Enhanced intracellular accumulation of recombinant HBsAg in CHO cells by dimethyl sulfoxide

2008 ◽  
Vol 43 (6) ◽  
pp. 690-695 ◽  
Author(s):  
Zheyong Ma ◽  
Xiaoping Yi ◽  
Yuanxing Zhang
Keyword(s):  
Dimethyl Sulfoxide ◽  
Cho Cells ◽  
Intracellular Accumulation ◽  
Recombinant Hbsag

Dimethyl sulfoxide protects against thermal and epithermal neutron-induced cell death and mutagenesis of Chinese hamster ovary (CHO) cells

2000 ◽  
Vol 47 (5) ◽  
pp. 1371-1378 ◽  
Author(s):  
Yuko Kinashi ◽  
Yoshinori Sakurai ◽  
Shinichiro Masunaga ◽  
Minoru Suzuki ◽  
Mitsuhiko Akaboshi ◽  
...  
Keyword(s):  
Cell Death ◽  
Dimethyl Sulfoxide ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Epithermal Neutron ◽  
Chinese Hamster

scholarly journals Induction and inhibition of Friend erythroleukemia cell differentiation by pyrimidine analogs: analysis of the requirement for intracellular accumulation and incorporation into DNA.

1982 ◽  
Vol 2 (8) ◽  
pp. 1020-1024 ◽  
Author(s):  
J A Bilello ◽  
K K Gauri ◽  
J Kühne ◽  
G Warnecke ◽  
G Koch
Keyword(s):  
Cell Differentiation ◽  
Dimethyl Sulfoxide ◽  
Dna Analysis ◽  
Erythroid Differentiation ◽  
Intracellular Accumulation ◽  
Induced Differentiation ◽  
Alkyl Chains ◽  
Erythroleukemia Cells ◽  
Erythroleukemia Cell ◽  
Pyrimidine Analogs

Alkyldeoxyuridines which differ from thymidine by a C5 substitution of straight or branched alkyl chains of two to six carbon atoms have been tested for their ability to be taken up, phosphorylated, and incorporated into DNA. Analysis of the uptake of 5-ethyl-2'-deoxyuridine and 5-propyl-2'-deoxyuridine (n-PrdU)--similar to both thymidine and 5-bromo-2'-deoxyuridine--indicates that transport is dependent upon a functional cellular thymidine kinase. All of the aforementioned pyrimidines with the exception of n-PrdU are phosphorylated to the triphosphate and incorporated into DNA. The homologs 5-iso-propyl-2'-deoxyuridine (iso-PrdU) and 5-hexyl-2'-deoxyuridine are neither transported into the cell, phosphorylated, nor incorporated into DNA. These analogs were tested (i) for their ability to induce in the absence of dimethyl sulfoxide and (ii) to determine whether they enhance or inhibit dimethyl sulfoxide-induced differentiation of Friend erythroleukemia cells. Inhibition of erythroid differentiation appears to require the incorporation of thymidine analogs into DNA, and thus only 5-ethyl-2'-deoxyuridine and 5-bromo-2'-deoxyuridine were effective in inhibiting dimethyl sulfoxide-induced differentiation. The observation that iso-PrdU, and to a lesser extent n-PrdU and 5-propyldeoxyuridine monophosphate, induce differentiation under conditions in which they are not detectable intracellularly is strong evidence that this class of inducer acts at the cell membrane.

scholarly journals UBR E3 ligases and the PDIA3 protease control degradation of unfolded antibody heavy chain by ERAD

2020 ◽  
Vol 219 (7) ◽  
Author(s):  
Danming Tang ◽  
Wendy Sandoval ◽  
Cynthia Lam ◽  
Benjamin Haley ◽  
Peter Liu ◽  
...  
Keyword(s):  
Cho Cells ◽  
Cell Engineering ◽  
Intracellular Accumulation ◽  
Cho Cell ◽  
E3 Ligases ◽  
Production Phase ◽  
Enhanced Secretion ◽  
Antibody Productivity ◽  
Antibody Heavy Chain ◽  
Dependent Pathway

Accumulation of unfolded antibody chains in the ER triggers ER stress that may lead to reduced productivity in therapeutic antibody manufacturing processes. We identified UBR4 and UBR5 as ubiquitin E3 ligases involved in HC ER-associated degradation. Knockdown of UBR4 and UBR5 resulted in intracellular accumulation, enhanced secretion, and reduced ubiquitination of HC. In concert with these E3 ligases, PDIA3 was shown to cleave ubiquitinated HC molecules to accelerate HC dislocation. Interestingly, UBR5, and to a lesser degree UBR4, were down-regulated as cellular demand for antibody expression increased in CHO cells during the production phase, or in plasma B cells. Reducing UBR4/UBR5 expression before the production phase increased antibody productivity in CHO cells, possibly by redirecting antibody molecules from degradation to secretion. Altogether we have characterized a novel proteolysis/proteasome-dependent pathway involved in degradation of unfolded antibody HC. Proteins characterized in this pathway may be novel targets for CHO cell engineering.

Induction and inhibition of Friend erythroleukemia cell differentiation by pyrimidine analogs: analysis of the requirement for intracellular accumulation and incorporation into DNA

1982 ◽  
Vol 2 (8) ◽  
pp. 1020-1024
Author(s):  
J A Bilello ◽  
K K Gauri ◽  
J Kühne ◽  
G Warnecke ◽  
G Koch
Keyword(s):  
Cell Differentiation ◽  
Dimethyl Sulfoxide ◽  
Dna Analysis ◽  
Erythroid Differentiation ◽  
Intracellular Accumulation ◽  
Induced Differentiation ◽  
Alkyl Chains ◽  
Erythroleukemia Cells ◽  
Erythroleukemia Cell ◽  
Pyrimidine Analogs

Alkyldeoxyuridines which differ from thymidine by a C5 substitution of straight or branched alkyl chains of two to six carbon atoms have been tested for their ability to be taken up, phosphorylated, and incorporated into DNA. Analysis of the uptake of 5-ethyl-2'-deoxyuridine and 5-propyl-2'-deoxyuridine (n-PrdU)--similar to both thymidine and 5-bromo-2'-deoxyuridine--indicates that transport is dependent upon a functional cellular thymidine kinase. All of the aforementioned pyrimidines with the exception of n-PrdU are phosphorylated to the triphosphate and incorporated into DNA. The homologs 5-iso-propyl-2'-deoxyuridine (iso-PrdU) and 5-hexyl-2'-deoxyuridine are neither transported into the cell, phosphorylated, nor incorporated into DNA. These analogs were tested (i) for their ability to induce in the absence of dimethyl sulfoxide and (ii) to determine whether they enhance or inhibit dimethyl sulfoxide-induced differentiation of Friend erythroleukemia cells. Inhibition of erythroid differentiation appears to require the incorporation of thymidine analogs into DNA, and thus only 5-ethyl-2'-deoxyuridine and 5-bromo-2'-deoxyuridine were effective in inhibiting dimethyl sulfoxide-induced differentiation. The observation that iso-PrdU, and to a lesser extent n-PrdU and 5-propyldeoxyuridine monophosphate, induce differentiation under conditions in which they are not detectable intracellularly is strong evidence that this class of inducer acts at the cell membrane.

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.

The Effect of Dimethyl Sulfoxide on In Vitro Platelet Function

1976 ◽  
Vol 36 (01) ◽  
pp. 221-229 ◽  
Author(s):  
Charles A. Schiffer ◽  
Caroline L. Whitaker ◽  
Morton Schmukler ◽  
Joseph Aisner ◽  
Steven L. Hilbert
Keyword(s):  
Platelet Count ◽  
Platelet Aggregation ◽  
Dimethyl Sulfoxide ◽  
Platelet Function ◽  
Platelet Volume ◽  
Short Term ◽  
Platelet Factor ◽  
Short Term Exposure ◽  
Structural Abnormalities

SummaryAlthough dimethyl sulfoxide (DMSO) has been used extensively as a cryopreservative for platelets there are few studies dealing with the effect of DMSO on platelet function. Using techniques similar to those employed in platelet cryopreservation platelets were incubated with final concentrations of 2-10% DMSO at 25° C. After exposure to 5 and 10% DMSO platelets remained discoid and electron micrographs revealed no structural abnormalities. There was no significant change in platelet count. In terms of injury to platelet membranes, there was no increased availability of platelet factor-3 or leakage of nucleotides, 5 hydroxytryptamine (5HT) or glycosidases with final DMSO concentrations of 2.5, 5 and 10% DMSO. Thrombin stimulated nucleotide and 5HT release was reduced by 10% DMSO. Impairment of thrombin induced glycosidase release was noted at lower DMSO concentrations and was dose related. Similarly, aggregation to ADP was progressively impaired at DMSO concentrations from 1-5% and was dose related. After the platelets exposed to DMSO were washed, however, aggregation and release returned to control values. Platelet aggregation by epinephrine was also inhibited by DMSO and this could not be corrected by washing the platelets. DMSO-plasma solutions are hypertonic but only minimal increases in platelet volume (at 10% DMSO) could be detected. Shrinkage of platelets was seen with hypertonic solutions of sodium chloride or sucrose suggesting that the rapid transmembrane passage of DMSO prevented significant shifts of water. These studies demonstrate that there are minimal irreversible alterations in in vitro platelet function after short-term exposure to DMSO.

Preservation of Morphological Integrity and Clot Retraction Activity of Human Platelets After Freezing

1964 ◽  
Vol 11 (01) ◽  
pp. 222-229 ◽  
Author(s):  
Isaac Djerassi ◽  
Albert Roy ◽  
Jorge Alvarado ◽  
Keyword(s):  
Dimethyl Sulfoxide ◽  
Liquid Nitrogen ◽  
Human Platelets ◽  
Slow Freezing ◽  
Critical Conditions ◽  
Plasma Medium ◽  
Clot Retraction ◽  
Controlled Freezing ◽  
Direct Immersion

SummaryHuman platelets frozen at −195° C (liquid nitrogen) retain their morphological integrity and ability to promote clot retraction when 5% dimethyl-sulfoxide and 5% dextrose are added to the suspending plasma medium. Slow freezing was more effective than direct immersion in the liquid nitrogen. Although similar results may be achieved with dimethylsulfoxide alone with rigidly controlled freezing rates, the addition of sugars may permit freezing under less critical conditions.Dimethylsulfoxyd und 5% Dextrose dem Plasmamilieu hinzugefügt werden. Das langsame Einfrieren ist effektiver als das direkte Eintauchen in flüssigen Stickstoff. Obschon ähnliche Resultate mit Dimethylsulfoxyd allein unter exakter Kontrolle der Einfrierungsgeschwindig-keit erreicht werden können, erlaubt die Zugabe von Dextrose ein Einfrieren unter weniger kritischen Bedingungen.

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