scholarly journals Internalization of ricin in Chinese hamster ovary cells.

1981 ◽  
Vol 1 (6) ◽  
pp. 544-551 ◽  
Author(s):  
B Ray ◽  
H C Wu
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Combined Treatment ◽  
Sodium Dodecyl ◽  
Chinese Hamster ◽  
Resistant Cell ◽  
Proteolytic Processing ◽  
Ph Optimum ◽  
Ovary Cells

Internalization of ricin into Chinese hamster ovary cells has been investigated. Combined treatment with galactose and pronase at 0 degrees C resulted in a complete release of surface-bound [125I]ricin into the media. Galactose-pronase-resistant cell-bound [125I]ricin represents internalized ricin molecules inside the cells. The internalization process is time, temperature, and concentration dependent. The pH optimum of internalization of ricin is about pH 7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis has revealed that intact ricin molecules are internalized. Neither reduction nor proteolytic processing of ricin is required for the entry of ricin into Chinese hamster ovary cells.

Internalization of ricin in Chinese hamster ovary cells

1981 ◽  
Vol 1 (6) ◽  
pp. 544-551
Author(s):  
B Ray ◽  
H C Wu
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Combined Treatment ◽  
Sodium Dodecyl ◽  
Chinese Hamster ◽  
Resistant Cell ◽  
Proteolytic Processing ◽  
Ph Optimum ◽  
Ovary Cells

Internalization of ricin into Chinese hamster ovary cells has been investigated. Combined treatment with galactose and pronase at 0 degrees C resulted in a complete release of surface-bound [125I]ricin into the media. Galactose-pronase-resistant cell-bound [125I]ricin represents internalized ricin molecules inside the cells. The internalization process is time, temperature, and concentration dependent. The pH optimum of internalization of ricin is about pH 7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis has revealed that intact ricin molecules are internalized. Neither reduction nor proteolytic processing of ricin is required for the entry of ricin into Chinese hamster ovary cells.

Isolation of a cDNA clone for human threonyl-tRNA synthetase: amplification of the structural gene in borrelidin-resistant cell lines

1989 ◽  
Vol 9 (5) ◽  
pp. 1832-1838
Author(s):  
K J Kontis ◽  
S M Arfin
Keyword(s):  
Cdna Clone ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Mrna Translation ◽  
Resistant Cell ◽  
Trna Synthetase ◽  
Expression Library ◽  
Hybridization Probe ◽  
Ovary Cells

A cDNA for threonyl-tRNA synthetase was isolated from a human placental cDNA lambda gt11 expression library by immunological screening, and its identity was confirmed by hybrid-selected mRNA translation. With this cDNA used as a hybridization probe, borrelidin-resistant Chinese hamster ovary cells that overproduced threonyl-tRNA synthetase were shown to have increased levels of threonyl-tRNA synthetase mRNA and gene sequences. Amplification of the gene did not appear to have been accompanied by any major structural reorganizations.

scholarly journals Selection of mutant Chinese hamster ovary cells altered glycoproteins by means of tritiated fucose suicide.

1981 ◽  
Vol 1 (10) ◽  
pp. 902-909 ◽  
Author(s):  
C B Hirschberg ◽  
R M Baker ◽  
M Perez ◽  
L A Spencer ◽  
D Watson
Keyword(s):  
Chinese Hamster Ovary ◽  
Gel Electrophoresis ◽  
Trichloroacetic Acid ◽  
Chinese Hamster Ovary Cells ◽  
Sodium Dodecyl ◽  
Chinese Hamster ◽  
Wild Type ◽  
Ovary Cells ◽  
Replica Plating ◽  
Selection Of

Mutant Chinese hamster ovary cells altered in glycoproteins have been isolated by selecting for ability to survive exposure to [6-3H]fucose. Mutagenized wild-type cells were permitted to incorporate [3H]fucose to approximately 1 cpm of trichloroacetic acid-insoluble radioactivity per cell and then frozen for several days to accumulate radiation damage. The overall viability of the population was reduced by 5- to 50-fold. Four consecutive selection cycles were carried out. The surviving cells were screened by replica plating-fluorography for clones showing decreased incorporation of fucose into trichloroacetic acid-insoluble macromolecules. Considerable enrichment for cells deficient in fucose uptake or incorporation into proteins (or both) was found in populations surviving the later selection cycles. Two mutant clones isolated after the fourth selection cycle had the same doubling time as the wild type, but contained only 30 to 40% as much fucose bound to proteins as the wild type. Sialic acid contents of the mutants and the wild type were similar. The mutants differed quantitatively and qualitatively from the wild type and from each other with respect to total glycoprotein profiles as visualized by sodium dodecyl sulfate gel electrophoresis. Differences were also found in resistances to cytotoxicity of lectins such as concanavalin A and wheat germ agglutinin.

scholarly journals Identification and characterization of a third complementation group of emetine-resistant Chinese hamster cell mutants.

1981 ◽  
Vol 1 (1) ◽  
pp. 58-65 ◽  
Author(s):  
J J Wasmuth ◽  
J M Hill ◽  
L S Vock
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Chinese Hamster Cell ◽  
Resistant Cell ◽  
Complementation Group ◽  
Ovary Cells ◽  
Complementation Groups ◽  
Resistant Mutants ◽  
Biosynthetic Machinery

We have isolated emetine-resistant cell lines from Chinese hamster peritoneal fibroblasts and have shown that they represent a third distinct class or complementation group of emetine-resistant mutants, as determined by three different criteria. These mutants, like those belonging to the two other complementation groups we have previously defined, which were isolated from Chinese hamster lung and Chinese hamster ovary cells, have alterations that directly affect the protein biosynthetic machinery. So far, there is absolute cell line specificity with respect to the three complementation groups, in that all the emetine-resistant mutants we have isolated from Chinese hamster lung cells belong to one complementation group, all those we have isolated from Chinese hamster ovary cells belong to a second complementation group, and all those isolated from Chinese hamster peritoneal cells belong to a third complementation group. Thus, in cultured Chinese hamster cells, mutations in at least three different loci, designated emtA, emtB, and emtC, encoding for different components of the protein biosynthetic machinery, can give rise to the emetine-resistant phenotype.

scholarly journals Isolation of a novel cell-attachment and spreading-promoting protein from human serum

1985 ◽  
Vol 227 (2) ◽  
pp. 421-427 ◽  
Author(s):  
M Vuento ◽  
M Korkolainen ◽  
P Kuusela ◽  
E Hölttä
Keyword(s):  
Affinity Chromatography ◽  
Human Serum ◽  
Isoelectric Focusing ◽  
Chinese Hamster Ovary ◽  
Gel Electrophoresis ◽  
Cell Attachment ◽  
Chinese Hamster Ovary Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Chinese Hamster ◽  
Ovary Cells

A protein with potent cell-attachment and spreading-promoting activity was isolated from fibronectin-free human serum. The purification steps included affinity chromatography on heparin-agarose and preparative isoelectric focusing. The purified protein was homogeneous as judged from dodecyl sulphate/polyacrylamide-gel electrophoresis. It had an isoelectric point of 5.0 and an Mr of 52 000. The protein promoted the spreading of Chinese-hamster ovary cells to plastic in a manner similar to that observed with fibronectin.

scholarly journals Comparative studies on the carbohydrate-containing membrane components of normal and adenosine 3′:5′-cyclic monophosphate-treated Chinese-hamster ovary cells

1973 ◽  
Vol 134 (1) ◽  
pp. 329-339 ◽  
Author(s):  
M. Mansoor Baig ◽  
R. M. Roberts
Keyword(s):  
Cyclic Amp ◽  
Cell Surface ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Sodium Dodecyl ◽  
Chinese Hamster ◽  
Double Labelling ◽  
Dibutyryl Cyclic Amp ◽  
Ovary Cells ◽  
Cyclic Monophosphate

1. We investigated some of the changes in plasma-membrane composition that accompany the alteration in cell growth and morphology induced by treating Chinese-hamster ovary cells with dibutyryladenosine 3′:5′-cyclic monophosphate (dibutyryl cyclic AMP). 2. A double-labelling technique was employed in which normal cells were given 14C-labelled precursor, and those treated with dibutyryl cyclic AMP were given 3H label. l-Leucine, d-glucosamine, and l-fucose were used to label the membranes. 3. After 3 days growth, the two populations of cells were harvested by trypsin treatment, the cells were pooled, and plasma membranes isolated. Proteins and glycoproteins of the membranes were separated by electrophoresis on sodium dodecyl sulphate–polyacrylamide gels, and the radioactive profiles for 14C and 3H and the staining patterns with Amido Black were compared. 4. Although certain components of the membrane from treated cells showed marked quantitative changes, there was neither major addition nor major deletions of components. 5. Complete proteolysis of the mixed membranes, of the material released from the cell surface by trypsin, and of the glycoproteins released from the cells into the medium, gave a series of radioactive glycopeptides when either fucose or glucosamine was employed as precursor. 6. After such glycopeptides were fractionated on columns of Sephadex G-50, marked differences in the elution profiles of 3H and 14C were noted. Dibutyryl cyclic AMP evidently causes alterations in the overall composition of the carbohydrate components of the cell surface. It was not possible to decide whether this was solely the result of the same glycoproteins being formed but in different proportions, or the result of modifications of oligosaccharide side chains on some of the glycoproteins. 7. Some of the changes were not unlike the reverse of those that accompany the transformation of fibroblasts by oncogenic viruses, and our results lend credence to the idea that the lowered amount of cyclic AMP noted in transformed cells is responsible for their altered surface properties.

scholarly journals Naphthol AS-BI (7-bromo-3-hydroxy-2-naphtho-o-anisidine) phosphatase and naphthol AS-BI beta-D-glucuronidase in Chinese hamster ovary cells: biochemical and flow cytometric studies.

1979 ◽  
Vol 27 (1) ◽  
pp. 120-124 ◽  
Author(s):  
F A Dolbeare ◽  
W Phares
Keyword(s):  
Flow Cytometry ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fluorescent Product ◽  
Ph Optimum ◽  
Intact Cells ◽  
Ovary Cells ◽  
Glucuronidase Activity ◽  
Flow Cytometric

Conditions for the biochemical and flow cytometric assay of 7-bromo-3-hydroxy-2-naphtho-o-anisidine phosphatase and beta-D-glucuronidase activities in Chinese hamster ovary cells were studied. In the biochemical assay, the pH optimum for the phosphatase activity was pH 4.6 with a Km of 10(-5) M; the pH optimum for beta-D-glucuronidase activity was pH 5.0 with a Km of 2 x 10(-5) M. For intact cells the derived constants were 3 to 10 times higher. The rate of hydrolysis of both substrates was also examined by flow cytometry. Cellular fluorescence increased linearly for only about 15 min. Diffusion of the fluorescent product probably caused nonlinearity of the fluorescence increase and was demonstrated by mixing cells incubated with substrate with those that had not been incubated. After 15 min, cells that had not been exposed previously to product or substrate contained the fluorescent product. Cells fractionated into size classes by centrifugal elutriation also were analyzed by flow cytometry for beta-D-glucuronidase activity. The activity increased linearly with the increase in cell size corresponding to the progression from G1 through S and into G2-M phases of the cell cycle.

Amplification of the gene for histidyl-tRNA synthetase in histidinol-resistant Chinese hamster ovary cells

1985 ◽  
Vol 5 (9) ◽  
pp. 2381-2388
Author(s):  
F W Tsui ◽  
I L Andrulis ◽  
H Murialdo ◽  
L Siminovitch
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fold Increase ◽  
Resistant Cell ◽  
Trna Synthetase ◽  
Synthetase Activity ◽  
Wild Type ◽  
Ovary Cells ◽  
Resistant Cells

Histidinol-resistant (HisOHR) mutants with up to a 30-fold increase in histidyl-tRNA synthetase activity have been isolated by stepwise adaptation of wild-type Chinese hamster ovary (CHO) cells to increasing amounts of histidinol in the medium. Immunoprecipitation of [35S]methionine-labeled cell lysates with antibodies to histidyl-tRNA synthetase showed increased synthesis of the enzyme in histidinol-resistant cells. The histidinol-resistant cell lines had an increase in translatable polyadenylated mRNA for histidyl-tRNA synthetase. A cDNA for CHO histidyl-tRNA synthetase has been cloned, using these histidyl-tRNA synthetase-overproducing mutants as the source of mRNA. Southern blot analysis of wild-type and histidinol-resistant cells with this cDNA showed that the histidyl-tRNA synthetase DNA bands were amplified in the resistant cells. These HisOHR cells owed their resistance to histidinol to amplification of the gene for histidyl-tRNA synthetase.

Selection of mutant Chinese hamster ovary cells altered glycoproteins by means of tritiated fucose suicide

1981 ◽  
Vol 1 (10) ◽  
pp. 902-909
Author(s):  
C B Hirschberg ◽  
R M Baker ◽  
M Perez ◽  
L A Spencer ◽  
D Watson
Keyword(s):  
Chinese Hamster Ovary ◽  
Gel Electrophoresis ◽  
Trichloroacetic Acid ◽  
Chinese Hamster Ovary Cells ◽  
Sodium Dodecyl ◽  
Chinese Hamster ◽  
Wild Type ◽  
Ovary Cells ◽  
Replica Plating ◽  
Selection Of

Mutant Chinese hamster ovary cells altered in glycoproteins have been isolated by selecting for ability to survive exposure to [6-3H]fucose. Mutagenized wild-type cells were permitted to incorporate [3H]fucose to approximately 1 cpm of trichloroacetic acid-insoluble radioactivity per cell and then frozen for several days to accumulate radiation damage. The overall viability of the population was reduced by 5- to 50-fold. Four consecutive selection cycles were carried out. The surviving cells were screened by replica plating-fluorography for clones showing decreased incorporation of fucose into trichloroacetic acid-insoluble macromolecules. Considerable enrichment for cells deficient in fucose uptake or incorporation into proteins (or both) was found in populations surviving the later selection cycles. Two mutant clones isolated after the fourth selection cycle had the same doubling time as the wild type, but contained only 30 to 40% as much fucose bound to proteins as the wild type. Sialic acid contents of the mutants and the wild type were similar. The mutants differed quantitatively and qualitatively from the wild type and from each other with respect to total glycoprotein profiles as visualized by sodium dodecyl sulfate gel electrophoresis. Differences were also found in resistances to cytotoxicity of lectins such as concanavalin A and wheat germ agglutinin.

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