH derives from a larger protein, proopiomelanocortin (POMC), which is also the precursor to lipotropin, endorphin, melanotropin, and other peptides. The sequence of steps by which POMC is processed to ACTH has been delineated in detail in cultured mouse tumor cells, but little is known about these steps in human pituitary adenomas. Synthesis and glycosylation of POMC were studied in a pituitary adenoma causing Cushing disease and in adjacent tissue by incubating intact tissue explants in medium containing [35S]methionine or [3H]glucosamine. Labeled tissue was analyzed by two-dimensional gel electrophoresis, and a spot cut from these gels was analyzed by paper electrophoresis of tryptic digests. The location of the spot and the patterns of its [35S]methionine-labeled tryptic fragments resembled those found previously in other ACTH-secreting tissues and hence this spot appears to be POMC. Both the tumor and the adjacent pituitary produced this POMC, although tumor production of POMC was greater. The electrophoretic patterns of the putative POMC tryptic peptides labeled with [35S]methionine from this tumor are similar to the patterns of POMC tryptic peptides from a Nelson tumor and an ectopic ACTH- producing tumor when analyzed by the same techniques. This suggests that the excess ACTH in all three diseases arises from the same precursor POMC molecule. Two-dimensional gel electrophoresis of [3H]glucosamine-labeled tumor suggests that the first proteolytic cleavage occurs between the alpha ACTH-(1-39) and beta-lipotropin sequences and that a subsequent cleavage occurs between the N-terminal region of POMC and ACTH. Paper electrophoresis of [3H]glucosamine-labeled tryptic glycopeptides reveals at least three radioactive peaks, suggesting that the two known glycosylation sites in human POMC may be variably glycosylated.

Adenoma↗
PubMed1981

mRNA for ovine follicle-stimulating hormone beta-chain. An in vitro translational assay.

Polyadenylated mRNA from pituitary glands of castrate male sheep was used to demonstrate the presence of an mRNA coding for the beta-subunit of follicle-stimulating hormone (FSH). The mRNA fraction was translated in a wheat germ membrane-free system in the presence of [35S]cysteine. Affinity-purified antibodies, raised against the reduced and carbamylmethylated beta-subunit of ovine FSH (RCM-oFSH beta), were used to extract an in vitro translation product of Mr approximately equal to 15,000, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibody binding of the product was readily blocked by unlabeled RCM-oFSH beta, but not by the RCM derivatives of the alpha- or beta-subunits of ovine luteinizing hormone. Tryptic peptide analysis using reversed phase high performance liquid chromatography yielded very similar patterns for the immunospecific translation product and authentic oFSH beta. FSH beta translation product was found to account for approximately 0.1-0.4% of the total isotope incorporation when mRNA preparations from castrate male sheep were used. Backgrounds for the immunoextraction procedure were kept at approximately 0.02% by using a unique two-cycle specific extraction method.

Animals↗
PubMed1981

Cloning of bovine prolactin cDNA and evolutionary implications of its sequence.

Prolactin, growth hormone, and chorionic somatomammotropin (placental lactogen) constitute a set of related polypeptides believed to derive from a common evolutionary ancestor protein. We have cloned and sequenced DNA complementary to the mRNA coding for bovine prolactin. This cDNA contains 702 bases corresponding to 10 amino acids in the leader peptide, all 199 amino acids of the hormone, and 75 nucleotides in the 3' untranslated region of the mRNA. Nucleotide sequence analysis of this cDNA permitted the identification of 10 amino acids in the signal peptide, plus the correction or elucidation of amino acid assignments at 16 sites where aspartic and glutamic acids had not been distinguished from their amides by amino acid sequencing. Codon usage in bovine prolactin mRNA is nonrandom, but, similarly to rat and human prolactins, it does not exhibit the strong preference for G or C in codon third positions seen in bovine, rat, and human growth hormone mRNAs. The translational termination signal in bovine prolactin in UAA, also the same as in rat and human prolactins and differing from the UAG "stop" codon used in bovine, rat, and human growth hormones and human chorionic somatomammotropin. The amino acid and mRNA nucleotide sequences of bovine, rat, and human prolactins and growth hormones were compared by several techniques based on various theories of molecular evolution. The comparison of prolactin to growth hormone is consistent in all three species, suggesting that the genes for these two hormones diverged about 350 million years ago. However, comparisons among the three prolactins or among the three growth hormones to determine the times of evolutionary divergence of the three species generated values that were inconsistent with each other and with the fossil record. Analysis of these discrepancies suggests that the genes for prolactin and growth hormone may now be evolving by different mechanisms.

Amino Acid Sequence↗
PubMed1981

Antiestrogens and ovine gonadotrophs: antagonism of estrogen-induced changes in gonadotropin secretions.

17 beta-Estradiol (E2) was found to affect gonadotrophs in long term ovine pituitary cell culture in several ways which were easily measured. It inhibited basal FSH secretion and, concomitantly, stimulated LH secretion. High physiological levels of E2 (10(-10) M; 27 pg/ml) were able to elicit about 70% of the maximal responses, and the effects were developed fully between 6-24 h after the addition of E2. In culture, the antiestrogens (AEs) tamoxifen, nafoxidine, and CI-680 antagonized both the inhibitory and stimulatory effects of E2 (10(-10) M) on gonadotropin secretion in a dose-dependent manner, and 10(-6) M AE was fully effective. At concentrations of 10(-6) M or lower, the AEs showed no significant intrinsic estrogenicity. At higher concentrations, the AEs progressively altered gonadotropin secretion in an apparently nonspecific fashion. Preincubations of cultures with AEs (3.3 X 10(-6) M) antagonized E2 action (10(-10) M) for approximately 18-24 h after the removal of AEs. Tamoxifen appeared to be the most potent AE tested. In conclusion, long term ovine pituitary cell cultures have provided a useful in vitro system for testing the biological potencies of AE analogs. AEs appeared to act as pure estrogen antagonists in ovine gonadotrophs.

Animals↗
PubMed1981

Estrogen regulation of follicle-stimulating hormone production in vitro: species variation.

Pituitary cell cultures were used to investigate factors, such as species, sex, and age, which might be associated with differences in FSH regulation by estrogens. 17 beta-Estradiol at 10(-9) M inhibited spontaneous secretion of FSH in sheep, pig, and cow cell cultures (65-95%), had no detectable effect on rabbit cell cultures, and stimulated spontaneous secretion of FSH in rat cell cultures (1.5- to 5-fold). These differential effects of 17 beta-estradiol were not due to changes in cell division; gonadotrophs did not divide in culture, and general cell division was unaffected by 17 beta-estradiol. Differences in 17 beta-estradiol action on FSH secretion were not associated wih age or sex. Spontaneous secretion of FSH from pituitary cell cultures also varied according to species and was ranked: rabbit approximately equal to sheep > pig > rat > cow. Secretion and synthesis of FSH were tightly coupled in at least three of these species. Nonestrogenic steroids, such as androgens, progestins, and corticosterone, exhibited only minor effects on spontaneous secretion of FSH in either male or female sheep cell culture. These results clearly show that estrogens are capable of altering FSH secretion and synthesis differentially in pituitary cell cultures from different species. It is possible that the differences noted in pituitary cell cultures from various species also exist in vivo.

Age Factors↗
PubMed1980

Molecular cloning of DNA complementary to bovine growth hormone mRNA.

We have cloned DNA complementary to mRNA coding for bovine growth hormone (bGH). Double-stranded DNA complementary to bovine pituitary mRNA was inserted into the Pst I site of plasmid pBR322 by the dC x dG tailing technique and amplified in E. coli x 1776. A recombinant plasmid containing bGH cDNA ws identified by hybridization to cloned rat growth hormone cDNA. It contains the entire coding and 3'-untranslated regions and 31 bases in the 5'-untranslated region. Nucleotide sequence analysis determined the sequence of the 26-amino acid signal peptide and confirmed the published amino acid sequence of the secreted hormone at all but 2 residues. Codon usage is nonrandom, with 81.7% of the codons ending in G or C. The nucleotide sequence of bGH mRNA is 83.9% homologous with rat GH mRNA and 76.5% homologous with human GH mRNA, while the respective amino acid sequence homologies are 83.5% and 66.8%.

Amino Acid Sequence↗
PubMed1980

Processing of the precursor to corticotropin and beta-lipotropin in humans.

The biosynthesis of human corticotropin (ACTH) was studied in organ culture of pituitary adenomas and by translating mRNA from an ectopic ACTH-producing tumor in a cell-free system. Peptides similar to human ACTH, beta-lipotropin, and the amino-terminal glycopeptide are cleaved from a common precursor with an apparent molecular weight of 35,000 on sodium dodecyl sulfate/polyacrylamide gels. The precursors synthesized in pituitary and ectopic ACTH-producing tissues are indistinguishable. The cleavage sites of the peptide chain appear to be similar to those previously deduced for murine and bovine ACTH. Immunoprecipitation studies suggest that the primary structure of the precursor peptide is similar in all three species. However, glycosylation is different in the human and murine precursors: the precursor to human ACTH appears to be glycosylated only in the amino-terminal fragment, not in the ACTH or beta-lipotropin sequences. Studies with an autopsied normal human pituitary suggest that neither normal nor adenomatous pituitary tissue glycosylates the ACTH sequence.

Adrenocorticotropic Hormone↗
PubMed1980

Cloning of DNA complementary to bovine prolactin mRNA.

We have cloned DNA complementary to mRNA coding for bovine prolactin (bPrl). Double-stranded cDNA prepared from bovine pituitary mRNA was inserted into the Pst I site of plasmid bPR322 by the dC x dG tailing technique and amplified in E. coli chi 1776. A recombinant plasmid containing bPrl cDNa was identified by hybridization to cloned rat Prl cDNA. It contains cDNA corresponding to the region of the mRNA coding for the carboxy terminal 101 amino acids of bPrl, as well as 42 nucleotides in the 3' untranslated region of the mRNA. Nucleotide sequencing confirmed the amino acid sequencing of this region of bPrl, and permitted the assignment of asparagine or glutamic acid at seven previously equivocal loci. Codon use in bPrl mRNA is comparable to that found in rat and human Prl mRNA's and differs from that in bovine, rat, and human growth hormone mRNA's.

Amino Acid Sequence↗
PubMed1980

Casework and the medical metaphor.

After discussing metaphors in general, the author describes the medical metaphor--or medical model--that was for many years the basis of casework. The author suggests that the medical model has had a detrimental influence on the profession of social work, and he offers alternative models.

Decision Making↗
PubMed1980

Synthesis and biological activity of 9-deoxo-9-methylene and related prostaglandins.

A number of PGE analogs have been synthesized in which the C-9 carbonyl group has been replaced by an exo-methylene group. These chemically stable 9-deoxo-9-methylene-PGEs exhibit biological profiles very similar to their less stable PGE relatives. 9-Deoxo-16,16-dimethyl-9-methylene-PGE2 (7) retains the useful uterine-stimulating potency of 16,16-dimethyl-PGE2 but is approximately 300 times less enteropooling in the rat. In preliminary clinical trials, 7 has shown efficacy for pregnancy termination by the oral and vaginal routes of administration, as well as relative freedom from gastrointestinal side effects.

16,16-Dimethylprostaglandin E2↗
PubMed1979

Prostaglandin prodrugs. I: Stabilization of dinoprostone (prostaglandin E2) in solid state through formation of crystalline C1-phenyl esters.

Dinoprostone para-substituted phenyl esters were synthesized in attempt to improve the solid-state stability of the parent prostaglandin. A phenol series covering a wide melting-point range was employed, and a linear relationship was observed between the phenol melting points and the resulting prostaglandin C1-ester melting points. The crystalline esters showed improved solid-state stability over the parent compound, and many esters were biologically active.

Animals↗
PubMed1979

Prostaglandin prodrugs. II: New method for synthesizing prostaglandin C1-aliphatic esters.

A new method for synthesizing C1-aliphatic esters of dinoprost and dinoprostone without using hydroxyl protective groups is described. Reaction of the prostaglandin with an alkyl halide in the presence of the sterically hindered amine N,N-diisopropylethylamine proceeds smoothly to give C1-esters in various solvents at ambient or slightly elevated temperatures. Polar solvents were strongly catalytic, and even the hindered tert-butyl esters were synthesized by employing solvents such as dimethylformamide or dimethyl sulfoxide. Biological evaluation in the hamster antifertility assay showed that some esters maintained high bioactivity.

Animals↗