Biomedical subjects
Publications and source records attributed to T Demura.
X-linked spinal and bulbar muscular atrophy (SBMA) is usually associated with feminization and hypogonadism. We were unable to find androgen receptor (AR) in the scrotal skin of three patients with SBMA, and propose that AR abnormality is the cause of the disease.
Previously, we established an anti-androgen receptor (AR) monoclonal antibody. Using the antibody, we investigated immunohistological AR localization in human testes, epididymides, seminal vesicles and scrotal skins. The testes, epididymides and scrotal skins were obtained from a prostate cancer patient without pre-hormonal therapy undergoing bilateral orchiectomy. The seminal vesicles were obtained from a bladder cancer patient undergoing radical cystectomy. The tissues were immediately frozen in liquid nitrogen and kept at -80 degrees C until used. Cryostat-frozen sections were cut at 5 microns and stained by an indirect method. We obtained the following results. 1) In the testes, nuclei of Leydig cells were stained though Sertoli cells were not stained. AR localization in Leydig cells which produce testosterone suggests autocrine or intracrine mechanism in the testis. 2) In the epididymides, nuclei of epithelial cells of epididymal ducts were stained, while muscles and connective tissues were not stained. In the seminal vesicles, nuclei of glandular epithelial cells were stained. 3) In the scrotal skins, the cells of squamous cell layer have positive stainings. The cells in the upper portion of squamous cell layer were stained more intensely than the cells in the lower portion. The basal layer was not stained. The cells of the outer root sheath of hair follicles in the scrotal skins were also stained. 4) In androgen target organs, AR-positive cells and AR-negative cells were mixed in the epithelium of a glandular duct, which suggests heterogeneity of AR localization in the androgen target organs.
A case of urinary undiversion in a 19 year old man with high ileal loop, who at the age of 1 year was inadvertently cystectomized for probable acute urinary retention masquerading as acute abdomen, is reported. The ileo-ceco-colonic segment was isolated and the colon was partially detubularized. After ileocecal intussusception and stabilization to the cecal wall (Hendren), a segment of ileal patch was applied to form an Indiana type pouch. The ileal loop was free from the abdominal wall and its distal end was anastomosed to the terminal ileum of the pouch. Undiversion was completed by connecting the pouch at its dependent portion with the remnant prostatic urethra. At 4 months postoperatively the pouch functions quite satisfactorily as a low pressure and good volume reservoir that empties well without reflux. His erectile and ejaculatory function have also been maintained.
Using autoradiography, localization of alpha 1-adrenoceptor in human prostate was examined with particular reference to zonal difference. Prostatic tissues were obtained from total cystoprostatectomy specimens of six male bladder tumor patients. Peripheral zone (PZ), central zone (CZ) and preprostatic region which contained preprostatic sphincter and periurethral gland were dissected from the tissues. [125I]-HEAT was used as radioligand for autoradiography. Autoradiographic distribution of alpha 1-adrenoceptor was quantitated by grain counting using a light microscope equipped with a micrometer. Specific binding was demonstrated predominantly in fibromuscular stroma of PZ, fibromuscular stroma of CZ and preprostatic sphincter. No specific binding was demonstrated in glandular epithelium of PZ and periurethral gland. Glandular epithelium of CZ showed a few specific bindings. Among fibromuscular portion, specific bindings in both CZ and preprostatic sphincter were significantly higher than in PZ. Specific binding in glandular epithelium of CZ was significantly less than in fibromuscular stroma of PZ. These results show that the human prostate has a zonal difference in the distribution of alpha 1-adrenoceptors, probably reflecting some functional difference in these regions.
Nuclei of the guinea pig adrenal cortex contain a protein that specifically binds progesterone and that, biochemically, is clearly distinct from the classical progesterone receptor. The adrenocortical nuclear progesterone-binding protein has now been purified more than 2000-fold by steroid-affinity chromatography with a 75% yield. The purified protein preparation demonstrated three major bands on sodium dodecyl sulfate-polyacrylamide gel of 79K, 74K, and 50K. To determine which of the three might represent the progesterone-binding protein, steroid photoaffinity labeling was performed which resulted in the specific and exclusive labeling of a 50K band. Thus, the adrenocortical nuclear progesterone-binding protein appears to be distinct from the classical progesterone receptor not only biochemically, but also on the basis of molecular size. To test whether the adrenocortical nuclear progesterone-binding protein can be hormonally stimulated, guinea pigs were treated with ACTH. The chronic administration of ACTH caused a 4- to 6-fold increase in the specific progesterone binding capacity without a change in the binding affinity. There appeared to be no significant difference in nuclear progesterone binding between the zona fasciculata and zona reticularis. This finding suggests a mediating role for the progesterone-binding protein in ACTH action. In addition, the nuclear progesterone-binding protein bound to nonspecific DNA sequences, further suggesting a possible transcriptional regulatory role.
The binding of [3H]R1881 to cytosol fractions was determined for the spongy tissues of horse, porcine and human penis. Binding of [3H]R1881 to cytosol fractions from these spongy tissues was found to be specific with high affinities (mean of Kd; 1.8 X 10(-10) M) but with equally low binding capacities (mean of 4.66 fmol/mg. protein). Saturation analysis revealed that the binding capacity was similar for both corpus cavernosum and corpus spongiosum of horse penis. We conclude that a trace amount of androgen receptor is present in spongy tissue of the penis and that there is no difference in cytosolic receptors between corpus cavernosum and corpus spongiosum in adult mammalian penis.
Progesterone, which is normally produced in the endoplasmic reticulum, was found to be rapidly degraded in the cytosolic fraction of the guinea pig adrenal cortex in vitro. Assuming this finding reflects what happens in vivo raises a question as to the source of progesterone for interacting with a nuclear progesterone-binding protein (P4-BP) that exists in this model system. It was subsequently found that pregnenolone, which in contrast to progesterone is relatively stable in the cytosol, was converted to progesterone by endogenous nuclear 3 beta-ol dehydrogenase. It was also determined that the nuclear-derived progesterone specifically bound to the nuclear P4-BP which is distinct from the classical progesterone receptor. The guinea pig adrenocortical cytosol contains a specific pregnenolone-binding protein (P5-BP) that could be virtue of its pregnenolone binding activity regulate the conversion of pregnenolone to progesterone in the nuclear compartment and thereby reduce the binding of progesterone to the nuclear P4-BP. A partially purified P5-BP preparation markedly inhibited the nuclear conversion of pregnenolone to progesterone and reduced the binding of progesterone to the nuclear P4-BP (P5-BP did not directly inhibit binding of progesterone to the nuclear P4-BP). The ability of P5-BP to inhibit the conversion of pregnenolone to progesterone was destroyed by heat and alkaline phosphatase treatment. The binding of pregnenolone to the P5-BP, as previously reported, is regulated by phosphorylation/dephosphorylation, and alkaline phosphatase-treated P5-BP loses the ability to bind pregnenolone; this process can be reversed by a cytosolic kinase. This provides a mechanism for controlled release of bound steroid. These results suggest that P5-BP regulates the nuclear conversion of pregnenolone to progesterone and thus the binding of progesterone to the nuclear P4-BP.
Hybrid cell lines were prepared by the fusion of BALB/c myeloma NS-1 cells with the lymphocytes of BALB/c mice that were immunized with partially purified androgen receptor (AR) from human prostates. Nine clones of the hybrid progeny were determined for the production of antibodies against AR by immunoprecipitation assay. One of the clones, referred to as "5F4", was chosen for analysis of the detailed specificity. The clone "5F4" secreted IgM class antibodies against AR. Competition study demonstrated that "5F4" antibody inhibited androgen binding of AR, suggesting that the antibody identifies androgen binding site of AR. Immunoblotting analysis showed that the antibody identified the ARs as two proteins, 95 kD and 41 kD proteins, on a sodium dodecyl sulfate polyacrylamide gel. It is suspected that a 95 kD protein should be a monomeric AR and a 41 kD protein is a proteolytic fragment of AR. Immunohistochemical analysis demonstrated that androgen-dependent tissues--human prostatic hypertrophy tissues, an AR abundant prostatic cancer tissue and fibroblast cells from human genital skin--were stained intensely with "5F4" monoclonal antibody, while androgen-independent tissues--fibroblast cells from lymph nodes, an AR deficient prostatic cancer tissue and human prostatic cancer cell line, PC-3--showed no staining. These results also support the specificity of the antibody for AR.
Nuclei purified from the guinea pig adrenal cortex contain a specific progesterone-binding activity which, based on enzyme degradation studies, appears to be proteinaceous. Saturation analysis revealed a Kd of about 15 nM and a binding capacity of about 33 pmol/mg DNA. The activity of the nuclear binding protein was specific essentially for progestational steroids; the two most potent progesterone competitors were 5 alpha-pregnane-3,20-dione and medroxyprogesterone (17 alpha-hydroxy-6 alpha-methylprogesterone), while 17 beta-estradiol, testosterone, cortisol, and other related steroids were poor competitors. The adrenocortical nuclear progesterone-binding protein was present to an equal extent in both male and female guinea pigs. The adrenocortical nuclear progesterone-binding protein differed from the classical progesterone receptor in that 1) the affinity of the adrenocortical binding protein for progesterone is an order of magnitude lower; 2) the potent synthetic progestin R5020 binds less tightly to the adrenocortical progesterone-binding protein; 3) the adrenocortical progesterone-binding protein is not modulated by estrogenic activity; 4) the adrenocortical progesterone-binding protein is more stable at 37 C; 5) the adrenocortical nuclear progesterone-binding protein is not salt extractable; and 6) Western blot analysis has revealed that an antiprogesterone receptor monoclonal antibody, which recognizes the guinea pig uterine classical nuclear progesterone receptor, does not recognize the adrenocortical nuclear progesterone-binding protein. Thus, the guinea pig adrenocortical nucleus contains a type of progesterone-binding protein that appears to be clearly different from the classical progesterone receptor.
Serum beta-HCG and urinary HCG were measured in 17 patients with histologically proved pure seminoma of the testis. The patients have been followed to date for 10 to 74 months after orchiectomy, and have no residual disease or recurrence of their disease. Preoperative urinary HCG levels were abnormal in 9 patients (53%). Serum beta-HCG levels were slightly elevated in 6 out of 12 patients evaluated, and were reduced after orchiectomy even in patients with preoperative normal beta-HCG levels (P less than 0.05). Preoperative levels of the tumour marker in seminoma were far lower than in patients with non-seminomatous germ cell tumours, but it would not be rare to see a small amount of HCG produced by the tumour cells in the seminoma.
Hybrid cell lines were prepared by the fusion of BALB/c myeloma NS-1 cells with the lymphocytes of BALB/c mice that were immunized with partially purified androgen receptor (AR) from human prostates. One of the clones, 5F4, was chosen for detailed specificity analysis. The avidin-biotin-peroxidase complex (ABC) procedure was used for immunohistochemical staining of the 5F4 monoclonal antibody. In human benign prostatic hypertrophy (BPH) tissues, cytoplasm and nuclei were stained. Of 16 prostatic cancer tissues, 2 were composed of AR-positive cells exclusively, 7 were composed of AR-negative cells, and 7 contained both AR-positive cells and AR-negative cells (mixed). Of nine cases that were AR-positive or mixed, seven cases responded to the hormone therapy, and two were not determined for responsiveness because the patients died early of other diseases. Of seven AR-negative cases, all but one inestimable case had no response to the hormone therapy. Immunohistochemical analysis of AR by using the monoclonal antibody 5F4, was a useful tool for determining androgen dependency of prostatic cancers.
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The prostate is one of the target organs of androgen. This fact is the foundation of the anti-androgen therapy for the treatment of prostatic of cancer as there are many prostatic cancers which show no response to hormone therapy. Since androgen acts via an androgen-androgen receptor complex in cytosol, trials to predict the effectiveness of the anti-androgen therapy for the treatment of prostatic cancers by means of the measurement of androgen receptor (AR) have been done. However some investigators emphasize that the response to hormone therapy and the contents of AR are not necessarily parallel. This discrepancy is probably due to the inaccurate measurement of AR. AR is measured by a radio-receptor-assay method, but it is possible that androgen binding proteins other than AR are also measured by the radioreceptor-assay method. In the human prostatic tissues, there are several androgen binding proteins other than AR. These are mainly testosterone-binding globulin (TeBG) and progesterone-binding protein (PBP). In this study, I attempted to separate and characterize androgen-binding proteins in human prostates. 500 g of benign prostatic hyperplasia (BPH) tissues obtained from patients undergoing retropublic or suprapubic prostatectomy were used. In order to separate these androgen-binding proteins, DEAE-cellulose ion exchange chromatography was used. Prostatic cytosol was applied on the DEAE-cellulose column. Two 3H-R1881 binding peaks were eluted: one was a flow-through fraction (peak I), and the other was an 0.1M KCl eluted fraction (peak II). When human serum was applied to the column, one 3H-DHT binding peak flowed through the column (peak III). It was thought that peak III contained TeBG. In order to ascertain whether AR was composed in peak I or peak II, an inhibition test by various non-radioactive steroids, R1881, DHT, testosterone, progesterone and estradiol was performed. 3H-R1881 binding of peak I was not inhibited by DHT or testosterone, while 3H-R1881 binding of peak II was inhibited by DHT and testosterone. The androgen-binding affinity of the peak I and peak II fraction was also measured using Scatchard's analysis. The dissociation constant of peak I was 6.25 nM, and that of peak II was 0.76 nM. Judging from these measurements the androgen-binding protein of peak II contained a specificity and high affinity to androgen. Consequently it was thought that AR was composed in peak II. Peak II fraction was applied on a Sephadex G200 gel chromatography column. A single androgen binding peak eluted in the void fraction.(ABSTRACT TRUNCATED AT 400 WORDS)
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