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E M Wilson

Publications and source records attributed to E M Wilson.

At least 109 records · Page 6Linked to original sources
PubMed1991

Calcium/calmodulin regulation of the rat prolactin gene is conferred by the proximal enhancer region.

The effects of the calcium/calmodulin signaling system on expression of the rat PRL gene were studied in rat pituitary GH3 cells using two specific naphthalene sulfonamide calmodulin (CaM) antagonist drugs, W7 and a more potent and more highly specific iodo-derivative, 5-iodo-1-C8. PRL (but not GH) mRNA accumulation was markedly inhibited by W7, which in coincubations abolished the stimulation normally seen with TRH. Transient transfection assays showed that expression of the reporter gene chloramphenicol acetyl transferase (CAT) linked to 5'-flanking sequences from the PRL gene was inhibited by the calcium-channel blocker verapamil and by the two CaM antagonists. The calcium effects showed partial promoter specificity, in that transcription of PRL-CAT constructs was markedly inhibited by verapamil, but the Rous sarcoma virus-CAT construct also showed significant inhibition, whereas the pBL-CAT2 construct was unaffected. Three hundred ninety five base pairs were sufficient to confer the full inhibitory effect of calcium channel blockade or CaM antagonist seen with longer constructs. The data indicate that CaM is important for PRL gene transcription, and that the effects of CaM are exerted on DNA sequences within the proximal 395bp of prolactin 5'-flanking DNA.

Animals↗
PubMed1991

A frameshift mutation destabilizes androgen receptor messenger RNA in the Tfm mouse.

A composite mouse androgen receptor DNA sequence was obtained by amplifying genomic DNA or cDNA using the polymerase chain reaction. The open reading frame was 2,697 basepairs, encoding a polypeptide of 899 amino acids (98,204 mol wt). Amino acid sequence comparisons indicated that the mouse androgen receptor (AR) is 97% homologous with rat AR and 83% with human AR. The amino acid sequences of the three receptors are identical within the DNA- and steroid-binding domains. Northern blot analysis revealed the predominant mouse AR mRNA to be 10 kilobases (kb). A 1.7-kb mRNA species was detected in mouse kidney using a cDNA probe containing only 5' untranslated AR sequence. Lack of hybridization with AR-coding sequence probes suggested that the 1.7-kb mRNA was not a truncated form of AR mRNA. Sequencing of genomic DNA isolated from testicular feminized (Tfm) mice revealed a single base deletion in the N-terminal domain, resulting in a frameshift mutation. Cycloheximide treatment caused a dramatic increase in AR mRNA in kidneys of Tfm mice, but not wild-type mice, suggesting that the Tfm mutation results in an unstable AR mRNA.

Amino Acid Sequence↗
PubMed1990

A single base mutation in the androgen receptor gene causes androgen insensitivity in the testicular feminized rat.

The complete form of androgen insensitivity is an inherited X-linked syndrome in which genetic males fail to undergo masculinization in utero due to defective functioning of the androgen receptor (AR). The molecular basis of androgen insensitivity was investigated in the testicular feminized (Tfm) rat with this syndrome. AR mRNA size and amount, as well as nuclear AR protein revealed by immunocytochemistry, suggested normal expression of the AR gene in the Tfm rat. Sequence analysis of the AR coding region from Tfm and wild-type littermate male rats revealed a single transition mutation, guanine to adenine, within exon E, changing arginine 734 to glutamine within the steroid-binding domain of the AR. This arginine is highly conserved among the family of nuclear receptors and may be part of a phosphorylation recognition site. A recreated mutant AR (Arg734----Gln) expressed in COS cells had only 10-15% of the androgen-binding capacity of wild-type AR; the reduced androgen-binding capacity was similar to that of AR in tissue extracts of the Tfm rat. Stimulation of transcriptional activity by the recreated mutant AR was reduced relative to wild-type AR in cotransfection assays in CV1 cells using as reporter plasmid the mouse mammary tumor virus promoter linked to the chloramphenicol acetyltransferase gene. Thus, arginine 734 appears essential for normal AR function both in androgen binding and transcriptional activation. Absence of these functions results in androgen insensitivity and lack of male sexual development.

Amino Acid Sequence↗
PubMed1990

Expression and localization of androgen receptor in the R-3327 Dunning rat prostatic adenocarcinoma.

The Dunning R-3327 rat prostatic adenocarcinoma and its sublines have been developed as a model system to study prostate tumor progression. We have used this system to study the changes in androgen receptor (AR) and AR mRNA expression which occur during tumor progression from androgen dependent to androgen independent growth. Dorsal prostate and all tumor sublines contained a 10-kilobase AR mRNA on Northern blot analysis. The levels of AR mRNA in each subline compared to dorsal prostate (100%) were: H (75%) greater than G (48%) greater than HI (25%) greater than HI-F = AT-1 = AT-3 = MAT-Lu = MAT-Ly-Lu = less than 5%. Immunocytochemistry showed AR predominantly in acinar epithelial cells of dorsal prostate and the androgen sensitive H subline. In the H subline, both acinar epithelial cells and locally invasive adenocarcinoma cells within the stroma showed positive immunostaining. The androgen responsive, anaplastic G subline also showed strong positive immunostaining. The androgen resistant AT-1 and MAT-Lu sublines lacked immunostaining for the AR. Steroid autoradiography revealed a similar cellular distribution of AR. These data suggest that in the Dunning system the loss of androgen binding and responsiveness is primarily due to selective changes in gene expression and not to gene rearrangements or posttranscriptional or translational modification of the AR mRNA or protein.

Adenocarcinoma↗
PubMed1990

Augmentation of glutathione in the fluid lining the epithelium of the lower respiratory tract by directly administering glutathione aerosol.

Glutathione (GSH), a cysteine-containing tripeptide, functions as an antioxidant, provides cells with cysteine, and is required for optimal function of the immune system. Because the epithelial-lining fluid (ELF) of the lower respiratory tract normally contains high GSH levels and lung ELF GSH deficiency states can exist, we evaluated the feasibility of augmenting lung ELF GSH levels by (i) administering GSH to sheep i.v. and by direct aerosolization and then (ii) measuring the GSH levels in lung ELF, lung lymph, venous plasma, and urine. When GSH (600 mg) was administered i.v. (n = 11), GSH levels in venous plasma, lung lymph, and ELF rose, but only transiently, suggesting the i.v. route would not deliver adequate GSH to the alveolar epithelial surface. For directly administering GSH to the lung by the aerosol route, in vitro studies were first conducted to show that greater than 50% of a GSH solution could be converted to droplets less than 3 microns in aerodynamic diameter without oxidizing the GSH. To target functional GSH to the lower respiratory tract, an aerosolized solution of GSH (600 mg) was administered to sheep (n = 12). Significantly, the GSH level in ELF increased 7-fold at 30 min (preaerosol, 45.7 +/- 10 microM; 30-min postaerosol, 337 +/- 64 microM; P less than 0.001). The ELF GSH levels remained above baseline at 1 hr (P less than 0.01), returning toward baseline over a 2-hr period. In contrast, GSH levels in lung lymph, venous plasma, and urine were not significantly increased during the period--i.e., aerosol therapy selectively augmented the GSH levels only at the lung epithelial surface. Thus, functional GSH can be delivered by aerosol to directly augment the ELF GSH levels of the lower respiratory tract. Such an approach may prove useful in treating a variety of lung disorders.

Aerosols↗
PubMed1990

Use of a cell-free system to identify the vaccinia virus L1R gene product as the major late myristylated virion protein M25.

A 25-kDa vaccinia virus (VV) virion protein, designated M25, is modified in vivo by covalent addition of myristic acid. The predicted amino acid sequences of all VV open reading frames which have been reported were searched for the sequence M-G-X-X-X-(S/T/A), which has been proposed to be the consensus recognition signal for cotranslational modification of proteins by N-myristyltransferase. This conserved signal was found at the amino terminus of a single locus, which corresponded to the leftmost rightward-reading open reading frame (L1R) initiating within the VV HindIII L DNA fragment. By using synthetic oligonucleotides in concert with polymerase chain reaction techniques, a chimeric gene consisting of open reading fram L1R fused to a bacteriophage T7 promoter was constructed and cloned into a plasmid vector. Transcripts derived from the wild-type expression plasmid (designated pL1G1) were translated in vitro in a wheat germ extract to yield a polypeptide with an apparent molecular mass of 25 kDa. This polypeptide was labeled with either [35S]methionine or [3H]myristic acid and comigrated with in vivo-labeled protein M25 on sodium dodecyl sulfate-polyacrylamide gels. Polyclonal antiserum generated in rabbits against a trpE:L1R fusion protein immunoprecipitated a 25-kDa protein labeled either in vitro (the L1R gene product, designated protein L1) or in vivo (from purified VV, protein M25), identifying the M25 protein as the gene product of open reading frame L1R. Chromatographic analysis of the protein L1-bound fatty acid moieties liberated after acid methanolysis resulted in recovery of greater than 99% of the fatty acid as myristate-associated label. Cell-free translation of proteins derived from a set of deletions from the carboxy terminus of the open reading frame L1R suggested that the site of myristylation maps near the amino terminus of protein L1. This hypothesis was supported by cell-free translation of mutant L1R transcripts in which the penultimate glycine codon had been altered by site-directed mutagenesis to encode either an aspartic acid (pL1D1) or alanine (pL1A1) residue. In both cases, the mutant transcripts were translated into a 25-kDa protein which could be labeled in vitro with [35S]methionine but not with [3H]myristic acid. These data demonstrate that VV open reading frame L1R encodes a myristylated protein and provide evidence that the site of modification of protein L1 is the amino-terminal glycine residue.

Amino Acid Sequence↗
PubMed1990

Immunohistochemical localization of the androgen receptor in rat and human tissues.

Immunohistochemical localization of the androgen receptor (AR) was performed in reproductive tissues, submaxillary gland, pituitary, and brain of the rat and in human prostate. AR was visualized using either of two polyclonal antibodies raised against peptides with sequences derived from rat and human AR. Tissue sections of 6-8 microns, frozen in isopentane and fixed in paraformaldehyde, were stained using immunoglobulin G fractions of immune, preimmune, and peptide-adsorbed immune sera in the avidin-biotin peroxidase procedure. AR was prominent in nuclei of acinar epithelial cells of epididymis, ventral prostate, seminal vesicle, and ductus deferens from the intact rat. Androgen withdrawal, 3 days after castration, resulted in the loss of receptor immunostaining, which was restored within 15 min of androgen administration. Stromal cell staining was absent or weak in the ventral prostate of intact rats, but was more evident in the epididymis. AR was confined to nuclei of cells within and bordering the interstitial compartment of the testis, including Sertoli cells, peritubular myoid cells, and interstitial cells, and was undetectable in germ cells. Submaxillary gland epithelial cells and a population of rat anterior pituitary cells showed strong nuclear staining of AR. In rat brain, AR was present in the medial preoptic, arcurate, and ventromedial nuclei of the hypothalamus, the medial nucleus of the amygdala, the CA-1 hippocampus, and the cortex. AR was prominent in acinar epithelial cells in human benign prostatic hyperplasia and was also present in stroma of fibromuscular benign hyperplasia. Heterogeneous staining was observed in stromal and epithelial cells of prostatic adenocarcinoma. The results of these studies indicate that AR can be detected immunohistochemically in a variety of tissues and cell types using antipeptide polyclonal antibodies. The presence of AR in tissues correlated with their known androgen responsiveness.

Animals↗
PubMed1990

Autologous down-regulation of androgen receptor messenger ribonucleic acid.

Autoregulation of androgen receptor (AR) mRNA was investigated using Northern blot analysis with AR cDNA fragments as probes. The amount of AR mRNA increased 2- to 10-fold with androgen withdrawal and decreased below control levels after androgen stimulation in rat ventral prostate, coagulating gland, epididymis, seminal vesicle, kidney, and brain, and in a human prostate cancer cell line, LNCaP. In rat ventral prostate, AR mRNA increased 2- to 3-fold within 24 h after castration and remained elevated for 4 days. Treatment with testosterone propionate beginning 24 h after castration reduced ventral prostate AR mRNA 4-fold within 8 h of androgen replacement. Administration of estradiol 24 h after castration had no significant effect on prostatic AR mRNA. Androgens, including testosterone and the synthetic androgen methyltrienolone (R1881), or the antiandrogen cyproterone acetate down-regulated AR mRNA in vitro in LNCaP cells, whereas estradiol was without effect. Administration of testosterone propionate to rats with androgen insensitivity did not decrease AR mRNA. Down-regulation of AR mRNA by androgen is therefore a receptor-mediated process which occurs in vivo in rat tissues that differ in androgen responsiveness and in cultured human prostate cells.

Animals↗
PubMed1990

Functional characterization of naturally occurring mutant androgen receptors from subjects with complete androgen insensitivity.

Mutations in the androgen receptor (AR) are thought to cause complete androgen insensitivity (CAIS) in 46,XY human subjects who have a female phenotype despite normal adult male concentrations of plasma testosterone. Assays of AR binding in cultured skin fibroblasts from subjects with CAIS show either an apparent absence of AR (AR-) or normal levels of AR (AR+) binding. In several subjects with CAIS, AR-, no gross AR mutation was detected by Southern blot analyses of genomic DNA and normal sized 10 kilobase mRNA was present on Northern blots of poly(A+) RNA from cultured genital skin fibroblasts. We have used the polymerase chain reaction to amplify individual exons within the human AR gene of subjects with CAIS and have identified point mutations in three subjects. In one AR- subject (R774C), amino acid 774 was changed from arginine (CGC) to cysteine (TGC), in another AR- subject (R831Q), arginine (CGA) was changed to glutamine (CAA) at position 831, and in an AR+ subject (V866M) a methionine (ATG) was substituted for valine (GTG) at position 866. Transfection of wild type and mutant AR cDNA clones into COS cells results in detection of AR protein by immunoblotting. AR ligand binding activity is absent in cells transfected with AR mutants R774C and R831Q, but present with AR mutant V866M. Androgen binding in cells transfected with AR mutant V866M has a 6-fold lower apparent binding affinity than that of wild-type AR. Transcriptional activation of the MMTV-CAT reporter gene was androgen dependent and specific and nearly maximal at physiological concentrations (10(-10) M) of androgen when wild-type AR was transfected into cells, whereas neither AR mutants R774C nor R831Q were able to stimulate CAT activity even at 10(-8) M androgen. AR mutant V866M was able to stimulate CAT activity but the androgen dose dependency was shifted toward pharmacological concentrations of steroid that exceed in vivo levels. The molecular basis of CAIS in humans exhibits genetic heterogeneity. Our study shows that some cases of CAIS are explained by an inability to form a functional AR-steroid complex and hence, the AR is unable to activate transcription of genes essential for male sex differentiation during fetal development.

Amino Acid Sequence↗
PubMed1990

Expression of recombinant androgen receptor in cultured mammalian cells.

Full-length rat and human androgen receptor (AR) cDNA clones were expressed in COS-7 and CV1 monkey kidney cells to analyze the AR protein using immunological and cotransfection techniques. The studies were aided by the development of two rabbit polyclonal antibodies, designated AR32 and AR52, directed against epitopes within the N-terminal region of AR. Each antibody recognizes native AR by sucrose gradient analysis and detects a 114-kilodalton protein in COS cells transfected with human or rat AR cDNA. Covalent binding of the synthetic androgen [3H]methyltrienolone (R1881) to the 114-kDa protein was saturable. The endogenous native AR was similarly 114 kDa on immunoblots of a human prostate adenocarcinoma cell line, LNCaP, and rat sex accessory gland extracts. AR was localized in nuclei of transfected COS cells and in LNCaP cells by immunocytochemical staining. Androgen induction of CAT activity was dose dependent in CV1 cells cotransfected with the AR expression vector and a reporter plasmid containing the mouse mammary tumor virus promoter linked to the chloramphenicol acetyltransferase gene. It is concluded that antipeptide antibodies are useful reagents in characterizing both native and denatured forms of the AR protein. The 114-kDa protein expressed transiently in cultured cells represents the full-length AR protein, has a molecular size equivalent to that of endogenous AR, and mediates androgen-dependent transcriptional activation in CV1 cells.

Amino Acid Sequence↗
PubMed1989

Primary structure and androgen regulation of a 20-kilodalton protein specific to rat ventral prostate.

Nuclear and cytosolic forms of a 20-kdalton rat ventral prostate protein were purified and partially sequenced from their N-termini. Isolated nuclei were treated with micrococcal nuclease and extracted in 0.6 M NaCl, and proteins were separated by affinity chromatography on Matrex gel green A, ammonium sulfate fractionation, and fast protein liquid chromatography on Superose 12. The 43 amino acid N-terminal sequence of the nuclear 20-kdalton protein was identical with the cytosolic protein except it lacked 7 N-terminal amino acids present in the cytosolic form. The DNA sequence of a full-length complementary DNA clone isolated from a ventral prostate gt11 library extended the N-terminal sequence of the cytosolic form by an additional nine amino acids from the predicted initiation methionine. The cDNA included the nucleotide sequence for the 43 amino acid N-terminal sequence of the purified 20-kdalton protein and predicted molecular weights of 16,686, 17,521, and 18,650, respectively, for the nuclear, cytoplasmic, and nonprocessed proteins. Northern blot analyses of reproductive tract tissue RNAs using the 20-kdalton protein cDNA as probe revealed a single mRNA species of 0.92 kb detectable only in extracts of rat ventral prostate. Expression of the 0.92-kb mRNA was androgen dependent since the mRNA was undetectable in extracts obtained 4 days after castration and was restored 16 h after restimulation with androgen.

Amino Acid Sequence↗
PubMed1989

Expression vector pT7:TKII for the synthesis of authentic biologically active RNA encoding vaccinia virus thymidine kinase.

A transcription vector, pT7: TKII, was constructed by a novel application of the polymerase chain reaction. Chimeric oligodeoxynucleotides were used to direct the synthesis of a DNA fragment which consisted of a truncated bacteriophage T7 promoter element fused to the vaccinia virus (VV) thymidine kinase gene (tk). This fragment was cloned into a pUC118 plasmid and sequenced to ensure no mutations had occurred during its synthesis. When linearized at the 3' end of the VV tk gene at the BamHI site located in the polylinker region of the vector, pT7:TKII was efficiently transcribed by T7 RNA polymerase into a 595 nucleotide transcript whose 5' end was identical to that found on authentic nascent VV tk mRNA. When translated in a rabbit reticulocyte lysate system, the synthetic VV tk RNA was shown to be biologically active in that it directed the synthesis of a 20-kDa protein which assembled into an enzymatically active 80-kDa tetrameric complex which was indistinguishable from VV thymidine kinase (TK) enzyme isolated from VV-infected cells. The pT7:TKII vector provides a powerful approach with which: (i) to investigate the translational and posttranslational regulation of the VV tk gene; (ii) to use directed genetics to identify potential cis-acting regulatory sequences or structures present within the VV tk RNA; and (iii) to apply protein engineering procedures to identify the catalytic, allosteric and subunit interactive domains of the VV TK enzyme. As an example, the translational effects of adding a m7G cap structure to the pT7:TKII-derived VV tk RNA are presented.

Cell-Free System↗
PubMed1989

A form of basic fibroblast growth factor is secreted into the adluminal fluid of the rat coagulating gland.

Rat coagulating gland and its secreted fluid contain a mitogenic substance that, by numerous criteria, appears to be similar to basic fibroblast growth factor (basic FGF). It is a potent competence factor in the BALB/c3T3 mouse embryo fibroblast assay, elutes from heparin-agarose at concentrations of NaCl greater than 1.0 M, and has an isoelectric point (pI) between 8 and 9. In coagulating gland fluid, its molecular weight was 20,000 +/- 5,000, as indicated by gel filtration on Sephadex G-200. Western blot analysis of purified factor from coagulating gland cytosol and fluid, respectively, revealed immunoreactive bands of 16,000 and 17,000 relative molecular weight similar to that of purified bovine basic FGF.

Animals↗
PubMed1989

Sequence of the intron/exon junctions of the coding region of the human androgen receptor gene and identification of a point mutation in a family with complete androgen insensitivity.

Androgens act through a receptor protein (AR) to mediate sex differentiation and development of the male phenotype. We have isolated the eight exons in the amino acid coding region of the AR gene from a human X chromosome library. Nucleotide sequences of the AR gene intron/exon boundaries were determined for use in designing synthetic oligonucleotide primers to bracket coding exons for amplification by the polymerase chain reaction. Genomic DNA was amplified from 46,XY phenotypic female siblings with complete androgen insensitivity syndrome. AR binding affinity for dihydrotestosterone in the affected siblings was lower than in normal males, but the binding capacity was normal. Sequence analysis of amplified exons demonstrated within the AR steroid-binding domain (exon G) a single guanine to adenine mutation, resulting in replacement of valine with methionine at amino acid residue 866. As expected, the carrier mother had both normal and mutant AR genes. Thus, a single point mutation in the steroid-binding domain of the AR gene correlated with the expression of an AR protein ineffective in stimulating male sexual development.

Alleles↗
PubMed1989

Identification of androgen receptors in normal human osteoblast-like cells.

The sex steroids, androgens and estrogens, are major regulators of bone metabolism. However, whether these hormones act on bone cells through direct or indirect mechanisms has remained unclear. A nuclear binding assay recently used to demonstrate estrogen receptors in bone [Eriksen, E.F., Colvard, D.S., Berg, N.J., Graham, M.L., Mann, K.G., Spelsberg, T.C. & Riggs, B.L. (1988) Science 241, 84-86] was used to identify specific nuclear binding of a tritiated synthetic androgen, [3H]R1881 (methyltrienolone), in 21 of 25 (84%) human osteoblast-like cell strains and a concentration of bound steroid receptors of 821 +/- 140 (mean +/- SEM) molecules per cell nucleus. Binding was saturable and steroid-specific. Androgen receptor gene expression in osteoblasts was confirmed by RNA blot analysis. Relative concentrations of androgen and estrogen receptors were compared by measuring specific nuclear estrogen binding. Nuclear binding of [3H]estradiol was observed in 27 of 30 (90%) cell strains; the concentration of bound estradiol receptor was 1537 +/- 221 molecules per cell nucleus. The concentrations of nuclear binding sites were similar in males and females for both [3H]R1881 and [3H]estradiol. We conclude that both androgens and estrogens act directly on human bone cells through their respective receptor-mediated mechanisms.

Binding, Competitive↗
PubMed1989

Regulation of c-fos messenger ribonucleic acid by fibroblast growth factor in cultured Sertoli cells.

The data reported here demonstrate that basic FGF and Sertoli-cell-derived FGF have rapid stimulatory effects on c-fos mRNA levels. Basic FGF appears to utilize a signal transduction pathway that is distinct from that used by FSH and serum but similar in its potency and transiency. This is consistent with other reports in the literature on FGFs mechanism of action. For example, a monoclonal antibody to phosphatidyl 4,5-biphosphate will block the effects of PDGF on thymidine incorporation into NIH 3T3 cells but has no effect on basic FGF. Our data support the emerging possibility of a novel pathway mediating FGF actions. A similar precedent has been established for serotonin-induced DNA synthesis in smooth muscle cells. The rat Sertoli cell, with both FSH and FGF rapidly inducing c-fos, is an excellent model system for addressing the mechanism of action of basic FGF, the specificity of the c-fos response and the role of FGF in the reproductive system. Stimulation of c-fos mRNA by basic FGF in the cultured Sertoli cell presents questions regarding the role of FGF and c-fos in the male reproductive system. Basic FGF has been shown to stimulate cell division and plasminogen activator activity in cultured immature porcine Sertoli cells. Plasminogen activator may play a critical role in the tissue remodeling required for spermiogenesis. Interestingly, fos has been shown to be expressed preferentially in pachytene spermatocytes in the mouse. These observations taken together with the finding that basic FGF mRNA levels in Xenopus oocytes are markedly elevated, suggests that basic FGF may be important for Sertoli cell function, spermatogonial cell proliferation and subsequent meiotic and sperm maturational events.

Animals↗