The effect of inhibitors of RNA and protein synthesis on dibutyryl cyclic AMP mediated morphological transformations of Chinese hamster ovary cells in vitro

1973 ◽  
Vol 50 (2) ◽  
pp. 566-573 ◽  
Author(s):  
David Patterson ◽  
Charles A. Waldren
Keyword(s):  
Protein Synthesis ◽  
Cyclic Amp ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Dibutyryl Cyclic Amp ◽  
Ovary Cells ◽  
Rna And Protein Synthesis ◽  
Morphological Transformations ◽  
Effect Of Inhibitors

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 Evidence that the dephosphorylation of Ser535 in the ∊-subunit of eukaryotic initiation factor (eIF) 2B is insufficient for the activation of eIF2B by insulin

2002 ◽  
Vol 367 (2) ◽  
pp. 475-481 ◽  
Author(s):  
Xuemin WANG ◽  
Maarten JANMAAT ◽  
Anne BEUGNET ◽  
Fiona E.M. PAULIN ◽  
Christopher G. PROUD
Keyword(s):  
Protein Synthesis ◽  
Glycogen Synthase ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Nucleotide Exchange ◽  
Ovary Cells

Eukaryotic initiation factor (eIF) 2B is a guanine-nucleotide exchange factor that plays a key role in the regulation of protein synthesis. It is activated by insulin, serum and other agents that stimulate general protein synthesis. The largest (∊) subunit of eIF2B is a substrate for glycogen synthase kinase (GSK)-3 in vitro, and phosphorylation by GSK3 inhibits the activity of eIF2B. The site of phosphorylation has previously been identified as Ser535. GSK3 is inactivated by phosphorylation in response to insulin or serum. In Chinese-hamster ovary cells, insulin and serum bring about the dephosphorylation of Ser535in vivo, concomitantly with the phosphorylation of GSK3, and these effects are mediated through signalling via phosphoinositide 3-kinase. We have made use of inhibitors of GSK3 to determine whether GSK3 is responsible for phosphorylation of Ser535in vivo and to explore the role of phosphorylation of Ser535 in the regulation of eIF2B. Treatment of cells with LiCl or with either of two recently developed GSK3 inhibitors, SB-415286 and SB-216763, brought about the dephosphorylation of Ser535, which strongly indicates that this site is indeed a target for GSK3 in vivo. However, these compounds did not elicit significant activation of eIF2B, indicating, consistent with conclusions from one of our previous studies, that additional inputs are required for the activation of eIF2B. Our results also show that each of the inhibitors used affects overall protein synthesis and have additional effects on translation factors or signalling pathways apparently unrelated to their effects on GSK3, indicating that caution must be exercised when interpreting data obtained using these compounds.

scholarly journals Peptide Antagonists That Inhibit Sin Nombre Virus and Hantaan Virus Entry through the β3-Integrin Receptor

2005 ◽  
Vol 79 (12) ◽  
pp. 7319-7326 ◽  
Author(s):  
Richard S. Larson ◽  
David C. Brown ◽  
Chunyan Ye ◽  
Brian Hjelle
Keyword(s):  
Viral Entry ◽  
Sequence Similarity ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Phage Display Library ◽  
Hantaan Virus ◽  
Sin Nombre Virus ◽  
Fab Fragment ◽  
Ovary Cells

ABSTRACT Specific therapy is not available for the treatment of hantavirus cardiopulmonary syndrome caused by Sin Nombre virus (SNV). The entry of pathogenic hantaviruses into susceptible human cells is dependent upon expression of the αvβ3 integrin, and transfection of human β3 integrin is sufficient to confer infectibility onto CHO (Chinese hamster ovary) cells. Furthermore, pretreatment of susceptible cells with anti-β3 antibodies such as c7E3 or its Fab fragment ReoPro prevents hantavirus entry. By using repeated selection of a cyclic nonamer peptide phage display library on purified αvβ3, we identified 70 peptides that were competitively eluted with ReoPro. Each of these peptides was examined for its ability to reduce the number of foci of SNV strain SN77734 in a fluorescence-based focus reduction assay according to the method of Gavrilovskaya et al. (I. N. Gavrilovskaya, M. Shepley, R. Shaw, M. H. Ginsberg, and E. R. Mackow, Proc. Natl. Acad. Sci. USA 95:7074-7079, 1998). We found that 11 peptides reduced the number of foci to a greater extent than did 80 μg/ml ReoPro when preincubated with Vero E6 cells. In addition, 8 of the 70 peptides had sequence similarity to SNV glycoproteins. We compared all 18 peptide sequences (10 most potent, 7 peptides with sequence similarity to hantavirus glycoproteins, and 1 peptide that was in the group that displayed the greatest potency and had significant sequence similarity) for their abilities to inhibit SNV, Hantaan virus (HTNV), and Prospect Hill virus (PHV) infection. There was a marked trend for the peptides to inhibit SNV and HTNV to a greater extent than they inhibited PHV, a finding that supports the contention that SNV and HTNV use β3 integrins and PHV uses a different receptor, β1 integrin. We then chemically synthesized the four peptides that showed the greatest ability to neutralize SNV. These peptides inhibited viral entry in vitro as free peptides outside of the context of a phage. Some combinations of peptides proved more inhibitory than did individual peptides. In all, we have identified novel peptides that inhibit entry by SNV and HTNV via β3 integrins and that can be used as lead compounds for further structural optimization and consequent enhancement of activity.

Persistence of sister chromatid exchange by 9-β-D-arabinofuranosyladenine in Chinese hamster ovary cells cultivated in vitro

Mutagenesis ◽  
1993 ◽  
Vol 8 (5) ◽  
pp. 445-448 ◽  
Author(s):  
Paolo Perticone ◽  
Marco Linguardo ◽  
Renata Cozzi ◽  
Rosa Maria Corbo ◽  
Stefania Polani
Keyword(s):  
Sister Chromatid ◽  
Chinese Hamster Ovary ◽  
Sister Chromatid Exchange ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells

In vitro biosynthesis of diphthamide, studied with mutant Chinese hamster ovary cells resistant to diphtheria toxin

1984 ◽  
Vol 4 (4) ◽  
pp. 642-650
Author(s):  
T J Moehring ◽  
D E Danley ◽  
J M Moehring
Keyword(s):  
Chinese Hamster Ovary ◽  
Diphtheria Toxin ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Elongation Factor ◽  
Synthetase Activity ◽  
Post Translational Modification ◽  
Ovary Cells ◽  
Elongation Factor 2

Diphthamide, a unique amino acid, is a post-translational derivative of histidine that exists in protein synthesis elongation factor 2 at the site of diphtheria toxin-catalyzed ADP-ribosylation of elongation factor 2. We investigated steps in the biosynthesis of diphthamide with mutants of Chinese hamster ovary cells that were altered in different steps of this complex post-translational modification. Biochemical evidence indicates that this modification requires a minimum of three steps, two of which we accomplished in vitro. We identified a methyltransferase activity that transfers methyl groups from S-adenosyl methionine to an unmethylated form of diphthine (the deamidated form of diphthamide), and we tentatively identified an ATP-dependent synthetase activity involved in the biosynthesis of diphthamide from diphthine. Our results are in accord with the proposed structure of diphthamide (B. G. VanNess, et al., J. Biol. Chem. 255:10710-10716, 1980).

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