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Biomedical subjects

W I Mainwaring

Publications and source records attributed to W I Mainwaring.

At least 19 recordsLinked to original sources
PubMed1989

A possible explanation for the peripheral selectivity of a novel non-steroidal pure antiandrogen, Casodex (ICI 176,334).

The in vivo antiandrogenicity of Casodex has been confirmed and characterised. Androgen receptor (AR) binding assays of rat ventral prostate gland cytosols revealed a relative binding affinity (RBA) for the AR of 0.267 and a k1 of 1.25 x 10(-7) M for Casodex. In addition, the peripheral selectivity of Casodex relative to other non-steroidal antiandrogens was confirmed in that daily treatment of non-castrated rats with Casodex (25 mg kg-1) did not elicit any changes in serum LH and testosterone concentrations relative to vehicle-treated controls, whereas elevated serum LH and testosterone were observed in rats treated with flutamide (25 mg kg-1). The peripheral selectivity of Casodex in the intact male rat was related to the distribution of radiolabelled antiandrogen following intravenous injection. All tissues with the exception of the hypothalamus and cerebral cortex (CC) sequestered radioactivity such that the tissue:serum ratio (TSR) for the drug was greater than unity. In the testis, the TSR was less than unity 1 h after injection but approached unity 5 h after injection and was greater than unity 10 h after injection. This may be explained by the presence of a blood-testis barrier for the drug, resulting in delayed equilibration between the blood and testis tissue. By comparison, an order of magnitude lower amounts of radioactivity in the hypothalamus and CC were maintained for the 10 h period after injection. These data, together with known physicochemical properties of Casodex suggest that a blood-brain barrier exists for the drug which results in exclusion of this antiandrogen from central sites of androgen negative feedback and that this accounts for its peripherally selective antihormonal profile.

Androgen Antagonists↗
PubMed1984

Limitations in the use of tritiated methyltrienolone for the photoaffinity labelling of androgen receptor proteins.

In the absence of photoexcitation and under conditions of low ionic strength, the native form of the androgen receptor in rat prostate sediments as a large, 9.2S complex with tritiated androgens, including [3H]methyltrienolone. On photoexcitation, the configuration of labelled receptor complexes changes to a form of lower sedimentation coefficient, 4.2S. Initial experiments indicated that photoaffinity labelling of the androgen receptor protein may be readily achieved and with extensive covalent attachment of [3H]methyltrienolone. However, from ultracentrifugation analyses conducted under denaturing conditions it was established that, at best, only 5-8% of available [3H]methyltrienolone is covalently attached to the receptor protein. The remaining [3H]methyltrienolone is presumably adsorbed or entrapped within the receptor protein and may resist extraction into organic solvents, but it is not authentically bound in an irreversible, covalent manner. Our findings raise doubts concerning the efficiency and usefulness of [3H]methyltrienolone as a photoaffinity reagent for androgen receptor proteins. An additional problem is that photoillumination of [3H]methyltrienolone leads to the attachment of radioactivity to non-receptor proteins present in human plasma.

Affinity Labels↗
PubMed1983

Enhanced transcription of rRNA genes by purified androgen receptor complexes in vitro.

Androgens stringently regulate the concentrations of the polyamines, spermine and spermidine, in rat ventral prostate. The intracellular distribution of the polyamines is also dependent on the androgenic milieu; a small proportion of polyamines is associated tightly with the nucleus. Soluble nucleolar chromatin contains RNA polymerase A and protein kinase activities, both of which are regulated by androgens and markedly stimulated by polyamines. It remains to be established whether the polyamines modulate these enzyme moieties directly, or whether the phosphorylation of other nucleolar proteins plays a crucial role in the androgenic regulation of rRNA synthesis. Using a protocol centered on affinity chromatography, the purification of the androgen receptor protein to a state approaching homogeneity has been accomplished. The purified receptor stimulates the RNA polymerase A and protein kinase activities associated with prostate nucleolar chromatin. The significance of these findings in the light of correct concepts of steroid hormone action is discussed.

Animals↗
PubMed1980

Aqueous two-phase partition studies of the glucocorticoid receptor: activation and pH-dependent changes of rat liver glucocorticoid-receptor complex in partly purified preparations.

1. The activation of the glucocorticoid receptor of rat liver cytosol was studied by the use of partitioning in an aqueous dextran-poly(ethylene glycol) two-phase system. a, Incubation unde conditions generally found to stimulate the activation results in a time-dependent increase of the partition coefficient of the glucocorticoid-receptor complex in the two-phase system. b, This increase was found to result from the fact that the activated form of the complex has a higher partition coefficient than the nonactivated one, since the two forms are eluted from DNA-Sepharose columns as clearly separated peaks with different partition coefficients, and a redistribution of complex between the peaks occurs without any changes of the partition coefficient of each single peak. c, The partition coefficient of a partly purified nonactivated complex was found to follow the same time course towards a higher value under 'activating' incubation conditions as the complex in whole cytosol. 2. The dependence of the partition coefficient of the glucocorticoid-receptor complex on the pH of the two-phase system revealed a pH-dependence of the state of the complex. Experiments with partly purified preparations of the complex showed that this change was either a conformational change or an aggregation of high affinity of the complex with itself or another DNA-binding substance.

Animals↗
PubMed1978

Testosterone regulates the synthesis of major proteins in rat ventral prostate.

The presence of three major proteins alpha, beta and gamma in rat ventral prostate was demonstrated by electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. Their regulation by androgens was studied by measuring the rates of synthesis of the proteins in minced prostatic tissue by using L-[35S]methionine. The three proteins account for 30-40% of the proteins synthesized in the gland. After castration, their rates of synthesis rapidly decline to about 1% that of normal animals, and this cannot be accounted for by the accompanying decrease in general protein synthesis. Testosterone reverses these changes in castrated animals, so that after 4 days normal synthesis is restored. The regulation is specific for androgens, since cyproterone acetate, an anti-androgen, is inhibitory and oestradiol-17beta and corticosterone are without effect. Preliminary characterization of the proteins indicates that protein alpha (mol.wt. 22000, pI unknown) is a glycoprotein containing glucose and/or mannose residues and occurs in both the mitochondrial and cytosol fractions. Protein beta (mol.wt. 12000, pI5.4) is also a glycoprotein, but is found exclusively in the cytosol fraction. Protein gamma (mol.wt. 8000, pI5.4) is also a glycoprotein, but is found exclusively in the cytosol fraction. Protein gamma (mol.wt. 8000, pI5.4) is also found exclusively in the cytosol fraction.

Animals↗
PubMed1978

The nuclear uptake of androgen by human benign prostate in vitro: action of antiandrogens.

An in vitro test system suitable to assess the potency of putative antiandrogens has been developed, using the human benign prostatic tissue obtained at operation. The system circumvents some problems associated with using human tissue, such as the presence of endogenous steroid and contamination with plasma proteins (particularly sex hormone binding globulin). Slices of tissue were incubated in the presence of 3H-testosterone and the uptake into nuclei was determined. The nature of the nuclear radioactivity and the steroid specificity indicates a mechanism similar to the established in the rat ventral prostate. The action of antiandrogens (cyproterone acetate, diethylstilbestrol, flutamide, hydroxylated flutamide and gestonorone capronate) has been studied at various concentrations.

Androgen Antagonists↗
PubMed1977

Effects of androgens on the complexity of poly(A) RNA from rat prostate.

Poly(A)-containing RNA, isolated from rat ventral prostate, has been analyzed for its base sequence complexity. The kinetics of hybridization of total cellular poly(A)-containing RNA with its complementary DNA (cDNA) from normal and castrated animals are markedly different. RNA from normal animals consists of three abundance classes, about 36% comprises one or two highly abundant RNA sequences, 29% consists of about 24 sequences and the remainder is a scarce class of approximately 8200 sequences. In contrast, the hybridization kinetics of prostatic RNA from castrated animals demonstrate that there is a moderate abundance class of 53 sequences and a scarce class of about 7800 sequences, but that a class of abundant sequences is not present. Using normal prostatic cDNA as a probe, we showed that the abundant sequences were not absent but reduced 10 fold following a 3-day castration period and 100 fold after 100 fold after 7 days. Such heterologous hybridization experiments also suggest that there is significant sequence homology in the RNA sequences present in the prostate irrespective of the hormonal status of the animals. The major effect of testosterone appears to be the regulation of the abundance of specific RNA sequences.

Animals↗
PubMed1976

Testosterone control of nucleic acid content and proliferation of epithelium and stroma in rat seminal vesicles.

Tissue wet weight, nucleic acid content and epithelial and stromal cell numbers were measured in the seminal vesicles of sexually mature male rats. After castration, tissue weight and RNA decreased rapidly and in aprallel to reach, after 14 days, values only 15-20% of those in control (not castrated) animals. During this period, DNA decreased to a much lesser extent (by about 40%), but this change in DNA correlates well with the observed loss of cells from the epithelium. Testosterone in vivo promoted an immediate resynthesis of RNA, the value characteristic of control animals being reached within 80h. Delays occurred in the hormone-induced regain of tissue weight (30h) and DNA (40h), each of which preceded proliferation of the epithelium (40--50h). The cells of the stroma were unaffected by these changes in the androgenic statls of the animal. It is suggested that these proliferative changes in the epithelium cannot account for the previously reported induction by testosterone of basic secretory proteins in this tissue.

Animals↗
PubMed1976

Androgen-dependent synthesis of basic secretory proteins by the rat seminal vesicle.

1. Two basic proteins were purified from secretions of rat seminal vesicles by using Sephadex G-200 chromatography and polyacrylamide-gel electrophoresis under denaturing conditions. 2. It is not certain that these two proteins are distinct species and not subunits of a larger protein, but their properties are similar. Highly basic (pI = 9.7), they migrate to the cathode at high pH and their amino acid composition shows them to be rich in basic residues and serine. Threonine and hydrophobic residues are few. Both proteins are glycoproteins and have mol.wts. of 17000 and 18500. 3. Together these two proteins account for 25-30% of the protein synthesized by the vesicles, but they are absent from other tissues. 4. Changes in androgen status of the animal markedly affect these proteins. After castration, a progressive decrease in the basic proteins is observed and the synthesis of the two proteins as measured by [35S]methionine incorporation in vitro is is decreased. Testosterone administration in vivo rapidly restores their rates of synthesis. 5. These effects on specific protein synthesis are also observed for total cellular protein, and it is suggested that testosterone acts generally on the total protein-synthetic capacity of the cell and not specifically on individual proteins. Proliferative responses in the secretory epithelium may also be involved. 6. The extreme steroid specificity of the induction process suggests that the synthesis of these basic proteins is mediated by the androgen-receptor system. 7. The biological function of these proteins is not clear, but they do not appear to be involved in the formation of the copulatory plug.

Amino Acids↗
PubMed1976

The androgenic regulation of prostate proteins with a high affinity for deoxyribonucleic acid. Evidence for a prostate deoxyribonucleic acid-unwinding protein.

1. When testosterone is injected into castrated rats in vivo, a significant increase in the incorporation of [35S]methionine into prostate proteins may be detected under conditions in vitro. 2. Studies based on DNA-cellulose chromatography show that the synthesis of prostate proteins with a high affinity for DNA is particularly enhanced by androgenic stimulation. 3. These changes in protein synthesis are negated when the anti-androgen, cyproterone acetate, is administered concomitantly with testosterone in vivo. 4. Two assays were developed for measuring the strand separation of prostate DNA; first, the retention of 3H-labelled native DNA on nitrocellulose membranes, and second, the activation of native DNA as a template for 9S prostate DNA polymerase. On the basis of these criteria, DNA-unwinding activity is present in the prostate gland and it is regulated by androgens in a steroid-and tissue-specific manner. 5. The results are discussed in the context of the mechanism of action of androgens, particularly since the changes provoked in DNA-unwinding activity by androgens precede the onset of DNA replication and mitosis.

Animals↗
PubMed1976

Nuclear components responsible for the retention of steroid--receptor complexes, especially from the standpoint of the specifcity of hormonal responses.

1. By covalently linking nuclear components from hormone-sensitive cells to Sepharose 2B, it is possible to investigate the interaction between nuclear components and cytoplasmic receptor-steroid complexes by affinity chromatography. 2. Many factors are implicated in the specifity of nuclear-cytoplasmic interactions, including the nature of the nuclear components, the presence of the cytoplasmic receptor protein and the provision of the appropriate steroid ligand. 3. Two distinct sets of binding sites are present in nuclear extracts immobilized to Sepharose 2B, namely a small number of specific high-affinity sites and a larger number of non-specific low affinity-sites. 4. Considerable evidence supports the importance of the high-affinity binding sites in the manifestation of hormonal specificity in different tissues. Although the study has centred largely on androgenresponsive systems, the findings are germane to cytoplasmic-nuclear interactions in general. 5. The high-affinity or acceptor sites in nuclear extracts reside in the basic but non-histone protein fraction. 6. Hormonal specificity is seemingly maintained by both the cytoplasmic and nuclear components, and the results are discussed in the context of the mechanism of action of steroid hormones.

Animals↗