THE INTERACTION OF THE LONG-ACTING THYROID STIMULATOR (LATS) WITH THYROID TISSUE IN VITRO

1970 ◽  
Vol 46 (1) ◽  
pp. 45-54 ◽  
Author(s):  
B. R. SMITH
Keyword(s):  
Molecular Weight ◽  
Serum Proteins ◽  
Soluble Fraction ◽  
Thyroid Tissue ◽  
Gel Filtration ◽  
Human Thyroid ◽  
Salt Precipitation ◽  
Specific Antigens ◽  
Long Acting

SUMMARY Most of the long-acting thyroid stimulator (LATS)-absorbing activity of human thyroid homogenates was shown to be in a soluble fraction containing several components including thyroglobulin, haemoglobulin and serum proteins. Salt precipitation and gel filtration studies indicated that the absorbing activity was not associated with thyroglobulin of high molecular weight, but with a 4s fraction. This 4s fraction has been shown to contain thyroid specific antigens and these may be important in LATS absorption.

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.

HEMODIALYSIS OF THE RAT

1966 ◽  
Vol 44 (5) ◽  
pp. 849-859 ◽  
Author(s):  
Sumner M. Robinson ◽  
David A. Hurwitz ◽  
Robert Louis-Ferdinand ◽  
William F. Blatt
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Low Molecular Weight

A technique is described for hemodialysis of either anesthetized or non-restrained rats. In the apparatus the dialysis plates of an autoanalyzer system are used with only minor modification. The efficiency of this method has been evaluated with regard to the clearance of saccharides, both in vitro and in vivo, as well as the extraction of nitrogenous low molecular weight moieties from circulating blood. Approximately 50% of the dialyzable material was obtained in a 1-hour dialysis. Further fractionation of the dialyzate was accomplished by gel filtration (Sephadex G-25).

scholarly journals Isolation and Characterization of Two Proteins fromMoraxella catarrhalis That Bear a Common Epitope

1998 ◽  
Vol 66 (9) ◽  
pp. 4374-4381 ◽  
Author(s):  
John C. McMichael ◽  
Michael J. Fiske ◽  
Ross A. Fredenburg ◽  
Deb N. Chakravarti ◽  
Karl R. VanDerMeid ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Bacterial Attachment ◽  
Vaccine Candidates ◽  
Isolation And Characterization ◽  
Sds Page

ABSTRACT The UspA1 and UspA2 proteins of Moraxella catarrhalisare potential vaccine candidates for preventing disease caused by this organism. We have characterized both proteins and evaluated their vaccine potential using both in vitro and in vivo assays. Both proteins were purified from the O35E isolate by Triton X-100 extraction, followed by ion-exchange and hydroxyapatite chromatography. Analysis of the sequences of internal peptides, prepared by enzymatic and chemical cleavage of the proteins, revealed that UspA1 and UspA2 exhibited distinct structural differences but shared a common sequence including an epitope recognized by the monoclonal antibody 17C7. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), purified UspA1 exhibited a molecular weight of approximately 350,000 when unheated and a molecular weight of 100,000 after being heated for 10 min at 100°C. In contrast, purified UspA2 exhibited an apparent molecular weight of 240,000 by SDS-PAGE that did not change with the length of time of heating. Their sizes as determined by gel filtration were 1,150,000 and 830,000 for UspA1 and UspA2, respectively. Preliminary results indicate the proteins have separate functions in bacterial pathogenesis. Purified UspA1 was found to bind HEp-2 cells, and sera against UspA1, but not against UspA2, blocked binding of the O35E isolate to the HEp-2 cells. UspA1 also bound fibronectin and appears to have a role in bacterial attachment. Purified UspA2, however, did not bind fibronectin but had an affinity for vitronectin. Both proteins elicited bactericidal antibodies in mice to homologous and heterologous disease isolates. Finally, mice immunized with each of the proteins, followed by pulmonary challenge with either the homologous or a heterologous isolate, cleared the bacteria more rapidly than mock-immunized mice. These results suggest that UspA1 and UspA2 serve different virulence functions and that both are promising vaccine candidates.

scholarly journals Characterization of proteins from the cytoskeleton of Giardia lamblia

1983 ◽  
Vol 59 (1) ◽  
pp. 81-103 ◽  
Author(s):  
R. Crossley ◽  
D.V. Holberton
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Giardia Lamblia ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Molecular Weights

Proteins from the axonemes and disc cytoskeleton of Giardia lamblia have been examined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. In addition to tubulin and the 30 X 10(3) molecular weight disc protein, at least 18 minor components copurify with the two major proteins in Triton-insoluble structures. The most prominent minor bands have the apparent molecular weights of 110 X 10(3), 95 X 10(3) and 81 X 10(3). Protein of 30 X 10(3) molecular weight accounts for about 20% of organelle protein on gels. In continuous 25 mM-Tris-glycine buffer it migrates mostly as a close-spaced doublet of polypeptides, which are here given the name giardins. Giardia tubulin and giardin have been purified by gel filtration chromatography in the presence of sodium dodecyl sulphate. Well-separated fractions were obtained that could be further characterized. Both proteins are heterogeneous when examined by isoelectric focusing. Five tubulin chains were detected by PAGE Blue 83 dye-binding after focusing in a broad-range ampholyte gel. Giardin is slightly less acidic than tubulin. On gels it splits into four major and four minor chains with isoelectric points in the pI range from 5.8 to 6.2. The amino acid composition of the giardin fraction has been determined, and compared to Giardia tubulin and a rat brain tubulin standard. Giardins are rich in helix-forming residues, particularly leucine. They have a low content of proline and glycine; therefore they may have extensive alpha-helical regions and be rod-shaped. As integral proteins of disc microribbons, giardins in vivo associate closely with tubulin. The properties of giardins indicate that in a number of respects - molecular size, charge, stoichiometry - their structural interaction with tubulin assemblies will be different from other tubulin-accessory protein copolymers studied in vitro.

Gel Filtration of Plasma from Rabbits Injected with Honomeric DES-AB 125I-Fibrin and 131I–Fibrinogen

1979 ◽  
Author(s):  
I. Kröhnke ◽  
I. Hahn ◽  
W. Krell ◽  
G. Müller-Berghaus
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Gel Filtration ◽  
Void Volume ◽  
Molecular Configuration ◽  
Chromatographic Behaviour ◽  
Fibrin Monomer ◽  
Deutsche Forschungsgemeinschaft

We intended to study the chromatographic behaviour of soluble des-AB fibrin prepared in vitro and injected into rabbits. To prepare des-AB 1251-fibrin, purified rabbit 125I-fibrinogen was clotted by thrombin and the formed clot dissolved in Tris-buffered 3 M urea. Gel filtration of des-AB fibrin in urea through sepharose-CL 6B columns equilibrated with buffered 3 M. urea revealed monomeric fibrin. Rabbits received 131I-fibrinogen and 5 min later monomeric des-AB 125I-fibrin in urea. 30 min after injection blood was drawn and the plasma obtained filtered through sepharose-CL 6B columns eguilibrated with buffered plasma. At 20°C as well as at 37°C des-AB 125I-fibrin was eluted in the void volume in front of the 131I-fibrinogen peak. The data demonstrate that monomeric des-AB 125I-fibrin in urea injected into rabbits remains soluble in plasma. Possibly, monomerJ fibrin is converted to circulating soluble high-molecular weight fibrin aggregates or fibrin monomer changes its molecular configuration, thus showing a different gel filtration behaviour.(Supported by the Deutsche Forschungsgemeinschaft).

In Vitro and in Vivo Antigen-Induced Release of High-Molecular Weight Neutrophil Chemotactic Activity from Human Nasal Tissue

1988 ◽  
Vol 2 (1) ◽  
pp. 7-11 ◽  
Author(s):  
T. Nagakura ◽  
T. Onda ◽  
Y. likura ◽  
T. Endo ◽  
H. Nagakura ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Nasal Polyps ◽  
Gel Filtration ◽  
Chemotactic Activity ◽  
Nasal Tissue ◽  
Nasal Secretions ◽  
Neutrophil Chemotactic Activity

High molecular weight neutrophil chemotactic activity has been identified in resected human nasal polyps, inferior turbinates, and nasal secretions following antigen challenge. The estimated molecular weight, by gel filtration chromatography, was approximately 600,000. However, a heterogeneity of molecular weight in some patients was recognized. Our results suggest a possible role for high molecular weight-neutrophil chemotactic activity in the pathogenesis of hypersensitivity in the human nasal cavity.

scholarly journals A novel 115-kD peripheral membrane protein is required for intercisternal transport in the Golgi stack.

1992 ◽  
Vol 118 (5) ◽  
pp. 1015-1026 ◽  
Author(s):  
M G Waters ◽  
D O Clary ◽  
J E Rothman
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Protein Transport ◽  
Gel Filtration ◽  
Bovine Liver ◽  
Peptide Sequence ◽  
Golgi Stack ◽  
Transport Assay ◽  
Weight Protein

We have used an in vitro Golgi protein transport assay dependent on high molecular weight (greater than 100 kD) cytosolic and/or peripheral membrane proteins to study the requirements for transport from the cis- to the medial-compartment. Fractionation of this system indicates that, besides the NEM-sensitive fusion protein (NSF) and the soluble NSF attachment protein (SNAP), at least three high molecular weight protein fractions from bovine liver cytosol are required. The activity from one of these fractions was purified using an assay that included the second and third fractions in a crude state. The result is a protein of 115-kD subunit molecular mass, which we term p115. Immunodepletion of the 115-kD protein from a purified preparation with mAbs removes activity. Peptide sequence analysis of tryptic peptides indicates that p115 is a "novel" protein that has not been described previously. Gel filtration and sedimentation analysis indicate that, in its native state, p115 is a nonglobular homo-oligomer. p115 is present on purified Golgi membranes and can be extracted with high salt concentration or alkaline pH, indicating that it is peripherally associated with the membrane. Indirect immunofluorescence indicates that p115 is associated with the Golgi apparatus in situ.

IN VITRO EFFECTS OF LATS AND TSH ON PHOSPHODIESTERASE ACTIVITY IN HUMAN THYROID HOMOGENATES

1971 ◽  
Vol 67 (2) ◽  
pp. 209-215 ◽  
Author(s):  
K. Miyai ◽  
N. Amino ◽  
M. Azukizawa ◽  
Y. Kumahara
Keyword(s):  
Enzyme Activity ◽  
Adenosine Monophosphate ◽  
Human Thyroid ◽  
Phosphodiesterase Activity ◽  
Significant Difference ◽  
In Vitro Effects ◽  
Long Acting ◽  
Bovine Tsh

ABSTRACT The in vitro effects of long-acting thyroid stimulator (LATS) and thyrotrophin (TSH) on phosphodiesterase activity in human thyroid homogenates were studied. The enzyme activity was estimated by the destruction of 3H-cyclic 3′,5′-adenosine monophosphate. Bovine TSH had no effect on the enzyme activity. Five experiments were carried out using different batches of LATS-IgG and normal IgG which were preincubated with thyroid homogenates or not. There was no significant difference between the enzyme activity with LATS-IgG and that with normal IgG, while the activity was mostly inhibited by theophylline. These data are not consistent with the hypothesis that LATS acts as an antibody to phosphodiesterase.

A comparison of porcine and human thyroid tissue in the assay of thyroid stimulating immunoglobulins

1986 ◽  
Vol 113 (3) ◽  
pp. 335-339
Author(s):  
S. de Rave ◽  
H. M.J. Goldschmidt ◽  
Y. T.J. Somers-Pijnenburg ◽  
B. Bravenboer ◽  
J. H. M. Lockefeer
Keyword(s):  
Medical Treatment ◽  
Thyroid Tissue ◽  
Human Thyroid ◽  
Graves Disease ◽  
Clinical Use ◽  
Human Thyroid Tissue ◽  
Thyroid Stimulating Immunoglobulins ◽  
Camp Generation

Abstract. The central role of Thyroid Stimulating Immunoglobulins (TSI) in the pathogenesis of the hyperthyroidism of Graves' disease has become generally accepted and a wide variety of assays for the detection of these antibodies has been developed. The dependence on the availability of human thyroid tissue makes most of these assays unsuitable for routine clinical use, a problem circumvented by the use of nonhuman thyroid tissue in some TSI assays. We therefore compared porcine and human thyroid tissue in a TSI assay based on in vitro cAMP generation. No major differences in within and between run variation were found and, with some notable exceptions, a reasonable correlation could be demonstrated between the results in both assays (R = 0.89, P < 0.001). However, the sensitivity of the porcine TSI assay is only 60% of the estimated sensitivity of the human TSI assay. In spite of the practical advantages this porcine TSI assay, and possibly also other TSI assays using non-human thyroid tissue, cannot totally replace human TSI assays. The value of these assays in predicting the outcome of medical treatment of Graves' disease remains to be established.

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