Differential fate of glycoproteins carrying a monoglucosylated form of truncated N-glycan in a new CHO line, MadIA214214, selected for a thermosensitive secretory defect

1997 ◽  
Vol 110 (3) ◽  
pp. 323-336
Author(s):  
M. Ermonval ◽  
R. Cacan ◽  
K. Gorgas ◽  
I.G. Haas ◽  
A. Verbert ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Initial Step ◽  
Chain Elongation ◽  
Temperature Sensitive ◽  
Placental Alkaline Phosphatase ◽  
Ovary Cells ◽  
Human Placental Alkaline Phosphatase ◽  
Glycoprotein Degradation

A temperature sensitive secretory line, MadIA214, was selected from mutagenized Chinese hamster ovary cells that express two heterologous export marker proteins: a secretory form of the human placental alkaline phosphatase (SeAP), and the Kd heavy chain of mouse MHC class I. SeAP secretion in MadIA214 was extremely reduced at elevated temperature (40 degrees C), while the export of functional H-2Kd molecules to the plasma membrane was only slightly affected. This mutant constitutively transferred onto newly synthesized proteins a truncated oligosaccharide core, Man5GlcNAc2, which was monoglucosylated in the protein-bound form. Nevertheless, the final oligosaccharide-structures associated to mature SeAP and H-2Kd were similar in mutant and wild-type glycoproteins. The inaccessibility in MadIA214 endoplasmic reticulum (ER) of one or more components required for oligosaccharide chain elongation is supported by the reconstitution of a correct core structure, obtained after disruption of cellular compartments, but not after cell permeabilisation or blocking ER-to-Golgi transport. The increased association of the ER-chaperone BiP with immature SeAP correlated with the thermodependent decrease in SeAP secretion. The retention of incompletely folded polypeptides in MadIA214 parallels both a marked ER-dilation and an important glycoprotein degradation documented by the formation of soluble oligomannosides with one GlcNAc residue. Our data provide the first in vivo evidence that the initial step in N-glycosylation differentially governs glycoprotein maturation, transport and degradation.

Activity and stability of centrosomes in Chinese hamster ovary cells in nucleation of microtubules in vitro

1984 ◽  
Vol 66 (1) ◽  
pp. 277-295
Author(s):  
R. Kuriyama
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Temperature Sensitive ◽  
Ovary Cells ◽  
Protease Digestion ◽  
The Difference

Mitotic centrosomes were prepared from Chinese hamster ovary cells and their capacity to nucleate microtubules in vitro was demonstrated by incubation with exogenous brain microtubule protein. The number of microtubules polymerized onto centrosomes was directly counted by electron microscopy of whole-mount preparations. This simple and accurate quantitative assay has permitted characterization of the microtubule nucleating activity of centrosomes in vitro. The number of microtubules polymerized onto centrosomes varied according to the structure of the centrosome. The activity was roughly proportional to the centriole number. The number and length of microtubules nucleated by centrosomes depended both on the concentration of tubulin and the incubation time with tubulin. Under saturating conditions, an average of 200–250 microtubules were initiated by single centrosomes. Centrosomal activity is unstable (t 1/2 = 8 h) and could easily be irreversibly disrupted by a medium of high ionic strength. The activity is stabilized by the addition of glycerol. Centrosomes can be stored at −80 degrees C. The optimum pH for microtubule nucleation is 6.8. Activity is sensitive to protease digestion, but neither DNase or RNase affected the nucleating activity of centrosomes. The activity is temperature-sensitive, but addition of phenylmethylsulphonyl fluoride (PMSF) induces thermal stability. At an optimal concentration of 0.5 mg/ml, this drug increased the half-life of the activity (t 1/2 = 95 h) and made it resistant to salt extraction. Protease inhibitors other than PMSF or dansyl fluoride did not have any stabilizing effect on the activity. The difference between the centrosomal structure of polymerized microtubules in vivo and in vitro is discussed.

Formycin B-resistant mutants of Chinese hamster ovary cells: novel genetic and biochemical phenotype affecting adenosine kinase

1983 ◽  
Vol 3 (8) ◽  
pp. 1468-1477
Author(s):  
K D Mehta ◽  
R S Gupta
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Complex Form ◽  
Single Step ◽  
Adenosine Kinase ◽  
Cross Resistance ◽  
Ovary Cells ◽  
Cell Extracts

Stable mutants which are approximately three- and eightfold resistant to the pyrazolopyrimidine nucleosides formycin A and formycin B (FomR) have been selected in a single step from mutagenized Chinese hamster ovary cells. In cell extracts, the two FomR mutants which were examined were both found to contain no measurable activity of the enzyme adenosine kinase (AK). However, cross-resistance studies with other adenosine analogs such as toyocamycin and tubercidin show that these mutants are distinct from toyocamycin or tubercidin resistant (Toyr) mutants which also contain no measurable AK activity in cell extracts. Studies on the uptake and incorporation of [3H]adenosine and [3H]tubercidin by various mutants and parental cell lines show that unlike the Toyr mutants, which are severely deficient in the phosphorylation of these compounds, the FomR mutants possess nearly normal capacity to phosphorylate these compounds and incorporate them into cellular macromolecules. These results suggest that the FomR mutants contain normal levels of AK activity in vivo. In cell hybrids formed between FomR X FomS cells and FomR X Toyr cells, the formycin-resistant phenotype of both of the FomR mutants behaved codominantly. However, the extracts from these hybrid cells contained either congruent to 50% (FomR X FomS) or no measurable (FomR X Toyr) AK activity, indicating that the lesion in these mutants neither suppresses the wild-type AK activity nor complements the AK deficiency of the Toyr mutants. The presence of AK activity in the FomR mutants in vivo, but not in their cell extracts, along with the codominant behavior of the mutants in hybrids, indicates that the lesions in the FomR mutant are of a novel nature. It is suggested that the genetic lesion in these mutants affects AK activity indirectly and that it may involve an essential cellular function which exists in a complex form with AK. Some implications of these results regarding the mechanism of action of formycin B are discussed.

scholarly journals Recombinant human GM-CSF induces leukocytosis and activates peripheral blood polymorphonuclear neutrophils in nonhuman primates

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 206-213 ◽  
Author(s):  
P Mayer ◽  
C Lam ◽  
H Obenaus ◽  
E Liehl ◽  
J Besemer
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
White Blood Cells ◽  
Chinese Hamster ◽  
Coli Strain ◽  
Polymorphonuclear Neutrophils ◽  
Dependent Manner ◽  
Ovary Cells ◽  
Gm Csf ◽  
Cell Counts

The in vivo efficacy of glycosylated and nonglycosylated recombinant human granulocyte macrophage colony-stimulating factor (rh GM-CSF) expressed in Chinese hamster ovary cells and Escherichia coli respectively was studied in rhesus monkeys following a daily subcutaneous (SC; three times) or intravenous (IV; over six hours) dose for seven consecutive days. The monkeys responded to the rh GM-CSF with a prompt (within 24 hours) rise in circulating white blood cells (WBCs). Thereafter the total cell counts increased steadily in a dose- dependent manner with repeated dosing to numbers six times over the pretreatment levels. Overall, granulocyte counts increased fivefold, lymphocytes twofold to fourfold, and monocytes threefold to fourfold. Platelets and erythrocytes were unaffected. Within 1 week after the end of treatment the leukocytosis had disappeared. Of the two routes of treatment, SC (three times daily)-administered rh GM-CSF was more effective than the same dose given by a six-hour IV infusion. In addition to inducing leukocytosis, parenterally administered rh GM-CSF primed mature circulating granulocytes for enhanced oxidative metabolism and killing of an E coli strain. These results show that exogenously administered glycosylated or nonglycosylated rh GM-CSF is both an effective stimulator of leukocytosis and a potent activator of the phagocytic function of mature granulocytes in monkeys.

scholarly journals Isolation of Chinese hamster ovary cells that overproduce asparaginyl-tRNA synthetase.

1984 ◽  
Vol 4 (9) ◽  
pp. 1939-1941
Author(s):  
R E Cirullo ◽  
J J Wasmuth
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Trna Synthetase ◽  
Temperature Sensitive ◽  
Ovary Cells ◽  
Resistant Phenotype ◽  
Lines Of Evidence

Temperature-resistant revertants, derived from the temperature-sensitive CHO asparaginyl-tRNA synthetase mutant, Asn-5, were isolated and characterized. Several lines of evidence indicate that the temperature-resistant phenotype of the revertants is due to their overproducing the same altered enzyme present in the Asn-5 parent.

Evaluation of the Toxicity of Intravenous Delivery of Auroshell Particles (Gold–Silica Nanoshells)

2012 ◽  
Vol 31 (6) ◽  
pp. 584-594 ◽  
Author(s):  
Shayne C. Gad ◽  
Kelly L. Sharp ◽  
Charles Montgomery ◽  
J. Donald Payne ◽  
Glenn P. Goodrich
Keyword(s):  
Water Solution ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Gold Nanoshells ◽  
Sprague Dawley ◽  
Ovary Cells ◽  
Good Laboratory ◽  
Chromosomal Aberration Assay

Gold nanoshells (155 nm in diameter with a coating of polyethylene glycol 5000) were evaluated for preclinical biocompatibility, toxicity, and biodistribution as part of a program to develop an injectable device for use in the photothermal ablation of tumors. The evaluation started with a complete good laboratory practice (GLP) compliant International Organization for Standardization (ISO)-10993 biocompatibility program, including cytotoxicity, pyrogenicity (US Pharmacopeia [USP] method in the rabbit), genotoxicity (bacterial mutagenicity, chromosomal aberration assay in Chinese hamster ovary cells, and in vivo mouse micronucleus), in vitro hemolysis, intracutaneous reactivity in the rabbit, sensitization (in the guinea pig maximization assay), and USP/ISO acute systemic toxicity in the mouse. There was no indication of toxicity in any of the studies. Subsequently, nanoshells were evaluated in vivo by intravenous (iv) infusion using a trehalose/water solution in a series of studies in mice, Sprague-Dawley rats, and Beagle dogs to assess toxicity for time durations of up to 404 days. Over the course of 14 GLP studies, the gold nanoshells were well tolerated and, when injected iv, no toxicities or bioincompatibilities were identified.

scholarly journals Formycin B-resistant mutants of Chinese hamster ovary cells: novel genetic and biochemical phenotype affecting adenosine kinase.

1983 ◽  
Vol 3 (8) ◽  
pp. 1468-1477 ◽  
Author(s):  
K D Mehta ◽  
R S Gupta
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Complex Form ◽  
Single Step ◽  
Adenosine Kinase ◽  
Cross Resistance ◽  
Ovary Cells ◽  
Cell Extracts

Stable mutants which are approximately three- and eightfold resistant to the pyrazolopyrimidine nucleosides formycin A and formycin B (FomR) have been selected in a single step from mutagenized Chinese hamster ovary cells. In cell extracts, the two FomR mutants which were examined were both found to contain no measurable activity of the enzyme adenosine kinase (AK). However, cross-resistance studies with other adenosine analogs such as toyocamycin and tubercidin show that these mutants are distinct from toyocamycin or tubercidin resistant (Toyr) mutants which also contain no measurable AK activity in cell extracts. Studies on the uptake and incorporation of [3H]adenosine and [3H]tubercidin by various mutants and parental cell lines show that unlike the Toyr mutants, which are severely deficient in the phosphorylation of these compounds, the FomR mutants possess nearly normal capacity to phosphorylate these compounds and incorporate them into cellular macromolecules. These results suggest that the FomR mutants contain normal levels of AK activity in vivo. In cell hybrids formed between FomR X FomS cells and FomR X Toyr cells, the formycin-resistant phenotype of both of the FomR mutants behaved codominantly. However, the extracts from these hybrid cells contained either congruent to 50% (FomR X FomS) or no measurable (FomR X Toyr) AK activity, indicating that the lesion in these mutants neither suppresses the wild-type AK activity nor complements the AK deficiency of the Toyr mutants. The presence of AK activity in the FomR mutants in vivo, but not in their cell extracts, along with the codominant behavior of the mutants in hybrids, indicates that the lesions in the FomR mutant are of a novel nature. It is suggested that the genetic lesion in these mutants affects AK activity indirectly and that it may involve an essential cellular function which exists in a complex form with AK. Some implications of these results regarding the mechanism of action of formycin B are discussed.

scholarly journals Properties of the kinetochore in vitro. I. Microtubule nucleation and tubulin binding.

1985 ◽  
Vol 101 (3) ◽  
pp. 755-765 ◽  
Author(s):  
T J Mitchison ◽  
M W Kirschner
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Lag Phase ◽  
Ovary Cells ◽  
Tubulin Binding ◽  
Mixed Polarity ◽  
Tubulin Immunofluorescence

We have isolated chromosomes from Chinese hamster ovary cells arrested in mitosis with vinblastine and examined the interactions of their kinetochores with purified tubulin in vitro. The kinetochores nucleate microtubule (MT) growth with complex kinetics. After an initial lag phase, MTs are continuously nucleated with both plus and minus ends distally localized. This mixed polarity seems inconsistent with the formation of an ordered, homopolar kinetochore fiber in vivo. As isolated from vinblastine-arrested cells, kinetochores contain no bound tubulin. The kinetochores of chromosomes isolated from colcemid-arrested cells or of chromosomes incubated with tubulin in vitro are brightly stained after anti-tubulin immunofluorescence. This bound tubulin is probably not in the form of MTs. It is localized to the corona region by immunoelectron microscopy, where it may play a role in MT nucleation in vitro.

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