Ryusuke Nakao

Kyushu University, Hukuoka, Fukuoka, Japan

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Publications (8)31.27 Total impact

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    ABSTRACT: Using a specific antiserum for the C-terminal glycine amide region of human pancreastatin (PST), pancreastatin-like immunoreactivity (PST-LI) was measured in cerebrospinal fluid (CSF) from 447 subjects (368 ± 10.8 pmol/l, mean ± S.E.M.) free from endocrine diseases. The CSF contents of PST-LI showed a mountain-shape type change which peaked at 40 years of age. The highest concentration was found in the group of ages 40–49 years old (412 ± 22.9 pmol/l) and the lowest concentration was found in the group of ages 80–89 years old (293.2 ± 45.2 pmol/l) among various age groups. Gel chromatographic examination revealed the presence of two major forms (MW 13,500 and 5,400) of PST-LI in CSF. Because of the character of this antibody, the large molecular form is possibly an N-terminally elongated PST and the other may be PST-52. This may be the first report on the unique age-related change of PST concentration in CSF.
    Neuroscience Letters 04/1994; 170(1-170):179-182. DOI:10.1016/0304-3940(94)90268-2 · 2.03 Impact Factor
  • R Nakao · T Yanase · Y Sakai · M Haji · H Nawata ·
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    ABSTRACT: The androgen receptor (AR) from a patient with Reifenstein syndrome (incomplete androgen insensitivity syndrome) was characterized. The patient's pubic skin fibroblasts had normal androgen binding. However, when incubated at 41 C, fibroblasts from the patient had a marked decrease in androgen binding as compared with normal fibroblasts. Analysis of coding sequences of the androgen receptor gene revealed a single nucleotide substitution in exon E, resulting in an amino acid change from glycine (GGG) to valine (GTG) at amino acid 743 within the steroid binding domain of AR. Reconstruction of this mutation by site-directed mutagenesis into a human AR complementary DNA followed by expression in COS1 cells led to production of a mutant AR with no significant difference in androgen binding when cells were incubated with androgen at room temperature. However, in contrast to wild type AR expressed in COS1 cells, the mutant AR had markedly lower androgen-binding affinity at 41 C. The mutant receptor could still stimulate a reporter gene at 37 C but this transcriptional stimulation was also decreased when compared with wild type AR receptor in a chloramphenicol acetyltransferase assay. These results suggest that partial androgen resistance in this patient with Reifenstein syndrome is due to a single point mutation in the steroid binding domain of the androgen receptor.
    Journal of Clinical Endocrinology &amp Metabolism 08/1993; 77(1):103-7. DOI:10.1210/jc.77.1.103 · 6.21 Impact Factor
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    Y Sakai · T Yanase · R Takayanagi · R Nakao · Y Nishi · M Haji · H Nawata ·
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    ABSTRACT: The mechanism of dissociated secretion between adrenal androgens and cortisol observed in several clinical situations remains unclear. We investigated whether the electron transfer systems NADPH-cytochrome P450 reductase and cytochrome b5, both of which had been shown to increase 17,20-lyase activity in vitro, were involved in the reaction selectivity between 17 alpha-hydroxylase and 17,20-lyase in adrenocortical adenomas obtained from eight patients with Cushing's syndrome producing different concentrations of adrenal androgen. In vitro enzyme assay using microsomal fraction of adenoma indicated that all adenomas from seven patients showed almost the same degree of 17 alpha-hydroxylase and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) activities. However, the 17,20-lyase activities of two adenomas producing high concentrations of adrenal androgens were 3-fold greater than those of other five adenomas producing low concentrations of adrenal androgens. The mRNA concentrations of cytochrome P45017 alpha and 3 beta HSD were approximately the same in all adenomas, whereas those of cytochrome b5 in two adenomas possessing high 17,20-lyase activities were greater than those in other adenomas. The increased levels of cytochrome b5 in the two adenomas were further confirmed at the protein level using Western blot analysis of the microsomal fraction. No significant expression of NADPH-cytochrome P450 reductase was detected in any of the adenomas by Northern blot analysis. These results suggest that the difference in the concentration of cytochrome b5 in adrenal adenomas from patients with Cushing's syndrome may partially account for the difference in the amount of adrenal androgens produced by the tumors.
    Journal of Clinical Endocrinology &amp Metabolism 06/1993; 76(5):1286-90. DOI:10.1210/jc.76.5.1286 · 6.21 Impact Factor
  • R Nakao · M Haji · T Yanase · A Ogo · R Takayanagi · T Katsube · Y Fukumaki · H Nawata ·
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    ABSTRACT: Androgen receptors (ARs) in two Japanese siblings with complete androgen insensitivity syndrome were characterized, and their molecular bases were investigated. Androgen binding was undetectable in cultured pubic skin fibroblasts from the patients by whole cell assay. Sequence analysis of exons B-H, which encode the DNA- and steroid-binding domains, of the AR gene from these patients using polymerase chain reaction revealed a single nucleotide substitution in exon F, resulting in an amino acid change at 786 from methionine (ATG) to valine (GTG) within the steroid-binding domain of AR. Reconstruction of this mutation by site-directed mutagenesis into human AR cDNA followed by expression in COS-1 cells led to production of the same amount and the same molecular mass of immunodetectable AR protein as those found with expression of the normal human AR cDNA. However, in contrast to wild-type AR expressed in COS-1 cells, the mutant AR showed markedly low affinity of androgen binding by whole cell assay. These results suggest that androgen resistance in these patients is due to the point mutation in the steroid-binding domain of the AR.
    Journal of Clinical Endocrinology &amp Metabolism 06/1992; 74(5):1152-7. DOI:10.1210/jc.74.5.1152 · 6.21 Impact Factor
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    ABSTRACT: We isolated a cDNA encoding type-1 angiotensin II receptor from a human liver cDNA library. The cDNA had an open reading frame encoding a protein of 359 amino acid residues with a relative Mr of 41,060. The deduced amino acid sequence of the human angiotensin II (Ang II) receptor was 95.3% and 94.2% identical to those of bovine and rat type-1 Ang II receptors, respectively, and had a significant similarity with the G protein-coupled receptor. The rank order of the binding to the receptor expressed in COS-7 cells was Ang II > Ang III > Ang I. The expression of the Ang II receptor mRNA was detected in human liver, lung, adrenal and adrenocortical adenomas but not in adrenomedullary tumor, pheochromocytoma, by Northern blot analysis.
    Biochemical and Biophysical Research Communications 04/1992; 183(2-183):910-916. DOI:10.1016/0006-291X(92)90570-B · 2.30 Impact Factor
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    ABSTRACT: Using two oligonucleotide probes which were synthesized based on the amino acid sequences of tryptic fragments obtained from purified bovine lung endothelin(ET) receptor, we isolated a cDNA encoding non-isopeptide-selective type of ET receptor (ETB) from a human liver cDNA library. The cDNA encoded a protein of 426 amino acid residues with putative seven transmembrane segments, exhibiting a significant similarity with the G-protein-coupled receptor superfamily. The deduced amino acid sequence of human ETB receptor was 88% and 64% identical to those of rat lung ETB receptor and human ET-1-specific(ETA) receptor, respectivaly. COS cells transfected with the cloned cDNA expressed the binding sites with high affinity for every ET or sarafotoxin(SRT) isopeptide. Modification of disulfide bridges, a N-terminal amino acid or C-terminal hydrophobic residues of the isopeptides markedly reduced their binding affinity for the expressed ETB receptor (Fig.), suggesting that ETB receptor recognizes the common structures for ET/SRT. The ETB mRNAs were significantly expressed in porcine aortic intima but not in the media, which supports the previous observation by us [FEBS Lett. (1991) 282 103–106].
    Clinical and Experimental Hypertension 01/1992; a14(4):741-765. DOI:10.3109/10641969209036219 · 1.23 Impact Factor
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    ABSTRACT: We elucidated the role of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) in human and bovine adrenocortical steroidogenesis. The urinary volume, sodium excretion and cylic GMP (cGMP) excretion and plasma cGMP were markedly increased by the synthetic α-human ANP (α-hANP) infusion in healthy volunteers. Plasma arginine vasopressin (AVP) and aldosterone levels were significantly suppressed. Both ANP and BNP inhibited aldosterone, 19-OH-androstenedione, cortisol and DHEA secretion dose-dependently and increased the accumulation of intracellular cGMP in cultured human and bovine adrenal cells. α-hANP significantly suppressed P450scc-mRNA in cultured bovine adrenal cells stimulated by ACTH. Autoradiography and affinitu labeling of [125I]hANP, and Scatchard plot demonstrated a specific ANP receptor in bovine and human adrenal glands. Purified ANP receptor from bovine adrenal glands identified two distinct types of ANP receptors, one is biologically active, the other is silent. A specific BNP receptor was also identified on the human and bovine adrenocortical cell membranes. The binding sites were displaced by unlabelled ANP as well as BNP. BNP showed an effect possibly via a receptor which may be shared with ANP. The mean basal plasma α-hANP level was 25 ± 5 pg/ml in young men. We confirmed the presence of ANP and BNP in bovine and porcine adrenal medulla. Plasma or medullary ANP or BNP may directly modulate the adrenocortical steroidogenesis. We demonstrated that the lack of inhibitory effect of α-hANP on cultured aldosterone-producing adenoma (APA) cells was due to the decrease of ANP-specific receptor, which caused the loss of suppression of aldosterone and an increase in intracellular cGMP.
    The Journal of Steroid Biochemistry and Molecular Biology 02/1991; 40(1-3-40):367-379. DOI:10.1016/0960-0760(91)90204-I · 3.63 Impact Factor
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    ABSTRACT: We studied the sequential changes of plasma levels of immunoreactive '7B2' (IR-7B2), a neuroendocrine polypeptide, after a subcutaneous injection of 50 micrograms of synthetic octapeptide somatostatin analogue (SMS 201-995) in seven patients with acromegaly due to GH-producing pituitary adenoma. Compared to the basal levels, mean plasma IR-7B2 and GH levels significantly decreased, until 5 and 10 h respectively after the administration of SMS 201-995. The mean (+/- SEM) nadir levels of plasma IR-7B2 and GH were 68.1 +/- 10.1 and 13.1 +/- 6.9%, respectively, compared to mean plasma levels before treatment (100%). Plasma IR-7B2 as well as GH levels did not change significantly when saline was administered subcutaneously to three acromegalic patients. In addition, plasma IR-7B2 levels did not change significantly after the administration of SMS 201-995 in normal subjects or in patients with primary hypothyroidism in whom SMS 201-995 induced a decrease of plasma TSH levels. These results strongly suggest that SMS 201-995 has an unequivocal suppressive effect on the synthesis and/or the secretion of 7B2 in human somatotroph adenoma cells.
    Clinical Endocrinology 02/1990; 32(1):49-55. DOI:10.1111/j.1365-2265.1990.tb03749.x · 3.46 Impact Factor