Biomedical subjects
Publications and source records attributed to E Mulder.
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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.
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Hypophysectomy of immature rats results after 5 days in a loss of LH responsiveness of Leydig cells. LH responsiveness can be partly maintained by treatment with FSH for 5 days. When estradiol benzoate was administered together with FSH to hypophysectomized rats the maintenance of LH responsiveness was not observed. The loss in LH responsiveness after hypophysectomy in terms of testosterone production could not be explained by either a change in the amount of Leydig cells present in the Leydig cell preparation or to a higher conversion of testosterone. The LH-stimulated cAMP production in cells from hypophysectomized rats was very low compared to cells from intact rats. There was no difference between cAMP production of Leydig cells from untreated, FSH-treated or FSH plus estradiol benzoate treated hypophysectomized rats. During the first 2 days after hypophysectomy LH responsiveness in both untreated and FSH-treated rats showed a comparable decrease. From day 2 after hypophysectomy LH responsiveness remained at a constant level in cells from rats treated with FSH, but declined further in cells from untreated rats. A single injection of estradiol benzoate to hypophysectomized rats treated with FSH counteracted the effect of FSH on LH responsiveness, but only when estradiol was administered at that time after hypophysectomy, when the effect of FSH on LH responsiveness was clear.
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The subnuclear distribution of 3H-oestradiol-receptor complexes was studied in uterine tissue of ovariectomized adult rats. Nuclei were sonically disrupted and 8 different subnuclear fractions were isolated by discontinuous sucrose density gradient centrifugation. 3H-Oestradiol-receptor complexes, measured by hydroxylapatite column chromatography, were localized in a light chromatin fraction as well as in a heavy chromatin fraction. Using the hydroxylapatite chromatography technique it was possible to demonstrate three classes of oestradiol-receptor complexes which differ in affinity for the chromatin. Oestradiol-receptor complexes with a high affinity for the chromatin were predominantly localized in the heavy chromatin fraction, whereas complexes with a lower affinity for their acceptor sites were present in the lighter chromatin fraction.
1. An [3H]oestradiol-exchange method was developed for the determination of oestradiol-receptor complexes in the nuclear fraction of immature rat testicular tissue. This method permits the determination of nuclear oestradiol-receptor sites in the presence of a relatively large amount of non-specific oestradiol binding present in testicular nuclei. After incubation of nuclei for 60min at 20 degrees C in the presence of [3H]oestradiol with or without a 1000-fold excess of non-radioactive diethylstilboestrol, specific binding can be determined quantitatively in the KCl-extractabe fraction, which contains 40% of the total receptor population. 2. The amount of receptor-bound steroid present in the 0.4m-KCl extract of testicular neclei remained constant during incubation at 20 degrees C. For uterine nuclei incubated with [3H]oestradiol at 37 degrees C a shift of specifically bound [3H]oestradiol occurred from the KCl-soluble fraction to the KCl-insoluble fraction. 3. In intact rat testis, about 20% of the total receptor concentration was present in its nuclear form. Hypophysectomy 5 days before measurement resulted in a twofold decrease in the amount of receptor, which was present mainly in the cytosol. After injection of choriogonadotropin to intact animals, the total receptor concentration increased threefold. 4. This nuclear exchange method might be useful for determination of occupied specific receptor sites in tissues with relatively low contents of specific receptors.
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Testosterone production in isolated Leydig cells from testes of immature and adult rats was stimulated by addition of LH in a dose dependent way. Hypophysectomy of adult rats had no influence on LH-stimulated testosterone production in isolated Leydig cells after 5 days. In contrast hypophysectomy of immature rats resulted after 5 days in an almost complete loss of LH sensitivity of isolated Leydig cells. Daily adminitration of FSH during 5 days starting immediately after hypophysectomy maintained LH responsiveness of isolated Leydig cells of immature rats. Also FSH administration starting on day 5 after hypophysectomy resulted in a restoration of LH responsiveness. Estradiol benzoate, injected simultaneously with FSH, abolished the FSH-induced LH responsiveness.
DNA/cell distributions were recorded by automated cytofluorometry (=pulse cytophotometry) in bone-marrow aspirates of leukaemia and lymphosarcoma patients subjected to chemotherapy. In most cases, early perturbations in DNA/cell histographs were observed, characteristically reflecting the known mode of action of the drugs. These changes in general preceded the clinical observation of drug response. In a series of 23 measurements in 19 patients, a positive correlation between early cytophotometric changes and clinical effects of chemotherapy was observed in 17 patients. Five patients were negative for both cytophotometric and clinical reactions and one patient was probably false-positive. The validity of the assay for early detection of drug resistance in acute leukaemia and related diseases is discussed.
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After administration of oestradiol-17beta to intact mature and immature rats, a decrease in the testicular concentration of specific oestradiol-binding sites was observed within 1 h. The binding capacity was replenished starting about 3 h after oestradiol administration and after 5 h the oestrogen receptor level had returned to control values. Exposure of intact animals to oestradiol-17beta for longer periods (up to 24 h) did not result in an increase of receptor levels in testicular cytosol. Mature animals which were hypophysectomized for periods of up to 10 days did not show a significant change in the number of specific oestradiol-binding sites in either total testicular tissue or dissected interstitial tissue. At 15 days or longer periods after hypophysectomy, an apparent increase in receptor concentrations in total testicular cytosol was observed due to a relative increase in the amount of interstitial tissue. A specific oestradiol-binding protein is present in plasma of immature male rats aged less than 30 days. This plasma protein could also be demonstrated in the cytosol of testes of immature rats. In contrast to the cytosol receptor, which shows a moderate affinity for diethylstilbestrol (DES), the plasma protein did not bind DES. The sedimentation values of the plasma protein and the oestradiol receptor were 4 S and 8 S respectively. These differences in characteristics made it possible to demonstrate the presence of the oestradiol receptor in addition to the binding protein in testicular cytosol of rats from 14 days of age onwards. The nuclear receptor for oestradiol-17beta could be demonstrated after incubation of testicular tissue of rats from 4 days of age onwards.
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