scholarly journals Interaction of phospholipid vesicles with cultured mammalian cells. II. Studies of mechanism.

1975 ◽  
Vol 67 (1) ◽  
pp. 49-60 ◽  
Author(s):  
R E Pagano ◽  
L Huang
Keyword(s):  
Cell Fusion ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Lipid Vesicles ◽  
Lipid Uptake ◽  
V79 Cells ◽  
Cell Lipid ◽  
Lipid Exchange ◽  
Exogenous Lipid ◽  
Sepharose 4B

The mechanism of interaction of artificially generated lipid vesicles (approximately 500 A diameter) with Chinese hamster V79 cells bathed in a simple balanced salt solution was investigated. The major pathways of exogenous lipid incorporation in vesicle-treated cells are vesicle-cell fusion and vesicle-cell lipid exchange. At 37 degrees C, the fusion process is dominant, while at 2 degrees C or with energy depleted cells, exchange of lipids between vesicles and cells is important. The fusion mechanism was demonstrated using vesicles of [14C]lecithin containing trapped [13H]inulin. Consistent with a fusion hypothesis, both components became cell associated at 37 degrees C in nearly the same proportions as they were present in the applied vesicles. Additional arguments in favor of vesicle-cell fusion and against phagocytosis or adsorption of intact vesicles are presented. At 2 degrees C or with inhibitor-treated cells, the [3H]inulin uptake was largely suppressed, while the lipid uptake was reduced to a lesser extent. Evidence for vesicle-cell lipid exchange was obtained using V79 cells grown on 3H precursors for cellular lipids. [14C]lecithin vesicles, incubated with such cells, showed no change in their elution properties when subjected to molecular sieve chromatography on Sepharose 4B. However, radioactivity and thin-layer chromatographic analyses revealed that a variety of cell lipiids had been exchanged into the uniamellar vesicles. Further evidence for the fusion and exchange processes was obtained using vesicles prepared from mixtures of [3H]lecithin and [14C]cholesterol. A two-step fusion mechanism consistent with the present findings is proposed as a working model for other fusion studies.

scholarly journals Interaction of phospholipid vesicles with cultured mammalial cells. I. Characteristics of uptake.

1975 ◽  
Vol 67 (1) ◽  
pp. 38-48 ◽  
Author(s):  
L Huang ◽  
R E Pagano
Keyword(s):  
Chinese Hamster ◽  
Lipid Vesicles ◽  
Metabolic Inhibitors ◽  
Lipid Vesicle ◽  
Lipid Phase ◽  
Lipid Uptake ◽  
V79 Cells ◽  
Prolonged Incubation ◽  
Exogenous Lipid ◽  
Effects Of Ph

The interaction of monolayer cultures of Chinese hamster V79 cells with artificially generated, unilamellar lipid vesicles (approximately 500 A diameter) was examined. Vesicles prepared from a variety of natural and synthetic radiolabeled phosphatidyl cholines (lecithins) were incubated with V79 cells bathed in a simple balanced salt solution. After incubation, the cells were analyzed for exogenous lipid incorporation. Large quantities (approximately 10(8) molecules/cell/h) of lecithin became cell associated without affecting cell viability. The effects of pH, charged lipids, and the influence of the vesicle lipid phase transition on the uptake process were examined. Glutaraldehyde fixation of cells before vesicle treatment, or incubation in the presence of metabolic inhibitors, failed to reduce the lecithin uptake by more than 25-50%, suggesting that the lipid uptake is largely energy independent. Cells in sparse culture took up about ten times more lipid than dense cultures. Prolonged incubation (greater than 15 h) of sparse cell cultures with lecithin vesicles resulted in significant cell death while no deleterious effect was found in dense cultures, or with 1:1 lecithin/cholesterol vesicles. When vesicle-treated cells were homogenized and fractionated, about 20-30% of the exogenous lipid was found in the plasma membrane fraction, with the remainder being distributed into intracellular fractions. Electron microscope radioautography further demonstrated that most of the internalized lipid was present in the cytoplasm, with little in the nucleus. These results are discussed in terms of possible modification of cell behavior by lipid vesicle treatment.

scholarly journals DNA strand specificity for UV-induced mutations in mammalian cells.

1989 ◽  
Vol 9 (3) ◽  
pp. 1277-1283 ◽  
Author(s):  
H Vrieling ◽  
M L Van Rooijen ◽  
N A Groen ◽  
M Z Zdzienicka ◽  
J W Simons ◽  
...  
Keyword(s):  
Dna Replication ◽  
Base Pair ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Parental Line ◽  
Induced Mutations ◽  
Hprt Gene ◽  
V79 Cells ◽  
H1 Cells ◽  
Double Base

The influence of DNA repair on the molecular nature of mutations induced by UV light (254 nm) was investigated in UV-induced hprt mutants from UV-sensitive Chinese hamster cells (V-H1) and the parental line (V79). The nature of point mutations in hprt exon sequences was determined for 19 hprt mutants of V79 and for 17 hprt mutants of V-H1 cells by sequence analysis of in vitro-amplified hprt cDNA. The mutation spectrum in V79 cells consisted of single- and tandem double-base pair changes, while in V-H1 cells three frameshift mutations were also detected. All base pair changes in V-H1 mutants were due to GC----AT transitions. In contrast, in V79 all possible classes of base pair changes except the GC----CG transversion were present. In this group, 70% of the mutations were transversions. Since all mutations except one did occur at dipyrimidine sites, the assumption was made that they were caused by UV-induced photoproducts at these sites. In V79 cells, 11 out of 17 base pair changes were caused by photoproducts in the nontranscribed strand of the hprt gene. However, in V-H1 cells, which are completely deficient in the removal of pyrimidine dimers from the hprt gene and which show a UV-induced mutation frequency enhanced seven times, 10 out of 11 base pair changes were caused by photoproducts in the transcribed strand of the hprt gene. We hypothesize that this extreme strand specificity in V-H1 cells is due to differences in fidelity of DNA replication of the leading and the lagging strand. Furthermore, we propose that in normal V79 cells two processes determine the strand specificity of UV-induced mutations in the hprt gene, namely preferential repair of the transcribed strand of the hprt gene and a higher fidelity of DNA replication of the nontranscribed strand compared with the transcribed strand.

DNA strand specificity for UV-induced mutations in mammalian cells

1989 ◽  
Vol 9 (3) ◽  
pp. 1277-1283
Author(s):  
H Vrieling ◽  
M L Van Rooijen ◽  
N A Groen ◽  
M Z Zdzienicka ◽  
J W Simons ◽  
...  
Keyword(s):  
Dna Replication ◽  
Base Pair ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Parental Line ◽  
Induced Mutations ◽  
Hprt Gene ◽  
V79 Cells ◽  
H1 Cells ◽  
Double Base

The influence of DNA repair on the molecular nature of mutations induced by UV light (254 nm) was investigated in UV-induced hprt mutants from UV-sensitive Chinese hamster cells (V-H1) and the parental line (V79). The nature of point mutations in hprt exon sequences was determined for 19 hprt mutants of V79 and for 17 hprt mutants of V-H1 cells by sequence analysis of in vitro-amplified hprt cDNA. The mutation spectrum in V79 cells consisted of single- and tandem double-base pair changes, while in V-H1 cells three frameshift mutations were also detected. All base pair changes in V-H1 mutants were due to GC----AT transitions. In contrast, in V79 all possible classes of base pair changes except the GC----CG transversion were present. In this group, 70% of the mutations were transversions. Since all mutations except one did occur at dipyrimidine sites, the assumption was made that they were caused by UV-induced photoproducts at these sites. In V79 cells, 11 out of 17 base pair changes were caused by photoproducts in the nontranscribed strand of the hprt gene. However, in V-H1 cells, which are completely deficient in the removal of pyrimidine dimers from the hprt gene and which show a UV-induced mutation frequency enhanced seven times, 10 out of 11 base pair changes were caused by photoproducts in the transcribed strand of the hprt gene. We hypothesize that this extreme strand specificity in V-H1 cells is due to differences in fidelity of DNA replication of the leading and the lagging strand. Furthermore, we propose that in normal V79 cells two processes determine the strand specificity of UV-induced mutations in the hprt gene, namely preferential repair of the transcribed strand of the hprt gene and a higher fidelity of DNA replication of the nontranscribed strand compared with the transcribed strand.

High Level Expression of Recombinant Human Tissue Factor in Chinese Hamster Ovary Cells as a Human Thromboplastin

1991 ◽  
Vol 65 (05) ◽  
pp. 521-527 ◽  
Author(s):  
Alnawaz Rehemtulla ◽  
Michael Pepe ◽  
Thomas S Edgington
Keyword(s):  
Tissue Factor ◽  
Mammalian Cells ◽  
Large Scale ◽  
Factor Viia ◽  
Affinity Purification ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Lipid Vesicles ◽  
Scale Production ◽  
Protein Chain

SummaryTissue factor (TF) is the high affinity transmembrane receptor and cofactor for cellular initiation of the plasma coagulation protease cascades by factor VIIa. We describe the synthesis of recombinant huTF by stably transfected CHO cell lines carrying integrated huTF DNA, and the isolation of huTF glycoprotein with specific functional activity equivalent to natural huTF, The expression vector (pCDM8), carrying the cytomegalovirus promoter to drive transcription of a partial cDNA construct encoding the complete huTF protein chain, was cotransfected with a plasmid containing the neomycin resistance gene for selection. These clones were further selected for level of expression of huTF protein. Optimal expression compatible with stability and cell growth was approximately 13.5 x 106 molecules per cell. To our knowledge, this is one of the highest levels of expression described for a recombinant transmembrane receptor in mammalian cells. Recombinant huTF protein was obtained by single-step immuno-affinity purification, and exhibits heterogeneity due to N-linked glycosylation. The protein was indistinguishable from natural huTF based on functional properties of the glycoprotein reconstituted in lipid vesicles, and expression of conformational epitopes. Large scale production of recombinant huTF is feasible to permit basic studies of protein structure as well as for design of huTF thromboplastin reagents.

scholarly journals Structural Changes in HPRT Gene of V79 Cells After Irradiation With Heavy Ions—Immediate and Delayed Effects

2021 ◽  
Vol 8 ◽  
Author(s):  
Pavel Bláha ◽  
Igor V. Koshlan ◽  
Nataliya A. Koshlan ◽  
Yulia V. Bogdanova ◽  
Daria V. Petrova ◽  
...  
Keyword(s):  
Heavy Ions ◽  
Mammalian Cells ◽  
Structural Changes ◽  
Chinese Hamster ◽  
Sharp Decrease ◽  
High Energy ◽  
Partial Deletion ◽  
V79 Cells ◽  
Delayed Effects ◽  
Radiobiological Effects

The radiobiological effects of accelerated ions with high charge and high energy (HZE) on mammalian cells and their propagation in time are still not sufficiently explained and attract great deal of attention. This work aims to compare the immediate and delayed effects with emphasis on the latter. As shown by our group, the dependence of mutant fraction on expression time after irradiation may have interesting, non-monotonic, character depending on LET (linear energy transfer) of the used heavy ions. We speculate that this phenomenon may occur due to the induced genomic instability. Another area of our research is the study of the DNA structural changes in these mutants induced at different expression times. Chinese hamster V79 cells were irradiated with accelerated ions 11B, 18O, 20Ne, and gamma radiation. The LET was ranging from 0.23 keV/μm of 60Co gamma rays up to 136 keV/μm of 20Ne ions. DNA of unique HPRT mutants was isolated, concentration measured, HPRT exons amplified, and analyzed at several different time points, up to about 40 days, after exposure. Over 1200 HPRT mutants were analyzed for deletions of exons and sorted into three main categories: partial deletion, PD—with deletion of one to eight exons; total deletions, TD—with all nine exons deleted; and no deletions—no change in the HPRT structure observed. In general, the number of samples with partial deletion was increasing with LET of the used radiation, suggesting that higher energy deposition to the cell nucleus is more likely to cause larger structural changes. In the case of total deletions, increase in their number with LET was observed up to LET ∼115 keV/μm followed by a sharp decrease. The samples were also analyzed for the distribution of deletions, in particular exons at various expression times, the so-called mutational patterns. Hypothesis of the mechanisms behind observed phenomena is given, and possible implications for further research are discussed.

scholarly journals Defective Brca2 influences topoisomerase I activity in mammalian cells.

2003 ◽  
Vol 50 (1) ◽  
pp. 139-144 ◽  
Author(s):  
Iwonna Rahden-Staroń ◽  
Maria Szumiło ◽  
Emilia Grosicka ◽  
Maria Kraakman van der Zwet ◽  
Małgorzata Z Zdzienicka
Keyword(s):  
Mammalian Cells ◽  
Topoisomerase I ◽  
Chinese Hamster ◽  
Brca2 Gene ◽  
Nuclear Extract ◽  
Chinese Hamster Cell ◽  
V79 Cells ◽  
Cell Mutant ◽  
Relaxation Activity ◽  
Nuclear Extracts

The Chinese hamster cell mutant V-C8 is defective in the Brca2 gene (Kraakman-van der Zwet et al., 2002, Cell Biol.; 22: 669). Here we report that V-C8 cells were 10-fold more sensitive to camptothecin, an inhibitor of topoisomerase I, than the parental V79 cells. The level of the relaxation activity of topoisomerase I in nuclear extracts was also lower (4-fold) in V-C8 than V79 cells, in spite of the fact that the level of the topoisomerase I protein was the same in these cells. The survival of V-C8 cells in the presence of camptothecin, the sensitivity of V-C8 topoisomerase I to camptothecin, and the level of the relaxation activity in V-C8 nuclear extract were almost completely restored by transfection of V-C8 cells with the murine Brca2 gene or by the transfer of human chromosome 13 providing the BRCA2 gene. These results indicate that the observed changes in the topoisomerase I activity in V-C8 are due to the defective function of the Brca2 gene.

Ultrastructural Changes in Three Different Mammalian Cells Following Ultraviolet Laser Irradiation

1972 ◽  
Vol 30 ◽  
pp. 350-351
Author(s):  
K. Shankar Narayan ◽  
Kailash C. Gupta ◽  
Tohru Okigaki
Keyword(s):  
Ultraviolet Light ◽  
Mammalian Cells ◽  
Biological Effects ◽  
Survival Rates ◽  
Chinese Hamster ◽  
Cell Types ◽  
Differential Sensitivity ◽  
Ultrastructural Changes ◽  
Ultraviolet Laser

The biological effects of short-wave ultraviolet light has generally been described in terms of changes in cell growth or survival rates and production of chromosomal aberrations. Ultrastructural changes following exposure of cells to ultraviolet light, particularly at 265 nm, have not been reported.We have developed a means of irradiating populations of cells grown in vitro to a monochromatic ultraviolet laser beam at a wavelength of 265 nm based on the method of Johnson. The cell types studies were: i) WI-38, a human diploid fibroblast; ii) CMP, a human adenocarcinoma cell line; and iii) Don C-II, a Chinese hamster fibroblast cell strain. The cells were exposed either in situ or in suspension to the ultraviolet laser (UVL) beam. Irradiated cell populations were studied either "immediately" or following growth for 1-8 days after irradiation.Differential sensitivity, as measured by survival rates were observed in the three cell types studied. Pattern of ultrastructural changes were also different in the three cell types.

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