scholarly journals Increased ouabain-sensitive 86Rubidium uptake after mitogenic stimulation of quiescent chicken embryo fibroblasts with purified multiplication-stimulating activity.

1977 ◽  
Vol 73 (3) ◽  
pp. 761-767 ◽  
Author(s):  
G L Smith
Keyword(s):  
Dna Synthesis ◽  
Chicken Embryo ◽  
Enzyme Assay ◽  
Specific Inhibitor ◽  
Early Event ◽  
Free Medium ◽  
Quiescent Cells ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts ◽  
Stimulation Of

Multiplication-stimulating activity (MSA), a protein which stimulates DNA synthesis and growth of chicken embryo fibroblasts, was purified from serum-free medium conditioned by the growth of a rat liver cell line. Purified MSA was shown to rapidly stimulate ouabain-sensitive Na+, K+-ATPase activity as measured by both enzyme assay and rate of 86Rubidium uptake. Labeled ouabain binding was also shown to increase after stimulation of quiescent cells by serum or purified MSA. Conditions which interfere with the ability of the cells to accumulate potassium, such as the presence of the specific inhibitor, ouabain; incubation in potassium-free medium; or the presence of the potassium ionophore, valinomycin, were all demonstrated to inhibit the stimulation of DNA synthesis by serum or purified MSA. These results suggest that an early event in the stimulation of DNA synthesis by purified MSA is an activation of membrane Na+, K+-ATPase with a resulting accumulation of potassium ions inside the cell.

Ethidium bromide-induced loss of mitochondrial DNA from primary chicken embryo fibroblasts

1985 ◽  
Vol 5 (5) ◽  
pp. 1163-1169
Author(s):  
P Desjardins ◽  
E Frost ◽  
R Morais
Keyword(s):  
Mitochondrial Dna ◽  
Ethidium Bromide ◽  
Chicken Embryo ◽  
Blot Hybridization ◽  
Cell Populations ◽  
Free Medium ◽  
Reassociation Kinetics ◽  
Embryo Fibroblasts ◽  
Drug Free ◽  
Chicken Embryo Fibroblasts

Chicken embryo fibroblasts in uridine-containing medium are inherently resistant to the growth-inhibitory effect of ethidium bromide. The drug was found to inhibit the incorporation of [3H]thymidine into mitochondrial DNA circular molecules. Mitochondrial DNA was quantitated by DNA-DNA reassociation kinetics with a probe of chicken liver mitochondrial DNA. A mean number of 604 copies of mitochondrial DNA per cell was found. This number decreased progressively in cells exposed to ethidium bromide, and by day 13 ca. one copy of mitochondrial DNA was detected per cell. When the cells were then transferred to drug-free medium, the number of copies increased very slowly as a function of time. On the other hand, analyses of DNA extracted from cell populations exposed to ethidium bromide for 20 or more days, with or without subsequent transfer to drug-free medium, revealed very little or no mitochondrial DNA by reassociation kinetics or by Southern blot hybridization of AvaI- or HindIII-digested total cellular DNA. As a result of the elimination of mitochondrial DNA molecules, the establishment of cell populations with a respiration-deficient phenotype was confirmed by measuring cytochrome c oxidase activity as a function of the number of cell generations and the absorption spectrum of mitochondrial cytochromes.

Tyrosine phosphorylation of specific proteins after mitogen stimulation of chicken embryo fibroblasts

1983 ◽  
Vol 3 (3) ◽  
pp. 380-390
Author(s):  
K D Nakamura ◽  
R Martinez ◽  
M J Weber
Keyword(s):  
Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Chicken Embryo ◽  
Sodium Dodecyl ◽  
Biological Response ◽  
Egf Receptors ◽  
Phosphorylation Of Proteins ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts ◽  
Stimulation Of

We found that stimulation of density-inhibited chicken embryo fibroblasts with serum, epidermal growth factor (EGF), platelet-derived growth factor, (PDGF), or multiplication-stimulating activity (MSA) leads to an increase in tyrosine phosphorylation of proteins in the region of Mr 40,000 (40K) to 42K. The increase in tyrosine phosphorylation after serum or EGF stimulation was transient, reaching a maximum at about 5 min and then declining. By fine-resolution analysis of proteins separated on sodium dodecyl sulfate-polyacrylamide gels, we found that after EGF stimulation, the major increase in phosphotyrosine content was in a 42K Mr protein, with a smaller increase in a 40K Mr protein. The increased phosphorylation in the 40K to 42K Mr region accounted for almost all of the increase in phosphotyrosine observed in these cells. These phosphotyrosine-containing proteins were different from the major phosphotyrosine-containing protein of Rous sarcoma virus-transformed chicken embryo fibroblasts, which migrates at an approximate Mr of 36K. Increased tyrosine phosphorylation of proteins of similar Mr was found in 3T3 cells treated with EGF, but not in NR-6 cells, which lack detectable EGF receptors. It is possible that the 40K to 42K Mr phosphotyrosine-containing proteins are involved in the integration of the biological response to a number of different growth factors.

scholarly journals Counter-transport in chick embryo fibroblasts. A significant factor in measurement of glucose entry

1982 ◽  
Vol 206 (2) ◽  
pp. 301-309 ◽  
Author(s):  
R J Gay ◽  
H Amos
Keyword(s):  
Chick Embryo ◽  
Glucose Transport ◽  
Cell Cultures ◽  
Chicken Embryo ◽  
Primary Cell ◽  
Primary Cell Cultures ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts ◽  
Stimulation Of

Enhanced rates of carrier-mediated 3-O-methyl-D-glucose (0.1 mM) transport were observed in primary cell cultures of chicken embryo fibroblasts deprived of glucose for 1 day. The addition of 5.5 mM-glucose, glucosamine or 2-deoxy-D-glucose for 15 min (37 degrees C) to glucose-starved cultures followed by washing and immediate measurement of 3-O-methyl-D-glucose transport resulted in an apparent further stimulation of transport. Transport stimulation increased with increasing concentrations of the added preincubation sugar and was observed at test concentrations ranging from 0.1 mM- to 10 mM-3-O-methyl-D-glucose. This enhancement occurred when the preloaded sugar was rapidly effluxing from cells and was eliminated by allowing cultures to incubate in buffer without sugar for 30 min (37 degrees C) after the removal of hexose and before measuring transport. A transient overshoot in the cumulative uptake of 3-O-methyl-D-glucose was observed in glucose-starved cultures that were pre-incubated in the presence of 55 mM-glucose or -glucosamine for 15 min (37 degrees C). These data suggest that counter-transport accounts for the apparent enhancement of glucose-transport capability observed in glucose-starved cells when they are briefly re-exposed to hexose.

scholarly journals Ethidium bromide-induced loss of mitochondrial DNA from primary chicken embryo fibroblasts.

1985 ◽  
Vol 5 (5) ◽  
pp. 1163-1169 ◽  
Author(s):  
P Desjardins ◽  
E Frost ◽  
R Morais
Keyword(s):  
Mitochondrial Dna ◽  
Ethidium Bromide ◽  
Chicken Embryo ◽  
Blot Hybridization ◽  
Cell Populations ◽  
Free Medium ◽  
Reassociation Kinetics ◽  
Embryo Fibroblasts ◽  
Drug Free ◽  
Chicken Embryo Fibroblasts

Chicken embryo fibroblasts in uridine-containing medium are inherently resistant to the growth-inhibitory effect of ethidium bromide. The drug was found to inhibit the incorporation of [3H]thymidine into mitochondrial DNA circular molecules. Mitochondrial DNA was quantitated by DNA-DNA reassociation kinetics with a probe of chicken liver mitochondrial DNA. A mean number of 604 copies of mitochondrial DNA per cell was found. This number decreased progressively in cells exposed to ethidium bromide, and by day 13 ca. one copy of mitochondrial DNA was detected per cell. When the cells were then transferred to drug-free medium, the number of copies increased very slowly as a function of time. On the other hand, analyses of DNA extracted from cell populations exposed to ethidium bromide for 20 or more days, with or without subsequent transfer to drug-free medium, revealed very little or no mitochondrial DNA by reassociation kinetics or by Southern blot hybridization of AvaI- or HindIII-digested total cellular DNA. As a result of the elimination of mitochondrial DNA molecules, the establishment of cell populations with a respiration-deficient phenotype was confirmed by measuring cytochrome c oxidase activity as a function of the number of cell generations and the absorption spectrum of mitochondrial cytochromes.

scholarly journals Tyrosine phosphorylation of specific proteins after mitogen stimulation of chicken embryo fibroblasts.

1983 ◽  
Vol 3 (3) ◽  
pp. 380-390 ◽  
Author(s):  
K D Nakamura ◽  
R Martinez ◽  
M J Weber
Keyword(s):  
Growth Factor ◽  
Tyrosine Phosphorylation ◽  
Chicken Embryo ◽  
Sodium Dodecyl ◽  
Biological Response ◽  
Egf Receptors ◽  
Phosphorylation Of Proteins ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts ◽  
Stimulation Of

We found that stimulation of density-inhibited chicken embryo fibroblasts with serum, epidermal growth factor (EGF), platelet-derived growth factor, (PDGF), or multiplication-stimulating activity (MSA) leads to an increase in tyrosine phosphorylation of proteins in the region of Mr 40,000 (40K) to 42K. The increase in tyrosine phosphorylation after serum or EGF stimulation was transient, reaching a maximum at about 5 min and then declining. By fine-resolution analysis of proteins separated on sodium dodecyl sulfate-polyacrylamide gels, we found that after EGF stimulation, the major increase in phosphotyrosine content was in a 42K Mr protein, with a smaller increase in a 40K Mr protein. The increased phosphorylation in the 40K to 42K Mr region accounted for almost all of the increase in phosphotyrosine observed in these cells. These phosphotyrosine-containing proteins were different from the major phosphotyrosine-containing protein of Rous sarcoma virus-transformed chicken embryo fibroblasts, which migrates at an approximate Mr of 36K. Increased tyrosine phosphorylation of proteins of similar Mr was found in 3T3 cells treated with EGF, but not in NR-6 cells, which lack detectable EGF receptors. It is possible that the 40K to 42K Mr phosphotyrosine-containing proteins are involved in the integration of the biological response to a number of different growth factors.

Kinetics of unscheduled DNA synthesis in UV-irradiated chicken embryo fibroblasts

1985 ◽  
Vol 146 (3) ◽  
pp. 305-310 ◽  
Author(s):  
L. Roza ◽  
M.H. Wade ◽  
G.P. van der Schans ◽  
P.H.M. Lohman ◽  
F. Berends
Keyword(s):  
Dna Synthesis ◽  
Chicken Embryo ◽  
Unscheduled Dna Synthesis ◽  
Embryo Fibroblasts ◽  
Kinetics Of ◽  
Chicken Embryo Fibroblasts
Sign in / Sign up

Export Citation Format

Share Document