Keizo Noma

Identification of immunoassayable estrogen receptor lacking hormone binding ability in tamoxifen-treated rat uterus

Using two different monoclonal antibodies to human estrogen receptor (ER), the enzymeimmunoassay was performed. The values of ER contents in human breast cancer and untreated rat uteri obtained by this procedure were correlated well with those by [3H] estradiol binding assay. When estradiol was injected to immature rats, the enzymeimmunoassay showed the uterine receptor dynamic pattern similar to those analyzed by exchange assays. In contrast, tamoxifen administration induced the immunoassayable but nonsteroid binding form of ER. This ER-like antigen was the heat-labile molecule with the sedimentation constant of 7 S while ER in untreated rat uterine cytosol sedimented at 9 S. These results suggest the presence of unique molecular state of ER induced by tamoxifen.

Serum prolactin levels in eighty patients with sarcoidosis

Abstract. Serum prolactin levels were measured by radio‐immunoassay in eighty patihts (thirty‐four males, forty‐six females) with sarcoidosis before treatment. In twelve patients (15%) serum prolactin levels were more than two standard deviations above the mean of normal subjects. Hyperprolactinaemia was found most frequently (22%) in patients with radiological stage 11; however, 14% of patients with stage I also had elevated serum prolactin levels.

INCREASED SERUM OESTRONE AND OESTRADIOL FOLLOWING SPIRONOLACTONE ADMINISTRATION IN HYPERTENSIVE MEN

The present study was undertaken to evaluate long‐term effects of spironolactone on basal serum oestrone, oestradiol, testosterone, LH and prolactin concentrations in hypertensive male patients. Serum prolactin response to TRH was also evaluated. Patients were divided into two groups: a conventional‐dosage group, consisting of six males with essential hypertension who took 75 to 150 mg of spironolactone daily for 12 weeks, and a high‐dosage group, consisting of two males with idiopathic hyperaldosteronism who took 300 mg of spironolactone daily for more than 40 weeks.

Chronic estrogen treatment causes an alteration in uterine estrogen receptor dynamics of rats.

Estrogen receptor content and dynamics in the uteri obtained from chronically estrogenized rats were analyzed. 12 day treatment with a subcutaneous implantation of a diethylstilbestrol pellet resulted in maximal stimulation of uteri with regard to wet tissue weight, DNA content, as well as progesterone receptor content without significant alteration of the estrogen receptor level. Estrogen receptor dynamics in just ovariectomized or ovariectomized and diethylstilbestrol-stimulated rats elicited by a single injection of estradiol were next examined using the exchange methods. The cytosol receptor content rapidly declined, with a small and temporary accumulation of the nuclear receptor in the uterus from rats continuously exposed to diethylstilbestrol during the preceding 12 days. A relatively rapid cytosol receptor replenishment was also observed in rats pretreated with diethylstilbestrol. This was accompanied by a rapid decrease in the nuclear receptor level to 70% of the preinjection value at 5 h after estradiol administration. These data are in contrast to findings on uteri of ovariectomized and nonestrogen-treated rats, in which a single injection of estradiol resulted in a prolonged nuclear receptor retention and a delayed cytosol receptor replenishment. Adrenalectomy did not result in a significant change of receptor dynamic patterns, suggesting that adrenal steroids do not play a role in the alteration of receptor dynamics elicited by continuous stimulation with diethylstilbestrol. These observations suggest that a continuous exposure of rat uteri to the estrogen causes an altered regulation of estrogen receptor dynamics by the homologous steroid compared to those in chronically estrogen-deprived rats.

Estrogen induces secretary proteins in transformed mouse Leydig cell in vivo, but not in vitro

The estrogen-induced proteins were analyzed in one of estrogen responsive mouse Leydig tumors. The incubation of cells freshly prepared from solid tumors with [35S] methionine resulted in the demonstration of estrogen-induced secretary protein with a molecular weight of 34,000. The additional minor estrogen-induced secretary protein (36,000) was also identified. An exposure of these cells to the culture condition for 48 hr caused the loss of their ability to synthesize these secretary proteins even in the presence of estrogen. In contrast, minced tumor tissue was observed to sustain the ability to synthesize these proteins at least for 48 hr. These results would suggest that some cellular arrangement is required for the synthesis of estrogen-induced proteins.

Detection of estrogen receptor-like high affinity components by exposure of cytosols from mouse leydig cell tumor or male rat liver to chaotropic salts☆

Abstract Modifications of steroid-macromolecule interaction induced by so-called chaotropic salts were studied. When the cytosol from one of estrogen-independent mouse Leydig cell tumors was incubated with [3H]-estradiol (E2), an unusual estrogen binding component was identified, with a relatively weak affinity for E2 (Kd 10−7 M). This binding could not be inhibited by diethylstilbestrol (DES). However, exposure of this cytosol to 0.4 M NaSCN resulted in the clear demonstration of high affinity binder for E2 (Kd 10−9 M). Furthermore, this high affinity binding with E2 was inhibited by DES in a competitive manner. The quantitation of the binding sites revealed that the binding capacity at low concentrations of [3H]-E2 (less than 10 nM) is higher in the presence of NaSCN than in the absence of NaSCN. Liver cytosol from noncastrated male rat was also found to contain E2 binding component with a low affinity (Kd 10−7 M) and a very high concentration of sites. The binding component with high affinity (Kd 10−9 M) as well as a concomitant loss of the low affinity sites became detectable in 0.4 M NaSCN. Moreover, this association with E2 was found to be competitively inhibited by DES (Ki 3 × 10−9 M). The similar changes were elicited by the other chaotropic salt, NaClO4. These observations suggest that the detection of receptor-like high affinity binder in chaotropic salts may be mediated by two different mechanisms: one is a formation of new high affinity binder, the other is a destruction of the unusual second binder. Furthermore, the easy demonstration of estrogen receptor in chaotropic salt which is not detectable in a conventional buffer system might indicate that careful consideration is required to decide the absence of receptor protein.

Von Recklinghausen's Disease with Pheochromocytoma and Nonmedullary Thyroid Cancer

Excerpt To the editor: It is well known that von Recklinghausens disease is inherited as an autosomal dominant trait and is often associated with various neoplasms, including pheochromocytoma and ...

STEROID RECEPTOR FORMS AND THEIR INTERACTION WITH CYTOPLASMIC MODULATORS

The cytoplasmic modulator affecting steroid receptor functions was studied. The diminution in the concentration of the low molecular weight substances in the cytosol caused the increased interaction of hormone-receptor complexes with nuclei (a step termed activation). Furthermore, dialysis of rat uterine cytosol in the absence of estrogens subsequently followed by incubation with isolated nuclei resulted in the demonstration of an appearance of unoccupied nuclear receptor which was found to be 4S form. The addition of the dialyzable compound into rat uterine estrogen receptor system caused the suppression of temperature-dependent activation while the already-activated estrogen receptor was not affected by the small molecules in relation to its nuclear binding ability. These results may indicate that this small molecule, so-called the low molecular weight inhibitor, is capable of interacting with nonactivated receptor. In one of the estrogen-independent Leydig cell tumor lines, unique low-affinity estrogen binder with a mol. wt approximately 36,000 was identified. This binder did not show appreciable nuclear binding ability even after conventional heat activation. However, removal of the small molecules from this cytosol resulted in a marked increase in affinity for estrogens with a concomittant alteration of the mol. wt to approximately 70,000. These changes also paralleled a dramatic enhancement of nuclear binding ability. This small molecule might be different from the low molecular weight inhibitor suppressing receptor activation, since this tumor cytosol containing unique estrogen binder had much less inhibitory activity against receptor activation when compared with those in the other cytosol containing usual estrogen receptor systems. In adult rat urine cytosol, a new modulator was identified to recognize only activated estrogen receptor but not glucocorticoid receptor and to induce receptor aggregation with a concomittant loss of its nuclear binding ability. These reactions were accelerated by the presence of physiological or higher KCl concentration and exposure to a relatively high temperature (20-30 degrees C). Interestingly, this factor was not identified in the immature rat uteri or liver.

Sodium molybdate converts the RNA-associated transformed, oligomeric form of the glucocorticoid receptor into the transformed, monomeric form.

The glucocorticoid receptor from rat liver cytosol prepared in 2 ml buffer/g tissue sedimented at approximately 10 S in low salt density gradient centrifugation without molybdate. When the receptor was heated at 25 degrees C, both approximately 10 S and approximately 7 S forms were seen in low salt gradient. The approximately 10 S form was not capable of binding to DNA-cellulose and was stabilized by sodium molybdate, namely it corresponded to untransformed receptor. The approximately 7 S form was capable of binding to DNA-cellulose and regarded as transformed receptor. On the other hand, partially-purified transformed receptor labeled with [3H]dexamethasone-21-mesylate sedimented at approximately 5 S, which migrated as a approximately 94 kDa species in SDS-polyacrylamide gel electrophoresis. The reconstitution analysis of this partially-purified approximately 5 S receptor and liver cytosol, showed the shift to approximately 7 S form. RNase A or T1 converted approximately 7 S transformed form into approximately 5 S but it did not affect approximately 10 S untransformed form. 5-20 mM sodium molybdate also shifted approximately 7 S to approximately 5 S. These results indicate that the approximately 7 S transformed form of the glucocorticoid receptor observed in low salt conditions might be an oligomer, probably including both approximately 5 S steroid-binding component and RNA/ribonucleoprotein, and that molybdate dissociates these interactions in a specific manner.