scholarly journals In vitro expression demonstrates impaired secretion of the γAsn319, Asp320 deletion variant fibrinogen

2005 ◽  
Vol 94 (07) ◽  
pp. 53-59 ◽  
Author(s):  
Satomi Kani ◽  
Fumiko Terasawa ◽  
Susan T. Lord ◽  
Minoru Tozuka ◽  
Hiroyoshi Ota ◽  
...  
Keyword(s):  
Culture Media ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fibrinogen Concentration ◽  
Ovary Cells ◽  
Cell Lysates ◽  
Sds Page ◽  
The Media ◽  
Kinetics Of

SummaryThe hypodysfibrinogenemia Otsu is caused by the two-residue deletion,γAsn319 and γAsp320. Analysis of plasma or purified fibrinogen from the heterozygous propositus revealed that the amount of variant γ-chain was lower than that of normal γ-chain. In order to examine the basis for this difference, we transfected Chinese hamster ovary cells and established stable cell lines that expressed both chains, γΔ/γN, only the normal chain, γN, and only the variant chain, γΔ. We measured fibrinogen concentration of confluent cultures by ELISA. We found the ratios of the concentrations in the media to the concentrations in the cell lysates of γΔ, γΔ/γN, and γN-cells were 0.42, 0.60, and 1.00, respectively. We measured the concentrations of the γΔ and γN chains by densitometric analysis of samples following separation by SDS-PAGE and found the fraction of γΔ-chains in cell lysates was always greater than the fraction in the respective culture media. We examined the kinetics of fibrinogen synthesis, assembly and secretion in pulse-chase experiments, and found that the γΔ-chain was assembled into intact fibrinogen at a rate similar to assembly of the γN-chain into normal fibrinogen, but was secreted into the medium at a slightly slower rate than normal fibrinogen. Considered together, these experiments indicate secretion of the variant fibrinogen was slightly impaired. These results suggest that the reduced level of γΔ319,320 fibrinogen in the plasma of the Otsu patient arises from modestly impaired secretion of this variant fibrinogen.

scholarly journals Polarity of microtubules nucleated by centrosomes and chromosomes of Chinese hamster ovary cells in vitro

1980 ◽  
Vol 84 (1) ◽  
pp. 151-159 ◽  
Author(s):  
LG Bergen ◽  
R Kuriyama ◽  
GG Borisy
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Internal Controls ◽  
Structural Explanation ◽  
Ovary Cells ◽  
Spindle Microtubules ◽  
Kinetics Of

The structural and growth polarities of centrosomal and chromosomal microtubules were studied by analyzing the kinetics of growth of these microtubules and those initiated by flagellar seeds. By comparing rates of elongation of centrosomal and flagellar-seeded microtubules, we determined whether the centrosomal microtubules were free to grow at their plus ends only, minus ends ony, or at both ends. Our results show that centrosomal microtubules elongate at a rate corresponding to the addition of subunits at the plus end only. The depolymerization rate was also equivalent to that for the plus end only. Chromosomal microtubule elongation was similar to the centrosome-initiated growth. Since the data do not support the hypothesis that both ends of these spindle microtubules are able to interact with monomer in solution, then growth must occur only distal or only proximal to the organizing centers, implying tha the opposite ends in unavailable for exchange of subunits. Experiments with flagellar-seeded microtubules serving as internal controls indicated that the inactivity of the minus end could not be accounted for by a diffusible inhibitor, suggesting a structural explanation. Since there is no apparent way in which the distal ends may be capped, whereas the proximal ends are embedded in the pericentriolar cloud, we conclude that centrosomal microtubules are oriented with their plus ends distal to the site of nucleation. A similar analysis for chromosomal microtubules suggests that they too must be oriented with their plus ends distal to the site of initiation.

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).

scholarly journals Novel Promoters Derived from Chinese Hamster Ovary Cells via In Silico and In Vitro Analysis

2019 ◽  
Vol 14 (11) ◽  
pp. 1900125 ◽  
Author(s):  
Ly N. Nguyen ◽  
Martina Baumann ◽  
Heena Dhiman ◽  
Nicolas Marx ◽  
Valerie Schmieder ◽  
...  
Keyword(s):  
Chinese Hamster Ovary ◽  
In Silico ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Vitro Analysis ◽  
In Vitro Analysis

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.

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