scholarly journals Involvement of the proteasome and antizyme in ornithine decarboxylase degradation by a reticulocyte lysate

1993 ◽  
Vol 295 (1) ◽  
pp. 305-308 ◽  
Author(s):  
Y Murakami ◽  
S Matsufuji ◽  
K Tanaka ◽  
A Ichihara ◽  
S Hayashi
Keyword(s):  
Tissue Culture ◽  
Ornithine Decarboxylase ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Complete Loss ◽  
Protein Inhibitor ◽  
Whole Cells ◽  
Main Pathway ◽  
Reticulocyte Lysate ◽  
Hepatoma Tissue

Ornithine decarboxylase (ODC) degradation in a freshly prepared reticulocyte lysate was examined. Immunodepletion of proteasomes from the reticulocyte lysate resulted in almost complete loss of ODC degradation. In contrast with the previously reported degradation in extracts of hepatoma tissue-culture (HTC) and Chinese-hamster ovary (CHO) cells or that by the purified 26 S proteasome, efficient degradation of ODC was observed in the lysate without exogenous antizyme, an ODC protein inhibitor induced by polyamines, owing to the presence of a significant amount of antizyme in the lysate. The degradation of ODC in the lysate was strongly suppressed on inactivation of antizyme in the lysate with antizyme inhibitor, a protein which binds to the antizyme and releases ODC from the ODC-antizyme complex. Thus the main pathway for ODC degradation in a reticulocyte lysate was essentially the same as that characterized previously in extracts of HTC and CHO cells, namely an ATP- and antizyme-dependent 26 S proteasome-catalysed pathway that is presumed to be responsible for ODC degradation in whole cells.

scholarly journals Cloning and expression of two ornithine decarboxylase forms from HMOA cells

1995 ◽  
Vol 312 (1) ◽  
pp. 13-16
Author(s):  
R Autelli ◽  
L Persson ◽  
F M Baccino
Keyword(s):  
Tissue Culture ◽  
Ornithine Decarboxylase ◽  
Half Life ◽  
Turnover Rate ◽  
Cloning And Expression ◽  
Single Amino Acid ◽  
Turnover Rates ◽  
Wild Type ◽  
First Time ◽  
Hepatoma Tissue

In HMOA cells [Mamont, Duchesne, Grove and Tardif (1978) Exp. Cell Res. 115, 387-393] the half-life of ornithine decarboxylase (ODC) is 8-14 h instead of 15 min as in the Hepatoma Tissue Culture parental cells, due to a single amino acid substitution [Miyazaki, Matsufuji, Murakami and Hayashi (1993) Eur. J. Biochem. 214, 837-844]. We demonstrate for the first time that HMOA cells possess two forms of ODC mRNA that are translated into two proteins differing greatly in turnover rates. We have cloned and transfected the cDNAs for the two ODC forms into COS-1 cells for a direct measurement of their turnover rate. The variant ODC form was much more stable than the wild-type protein, with a half-life of 14 h as compared with 2.5 h.

scholarly journals Accumulation of ornithine decarboxylase-antizyme complex in HMOA cells

1985 ◽  
Vol 225 (3) ◽  
pp. 689-697 ◽  
Author(s):  
Y Murakami ◽  
K Fujita ◽  
T Kameji ◽  
S Hayashi
Keyword(s):  
Tissue Culture ◽  
Complex Formation ◽  
Ornithine Decarboxylase ◽  
New Method ◽  
Htc Cells ◽  
Hepatoma Tissue

A new method was developed for the assay of ornithine decarboxylase (ODC)-antizyme complex, in which alpha-difluoromethylornithine (DFMO)-inactivated ODC was used to release active ODC competitively from the complex. ODC-antizyme complex was present in the extracts of hepatoma tissue-culture (HTC) cells and of ODC-stabilized variant HMOA cells, in much larger amounts in the latter. Cellular amounts of the complex fluctuated after a change of medium in a similar manner in HTC and HMOA cells, increasing during the period of ODC decay. After treatment with cycloheximide, the decay of ODC-antizyme complex in HMOA cells was more rapid than the decay of free ODC, but it was much slower than the decay of free ODC or complexed ODC in HTC cells. Administration of putrescine caused a rapid increase in the amount of ODC-antizyme complex in both HTC and HMOA cells, but nevertheless the decay of total ODC (free ODC plus ODC-antizyme complex) was more rapid with putrescine than with cycloheximide. These results suggested the possibility that ODC is degraded through complex-formation with antizyme. In contrast with complexed antizyme, free antizyme was not stabilized in HMOA cells.

scholarly journals Regulation of ExoS Production and Secretion byPseudomonas aeruginosa in Response to Tissue Culture Conditions

1999 ◽  
Vol 67 (2) ◽  
pp. 914-920 ◽  
Author(s):  
Amy J. Vallis ◽  
Timothy L. Yahr ◽  
Joseph T. Barbieri ◽  
Dara W. Frank
Keyword(s):  
Tissue Culture ◽  
Cho Cells ◽  
Type Iii Secretion ◽  
Time Course ◽  
Culture Medium ◽  
Chinese Hamster ◽  
Growth Conditions ◽  
Culture Conditions ◽  
Tissue Culture Medium ◽  
Type Iii

ABSTRACT This study was initiated to characterize the regulation and secretion of ExoS by Pseudomonas aeruginosa during contact with eukaryotic cells. The production of ExoS was monitored by a sensitive ADP-ribosyltransferase activity assay, and specific activities were calculated for supernatant and cell-associated fractions. Time course analysis indicated that ExoS was produced after a lag period, suggesting that induction of the regulon is necessary for the expression of detectable amounts of enzyme activity. Under tissue culture growth conditions, ExoS was induced when P. aeruginosa was in contact with Chinese hamster ovary (CHO) cells or after growth in tissue culture medium with serum. The serum induction of ExoS appeared to result in generalized type III secretion, while induction by contact with CHO cells appeared to result in polarized type III secretion. Mutants in the type III secretory system that express a null phenotype for ExoS production in bacteriological medium produced but did not secrete the enzyme when P. aeruginosa was grown under inducing conditions in tissue culture medium. These results suggest that both induction and secretion of ExoS may differ when the bacteria are exposed to different growth environments. The putative type III translocation proteins and secretion apparatus of P. aeruginosa were required for translocation of bacterial factors that mediate changes in CHO cell morphology during infection.

Metabolic engineering of CHO cells to prepare glycoproteins

2018 ◽  
Vol 2 (3) ◽  
pp. 433-442 ◽  
Author(s):  
Qiong Wang ◽  
Michael J. Betenbaugh
Keyword(s):  
Metabolic Engineering ◽  
Cho Cells ◽  
Genetic Manipulation ◽  
Chinese Hamster ◽  
Post Translational Modification ◽  
Effector Functions ◽  
Recombinant Glycoproteins ◽  
Production Platform ◽  
Chemical Inhibitors ◽  
Amino Acid Mutations

As a complex and common post-translational modification, N-linked glycosylation affects a recombinant glycoprotein's biological activity and efficacy. For example, the α1,6-fucosylation significantly affects antibody-dependent cellular cytotoxicity and α2,6-sialylation is critical for antibody anti-inflammatory activity. Terminal sialylation is important for a glycoprotein's circulatory half-life. Chinese hamster ovary (CHO) cells are currently the predominant recombinant protein production platform, and, in this review, the characteristics of CHO glycosylation are summarized. Moreover, recent and current metabolic engineering strategies for tailoring glycoprotein fucosylation and sialylation in CHO cells, intensely investigated in the past decades, are described. One approach for reducing α1,6-fucosylation is through inhibiting fucosyltransferase (FUT8) expression by knockdown and knockout methods. Another approach to modulate fucosylation is through inhibition of multiple genes in the fucosylation biosynthesis pathway or through chemical inhibitors. To modulate antibody sialylation of the fragment crystallizable region, expressions of sialyltransferase and galactotransferase individually or together with amino acid mutations can affect antibody glycoforms and further influence antibody effector functions. The inhibition of sialidase expression and chemical supplementations are also effective and complementary approaches to improve the sialylation levels on recombinant glycoproteins. The engineering of CHO cells or protein sequence to control glycoforms to produce more homogenous glycans is an emerging topic. For modulating the glycosylation metabolic pathways, the interplay of multiple glyco-gene knockouts and knockins and the combination of multiple approaches, including genetic manipulation, protein engineering and chemical supplementation, are detailed in order to achieve specific glycan profiles on recombinant glycoproteins for superior biological function and effectiveness.

Review of the current methods of Chinese Hamster Ovary (CHO) cells cultivation for production of therapeutic protein

2020 ◽  
Vol 17 ◽  
Author(s):  
Shazid Md. Sharker ◽  
Md. Atiqur Rahman
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Mass Production ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Therapeutic Protein ◽  
Specific Productivity ◽  
Ovary Cells ◽  
Further Development ◽  
Interesting Area

Most of clinical approved protein-based drugs or under in clinical trial have a profound impact in the treatment of critical diseases. The mammalian eukaryotic cells culture approaches, particularly the CHO (Chinese Hamster Ovary) cells are mainly used in the biopharmaceutical industry for the mass-production of therapeutic protein. Recent advances in CHO cell bioprocessing to yield recombinant proteins and monoclonal antibodies have enabled the expression of quality protein. The developments of cell lines are possible to upgrade specific productivity. As a result, it holds an interesting area for academic as well as industrial researchers around the world. This review will concentrate on the recent progress of the mammalian CHO cells culture technology and the future scope of further development for the mass-production of protein therapeutics.

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