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Volume 62, Issue 12
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[in Japanese]1986 Volume 62 Issue 12 Pages 1315-1325
Published: December 20, 1986
Released on J-STAGE: September 24, 2012
JOURNAL FREE ACCESS -
Sumitaka SAISHO, Kazuhiko SHIMOZAWA, Jun-ichi YATA1986 Volume 62 Issue 12 Pages 1326-1335
Published: December 20, 1986
Released on J-STAGE: September 24, 2012
JOURNAL FREE ACCESSTaking an advantage of the property of Δ4 -steroid that have a maximum absorbance around 250nm wave-length of ultraviolet, we devised an assay method for the determination of serum Δ4 -steroids concentration using a reverse phase high performance liquid chromatography (HPLC) -UV spectrometry.
The assay procedure was as follows : (1) A mixed solvent containing methanol, acetonitrile and water in 55/3/42 by volume was used as a mobile phase, and which was pumped at a constant flow rate of 1.5ml/min. (2) The main column and precolumn used were ERC-ODS-1161 (φ6mm × 10cm) and ERC-ODS-1652 (φ6mm × 3cm), respectively. (3) Two liquid-liquid extraction methods were used. One was a conventional method using dichloromethane for an extraction solvent, and the other was a simplified method using Extrelut column and ethylacetate. (4) Before a practical assay we examined the retention time of each steroid determined and its ratio of peak height to that of the internal standard (dexamethasone).
We found good correlations between the concentrations of cortisol (F), 17α-hydroxy-progesterone (17-OHP) and 21-deoxycortisol (21-DOF) estimated by this HPLC method and those by highly specific radioimmunoassay method.
The concentrations of cortisone (E) and F of eight umbilical venous blood specimens were 159.7 ± 26.3 (Mean SD) ng/ml and 93.3 ± 58.9 ng/ml, respectively, and 17-OHP was detected 7 of them and its concentration was 17.4 ± 12.4 ng/ml. On the other hand, 17-OHP and 21-DOF peaks could not be detected in 1 month old normal infants. In untreated patients with adrenal 21-hydroxylase deficiency (21-OHD), the concentrations of E and F were very low and those of 17-OHP and 21-DOF were both extremely high, which were 26.1 ± 18.5 ng/ml, 5.9 ± 4.3 ng/ml, 157.0 ± 122.6 ng/ml, 26.1 ± 10.0 ng/ml, respectively.
Our method is more simplified and has higher sensitivity than the methods previously reported, and several Δ4-steroids which are clinically important can be analysed in a brief time. It was concluded that we could make an easy and accurate diagnosis of disorders of steroid biosynthesis and metabolism including congenital adrenal hyperplasia by this method.View full abstractDownload PDF (803K) -
Tatsuo TAKAHASHI, Akio KUWAYAMA, Tetsuo KATOH, Naoki KAGEYAMA1986 Volume 62 Issue 12 Pages 1336-1351
Published: December 20, 1986
Released on J-STAGE: September 24, 2012
JOURNAL FREE ACCESSTwenty-eight patients with various types of pituitary adenomas were studied endocrinologically and neuroradiologically. We observed the changes of tumor size during bromocriptine treatment. After various periods of bromocriptine therapy, we operated on these tumors and examined then histologically.
One of 5 patients with nonfunctioning adenomas improved remarkably in his visual field and acuity after 7-month bromocriptine therapy. The pathological findings disclosed remarkable changes in tumors composed of shrunken island-like cell nests and acellular spaces. These shrunken island-like cell nests were composed of tumor cells whose cytoplasmic volume decreased and whose nuclear chromatin clumped. In acellular spaces, there were irreversibly destructed tumor cells, hyaline substances, tumor cell debris and collagen fibrils.
One of 8 cases of acromegalies showed a remarkably reduced tumor on CT with clinical improvement after treatment with bromocriptine for 10 months. This patient's serum growth hormone titer was raised by an abnormal response to intravenously injected TRH (thyrotropin releasing hormone), and his serum prolactin was abnormally high. Therefore, this tumor was thought to be a mixed adenoma with growth hormone secreting and/or prolactin secreting cells. Histological examinations disclosed cell shrinkage of tumor cells. Interestingly, there were scanty fibrotic changes in this tumor in spite of the long term bromocriptine therapy.
In 15 cases of prolactinomas, the larger the tumor size and the longer the period of the bromocriptine therapy, the more fibrosis was seen. Under a period of bromocriptine therapy longer than 3 months, the fibrotic changes of tumor progressed, and this made more difficulty in selective adenomectomy even in the case of intrasellar adenomas. Therefore we thought that transsphenoidal surgery could successfully be done within 3 months during continuation of bromocriptine therapy.View full abstractDownload PDF (4124K) -
Hiroya MATSUO, Takeshi MARUO, Makoto HOSHINA, Matsuto MOCHIZUKI1986 Volume 62 Issue 12 Pages 1352-1361
Published: December 20, 1986
Released on J-STAGE: September 24, 2012
JOURNAL FREE ACCESSIn order to elucidate a possible self-regulation for hCG synthesis in placenta, effects of estradiol on hCG production and secretion were evaluated by culturing early placental tissue in the presence or absence of estradiol. The cellular level of mRNAs encoding hCG (α, β) and hPL were estimated by mean grain count per syncytial nucleus on the placental sections hybridized in situ with labeled cDNA probes corresponding to these mRNAs. Immunoreactive hCG, hCGα and hPL in the media and explanted tissues were measured by the homologous RIAs. Addition of estradiol at concentration of 1-10ng/ml into the medium caused an increase in the cellular levels of hCGαmRNA after 24-hour cultured, and exhibited significant increases in immunoreactive hCGα levels in the media and explanted tissues after 72-hour culture. The addition of estradiol neither affected the cellular levels of mRNA encoding hCGβ and hPL nor immunoreactive hCG and hPL levels in the media and tissues. The appropriated concentration of estradiol (1-10ng/ml) used in above experiments was found to be similar to the tissue concentration in normal placenta.
These findings suggest that the physiological concentration of estradiol selectively stimulates hCGα synthesis and secretion by normal placenta. Thus, estradiol in placenta may be a factor responsible for the increase of hCGα in maternal serum and placental tissue with the progress of gestation.View full abstractDownload PDF (1868K) -
Yoshinobu SUZUKI1986 Volume 62 Issue 12 Pages 1362-1375
Published: December 20, 1986
Released on J-STAGE: September 24, 2012
JOURNAL FREE ACCESSUsing porcine thyroid cells of primary monolayer culture, this study was conducted to clarify the nature and characteristics of epidermal growth factor (EGF) receptors on porcine thyroid cells and also to investigate the effects of EGF, TSH and phorbol ester on DNA synthesis. Receptors for EGF on porcine thyroid cells exist in two forms which differ in both affinity and capacity. Scatchard analysis of saturation binding assay performed at 4°C for 6h indicates that there is a high affinity class of receptors with low capacity (K1 = 4.70 ± 0.90 × 10-9M and 8,600 ± 1,200 sites/cell) and low affinity receptors with high capacity (K2 = 2.24 ± 1.17 × 10-7M and 65,500 ± 18,000 sites/cell) on the cells cultured for 4 days in the absence of TSH. When thyroid cells were cultured in the presence of various concentrations of TSH (0-50 mU/ml) and for various times (0-96h) with TSH (10mU/ml), specific EGF binding to the cells increased dose- and time-dependently. On TSH (10mU/ml) -treated cells for 4 days, two kinds of EGF receptors, i.e. high affinity and low capacity (K1 = 5.39 ± 1.75 × 10-9M and 17,200 ± 2,500 sites/cell) and low affinity and high capacity (K2 = 1.70 ± 1.40 × 10-7M and 76,300 ± 17,900 sites/cells), were resolved. The results indicate that TSH can modulate EGF receptors by increasing the number of high affinity sites on porcine thyroid cells. Next, using [Me-3 H] thymidine incorporation into TCA precipitable materials for 48h, we studied the biological effects of EGF, TSH and phorbol myristate acetate (one of the potent phorbol esters) on DNA synthesis. Both EGF and PMA can promote [Me-3 H] thymidine incorporation. Maximal responses were obtained with EGF ranging from 10-9 to 10-7M and with PMA from 10-9 to 10-7M. In contrast, TSH inhibits [Me-3H] thymidine incorporation dose-dependently. Almost the same results were obtained by these agents on TSH (10mU/ml) -treated cells for 4 days, but EGF stimulated cell growth at a lower concentration of 10-11 M, which was.possibly related to an increase of high affinity receptor numbers. The growth promoting effect of EGF and PMA in combination was additive, and TSH suppressed the cell growth in the concomitant presence of EGF and PMA.
From the present study, it is indicated that TSH can modulate EGF receptors on porcine thyroid cells, and also that both EGF and PMA can stimulate DNA synthesis of porcine thyroid cells at the concentrations of 10-11-10-7M. However, TSH, itself, may inhibit DNA synthesis.View full abstractDownload PDF (1913K) -
Takashi MATOZAKI, Choitsu SAKAMOTO, Munehiko NAGAO, Shigeaki BABA1986 Volume 62 Issue 12 Pages 1376-1383
Published: December 20, 1986
Released on J-STAGE: September 24, 2012
JOURNAL FREE ACCESSThe effects of pretreatment with pancreatic secretagogues and subsequently activated cellular events on [125 I-Tyr1] somatostatin binding to acinar membranes were studied. Pretreatment of pancreatic acini with bombesin at increasing concentrations for 120 min reduced labeled somatostatin binding to the acinar membranes in a dose-dependent fashion with a maximal reduction of binding at 10-8M bombesin (44.3 ± 1.8% of control). The maximal inhibition of labeled somatostatin binding by pretreatment with bombesin was almost comparable to that with COOH-terminal octapeptide cholecystokinin (CCK8) or carbamylcholine (carbachol). Furthermore, pretreatment of acini with vasoactive intestinal peptide (VIP) as well as secretin resulted in a small, but significant decrease of subsequent labeled somatostatin binding. In addition, adenosine 3′, 5′ cyclic nucleotide derivatives or a phosphodiesterase inhibitor mimicked the effect of VIP or secretin. The effect of simultaneous pretreatment of acini with VIP and carbachol on subsequent labeled somatostatin binding appeared to be almost equal to the calculated additive value for each peptide.These results suggest that the binding of somatostatin to its receptors in the pancreatic acini may be regulated via two functionally distinct pathways.View full abstractDownload PDF (744K) -
Heterogeneity and Pathophysiological Significance of Antibodies to TSH ReceptorTadashi YOSHIDA1986 Volume 62 Issue 12 Pages 1384-1399
Published: December 20, 1986
Released on J-STAGE: September 24, 2012
JOURNAL FREE ACCESSRecent studies revealed that anti-TSH receptor autoantibodies are involved in the pathogenesis of both Graves' disease and a part of hypothyroidism, but precise mechanism of action of these antibodies remained to be studied. In order to delineate the heterogeneity of these antibodies and their pathophysiological significance, we produced monoclonal antibodies to TSH receptor and studied their characteristics.
Mouse monoclonal antibodies to TSH receptor were derived from spleen cells of mice immunized with partially purified human TSH receptor, which was prepared by TSHcoupled affinity chromatography of thyroid membrane solubilized with Triton X-100. By fusing spleen cells and mouse myeloma cells in the presence of polyethylene glycol and selecting with limiting dilution method, 5 hybridomas were obtained. Among 3 antibodies, which inhibited TSH binding to thyroid membrane (TSH displacing activity, TDA), 2 inhibited TSH stimulation of thyroid adenylate cyclase (AC) (human thyroid adenylate cyclase inhibitor activity, HTACI), and one showed no bioactivity. Among other 2 antibodies without TDA, 1 stimulated AC (human thyroid adenylate cyclase stimulator activity, HTACS) and the other inhibited TSH stimulation (HTACI). All activities of these antibodies were dependent on IgG concentration and disappeared by treatment of anti-mouse IgG antibodies.
In addition, 4 human-human hybridomas were established by fusing human peripheral lymphocytes of patients with Graves' disease and nongoitrous hypothyroidism with human lymphoblastoid cell line. Among 2 antibodies with TDA, one antibody inhibited TSH stimulation of AC, inhibiting TSH binding competitively and another antibody stimulated AC, inhibiting TSH binding noncompetitively. Among the other 2 antibodies, which did not inhibit TSH binding but were shown to bind to TSH receptor by immunoprecipitation, one stimulated AC and the other inhibited TSH stimulation of AC. Among 2 antibodies with HTACI, one antibody with positive TDA inhibited stimulation of AC by stimulative antibodies with positive TDA, but the other without TDA inhibited stimulation of AC by both antibodies with or without positive TDA. These inhibitory antibodies did not inhibit stimulation of AC by Forskolin and Gpp (NH) p, which are known to affect other parts of receptor-AC system than receptor unit.
These data suggest that anti-TSH receptor antibodies are heterogenous in the mode of binding to the receptor and in their bioactivities, and may be involved in the pathogenesis of both Graves' disease and a part of idiopathic hypothyroidism.View full abstractDownload PDF (1382K)
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