Comparison of human and porcine thyroid membranes for radioreceptor assay of bovine thyrotrophin and thyrotrophin-binding inhibiting immunoglobulins

1982 ◽  
Vol 93 (3) ◽  
pp. 371-380 ◽  
Author(s):  
Hazel Humphries ◽  
Susan M. Dirmikis ◽  
D. S. Munro
Keyword(s):  
Detection Limit ◽  
Detailed Comparison ◽  
Normal Subjects ◽  
Inhibitory Effect ◽  
Radioreceptor Assay ◽  
Human Thyroid ◽  
Affinity Constant ◽  
Scatchard Analysis ◽  
Maximal Inhibition ◽  
Long Acting

A detailed comparison between the use of human and porcine thyroid membranes for the radioreceptor assay (RRA) of bovine TSH (bTSH) and thyrotrophin-binding inhibiting immunoglobulins (TBIIg) is reported. Bovine thyroid membranes were also investigated but were found to be far less satisfactory than either human or porcine thyroid membranes. The affinity constant (Ka) of the interaction of bTSH with porcine thyroid membranes (Ka = 3·3 × 109l/mol) measured by Scatchard analysis was higher than with human thyroid membranes (Ka = 2·1 × 108l/mol). Porcine thyroid membranes were more sensitive for the assay of bTSH (detection limit 30 μu., half-maximal inhibition 0·3 mu.) than human thyroid membranes (detection limit 200 μu., half-maximal inhibition 7·4 mu.). Preincubation of membranes from either species with immunoglobulin rich in long-acting thyroid stimulator (LATS) inhibited the saturable binding of 125I-labelled TSH to a greater extent than did normal immunoglobulin. The binding of 125I-labelled TSH to porcine membranes was more sensitive to the inhibitory effect of LATS-immunoglobulin and was also less affected by normal immunoglobulin than was binding to human thyroid membranes. When assayed with each type of membrane preparation there was good correlation between the RRA of immunoglobulins prepared from patients with Graves's disease and from normal subjects (n = 18) (r = 0·85, P <0·001, n = 73). The incidence of positive TBIIg in untreated Graves's disease was greater for porcine than for human thyroid membranes.

Peripheral blood lymphocyte transformation in response to human thyroid fractions in patients with Graves' hyperthyroidism and ophthalmopathy

1980 ◽  
Vol 93 (4) ◽  
pp. 419-423 ◽  
Author(s):  
J. R. Wall ◽  
Anne Trewin ◽  
Diane M. Joyner
Keyword(s):  
Peripheral Blood Lymphocyte ◽  
Peripheral Blood ◽  
Blood Lymphocyte ◽  
Plasma Membranes ◽  
Soluble Fraction ◽  
Lymphocyte Transformation ◽  
Normal Subjects ◽  
Radioreceptor Assay ◽  
Human Thyroid ◽  
Cytosol Fraction

Abstract. Peripheral blood lymphocyte (PBL) transformation in response to human thyroid fractions was carried out in patients with Graves' hyperthyroidism and ophthalmopathy. The fractions used were, an extract, a soluble (cytosol) fraction, and membranes prepared by differential centrifugation at (i) 6500 × g (mitochondria), (ii) 10000 × g (plasma membranes as used in the radioreceptor assay for thyroid stimulating antibody (TSAb) and (iii) 40000 × g (microsomes). Results were expressed as stimulation indices (SI). Mean SI for patients were significantly increased compared with those for normals for cytosol and mitochondria but not the other fractions. Taking the upper limit of normal as mean + 2 sd for normal subjects for each fraction, positive tests were found in 12 of 35 patients tested. This was in response to extract in 3 of 32 patients, to the soluble fraction in 10 of 31 patients, to mitochondria in 2 of 13 patients, to plasma membranes in 1 of 33 patients, and to microsomes in 1 of 27 patients. Positive tests were associated with hyperthyroidism but not ophthalmopathy.

ADENYL CYCLASE ACTIVITY IN THE MOUSE THYROID GLAND

1972 ◽  
Vol 52 (3) ◽  
pp. 533-540 ◽  
Author(s):  
PAT KENDALL-TAYLOR
Keyword(s):  
Inhibitory Effect ◽  
Thyroid Stimulating Hormone ◽  
Adenyl Cyclase ◽  
Human Thyroid ◽  
Intact Mouse ◽  
Adenyl Cyclase Activity ◽  
Thyroid Glands ◽  
Long Acting ◽  
Rate Of Activation ◽  
Mouse Thyroid

SUMMARY The activation of adenyl cyclase in intact mouse thyroid glands was measured by the conversion of [3H]ATP to [3H]cyclic AMP. Untreated serum had an inhibitory effect. Human thyroid-stimulating hormone (TSH) and γG-globulin prepared from serum containing the long-acting thyroid stimulator (LATS), activated adenyl cyclase and the log-dose—response relationship did not deviate from parallelism. The rate of activation by LATS was slower and the peak response was delayed, compared with the response to TSH. The possible significance of this is discussed. Other hormones and neurotransmitters examined had no effect on thyroidal adenyl cyclase.

STUDIES ON THE INTERACTION OF LONG-ACTING THYROID STIMULATOR (LATS) WITH SOLUBLE THYROID FRACTIONS

1971 ◽  
Vol 68 (4) ◽  
pp. 625-644 ◽  
Author(s):  
N. Amino ◽  
K. Miyai ◽  
M. Azukizawa ◽  
Y. Kumahara
Keyword(s):  
Inhibitory Activity ◽  
Acid Treatment ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Inhibitory Effect ◽  
Human Thyroid ◽  
Thyroid Cell ◽  
Long Acting ◽  
Sepharose 4B

ABSTRACT The specificity, stability and reversibility of the in vitro interaction of LATS with soluble human thyroid fractions was studied. With regard to tissue specificity, the cell sap obtained from human liver, spleen, kidney, and muscle did not inhibit the LATS activity while the same amount of thyroid cell sap significantly inhibited it. When the LATS inhibitory activity in thyroid subcellular fractions was compared, the microsomal fraction was more active than cell sap or solubilized microsomes in terms of milligram of protein, but the cell sap had considerable activity as based on the original thyroid weight. Lyophilization of cell sap did not reduce the LATS inhibitory activity, but treatment with 2 m NaSCN and 6 m urea apparently destroyed this capacity. Acid treatment of cell sap at pH 2.5 and at 3.0 completely destroyed its ability to inhibit LATS activity. Inhibition of LATS activity was roughly proportional to the amount of thyroid cell sap. Human TSH, on the other hand, was not inhibited by cell sap which had a significant inhibitory effect on LATS. LATS activity was more effectively inhibited when a mixture of LATS-IgG and thyroid cell sap was incubated for 96 hours than for 12 hours. The inhibition of LATS activity by thyroid cell sap was partially but significantly reversed by acid treatment, as observed in experiment using microsomes. When thyroid cell sap was fractionated by gel filtration on Sepharose 4B, LATS inhibitory activity was distributed in all the fractions including the 27S to 4S proteins. In DEAE-cellulose column chromatography, LATS inhibitory activity tended to be eluted at a higher ionic strength. In each fraction of Sepharose 4B and DEAE-cellulose, LATS inhibitory activity was found to be unrelated to the thyroglobulin content. It is believed that the inhibition of LATS activity by thyroid cell sap is compatible with an antigen-antibody reaction and that the LATS inhibitor may not be a thyroglobulin itself but a more negatively charged heterogeneous substance.

Interaction of the mouse thyrotrophin receptor with thyrotrophin binding inhibitor immunoglobulins

1983 ◽  
Vol 96 (3) ◽  
pp. 481-488 ◽  
Author(s):  
B. M. Luttrell
Keyword(s):  
Plasma Membranes ◽  
Normal Subjects ◽  
Radioreceptor Assay ◽  
Species Specificity ◽  
Individual Values ◽  
Serum Immunoglobulins ◽  
Undetectable Serum ◽  
Long Acting ◽  
The Individual ◽  
A Minor

The species-specificity of thyrotrophin binding inhibitor immunoglobulin (TBII) for the thyroid TSH receptor was investigated using a preparation of thyroid plasma membranes (TPM) from propylthiouracil-treated mice, as well as from human glands. The interest in the mouse arose from its use as the bioassay animal for the long-acting thyroid stimulator (LATS). A comparison was made of the response in the two radioreceptor assays of serum immunoglobulins from ten normal subjects and twenty patients with Graves's disease, who had also been selected to have positive TBII activity in the assay based on human TPM. All the specimens from the patients with Graves's disease had detectable TBII activity in the mouse radioreceptor assay, inhibiting the binding of 125I-labelled TSH to a greater extent than did any of the specimens from normal subjects. There was evidence for a minor degree of species-specificity, since at least one of the specimens from the Graves' disease group had unexpectedly high activity in the assay based on mouse TPM and another had unexpectedly weak activity in that assay. However, this specificity appeared to be unrelated to the presence or absence of LATS. The effect of LATS on the response of serum immunoglobulins in the mouse radioreceptor assay was tested using nine patients with Graves's disease who had undetectable serum LATS and another eight patients with Graves's disease whose serum gave a positive LATS response. These patients had also all been selected to have positive TBII activity in their serum, as determined with human TPM. All samples from each of the LATS-positive and LATS-negative subgroups gave a positive TBII response in the radioreceptor assay based on mouse TPM, and there was extensive overlap between the individual values for the two subgroups. It is concluded that the failure of some TBII-positive serum immunoglobulins to stimulate the mouse thyroid gland and produce a positive LATS response is not due to species-specificity at the level of receptor binding.

Modulation of luteinizing hormone receptors: effect of an inhibitor of prolactin secretion, p-coumaric acid

1983 ◽  
Vol 98 (3) ◽  
pp. 307-311 ◽  
Author(s):  
Mridula Chowdhury ◽  
S. N. Kabir ◽  
A. K. Pal ◽  
Anita Pakrashi
Keyword(s):  
Hormone Receptors ◽  
Complete Recovery ◽  
Inhibitory Effect ◽  
Prolactin Secretion ◽  
Serum Prolactin ◽  
Affinity Constant ◽  
Scatchard Analysis ◽  
Coumaric Acid ◽  
Binding Data

Daily oral administration of p-coumaric acid (PCA) at a dose of 50 mg/kg body wt for 21 days to adult male mice caused a dramatic reduction in serum prolactin concentrations. A significant fall in testicular LH binding was also observed after PCA treatment. Complete recovery of testicular LH binding was obtained by daily administration of prolactin (500 μg/mouse) when given simultaneously from day 9 of PCA treatment. A lower daily dose of prolactin (250 μg) was found to be ineffective. Scatchard analysis of binding data suggested a decrease in the number of testicular LH binding sites after PCA treatment whereas the affinity constant was unchanged. These results provide direct evidence for an inhibitory effect of PCA on prolactin secretion and also provide additional evidence in favour of a role of prolactin in the modulation of LH receptors.

THE BINDING OF INSULIN AND SOMATOMEDIN A TO HUMAN PLACENTAL MEMBRANE

1975 ◽  
Vol 80 (1) ◽  
pp. 14-31 ◽  
Author(s):  
K. Takano ◽  
K. Hall ◽  
L. Fryklund ◽  
A. Holmgren ◽  
H. Sievertsson ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Binding Sites ◽  
Membrane Fraction ◽  
Specific Binding ◽  
Insulin Binding ◽  
Radioreceptor Assay ◽  
Affinity Constant ◽  
Scatchard Analysis ◽  
Mean Values ◽  
High Affinity

ABSTRACT A particulate membrane fraction from human placental membrane was shown to be rich in binding sites not only for insulin but also for somatomedin A. The binding of the 125I-labelled peptide was time and temperature dependent. Degrading activity present in the membrane fraction was negligible at +4°C. The Scatchard plot for insulin binding revealed two types of binding sites with an apparent high affinity constant of 3.8× 108 m−1 and with 5.4 × 10−9 moles of binding sites per mg of membrane protein. The Scatchard analysis of somatomedin A revealed two classes of binding sites with an apparent high affinity constant of 2.7 × 107 m−1 and with 1.9× 10−8 moles of binding sites per mg of membrane protein. In high concentrations insulin interfered with the specific binding sites for somatomedin A and vice versa. In comparison with insulin the somatomedin A preparation was one million times more potent in displacing labelled somatomedin A than in displacing labelled insulin from their respective binding sites. A radioreceptor assay utilizing particulate placental membrane and labelled somatomedin A purified on the membrane enabled the determination of somatomedin in unextracted serum. The mean values of somatomedin A in sera from patients with pituitary dwarfism and acromegaly were 0.57 and 3.2 U/ml, respectively by radioreceptor assay and 0.41 and 1.61 U/ml, respectively by bioassay. Various causes of this discrepancy between the methods are discussed.

Characterization of Thromboxane A2/Prostaglandin H2 Receptors in Porcine Coronary Artery -The Inhibitory Effect of a Novel Dibenzoxepin Derivative, KW-3635

1992 ◽  
Vol 67 (05) ◽  
pp. 582-584 ◽  
Author(s):  
Ichiro Miki ◽  
Akio Ishii
Keyword(s):  
Coronary Artery ◽  
Binding Sites ◽  
Thromboxane A2 ◽  
Inhibitory Effect ◽  
Scatchard Analysis ◽  
Single Class ◽  
Porcine Coronary Artery ◽  
Equilibrium Binding ◽  
H2 Receptors

SummaryWe characterized the thromboxane A2/prostaglandin H2 receptors in porcine coronary artery. The binding of [3H]SQ 29,548, a thromboxane A2 antagonist, to coronary arterial membranes was saturable and displaceable. Scatchard analysis of equilibrium binding showed a single class of high affinity binding sites with a dissociation constant of 18.5 ±1.0 nM and the maximum binding of 80.7 ± 5.2 fmol/mg protein. [3H]SQ 29,548 binding was concentration-dependently inhibited by thromboxane A2 antagonists such as SQ 29,548, BM13505 and BM13177 or the thromboxane A2 agonists such as U46619 and U44069. KW-3635, a novel dibenzoxepin derivative, concentration-dependently inhibited the [3H]SQ 29,548 binding to thromboxane A2/prosta-glandin H2 receptors in coronary artery with an inhibition constant of 6.0 ± 0.69 nM (mean ± S.E.M.).

Calcium and the Fc Receptor on Human Platelets

1987 ◽  
Vol 57 (03) ◽  
pp. 298-301
Author(s):  
William F Clark ◽  
Gerald J M Tevaarwerk ◽  
Bruce D Reid ◽  
Suzanne Hall ◽  
Anita Caveney ◽  
...  
Keyword(s):  
Specific Binding ◽  
Normal Subjects ◽  
Human Platelets ◽  
Fc Receptor ◽  
Normal Male ◽  
Scatchard Analysis ◽  
Normal Female ◽  
Apparent Difference ◽  
Binding Data ◽  
Direct Binding

SummaryWe have described the calcium dependence of the IgG Fc receptor (Fc-R) on human platelets by analyzing the direct binding of radiolabelled Fc fragments, monomers and dimers of IgG. Specific binding to platelets was undetectable at 37° C in a calcium-free preparation but readily detected when calcium was restored. Scatchard analysis of the binding data for the calcium-restored platelets permitted calculation of the available Fc-R and the Ka of binding for the different IgG ligands. The mean Ka of binding for 12 normal subjects varied from 107 to 108 L/M, with an equal receptor number measured by Fc fragments and dimers of IgG, but a lesser amount for monomeric IgG. There was no apparent difference in Fc-R number for platelets from 6 normal male versus 6 normal female subjects.At 4° C binding was detectable for dimers and polymers of IgG in a calcium-free preparation and this was markedly increased with recalcification. Thus, our data are consistent with an Fc receptor population on human platelets whose avidity for binding is significantly enhanced in a calcium-restored medium.

Synchronized Inhibition of the Phospholipid Mediated Autoactivation of Factor XII in Plasma by β2-glycoprotein I and Anti-β2-glycoprotein I

1995 ◽  
Vol 73 (05) ◽  
pp. 798-804 ◽  
Author(s):  
Inger Schousboe ◽  
Margit Søe Rasmussen
Keyword(s):  
Lupus Erythematosus ◽  
Lupus Anticoagulant ◽  
Response Curve ◽  
Inhibitory Effect ◽  
Factor Xii ◽  
High Antibody ◽  
Contact Activation ◽  
Maximal Inhibition ◽  
Lupus Anticoagulants ◽  
Glycoprotein I

SummaryLupus anticoagulants are a group of antibodies commonly found in patients with autoimmune diseases such as systemic lupus erythematosus. Lupus anticoagulants inhibit phospholipid dependent coagulation and may bind to negatively charged phospholipids. Recent studies have suggested an association between anti-β2-glycoprotein I and a lupus anticoagulant, whose activity is frequently dependent on the presence of β2-glycoprotein I. Based on these observations, the effect of anti-β2-glycoprotein I on the autoactivation of factor XII in plasma was investigated. Autoactivation initiated by the presence of negatively charged phospholipids, but not by sulfatide, was strongly inhibited by immunoaffinity purified anti-β2-glycoprotein I. The dose-response curve of anti-β2-gly coprotein I was identical with that of a precipitating antibody, showing no inhibition at low and high antibody dilutions and maximal inhibition at an intermediate dilution. At high antibody concentrations, an increased rate of factor Xlla activation was observed. This increase was of the same magnitude as the decreased rate observed in plasma supplemented with the same amount of β2-glycoprotein I as in the plasma itself. This confirms the inhibitory effect of β2-GP-I on the contact activation and shows that inhibition is effective on the autoactivation of factor XII in plasma. The inhibitory action of β2-glycoprotein I was independent of the inhibition caused by the anti- β2-glycoprotein I/β2-glycoprotein I complex suggesting a synchronized inhibition of factor XII autoactivation by β2-glycoprotein I and anti-β2-gly coprotein I. The inhibition caused by the antibody is suggested to be caused by a reduced availability of negatively charged phospholipids due to the binding of the anti-β2- GP-I/β2-GP-I complex. This complex may be a lupus anticoagulant.

VARIATIONS IN PLASMA RENIN ACTIVITY AND URINARY ALDOSTERONE EXCRETION IN NORMAL SUBJECTS AFTER SALT RESTRICTION AND ACUTE PITUITARY INHIBITION WITH 6-DEHYDRO-16-METHYLENE HYDROCORTISONE

1971 ◽  
Vol 67 (1) ◽  
pp. 159-173
Author(s):  
A. Peytremann ◽  
R. Veyrat ◽  
A. F. Muller
Keyword(s):  
Plasma Renin Activity ◽  
Salt Intake ◽  
Plasma Renin ◽  
Normal Subjects ◽  
Inhibitory Effect ◽  
Renin Activity ◽  
Sodium Balance ◽  
Experimental Conditions ◽  
Salt Restriction ◽  
Aldosterone Production

ABSTRACT Variations in plasma renin activity and urinary aldosterone excretion were studied in normal subjects submitted to salt restriction and simultaneous inhibition of ACTH production with a new synthetic steroid, 6-dehydro-16-methylene hydrocortisone (STC 407). At a dose of 10 mg t. i. d. this preparation exerts an inhibitory effect on the pituitary comparable to that of 2 mg of dexamethasone. In subjects maintained on a restricted salt intake, STC 407 does not delay the establishment of an equilibrium in sodium balance. The increases in endogenous aldosterone production and in plasma renin activity are also similar to those seen in the control subjects. A possible mineralocorticoid effect of STC 407 can be excluded. Under identical experimental conditions, the administration of dexamethasone yielded results comparable to those obtained with STC 407.

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