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E Mulder

Publications and source records attributed to E Mulder.

At least 73 records · Page 4Linked to original sources
PubMed1985

Photoaffinity labelling of androgen receptors with 17 beta-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one.

The synthetic androgen 17 beta-hydroxy-17 alpha-[3H]methyl-4,9,11-estratrien-3-one (R1881) has been used as photoaffinity label to characterize androgen receptors in calf uterus and rat prostate. Polyacrylamide gel electrophoresis under denaturing conditions showed that the DNA-binding form of the androgen receptor in calf uterus cytosol is a protein with a molecular mass of 98 kD. In rat prostate cytosol an androgen receptor with a molecular mass of 46 kD could be photoaffinity labelled with R1881. The photoaffinity labelling procedure described here provides a method for studying the hormone binding domain of androgen receptors in partial purified preparations.

Affinity Labels↗
PubMed1985

Rapid and sensitive detection of oestrogen receptors in cells and tissue sections by autoradiography with 125I-oestradiol.

The presence of receptors for steroid hormones in individual cells and tissue sections was assessed within 4-24 h using dry mount autoradiography with radio-iodinated oestradiol. Low affinity and nonspecific binding of steroids were significantly reduced by washing the cells or sections with diluted antiserum to oestradiol. For cells of the MCF-7 cell line variations in grain density were observed, indicating that cells of the MCF-7 cell line are heterogeneous with respect to their cellular receptor concentrations of oestrogen receptors. Receptor-negative cells, such as peritoneal macrophages, did not retain oestradiol label. In tissue sections of rat and calf uterus, predominant labelling was observed on the endometrial gland cells and stroma. Oestradiol receptor binding in the uterus cytosol for both radio-iodinated and tritiated oestradiol showed the same qualitative characteristics as determined by sucrose gradient sedimentation profiles and a comparable amount of binding sites was found for both labels. The relative binding affinity of 125I-oestradiol compared to [3H]oestradiol is about 70-80%. The dry mount autoradiographic technique as presented can be used for rapid screening of heterogeneity in oestrogen receptor distribution in cells and tissue sections, since this technique reveals differences in receptor concentrations on the single cell level.

Animals↗
PubMed1985

Characterization of steroid hormone receptors with ion-exchange fast protein liquid chromatography.

Androgen, estrogen and progesterone receptors have been characterized with anion exchange Fast Protein Liquid Chromatography (FPLC) on a Mono Q column (Pharmacia). In the presence of sodium molybdate androgen receptors in cytosols from rat prostate, rat epididymis and calf uterus eluted as a single sharp peak at 0.32 M NaCl with recoveries of approx 90%. The molybdate-stabilized form of the androgen receptor from rat prostate was purified about 75-fold. The receptor containing FPLC-peak fractions sedimented in high salt (0.4 M KCl) linear sucrose gradients at 3.6 S (prostate) and at 4.6 S (epididymis and calf uterus) respectively. Multiple forms of the androgen receptor were present in cytosols from rat prostate prepared in the absence of sodium molybdate, probably due to proteolytic breakdown of the native form. Calf uterine estradiol and progesterone receptors prepared in the presence of sodium molybdate (20 mM) eluted from the Mono Q column at 0.32 M NaCl. The molybdate-stabilized forms of the oestradiol and progesterone receptors were purified approx 70-fold and 30-fold respectively. In the absence of molybdate the estradiol receptor dissociated into two major forms eluting at 0.23 M NaCl and 0.37 M NaCl. After heat induced transformation (30 min at 25 degrees C) of the estradiol receptor one major peak was eluted at 0.42 M NaCl, indicating a change in the surface charge of the estradiol receptor as a result of the 4 S to 5 S transformation. It is concluded that the FPLC anion exchange system is a powerful, fast tool for characterization and partial purification of steroid receptors. In addition this technique could be applied as a rapid procedure for the quantitative estimation of steroid receptors in small biological samples.

Animals↗
PubMed1985

Changes of oestrogen receptor levels in Leydig cells from mice and rats during culture.

Two functional properties of Leydig cells in culture, i.e. LH-stimulated steroidogenesis and nuclear oestrogen receptor levels have been investigated. Leydig cells isolated from testes of immature rats and mature mice maintained their responsiveness to LH during 48-72 h of cell culture, although the mouse Leydig cells appeared to be less responsive to LH after 72 h of culture. In contrast, nuclear oestrogen receptor levels in both types of Leydig cells declined to 10-20% of the initial value after 24 h in culture. In the 48-72 h culture period nuclear oestrogen receptor levels recovered to 75% of the initial value only in Leydig cells from immature rats, whereas the nuclear oestrogen receptor levels in Leydig cells from mature mice remained low. These data demonstrate that during in vitro culture of Leydig cells, preservation of LH responsiveness does not necessarily warrant that other Leydig cell parameters e.g. nuclear oestrogen receptors also remain unaltered.

Animals↗
PubMed1984

Interaction of rat prostate androgen receptors with polynucleotides, RNA, DNA and cloned DNA fragments.

Androgen receptors were partially purified from prostates of mature (non-castrated) rats by chromatography on 2',5'-ADP-Sepharose and labelled by exchange with 5 alpha-[3H]dihydrotestosterone. The partially purified receptor preparation was free of DNAase activity and sedimented at approx. 3 S. The specificity of the interaction of this androgen receptor with nucleotides was investigated in a competitive binding assay using inhibition of binding of the steroid receptor complex to ADP-Sepharose. Certain polyribonucleotides were strongly bound (e.g., poly(UG), poly(AU), poly(G) and poly(U] and competed more effectively for the receptor binding sites than prostate RNA. Restriction fragments of genomic clones from the genes which code for prostatic binding protein showed only moderate affinity for the 3 S receptor form. These data suggest that the 3 S form of the androgen receptor lacks the specific domain or conformation necessary for specific interaction with DNA, but retains a high affinity for certain forms of RNA. Some potent inhibitors of proteolysis (diisopropylfluorophosphate, leupeptin) did not have any effect on the form of the receptor isolated from mature intact animals. A possible function of the 3 S form in post-transcriptional processing is discussed.

Adenosine Diphosphate↗
PubMed1984

Fluorescent androgen derivatives do not discriminate between androgen receptor-positive and -negative human tumor cell lines.

For the evaluation of histochemical procedures for detection of androgen receptors, three human tumor cell lines have been used: PC-93 and NHIK-3025, both biochemically characterized as androgen receptor-positive, and EB-33, biochemically characterized as androgen receptor-negative. The binding of three fluorescent ligands, testosterone-17 beta-hemisuccinate-bovine serum albumin-fluorescein isothiocyanate, testosterone-17 beta-hemisuccinate-fluoresceinamine, and 5 alpha-dihydrotestosterone-17 beta-hemisuccinate-fluoresceinamine, to the cells was evaluated. The relative binding affinities of the ligands for the androgen receptors were low (less than 5% when compared to methyltrienolone). Treatment of the cells with the androgen-fluoresceinamine derivatives resulted in a fluorescent labeling of the cytoplasm in both intact and "freeze-damaged" cells of the three cell lines. This staining was independent of the presence of receptors. Nuclei were not stained. Incubation of intact cels with the protein-linked conjugate did not result in significant cellular fluorescence. Only cells with damaged membranes showed a positive histochemical reaction, both in nucleus and cytoplasm, irrespective of the receptor content of the cells. The fluorescence intensity was not suppressed with excess 5 alpha-dihydrotestosterone or methyltrienolone, which are known to prevent binding of low affinity ligands to androgen receptors. From these results it is concluded that androgen receptors cannot be detected by these fluorescent ligands with low affinity for the receptor. The observed fluorescence of the cells is therefore due to binding of the ligands to other binding sites. The visualization/histochemical demonstration of these binding sites does not appear to be related to the presence of androgen receptors.

Adenocarcinoma↗
PubMed1984

Fluorescent ligands, used in histocytochemistry, do not discriminate between estrogen receptor-positive and receptor-negative human tumor cell lines.

A cell line containing estrogen receptors (MCF-7) and a cell line lacking estrogen receptors (PC-93) were used for a comparison of biochemical and histochemical procedures to detect estrogen receptors. We evaluated three different fluorescent estrogen derivatives: 17 beta-estradiol-6-carboxymethyloxime-bovine serum albumin-fluorescein isothiocyanate, 17 beta-estradiol-17-hemisuccinate-fluoresceinamine, and coumestrol. The main results were: The relative binding affinities of these ligands for the estrogen receptor were between 0.1 and 2% of the affinity of estradiol. Fluorescent staining of the cells showed no relation to the presence of estrogen receptors. Staining was not suppressed with excess estradiol-17 beta, which is known to prevent binding of low affinity ligands to estrogen receptors. Cells with intact membranes were not stained after treatment with the albumin-linked estrogen derivative; only cells with damaged cell membranes were stained. Treatment of cells with 17 beta-estradiol-17-hemisuccinate-fluoresceinamine resulted in a fluorescent labeling of the cytoplasm in intact and artificially damaged cells. Coumestrol caused only fluorescence of the cytoplasm in intact cells. It is concluded that estrogen receptors cannot be detected with these low affinity ligands. Fluorescence of these cells is probably due to binding of the ligands to low affinity binding sites. The presence of these low affinity binding sites appears not to be related to the presence or absence of estrogen receptors and can therefore not be used to discriminate between estrogen receptor-positive and receptor-negative tumor cells.

Breast Neoplasms↗
PubMed1983

DNA and ribonucleotide binding characteristics of two forms of the androgen receptor from rat prostates.

Androgen receptors (sedimentation value approximately 4S and Stokes radius 2.8 nm) present in the cytoplasmic fraction obtained from prostates of castrated rats bind to DNA-Sepharose and double stranded DNA. A receptor fragment (sedimentation value approximately 3S and Stokes radius 2.3 nm) obtained from rat prostates in the course of a purification procedure showed greatly diminished binding affinity for both DNA-Sepharose and soluble DNA. In contrast, both the 4S cytosol receptor and the 3S receptor form interacted with equal affinity with prostate RNA or poly(UG). These observations provide evidence that for DNA binding a different or additional part of the receptor molecule is required than for RNA and polyribonucleotide binding.

Animals↗
PubMed1983

Presence of progesterone receptors and absence of oestrogen receptors in human intracranial meningioma cytosols.

The occurrence of oestrogen and progestin receptors in cytosols from human intracranial meningiomas was studied with a dextran-coated charcoal assay and Scatchard plot analysis. [3H]-Oestradiol and [3H]-promegestone (17 alpha, 21-dimethyl-19-norpregna-4,9-diene-3,20-dione, R-5020) were used as tracers. Using this method, no high-affinity binding sites for oestradiol were observed, whereas progestin binding was identified in 18 out of 20 meningioma cytosols. The number of progestin binding sites was identical in meningioma cytosols obtained from female patients (192 +/- 57 fmol/mg protein, mean +/- S.E.M., n = 12) and those obtained from male patients (230 +/- 57 fmol/mg protein, n = 6), as was the dissociation constant of the complex (1.5 +/- 0.3 vs 1.4 +/- 0.3 nmol/l respectively). Only progestins (progesterone, R-5020 and megestrol acetate) competed successfully with tritiated R-5020. Oestrogens, androgens and cortisol showed no appreciable cross-reaction. It was concluded that the cytosols from human intracranial meningiomas contain progesterone receptors in the absence of oestrogen receptors. The presence of these progesterone receptors may indicate that (anti)-progestational treatment could be of potential value in cases which cannot be treated by surgery alone.

Adult↗
PubMed1983

Cyproterone acetate prevents translocation of the androgen receptor in the rat prostate.

The translocation of the androgen receptor in prostatic tissue has been studied under the influence of different ligands (testosterone, methyltrienolone and cyproterone acetate) in vivo and in vitro. Nuclear and cytoplasmic androgen receptors were estimated using an exchange assay with [3H]methyltrienolone ( [3H]R1881) 1 h and 16 h after injection in castrated rats of either 100 micrograms testosterone (T), 10 mg cyproterone acetate (CA) or the combination of T and CA. Within 1 h after T administration, nuclear receptor levels increased with a concomitant depletion of cytosol receptors. In the CA-treated rats nuclear receptor levels were not different from those of control castrated animals and there was no depletion of cytosol receptors. The combined treatment of T and CA resulted in a partial depletion of cytosol receptors and a simultaneous increase of nuclear receptors. The absence of an increase in nuclear androgen receptors in CA-treated animals cannot be explained by a delay in translocation, because even 16 h after CA injection, only a very small number of nuclear receptors were detectable. Incubation of minced prostatic tissue with [3H]CA or [3H]R 1881 resulted in receptor translocation only in the R1881 incubations and confirmed the in vivo results. Competition studies with different steroids and cytosol receptor (non-activated, 8S form in low salt gradient) or nuclear receptor (activated 3.6S form in high salt gradient) of prostatic tissue show that CA can compete with R1881 for specific androgen-binding sites with a similar relative binding affinity for both receptor preparations. The present results provide evidence that CA prevents translocation of the androgen receptor to the nucleus, although CA can be bound with similar affinities to the nuclear receptor and the cytoplasmic receptor. We propose that the anti-androgenic action of CA involves an inhibition of receptor translocation.

Androgen Antagonists↗
PubMed1981

Nuclear androgen receptors in human prostatic tissue. Extraction with heparin and estimation of the number of binding sites with different methods.

A procedure for the estimation of nuclear androgen receptors in benign prostatic hyperplastic tissue is described, which employs extraction of receptors from nuclei with buffers containing heparin. Extraction of a nuclear pellet with a heparin-containing (1 g/l) buffer appeared to have definite advantages over 0.4 mol/l KCl extraction. Heparin appeared to be twice as efficient in extracting androgen receptors. In addition aggregated receptor proteins, formed after storage at -80 degrees C, were partly deaggregated by heparin. Specific isolation of the androgen receptor was performed using either agar gel electrophoresis, protamine sulphate precipitation or LH-20 gel filtration. A comparison was made between the amounts of estimated receptors with these different techniques. Protamine sulphate precipitation resulted in the highest estimates of receptor-bound 5 alpha-[3H]dihydrotestosterone (3H-DHT). Treatment of the labelled nuclear extracts with a charcoal suspension prior to the receptor assay resulted in lower amounts of estimated androgen receptors. A method for routine evaluation of nuclear androgen receptors in prostatic tissue has been evaluated, which involves extraction of nuclear pellets with a heparin-containing (1 g/l) buffer, exchange labelling of the nuclear extracts for 20 h at 10 degrees C and quantification of the receptors with protamine sulphate precipitation.

Cell Nucleus↗
PubMed1981

Characterization and partial purification of androgen receptors from ram seminal vesicles.

An androgen receptor has been demonstrated in the cytosol and in the nuclear fraction of ram seminal vesicles. The cytosol receptor was stabilized by sodium molybdate and 2 distinct [3H]methyltrienolone-binding proteins, one sedimenting at 9S and one sedimenting at 3S, could be demonstrated by sucrose-gradient centrifugation in the presence of 50 mM molybdate. The slower sedimenting form could be partially purified by ADP-sepharose chromatography. The purified receptor still sedimented at 3S after centrifugation on sucrose gradients containing either 0.6 M KCl or 50 mM molybdate. The receptor was destroyed by heating at 50 degrees C for 30 min and its complex with [3H]methyltrienolone dissociated slowly at low temperatures. The apparent equilibrium-dissociation constant (KD) for the purified receptor was: 3.8 x 10(-10) M. The relative affinities for different steroids decreased in the following sequence: 5 alpha-dihydrotestosterone greater than or equal to methyltrienolone greater than testosterone much greater than estradiol greater than R5020 greater than progesterone greater than diethylstilbestrol. The nuclear androgen receptor sedimented at 3S on sucrose gradients containing 0.6 M KCl. At pH 7.4 it behaved as an acidic protein with an electrophoretic mobility towards the anodic region of the agar gel. Because of the relatively large content of cytoplasmic and nuclear androgen receptors and the availability of large amounts of tissue the ram seminal vesicles could be a suitable source for large-scale purification of these receptors.

Animals↗
PubMed1981

Extraction of nuclear androgen receptors from rat prostate with different reagents.

After in vitro labelling of androgen receptors in prostate tissue from castrated rats, about 70% of the labelled androgens in nuclei could be extracted with buffer solutions with 0.4 M KCl, or 10 mM pyridoxal phosphate, or 0.4 mM Cibacron blue, or heparin (0.2 mg/ml). In the nuclear extracts, 60-80% of the steroid was recovered as steroid-receptor complex. Sedimentation values of the receptors, on sucrose gradients containing 0.4 M KCl, were 3-4 S for the KCl extract and 4-5 S for pyridoxal phosphate and heparin extracts. The Cibacron blue extract contained an aggregated form of the receptor (5-7 S). The presence of the protease inhibitor di-isopropylfluorophosphate during isolation caused a small increase in S value of the receptors. However, the differences in sedimentation values between KCl, pyridoxal phosphate and heparin extracts remained. The receptors in the KCl extracts could be precipitated with protamine sulphate only after addition of 10 mM pyridoxal phosphate to the extracts. On the basis of these results it is concluded that the androgen receptors in rat prostate can be effectively extracted from nuclei by certain reagents which have in common a strongly negatively charged group and a less polar or hydrophobic region. These reagents form complexes with nuclear receptors and influence the sedimentation values and precipitability with protamine sulphate of these receptors.

Animals↗
PubMed1981

Oestrogen receptors in rat mammary tissue and plasma concentrations of prolactin during mammary carcinogenesis induced by oestrogen and ionizing radiation.

Female-Sprague-Dawley rats received a subcutaneous implant containing 2 mg oestradiol at the age of 7 weeks. One week later half of the rats treated with oestrogen and half of the rats in an untreated control group were irradiated with 2 Gy (200 rad) of X-rays. The content of oestrogen receptor of the mammary tissue and the concentration of prolactin in the plasma were studied at intervals of 2 months for a period of 14 months after this treatment. Oestrogen treatment resulted in a decrease in the content of oestrogen receptors in the mammary tissue of both irradiated and non-irradiated rats. In oestrogen-treated rats, plasma prolactin was raised 10-50 times and pituitary tumours were observed. Radiation had no additional effect on the oestrogen-receptor content of mammary tissue or the concentration of plasma prolactin. The changes in the oestrogen-receptor content of mammary tissue and the prolactin concentration of plasma preceded the development of mammary tumours. It is suggested that the synergistic action of oestrogen and radiation on rat mammary tumour development is the result of a stimulation by oestrogen and/or prolactin of the sensitivity of the mammary gland to ionizing radiation.

Animals↗
PubMed1980

Inhibition of nucleic acid and chromatin binding of the rat prostate androgen receptor by pyridoxal phosphate, heparin and Cibacron blue.

The androgen receptor from rat prostate binds 5 alpha-dihydrotestosterone and related androgenic steroids at a steroid binding site and in addition shows selective binding to structures related to nucleic acids (chromatin binding site). Cytoplasmic androgen receptor, labeled with the synthetic androgen methyltrienolone (R 1881) was readily bound at 40c by 2',5'-ADP-agarose, DNA-cellulose and phosphocellulose. The binding to ADP-agarose and DNA-cellulose was used as a model for study of the nucleic acid binding site of the receptor. Complete elution of androgen receptors from these matrices could be obtained with low concentrations of pyridoxal phosphate (10 mM), heparin (0.2 mg/ml) and Cibacron blue (0.4 mM). Sodium molybdate (10 mM) did not interfere significantly with binding of the androgen receptor to ADP-agarose and had little effect on elution of receptors from the gel. Pyridoxal phosphate, heparin and Cibacron blue in low ionic strength buffers were also shown to be very effective for the extraction of androgen receptors from nuclei of rat prostatic tissue. These results suggest gross similarities in the structure of the androgen receptor with activated forms of receptors for corticoids, estrogens and progestins with respect to nucleic acid binding.

Animals↗