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Biomedical subjects

E M Wilson

Publications and source records attributed to E M Wilson.

At least 127 records · Page 7Linked to original sources
PubMed1989

Developmental expression of an androgen-regulated epididymal protein.

Acidic epididymal glycoprotein (AEG), an androgen-regulated secretory protein of rat epididymis, was quantitated by RIA in epididymal extracts of rats of increasing age. Although detectable at 1 day of age, significant concentrations of AEG were not measured until 20 days; concentrations increased steadily, so that by 120 days of age, AEG represented 10% of total soluble protein. AEG mRNA was detected by Northern blot analysis of RNA from epididymides of 5-day-old animals and rapidly increased in amount between 20 and 35 days, reaching a maximum at 45 days. Using immunohistochemistry, AEG was localized in epididymal epithelial cells at 1 day of age. The number of cells staining for AEG increased markedly after 15 days. At 120 days, the immunoreactivity was predominantly localized to the lumen of the epithelial duct. To delineate factors that may influence AEG expression in the developing epididymis, we measured concentrations of androgen and androgen receptor mRNA in tissue extracts prepared from animals of various ages. Androgen receptor mRNA was detectable in epididymal extracts isolated from 1- to 90-day-old animals. Epididymal androgen concentrations were high at all ages (range, 6.0-31.2 ng/g tissue). The marked increase in AEG mRNA concentration at 20 days of age was not associated with an increase in either androgen or androgen receptor mRNA concentrations, suggesting that other factors may be necessary for AEG expression.

Aging↗
PubMed1989

Regulation of growth hormone secretion and messenger ribonucleic acid accumulation in human somatotropinoma cells in vitro.

GH secretion and mRNA levels were measured in cultured cells obtained from six human pituitary somatotroph tumors to investigate their hormonal and intracellular regulation. The responses were variable between tumors, but, in general, mRNA levels were less responsive than GH release to in vitro manipulation. GH-releasing factor [GRF-(1-29) amide; 10 nM] increased GH release and mRNA levels in three of four tumors tested to 30-97% above control values, but the fourth tumor was unresponsive. Somatostatin (1 microM) inhibited GH release significantly in four of the six cases, to 35-79% of control levels, but had no inhibitory effect on GH mRNA accumulation, in contrast to earlier studies on rat pituitary tissue. Bromocriptine (100 nM) likewise inhibited GH release (50-75% of control), but not GH mRNA levels, in the four tumors tested. Forskolin (10 microM; used to activate adenylate cyclase) stimulated GH release and mRNA levels in the two cases that responded most clearly to GRF, but had no significant effect in the other tumors; however, the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (100 nM) had no consistent effect on mRNA levels despite stimulating secretion in four of six cases. Thus, there was considerable variation in responses among the tumors tested; however, the responsiveness to GRF was approximately paralleled by that to forskolin, consistent with the suggestion that adenylate cyclase activity and responsiveness are variable among these tumors. Furthermore, the divergent effects of somatostatin on GH release and mRNA suggest uncoupling between its receptor and transcriptional regulatory mechanisms.

Adenoma↗
PubMed1989

Cortisol alters gene expression during involution of the rat ventral prostate.

The ability of high doses of cortisol to retard the involution process in the rat ventral prostate was related to alterations in the pattern of gene expression. Poly(A)+ RNA preparations from the prostates of noncastrated, castrated, and castrated rats injected daily for 7 days with cortisol were compared by Northern blot hybridizations for the relative expression of genes associated with cell differentiation and maintenance (the C1 prostatic steroid binding protein gene and alpha-tubulin), with cell death (TRPM-2, hsp 70, and c-fos), and with hormone regulation (the androgen and glucocorticoid receptors). As anticipated, the concentration of C1 mRNA in the prostate fell to less than 4% of that in the noncastrated controls within 4 days after castration and was nearly undetectable after 7 days. This decline was retarded by cortisol treatment of 7-day castrated animals which sustained the level of C1 transcripts at approximately 50% of control. While the pattern of expression of alpha-tubulin indicated some minor fluctuations, with the highest level occurring 7 days after castration, the prostates of the cortisol-treated group had essentially the same concentration of this mRNA as the noncastrates. Cortisol also modified the expression of genes associated with prostatic cell death. The large increase in prostatic TRPM-2 mRNA, seen 7 days after castration, was reduced by over 80% after treatment with the glucocorticoid. Although not as abundantly expressed as TRPM-2, the castration-induced levels of transcripts for both hsp 70 and the protooncogene c-fos were substantially reduced by cortisol.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

A rat Sertoli cell factor similar to basic fibroblast growth factor increases c-fos messenger ribonucleic acid in cultured Sertoli cells.

We have shown that the cultured Sertoli cell from the immature rat contains a fibroblast growth factor (FGF)-like factor. It behaves as a cationic peptide, is a potent competence factor for BALB/c3T3 mouse embryo fibroblasts, and displays a high affinity for heparin. Both bovine basic FGF and Sertoli cell FGF-like factor rapidly increase c-fos mRNA in cultured Sertoli cells. FSH, serum, and phorbol esters individually stimulate c-fos in cultured Sertoli cells whereas platelet-derived growth factor, epidermal growth factor, and insulin-like growth factor-I have little affect. However, unlike FSH, basic FGF does not stimulate an increase in cAMP and unlike either serum or phorbol esters, basic FGF does not stimulate phosphoinositol turnover or intracellular calcium changes. When Sertoli cell protein kinase C activity is suppressed by preexposure to phorbol ester, basic FGF continues to be a potent stimulator of c-fos, indicating that the calcium/phospholipid pathway is not involved in FGF induction. Basic FGF and FSH also increase jun-B mRNA levels in cultured Sertoli cells. In response to FGF, jun-B is more transiently increased than c-fos. In contrast, in response to FSH, jun-B persists longer than c-fos. These results indicate that cultured Sertoli cells contain a FGF-like factor that increases c-fos mRNA via a mechanism not involving cAMP and the calcium/phospholipid pathways. The different responsiveness of c-fos and jun-B to FSH and basic FGF may explain differences in the ultimate actions of these two ligands.

Animals↗
PubMed1989

Suppression of prolactin release and mRNA accumulation by two novel dopamine agonist agents.

Two novel dopamine agonist drugs, CV 205-502 and CQP 201-403, have been investigated to compare their effects on prolactin secretion and prolactin mRNA accumulation in cultured rat pituitary cells. Both drugs gave dose-dependent suppression of prolactin release over a 24 h incubation period: when each drug was used at 100 nmol/l CV 205-502 and CQP 201-403 induced suppression to 8.9 +/- 1.7 and 10.2 +/- 1.8% of control release, respectively, compared to 26.7 +/- 4.8% of control with 100 nmol/l bromocriptine. There was no consistent effect on growth hormone release. Cytoplasmic accumulation of prolactin mRNA was also inhibited by both drugs at this concentration, to 50.2 +/- 5.5% of control values by CV 205-502 and to 67.4 +/- 8% of control by CQP 201-403, and to a similar extent by 100 nmol/l bromocriptine (50.6 +/- 9.1% of control). None of the drugs had any significant effect on GH mRNA levels. These data suggest that the agents exert their effect at a pretranslational stage of prolactin synthesis, as well as at the level of hormone release.

Aminoquinolines↗
PubMed1989

Androgen receptor locus on the human X chromosome: regional localization to Xq11-12 and description of a DNA polymorphism.

The gene for the androgen receptor, mutations at which cause the X-linked androgen insensitivity syndrome, has been localized to the q11----q12 region of the human X chromosome by analysis, using a cloned cDNA for the androgen receptor, of somatic cell hybrid panels segregating portions of the X chromosome. A moderate-frequency HindIII RFLP has been found which should be useful in genetic linkage analysis of the various inherited forms of androgen insensitivity.

Animals↗
PubMed1988

Cloning of human androgen receptor complementary DNA and localization to the X chromosome.

The androgen receptor (AR) mediates the actions of male sex steroids. Human AR genomic DNA was cloned from a flow-sorted human X chromosome library by using a consensus nucleotide sequence from the DNA-binding domain of the family of nuclear receptors. The AR gene was localized on the human X chromosome between the centromere and q13. Cloned complementary DNA, selected with an AR-specific oligonucleotide probe, was expressed in monkey kidney (COS) cells and yielded a high-affinity androgen-binding protein with steroid-binding specificity corresponding to that of native AR. A predominant messenger RNA species of 9.6 kilobases was identified in human, rat, and mouse tissues known to contain AR and was undetectable in tissues lacking AR androgen-binding activity, including kidney and liver from androgen-insensitive mice. The deduced amino acid sequence of AR within the DNA-binding domain has highest sequence identity with the progesterone receptor.

Amino Acid Sequence↗
PubMed1988

Characterization of a binding factor that interacts with the sequences upstream of the vaccinia virus thymidine kinase gene.

A small 176 base-pair cloned DNA fragment, representing the nucleotide sequences proximal to the 5'-end of the vaccinia virus thymidine kinase (VV TK) gene, was radiolabeled and used in concert with gel retention assays to detect, partially purify, and characterize a promoter binding factor (PBF) extracted from vaccinia virions. The VV TK PBF was purified from solubilized virus particles by a combination of ion-exchange and DNA-affinity chromatographic procedures. The interaction between VV TK PBF and VV TK promoter sequences was relatively specific in that binding to the radiolabeled probe could be effectively inhibited by unlabeled VV TK promoter or VV TK promoter-specific oligonucleotides, but not by similar-sized fragments of control plasmid DNA. The VV TK PBF did, however, bind to other VV early-promoter elements. Glycerol gradient sedimentation provided an estimate of 130-140 kD for the native molecular weight of VV PBF. This correlated well with data from the purification of VV PBF from radiolabeled VV particles that revealed 2 polypeptides, with molecular weights of 70 and 68 kD that co-purified with VV TK PBF activity. Taken together, these results suggest that a heterodimeric promoter-binding factor, which is present within the cytoplasm of VV-infected cells, is capable of specifically interacting with VV early-promoter elements.

Chromatography, Affinity↗
PubMed1988

Expression of streptococcal M protein in mammalian cells.

The M protein encoded by group A streptococci is a cell-wall polypeptide that has the property of enabling these organisms to evade the phagocytic cells of the human host. Therefore, the M protein plays a major role in the pathogenesis of streptococcal diseases. As an initial step toward the use of this protein as a target antigen for the production of protective anti-streptococcal immunity, a live vaccinia virus recombinant containing the M-protein gene has been constructed (VV:M6 delta). The bacterial M-protein DNA sequence is stable within this genetic context and is actively transcribed by viral RNA polymerase. Furthermore, high levels of immunoreactive M protein were detected in vivo when the VV:M6 delta recombinant was used to infect mammalian cells in culture. Thus, in addition to providing a powerful approach for dissecting the immunodominant domains of the M protein, the VV:M6 delta recombinant appears to be an excellent candidate vaccine for animal trials.

Animals↗
PubMed1988

Deletion of the steroid-binding domain of the human androgen receptor gene in one family with complete androgen insensitivity syndrome: evidence for further genetic heterogeneity in this syndrome.

The cloning of a cDNA for the human androgen receptor gene has resulted in the availability of cDNA probes that span various parts of the gene, including the entire steroid-binding domain and part of the DNA-binding domain, as well as part of the 5' region of the gene. The radiolabeled probes were used to screen for androgen receptor mutations on Southern blots prepared by restriction endonuclease digestion of genomic DNA from human subjects with complete androgen insensitivity syndrome (AIS). In this investigation, we considered only patients presenting complete AIS and with the androgen receptor (-) form as the most probable subjects to show a gene deletion. One subject from each of six unrelated families with the receptor (-) form of complete AIS and 10 normal subjects (6 females and 4 males) were studied. In the 10 normal subjects and in 5 of the 6 patients, identical DNA restriction fragment patterns were observed with EcoRI and BamHI. In one affected individual, a partial deletion of the androgen receptor gene involving the steroid-binding domain was detected. Analysis of other members of this family confirmed the apparent gene deletion. Our data provide direct proof that complete AIS in some families can result from a deletion of the androgen receptor structural gene. However, other families do not demonstrate such a deletion, suggesting that point mutations (or small, undetectable deletions) may also result in the receptor (-) form of complete AIS, adding further to the genetic heterogeneity of this syndrome.

Blotting, Southern↗
PubMed1988

The production of antibodies against the conserved cysteine region of steroid receptors and their use in characterizing the avian progesterone receptor.

The most highly conserved feature of steroid receptor primary structure, the conserved cysteine region, corresponds to the DNA-binding domain of steroid receptors. This domain of chick progesterone receptor (PR) and other receptors was immunochemically characterized using site-directed antibodies. Eight peptides were synthesized corresponding to portion of the conserved cysteine region from PR, glucocorticoid, or estrogen receptor proteins. Polyclonal antibodies were obtained by immunizing rabbits with peptide conjugated to a protein carrier. The cross-reactivity of each antiserum was tested by Western blotting against chick and human PR, human glucocorticoid receptor, and human estrogen receptor. Of the several antisera positive by Western blots, only one cross-reacted with native chick PR. It was found that antibody bound to 4S transformed receptor, but not to 8S nontransformed receptor. At 50 mM KCl, antibody bound preferentially to transformed receptor form A, but at 400 mM KCl antibody bound equally well to either receptor form A or B. Binding of PR to DNA-cellulose was partially inhibited by the presence of cross-reacting peptide antibody. Thus, we have obtained at least one site-directed antibody against steroid receptors which is a useful structural probe to the DNA-binding functional domain of these gene regulatory proteins.

Amino Acid Sequence↗
PubMed1988

Molecular cloning of complementary deoxyribonucleic acid for an androgen-regulated epididymal protein: sequence homology with metalloproteins.

Acidic epididymal glycoprotein (AEG) is a 31,000 molecular weight secretory protein of the rat epididymis. Screening of a rat epididymal cDNA library with affinity-purified AEG antiserum yielded cDNA for AEG. Identity of the clones was verified by comparison of amino acid sequence of the purified protein with the sequence derived from the nucleotide sequence of the cDNA isolates. Two classes of AEG cDNA, approximately 1500 base pairs (bp) and 950 bp in length, differed by 538 bp in the 3'-untranslated region and by four single nucleotide mismatches, one of which was in the coding region. Northern blot hybridization of epididymal RNA revealed two species of AEG mRNA, corresponding in length to each type of cDNA. Analysis of RNA from individual animals provided evidence that the two mRNA species are the products of allelic genes. In vivo studies demonstrated that the level of total AEG mRNA is regulated by androgen. Amino acid sequence homology of AEG with metal-binding domains of several proteins suggests that AEG is a metalloprotein.

Amino Acid Sequence↗
PubMed1988

Antibodies to steroid receptor deoxyribonucleic acid binding domains and their reactivity with the human glucocorticoid receptor.

Functional properties of the DNA-binding domain of the human glucocorticoid receptor were investigated using high titer polyclonal antibodies produced against single synthetic peptides or a mixture of peptides whose sequences were derived from the DNA-binding domain of steroid receptor proteins. Three of seven antisera recognized both native and denatured forms of the glucocorticoid receptor, although considerably lower antisera dilutions were required for antibody binding to native receptor. Activation of the glucocorticoid receptor to its DNA-binding form was required for antibody recognition of the native receptor. Antisera to the second finger region of the DNA-binding domain caused a portion of the activated 4S glucocorticoid receptor to sediment as 7 or 9S in sucrose gradients containing 0.4 M KCl, but did not alter the sedimentation of the nontransformed 8S receptor. Specificity of the glucocorticoid receptor-antibody interaction was demonstrated by loss of reactivity after preabsorption with peptide antigens. Antisera that interacted specifically with the glucocorticoid receptor inhibited DNA binding of the activated receptor by as much as 80%. Thus, antibody probes directed against DNA-binding domain sequences provide immunological evidence that glucocorticoid receptor activation exposes the DNA-binding region of the receptor.

Amino Acid Sequence↗
PubMed1988

The human androgen receptor: complementary deoxyribonucleic acid cloning, sequence analysis and gene expression in prostate.

Androgenic hormones mediate their effects on male sex differentiation and development through a high affinity receptor protein. We report here cloning of the complete coding sequence of the human androgen receptor (hAR). By sequence homology hAR is a member of the nuclear receptor family, with closest sequence identity to the progesterone, mineralocorticoid, and glucocorticoid receptors. Regions of highest homology include the DNA-binding domain and a small region within the hydrophobic ligand-binding domain. Comparison of the deduced 919 amino acid sequence of hAR (98,999 mol wt) to the 902 amino acid sequence of rat AR (98,227 mol wt) reveals identical sequences in the DNA- and hormone-binding domains, with an overall homology of 85%. In human prostate, the major androgen receptor mRNA species is 10 kilobases while a less abundant mRNA is approximately 7 kilobases. Rabbit polyclonal antibodies were raised against a synthetic peptide from the N-terminal region of hAR. Immunocytochemical analysis of human prostate tissue demonstrated that AR is localized predominantly in nuclei of glandular epithelial cells.

Amino Acid Sequence↗
PubMed1988

The rat androgen receptor: primary structure, autoregulation of its messenger ribonucleic acid, and immunocytochemical localization of the receptor protein.

A composite androgen receptor DNA sequence 4,181 base pairs in length was determined from three cDNA clones isolated from a rat epididymal bacteriophage lambda gt11 library. An open reading frame of 902 amino acids encodes a protein of 98,227 mol wt. Structural domains characteristic of the steroid receptor family include an amino-terminal region with five repeated amino acid motifs, a central DNA-binding domain homologous with other steroid receptors, and a carboxyl-terminal steroid-binding region. A receptor cDNA probe used in Northern blot analysis hybridized with a predominant 10-kilobase androgen receptor mRNA in male reproductive tissues of the rat. Autoregulation of androgen receptor mRNA was indicated in rat ventral prostate by an increase in the level of 10-kilobase mRNA after castration and suppression of receptor mRNA upon androgen restimulation. A 15 amino acid peptide with sequence derived from the deduced androgen receptor sequence was synthesized and used as immunogen in raising receptor antibodies in rabbits. Antisera reacted with high titer against the synthetic peptide by enzyme-linked immunosorbent assay and against the native [3H]dihydrotestosterone-labeled androgen receptor as evidenced by an increase in receptor sedimentation rate determined by sucrose gradient centrifugation. Immunocytochemical staining localized the androgen receptor to epithelial cell nuclei in rat ventral prostate.

Amino Acid Sequence↗
PubMed1988

Calcium dependence of prolactin mRNA accumulation in GH3 rat pituitary tumour cells.

Stimulation of prolactin secretion by TRH probably involves mobilization of intracellular calcium to a greater extent than calcium influx, but less is known about the possible calcium-dependent mechanisms which may control prolactin gene transcription. We have studied this question in the rat pituitary tumour GH3 cell line by measuring prolactin mRNA accumulation by a cytoplasmic dot hybridization assay. Cobalt chloride, an intracellularly acting calcium antagonist, caused marked dose-dependent reductions in prolactin release and mRNA concentrations, whereas the calcium channel-blocking agent verapamil had no effect on prolactin release and had smaller effects on prolactin mRNA. Cobalt chloride abolished the stimulatory effect of TRH on prolactin mRNA levels, while verapamil caused only moderate inhibition. Growth hormone mRNA levels in the same cells were not significantly affected by TRH or verapamil, and only marginally reduced by cobalt. These data suggest that, as for prolactin release from normal rat pituitary lactotrophs, prolactin mRNA accumulation in GH3 cells appears to have a requirement for intracellular calcium which is only partly dependent upon calcium influx.

Calcium↗