Establishment and characterization of monoclonal antibody against androgen receptor

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.     

Hormonal effects on the development changes of mouse small intestinal glycolipids

The composition of intestinal glycosphingolipids during normal and hormone-perturbed development was investigated. The concentrations of glycosphingolipids of mouse small intestine were affected by the injection of thyroxine or cortisone during suckling and weaning periods. GDla was reduced by the hormonal treatment among major gangliosides, GM3, GM1 and GD1a, of mouse small intestine during the suckling period. In contrast, asialo GM1 was precociously produced by the treatment, which scarcely found in control suckling mouse small intestine. The results showed that these hormones were related to developmental alteration of small-intestinal glycolipids.     

Guest-induced conformational change of β-cyclodextrin capped with an environmentally sensitive chromophore

A capped cyclodextrin, 6-deoxy-6-(p-hydroxy-m-nitrophenacylthio)-β-cyclodextrin, was prepared in order to detect any conformational change of the host upon the guest binding. The association constant between the cyclodextrin and 1-adamantanecarboxylate, cyclohexanecarboxylate, p-methylbenzoate, 3,3-dimethylbutyrate, or 2,2-dimethylpropionate was enhanced 20, 5.3, 3.7, 2.3, or 2.0 times, respectively, by chromophore capping. The changes in the electronic, NMR, and circular dichroism spectra as well as pK_a of this cyclodextrin upon binding of the guest strongly indicate a conformational change around the chromophoric moiety of the cyclodextrin.     

The role of cyclic AMP in aldosterone production by isolated zona glomerulosa cells.

The role of cyclic AMP in the regulation of aldosterone production by adrenocorticotropic hormone (ACTH), angiotensin II (A II), potassium, and serotonin was examined in collagenase-dispersed adrenal glomerulosa cells. The ability of 8-bromo cyclic AMP and choleragen to stimulate maximum aldosterone production indicated that cyclic AMP could act as second messenger for certain of the aldosterone-stimulating factors. The actions of ACTH and choleragen on aldosterone and cyclic AMP production were correlated in dog and rat cells, and a similar relation was seen during stimulation of rat cells by serotonin. In contrast, A II and potassium did not cause changes in cyclic AMP formation while stimulating aldosterone production. Intracellular and receptor-bound cyclic AMP were increased 3-fold by 10(-7) M ACTH but not by A II. Addition of a phosphodiesterase inhibitor increased the magnitude of the cyclic AMP response to ACTH but did not change the lack of stimulation by A II or potassium. In dog cells, the effects of A II and potassium on aldosterone production were partially additive to those of ACTH, choleragen, and 8-bromo cyclic AMP. In contrast, no additivity was observed between A II and potassium, or between combinations of the cyclic AMP-dependent stimuli. These results indicate that the actions of ACTH on aldosterone secretion are mediated by cyclic AMP formation, whereas A II and potassium stimulate aldosterone production through an independent mechanism. The lack of additivity between steroid responses to A II and potassium suggests that these factors could share a common mode of action on steroidogenesis in zona glomerulosa cells.     

Freeze-Fracture images on filipin-sterol complexes in the thyroid follicle epithelial cell of mice with special regard to absence of cholesterol at the site of micropinocytosis

Summary In order to clarify the distribution of cholesterol in the plasma-and cyto-membranes of the thyroid follicle cell, freeze-fracture images of the filipin-treated tissues of normal and TSH-treated mice were observed. The filipin-sterol complexes, 25 to 30 nm protuberances or pits are distributed densely and almost homogeneously on the fractured plasma membrane, though the small depressions showing aggregates of intramembrane particles lack the complexes. Each depression corresponds to the coated pit, which might be an initial site for micropinocytosis of the luminal colloid. The limiting membranes of all the large colloid droplets reabsorbed are generally very rich in the complexes, but some small regions on the limiting membrane of the droplet are less in their density. The membranes of the rough endoplasmic reticulum, of the nucleus and of the Golgi apparatus are almost free from the complexes, though small clusters consisting of 2–5 complexes are rarely scattered. In thin sections, the membranes which are rich in the filipinsterol complexes become obscure in their fine structure after treatment with filipin for 12–14 h.This study was supperted by grants from the Japan Ministry of Education     

A membrane-bound metallo-endopeptidase from rat kidney hydrolyzing parathyroid hormone. Purification and characterization

A metallo-endopeptidase, which appears to be an integral membrane protein of rat kidney, was purified to homogeneity by a series of standard chromatographic procedures. This enzyme significantly hydrolyzed human parathyroid hormone [hPTH(1-84)] and a synthetic substrate Suc-Leu-Leu-Val-Tyr-Mec (Suc = succinyl, Mec = 4-methyl-coumarinyl-7-amide). The purified enzyme had apparent molecular masses of 250 kDa on gel filtration, and 88 kDa and 245 kDa on sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing and non-reducing conditions, respectively. Its pH optimum for activity was 8.0-8.5 and its isoelectric point was pH 4.9. Its activity was inhibited by EDTA, EGTA and o-phenanthroline, but not by phosphoramidon. The metal-depleted enzyme was reactivated by the addition of metal ions. The enzyme was also inhibited by chymostatin and eglin C, and by thiol compounds. Of the synthetic substrates examined, the enzyme hydrolyzed only Suc-Leu-Leu-Val-Tyr-Mec, one of the synthetic substrates for alpha-chymotrypsin. It did not hydrolyze synthetic substrates with less than four amino acid residues with tyrosine in the P1 position. The enzyme hydrolyzed hPTH and reduced hen egg lysozyme but did not hydrolyze azocasein or [3H]methyl-casein. NH2-terminal amino acid sequence analyses of the degradation products of hPTH(1-84) and reduced hen egg lysozyme by the purified enzyme revealed that the enzyme preferentially cleaved these peptides at peptide bonds flanked by hydrophilic amino acid residues. Amino acid analyses showed that the main degradation products of PTH were hPTH(17-29), hPTH(30-38) and hPTH(74-84). The ability of the enzyme to hydrolyze peptide bonds flanked by hydrophilic amino acid residues and its inability to degrade azocasein distinguish it from several other kidney endopeptidases reported, such as endopeptidase 24.11 and meprin.     

Comparison of osteoporosis and calcium intake between Japan and the United States.

The number of osteoporotic females in Japan with vertebral bone mineral density measured by dual energy x-ray absorptiometry, defined as less than -3 SD of the peak bone mass, is approximately 10,000,000, corresponding to 8% of the whole population of Japan. While this value approximately corresponds to the prevalence of low bone mineral density in the United States, the incidence of hip fracture appears to be much less in Japan than in the United States, 50,000 per 125,000,000 per year compared with 250,000 for a population twice as large. This seems to be paradoxical because of the lower bone mineral density and lower calcium intake in Japan, with 400-500 mg/day mainly as soybean products, small fish with bones, and vegetables. The difference in hip fracture incidence, however, may not actually be as wide as it seems when the larger number of bedridden elderly subjects in Japan is taken into consideration. In these bedridden subjects with severe immobilization osteoporosis, hip fracture is only prevented by the fact that they are not ambulatory. Life-style difference may also offer an explanation. Sitting on a tatami mattress on completely flexed knees with frequent standing up, along with other household work, in a narrow home space may ensure a marked development of hip musculature and also provide skill in balancing oneself to prevent fails. The difference in fracture incidence should be analyzed from various angles to obtain a firm ground for the future prevention of hip fracture due to osteoporosis. Although osteoporosis universally affects all races and nationalities, conspicuous differences may be encountered in the severity of its manifestations and complications.(ABSTRACT TRUNCATED AT 250 WORDS)     

Possible role of sodium-hydrogen antiport in acetylcholine-induced relaxation of rat aorta.

The decreased extracellular Na concentration (25mM) attenuated vasodilator effect of acetylcholine (ACh) in norepinephrine-treated aortic ring. This attenuation was greater in the low Na medium substituted by Li, which can exchange intracellular H through Na-H antiport, as compared with that substituted by choline, which cannot. 10 microM amiloride canceled the difference between the two low Na mediums. Thus the inhibition of Na-H antiport may counteract the suppressive effect of decreased intracellular Na on ACh vasodilation, suggesting a possible role of Na-H antiport in a release of vasoactive substance(s) from endothelial cells.