scholarly journals The Human Thyrotropin (TSH) Receptor in a TSH Binding Inhibition Assay for TSH Receptor Autoantibodies1

1997 ◽  
Vol 82 (7) ◽  
pp. 2129-2134
Author(s):  
Ayumu Kakinuma ◽  
Gregorio D. Chazenbalk ◽  
Juan Carlos Jaume ◽  
Basil Rapoport ◽  
Sandra M. McLachlan
Keyword(s):  
Intact Cell ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Tsh Receptor ◽  
Intact Cells ◽  
Ovary Cells ◽  
Large Numbers ◽  
Binding Inhibition ◽  
Excellent Source ◽  
Bovine Tsh

Seven years after the molecular cloning of the human TSH receptor (TSHR), the porcine TSHR remains in general use in the TSH binding inhibition (TBI) assay for autoantibodies to the TSHR. We compared porcine and recombinant human TSHR in two types of TBI assays: one using intact Chinese hamster ovary cells expressing the recombinant human TSHR on their surface, and the other using soluble receptors extracted from these cells with detergent. In the intact cell TBI assay, monolayers expressing large numbers of TSHR were less effective than cells expressing few receptors. These findings are consistent with the very low concentration of TSHR autoantibodies in serum. Binding of[ 125I]human TSH was about 5-fold lower than that of[ 125I]bovine TSH to the intact cells. Nevertheless, TBI values with the two ligands were similar for most sera. However, a few sera produced greater inhibition of human than of bovine TSH binding. In the solubilized human TSHR TBI assay, in contrast to the intact cell TBI assay, cells expressing very large number of TSHR were an excellent source for detergent extraction of soluble human TSHR, but only if the cells were extracted while still on the dish and not after scraping. A 10-cm diameter dish of cells provided TSHR for 100–200 replicate determinations when substituted for solubilized porcine TSHR in a commercial TBI kit. TBI values in serum from 30 individuals with suspected Graves’ disease correlated closely when tested with solubilized human and porcine TSHR (r = 0.954; P < 0.001). However, 2 sera that were negative with the porcine TSHR were positive with the human TSHR. TBI and thyroid-stimulating activity in these sera correlated weakly regardless of whether the TBI used human or porcine TSHR. These findings open the way to a practical TBI assay using recombinant human TSHR.

The insecticide 1,1,1-trichloro-2,2-bis(p-chlorophenyl) ethane (DDT) alters the membrane raft location of the TSH receptor stably expressed in Chinese hamster ovary cells

2011 ◽  
Vol 253 (2) ◽  
pp. 121-129 ◽  
Author(s):  
Francesca De Gregorio ◽  
Mario Pellegrino ◽  
Simona Picchietti ◽  
Maria C. Belardinelli ◽  
Anna Rita Taddei ◽  
...  
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Tsh Receptor ◽  
Membrane Raft ◽  
Ovary Cells

scholarly journals EVIDENCE FOR PLEIOTROPIC CHANGES IN LINES OF CHINESE HAMSTER OVARY CELLS RESISTANT TO CONCANAVALIN A AND PHYTOHEMAGGLUTININ-P

1973 ◽  
Vol 56 (3) ◽  
pp. 666-675 ◽  
Author(s):  
J. A. Wright
Keyword(s):  
Concanavalin A ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Single Step ◽  
Cross Resistance ◽  
Wild Type ◽  
Con A ◽  
Intact Cells ◽  
Ovary Cells ◽  
Resistant Lines

Lines of Chinese hamster ovary cells resistant to the lectins concanavalin A (Con A) and phytohemagglutinin-P (PHA-P) have been isolated and characterized. Lines were isolated by a stepwise, a single-step, or a cycling single-step procedure, from both mutagen-treated and untreated cultures. The resistant lines showed a higher efficiency of colony formation in the presence of the appropriate lectin than did the wild-type parental line. The cell lines resistant to Con A did not exhibit any detectable cross resistance to PHA-P, nor did the PHA-resistant cells exhibit cross resistance to Con A. The toxicity of Con A from the wild-type and Con A-resistant lines was reduced in the presence of methyl α-D-glucopyranoside; this effect was not seen with the PHA-resistant line. Using 125I-labeled Con A, it was found that Con A was bound preferentially to the surface of intact cells, and that the amount of labeled Con A bound to intact cells was similar for the wild-type and lectin-resistant lines. The Con A-resistant lines were found to be more susceptible to the toxic effects of a number of different compounds, including cyclic AMP and its dibutyryl derivative, sodium butyrate, high concentrations of glucose, phenethyl alcohol, phenol, ouabain, and testosterone. It appears that, in these lines, acquisition of resistance to Con A gave rise to pleiotropic effects which were detected by changes in the sensitivity of the cells to a variety of agents.

scholarly journals Evidence for both prelysosomal and lysosomal intermediates in endocytic pathways.

1984 ◽  
Vol 98 (1) ◽  
pp. 108-115 ◽  
Author(s):  
B Storrie ◽  
R R Pool ◽  
M Sachdeva ◽  
K M Maurey ◽  
C Oliver
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fluid Phase ◽  
Intact Cells ◽  
Ovary Cells ◽  
Chase Period ◽  
Percoll Gradients ◽  
Marker Distribution ◽  
Progressive Accumulation ◽  
Dense Vesicles

Horseradish peroxidase (HRP), an enzyme internalized by fluid phase pinocytosis, has been used to study the process by which pinosome contents are delivered to lysosomes in Chinese hamster ovary cells. Pinosome contents were labeled by allowing cells to internalize HRP for 3-5 min. Following various chase times, cells were either processed for HRP and acid phosphatase (AcPase) cytochemistry or homogenized and fractionated in Percoll gradients. In Percoll gradients, pinosomes labeled by a 3-5 min HRP pulse behaved as a vesicle population more dense than plasma membrane and less dense than lysosomes. In pulse-chase experiments, internalized HRP was chased rapidly (3-6 min chase) to a density position intermediate between the "initial" pinocytic vesicle population and lysosomes. With longer chase periods, a progressive accumulation of HRP in more dense vesicles was observed. Correspondence between the HRP distribution and lysosomal marker distribution was reached after a approximately 1-h chase. By electron microscope cytochemistry of intact cells, the predominant class of HRP-positive vesicles after pulse uptakes or a 3-min chase period was characterized by a peripheral rim of reaction product and was AcPase negative. After 10-120-min chase periods, the predominant class of HRP-positive vesicles was characterized by luminal deposits and HRP activity was frequently observed in multivesicular bodies. HRP-positive vesicles after a 10- or 30-min chase were AcPase-positive. No HRP activity was detected in Golgi apparatus. Together these observations indicate that progressive processing of vesicular components of the vacuolar apparatus occurs at both a prelysosomal and lysosomal stage.

scholarly journals CHANGES IN SURFACE MORPHOLOGY OF CHINESE HAMSTER OVARY CELLS DURING THE CELL CYCLE

1973 ◽  
Vol 57 (3) ◽  
pp. 815-836 ◽  
Author(s):  
Keith Porter ◽  
David Prescott ◽  
Jearl Frye
Keyword(s):  
Cell Cycle ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Confluent Culture ◽  
Large Numbers ◽  
Wide Range ◽  
Surface Changes

Synchronized populations of Chinese hamster ovary (CHO) cells in confluent culture have been examined by scanning electron microscopy and their surface changes noted as the cells progress through the cycle. During G1 it is characteristic for cells to show large numbers of microvilli, blebs, and ruffles. Except for the ruffles, these tend to diminish in prominence during S and the cells become relatively smooth as they spread thinly over the substrate. During G2 microvilli increase in number and the cells thicken in anticipation of rounding up for mitosis. It appears that the changes observed here reflect the changing capacity of CHO cells during the cycle to respond to contact with other cells in the population, because, as noted in the succeeding paper (Rubin and Everhart), CHO cells in sparse nonconfluent cultures do not show the same wide range of changes during the cell cycle. Normal, nontransformed cells of equivalent type in confluent culture are essentially devoid of microvilli, blebs, and ruffles. The relation of these surface configurations to the internal structure of the cell is discussed.

scholarly journals Organization of Mammalian Cytoplasm

2003 ◽  
Vol 23 (24) ◽  
pp. 9318-9326 ◽  
Author(s):  
Alice Hudder ◽  
Lubov Nathanson ◽  
Murray P. Deutscher
Keyword(s):  
Protein Synthesis ◽  
Mammalian Cells ◽  
Cell Function ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cellular Protein ◽  
Actin Binding ◽  
Intact Cells ◽  
Ovary Cells ◽  
Cytoplasmic Proteins

ABSTRACT Although the role of macromolecular interactions in cell function has attracted considerable attention, important questions about the organization of cells remain. To help clarify this situation, we used a simple protocol that measures macromolecule release after gentle permeabilization for the examination of the status of endogenous macromolecules. Treatment of Chinese hamster ovary cells with saponin under carefully controlled conditions allowed entry of molecules of at least 800 kDa; however, there were minimal effects on internal cellular architecture and protein synthesis remained at levels comparable to those seen with intact cells. Most importantly, total cellular protein and RNA were released from these cells extremely slowly. The release of actin-binding proteins and a variety of individual cytoplasmic proteins mirrored that of total protein, while marker proteins from subcellular compartments were not released. In contrast, glycolytic enzymes leaked rapidly, indicating that cells contain at least two distinct populations of cytoplasmic proteins. Addition of microfilament-disrupting agents led to rapid and extensive release of cytoplasmic macromolecules and a dramatic reduction in protein synthesis. These observations support the conclusion that mammalian cells behave as highly organized, macromolecular assemblies (dependent on the actin cytoskeleton) in which endogenous macromolecules normally are not free to diffuse over large distances.

scholarly journals Interactions between Newly Synthesized Glycoproteins, Calnexin and a Network of Resident Chaperones in the Endoplasmic Reticulum

1997 ◽  
Vol 136 (3) ◽  
pp. 555-565 ◽  
Author(s):  
Utpal Tatu ◽  
Ari Helenius
Keyword(s):  
Endoplasmic Reticulum ◽  
Chinese Hamster Ovary Cells ◽  
Gradient Centrifugation ◽  
Chinese Hamster ◽  
Cross Linking ◽  
Intact Cells ◽  
Ovary Cells ◽  
Influenza Hemagglutinin ◽  
Infected Cells ◽  
Chemical Cross Linking

Calnexin is a membrane-bound lectin and a molecular chaperone that binds newly synthesized glycoproteins in the endoplasmic reticulum (ER). To analyze the oligomeric properties of calnexin and calnexin-substrate complexes, sucrose velocity gradient centrifugation and chemical cross-linking were used. After CHAPS solubilization of Chinese Hamster Ovary cells, the unoccupied calnexin behaved as a monomer sedimenting at 3.5 S20,W. For calnexin-substrate complexes the S-values ranged between 3.5–8 S20,W, the size increasing with the molecular weight of the substrate. Influenza hemagglutinin, a well-characterized substrate associated with calnexin in complexes that sedimented at 5–5.5 S20,W. The majority of stable complexes extracted from cells, appeared to contain a single calnexin and a single substrate molecule, with about one third of the calnexin in the cell being unoccupied or present in weak associations. However, when chemical cross-linking was performed in intact cells, the calnexin-substrate complexes and calnexin itself was found to be part of a much larger heterogeneous protein network that included other ER proteins. Pulse-chase analysis of influenza-infected cells combined with chemical cross-linking showed that HA was part of large, heterogeneous, cross-linked entities during the early phases of folding, but no longer after homotrimer assembly. The network of weakly associated resident ER chaperones which included BiP, GRP94, calreticulin, calnexin, and other proteins, may serve as a matrix that binds early folding and assembly intermediates and restricts their exit from the ER.

scholarly journals The role of sphingomyelin in phosphatidylcholine metabolism in cultured human fibroblasts from control and sphingomyelin lipidosis patients and in Chinese hamster ovary cells

1990 ◽  
Vol 268 (3) ◽  
pp. 719-724 ◽  
Author(s):  
M W Spence ◽  
H W Cook ◽  
D M Byers ◽  
F B S C Palmer
Keyword(s):  
Chinese Hamster Ovary ◽  
Cultured Cells ◽  
Chinese Hamster Ovary Cells ◽  
Human Fibroblasts ◽  
Specific Radioactivity ◽  
Chinese Hamster ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Intact Cells ◽  
Ovary Cells

Human fibroblasts in culture take up exogenous [choline-Me-3H,32P]sphingomyelin (SM) from the medium and incorporate it into cellular SM and phosphatidylcholine [Spence, Clarke & Cook (1983) J. Biol. Chem. 258, 8595-8600]. The ratio of [3H]choline/[32P]Pi is similar in SM and phosphatidylcholine, indicating that the phosphocholine (P-Cho) moiety is transferred intact. Similar results are obtained with Niemann-Pick (NP) cells which are deficient in lysosomal sphingomyelinase activity, suggesting that the P-Cho transfer may not be mediated by the lysosomal sphingomyelinase and that alternative pathways of sphingomyelin catabolism are present in cultured cells. In this study we have shown that: (1) the P-Cho pool in control and NP cells incubated with exogenous labelled SM has a specific radioactivity intermediate between that of SM and PtdCho; (2) expansion of the intracellular P-Cho pool by incubation with exogenous choline reduces the incorporation of [3H]choline from SM into PtdCho; and (3) incorporation of P-Cho from SM into PtdCho is decreased at the non-permissive temperature in Chinese hamster ovary cells with a temperature-sensitive mutation in the cytidylyltransferase reaction. These results suggest that incorporation of P-Cho from SM into PtdCho involves a reaction sequence in which P-Cho is hydrolysed from SM by a sphingomyelinase, followed by incorporation of P-Cho into PtdCho via the cytidine pathway of biosynthesis (SM----P-Cho----CDP-Cho----PtdCho). The appreciable incorporation of P-Cho from SM into PtdCho in sphingomyelinase-deficient NP cells suggests a more substantial or effective lysosomal sphingomyelinase activity in intact cells than is measured in vitro, and/or a significant contribution by other sphingomyelinase activities in these cells.

scholarly journals Changes in insulin-receptor tyrosine, serine and threonine phosphorylation as a result of substitution of tyrosine-1162 with phenylalanine

1991 ◽  
Vol 274 (1) ◽  
pp. 173-179 ◽  
Author(s):  
J M Tavaré ◽  
M Dickens
Keyword(s):  
Insulin Receptor ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Serine Phosphorylation ◽  
Beta Subunit ◽  
Intact Cells ◽  
Ovary Cells ◽  
Tyrosine Residues

Previous studies, by ourselves and others, have shown that tyrosine residues 1158, 1162 and 1163 are very rapidly autophosphorylated on the human insulin receptor after insulin binding and that this is followed by the autophosphorylation of tyrosine residues 1328 and 1334. The autophosphorylation of these tyrosine residues, and their role in transmembrane signalling, were examined by using Chinese-hamster ovary cells transfected with either normal intact insulin receptors or receptors in which tyrosine residues 1162 or 1162/1163 were substituted with phenylalanine. These studies show the following. (1) Tyrosine-1158 could still be autophosphorylated when tyrosine-1162 and -1163 were substituted with phenylalanine. (2) Insulin-stimulated insulin-receptor tyrosine phosphorylation in intact cells was complete within 30 s and was accompanied, after a lag of 2-5 min, by a rise in serine and threonine phosphorylation the beta-subunit. (3) Replacement of tyrosine-1162 with phenylalanine blocked insulin-stimulated threonine phosphorylation of the insulin receptor in intact cells. (4) Insulin-stimulated serine phosphorylation of the beta-subunit was found in both intact cells and partially purified receptor preparations incubated with [gamma-32P]ATP and was still apparent after the replacement of tyrosine-1162 with phenylalanine. (5) Our data strongly suggest that insulin-stimulated insulin-receptor serine and threonine phosphorylations are initiated through two distinct pathways, with only the latter showing a strict dependence on autophosphorylation of tyrosine-1162.

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