scholarly journals The Interaction of TSH Receptor Autoantibodies with 125I-Labelled TSH Receptor

1999 ◽  
Vol 84 (10) ◽  
pp. 3797-3802 ◽  
Author(s):  
Jane Sanders ◽  
Yasuo Oda ◽  
Sara Roberts ◽  
Angela Kiddie ◽  
Tonya Richards ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cyclic Amp ◽  
Binding Site ◽  
Tsh Receptor ◽  
Thyroid Cells ◽  
Terminal Domain ◽  
Close Relationship ◽  
Immunoprecipitation Reaction ◽  
Stimulation Of

Abstract Detergent-solubilized porcine TSH receptor (TSHR) has been labeled with 125I using a monoclonal antibody to the C-terminal domain of the receptor. The ability of sera containing TSHR autoantibody to immunoprecipitate the labeled receptor was then investigated. Sera negative for TSHR autoantibody (as judged by assays based on inhibition of labeled TSH binding to detergent-solubilized porcine TSHR) immunoprecipitated about 4% of the labeled receptor, whereas sera with high levels of receptor autoantibody immunoprecipitated more than 25% of the labeled receptor. The ability to immunoprecipitate labeled TSHR correlated well with ability of the sera to inhibit labeled TSH binding to the receptor (r = 0.92; n = 63), and this is consistent with TSHR autoantibodies in these samples being directed principally to a region of the receptor closely related to the TSH binding site. Preincubation of labeled TSHR with unlabeled TSH before reaction with test sera inhibited the immunoprecipitation reaction, providing further evidence for a close relationship between the TSHR autoantibody binding site(s) and the TSH binding site. This was the case whether the sera had TSH agonist (i.e., thyroid stimulating) or TSH antagonist (i.e., blocking) activities, thus, providing no clear evidence for different regions of the TSHR being involved in forming the binding site(s) for TSHR autoantibodies with stimulating and with blocking activities. The ability of TSHR autoantibodies to stimulate cyclic AMP production in isolated porcine thyroid cells was compared with their ability to immunoprecipitate labeled porcine TSHR. A significant correlation was observed (r = 0.58; n = 50; P < 0.001) and the correlation was improved when stimulation of cyclic AMP production was compared with inhibition of labeled TSH binding to porcine TSHR (r = 0.76). Overall, our results indicate that TSHR autoantibodies bind principally to a region on the TSHR closely related to the TSH binding site, and this seems to be the case whether the autoantibodies act as TSH agonists or antagonists.

RELATION OP FIBRIN STIMULATION OF tPA MEDIATED PLASMINOGEN ACTIVATION AND FIBRIN BINDING TOWARDS FIBRONEKTIN AS REVEALED BY A MONOCLONAL ANTIBODY (MAB) AGAINST FCB-2 FIBRINOGEN FRAGMENTS

1987 ◽  
Author(s):  
S Meusburger ◽  
R Beckmann ◽  
J Wojta ◽  
B R Binder
Keyword(s):  
Monoclonal Antibody ◽  
Binding Site ◽  
Fluid Phase ◽  
Plasminogen Activation ◽  
Elisa System ◽  
Matrix Bound ◽  
Fibronectin Binding ◽  
Fibrin Monomers ◽  
Stimulation Of

Fibrin binds to the finger domain of fibronektin via the C-terminal end of the chain and it was reported previously by us that in a fluid phase assay fibronektin inhibits fibrin enhancement of plasminogen activation by tPA. However, other have shown that tPA binds to fibronectin thereby possibly mediating enhanced matrix bound plasmin formation. In the present study we tried to further characterize the interaction between fibronectin and fibrin in regard to fibrin dependent enhancement of plasminogen activation by tPA. For fibrin binding to fibronectin we have developed an ELISA system using fibronectin coated plates and antibodies against fibrin(ogen) to quantify bound fibrin. For determination of plasminogen activation we used a coupled spectrophotometric fluid phase assay with Glu-plasminogen as substrate and H-D-Val-Leu-Lys-pNA to quantify the formed plasmin. Fibrin binding to coated fibronectin was linear between 500ng and 1 mg/ml for fibrin monomers (reptilase), FCB-2 fragments and thrombin (3.3 U/ml) treated fibrinogen, respectively. A monoclonal antibody directed against the FCB-2 fibrinogen fragment which also could be shown to recognize fibirn but not fibrinogen did not recognize fibronectin bound fibrin and inhibited also the fibrin stimulatory effect on plasminogen activation indicating that the epitope against which the antibody is directed is closely related to both the fibronectin binding site and the site involved in t-PA stimulation.

scholarly journals Alterations in the binding site of the cyclic AMP receptor protein at the Escherichia coli galactose operon regulatory region

1988 ◽  
Vol 253 (3) ◽  
pp. 809-818 ◽  
Author(s):  
K Gaston ◽  
B Chan ◽  
A Kolb ◽  
J Fox ◽  
S Busby
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Binding Site ◽  
Consensus Sequence ◽  
Regulatory Region ◽  
Receptor Protein ◽  
Binding Assays ◽  
Cyclic Amp Receptor Protein ◽  
Stimulation Of

Gene manipulation techniques have been used to alter the binding site for the cyclic AMP-cyclic AMP receptor protein complex (cAMP-CRP) at the regulatory region of the Escherichia coli galactose (gal) operon. The effects of these changes on CRP-dependent stimulation of expression from the galP1 promoter in vivo have been measured, and gel binding assays have been used to measure the affinity of cAMP-CRP for the modified sites. Firstly we have deleted progressively longer sequences from upstream of the gal CRP site in order to locate the functional limit of the site. A deletion to -49, removing the first base that corresponds to the consensus sequence for a CRP binding site, is sufficient to reduce CRP binding and block CRP-dependent stimulation of P1. Secondly, we used synthetic oligonucleotides to invert the asymmetric nucleotide sequence at the gal CRP binding site or to make the sequence symmetric. Inversion of the site has little effect on CRP binding, the architecture of open complexes at P1 revealed by DNAase I footprinting, or the stimulation of transcription from P1. Making the site symmetric increases the affinity for CRP by over 50-fold and leads to increased transcription from P1, whilst hardly altering the DNAase I footprint of open complexes. Our results confirm that the strength of binding of CRP depends on the nature of the site and show that it is this that principally accounts for differences in CRP-dependent stimulation of transcription.

One Monoclonal Antibody to Human Thyrotropin (TSH) Receptor Agonist of TSH Hormone Stimulates the Proliferation of Human Thyroid Cells

1995 ◽  
Vol 161 (2) ◽  
pp. 262-269 ◽  
Author(s):  
Armelle Ropars ◽  
Lila Takorabet ◽  
Sandrine Marion ◽  
Jeannine Charreire
Keyword(s):  
Monoclonal Antibody ◽  
Receptor Agonist ◽  
Tsh Receptor ◽  
Human Thyroid ◽  
Thyroid Cells

Characterization of monoclonal antibodies directed against the TSH receptor, as revealed with the cytochemical bioassay

1987 ◽  
Vol 116 (1_Suppl) ◽  
pp. S173-S180 ◽  
Author(s):  
N.J. Marshall ◽  
L. D. Kohn ◽  
P. A. Ealey
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Tsh Receptor ◽  
Thyroid Cells ◽  
Essential Component ◽  
Potent Stimulator ◽  
Cytochemical Bioassay

Abstract. The monoclonal antibody 11E8 (Kohn et al. 1984) is a novel proof of the actions of thyroid stimulator, since it is not only a potent stimulator of thyroid cells but also an inhibitor for TSH-binding. This might reflect an interaction of 11E8 with a discrete domaine of the TSH receptor, which is an essential component for TSH but not for TSab actions. These unique properties of 11E8 can be applied to the characterization of other thyroid stimulators which may interact with different domaines of the receptor. Moreover, 'TSab-like' stimulators, such as the sheep anti-TSH antiserum, could potentially be identified by 11E8.

STIMULATION OF CYCLIC AMP PRODUCTION IN FRTL-5 THYROID CELLS BY CRUDE IMMUNOGLOBULIN FRACTIONS OF SERUM FROM PREGNANT WOMEN

1989 ◽  
Vol 31 (3) ◽  
pp. 267-275 ◽  
Author(s):  
K. KASAGI ◽  
A. HIDAKA ◽  
H. HATABU ◽  
Y. TOKUDA ◽  
T. MISAKI ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Pregnant Women ◽  
Thyroid Cells ◽  
Stimulation Of

Evaluation of the rat thyroid cell strain FRTL-5 as an in-vitro bioassay system for thyrotrophin

1984 ◽  
Vol 101 (3) ◽  
pp. 269-NP ◽  
Author(s):  
S. P. Bidey ◽  
L. Chiovato ◽  
A. Day ◽  
M. Turmaine ◽  
R. P. Gould ◽  
...  
Keyword(s):  
Cyclic Amp ◽  
Human Thyroid ◽  
Thyroid Cell ◽  
Tissue Slices ◽  
Iodide Uptake ◽  
Thyroid Cells ◽  
Rat Thyroid ◽  
Bovine Tsh ◽  
Stimulation Of

ABSTRACT The cyclic AMP response to bovine TSH was characterized in a strain of rat thyroid follicular cells (FRTL-5) maintained in continuous culture. Significant stimulation of intracellular cyclic AMP was attained at a TSH dose of 5 μu./ml. Cyclic AMP accumulation continued to increase, at higher TSH doses, with no evidence for attainment of a maximum level at the highest dose tested (5 mu./ml). The precision of TSH measurement was better than 10% over the range 50–5000 μu./ml, comparing favourably with that observed with analogous assays based on human cells, tissue slices or membrane preparations. Using sequential subcultures of FRTL-5 cells, the between-assay variation in response to a single dose of a standard preparation of bovine TSH (53/11; 370 μu./ml) was of the order of 20% which compared favourably with the between-assay variation observed with different cultures of human thyroid cells. Prolongation of the incubation of FRTL-5 cells with TSH to 3 h revealed a progressive increase in the extracellular accumulation of cyclic AMP. Addition of TSH to resting FRTL-5 cells resulted in a stimulation of inorganic iodide uptake with pronounced bell-shaped dose–response characteristics. Thus a maximum uptake was observed at a TSH dose of 100 μu./ml with a significant reduction at higher doses. Acute stimulation of cells with TSH (100 μu./ml) resulted in a rapid and marked alteration in cell morphology, with evidence of cellular retraction and surface ruffling. J. Endocr. (1984) 101, 269–276

Histamine and cyclic AMP in isolated canine parietal cells.

1979 ◽  
Vol 237 (5) ◽  
pp. E444 ◽  
Author(s):  
A H Soll ◽  
A Wollin
Keyword(s):  
Oxygen Consumption ◽  
Cyclic Amp ◽  
Parietal Cells ◽  
Histamine Stimulation ◽  
Close Relationship ◽  
The Relationship ◽  
Parallel Fashion ◽  
Stimulation Of

The relationship between cyclic AMP production and the response of isolated canine parietal cells to histamine has been examined. Histamine increased cyclic AMP generation, and this effect correlated with histamine stimulation of oxygen consumption and aminopyrine accumulation. Metiamide inhibited histamine-stimulated cyclic AMP generation and oxygen consumption in a parallel fashion. At concentrations below 100 microM, isobutyl AMP production and oxygen consumption in a similar fashion. However, with IMX above 100 microM, histamine caused no further increases in oxygen consumption, despite markedly enhanced cyclic AMP generation. Neither carbachol nor gastrin increased cyclic AMP production beyond that produced by IMX alone, and the combinations of histamine and carbachol and of histamine and gastrin produced no greater cyclic AMP generation than produced by histamine. These findings support a close relationship between cyclic AMP production and the action of histamine but not of carbachol or gastrin on isolated parietal cells. The mechanisms underlying the potentiating interactions between histamine, carbachol, and gastrin involve step(s) beyond stimulation of cyclic AMP generation.

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