Studies on the biochemical properties of surface component of normal and SV-40 transformed 3T3 mouse cells

1970 ◽  
Vol 48 (8) ◽  
pp. 851-857 ◽  
Author(s):  
Rose Sheinin ◽  
Kazukiyo Onodera
Keyword(s):  
Biochemical Properties ◽  
3T3 Cells ◽  
Surface Component ◽  
Mouse Cells ◽  
Marked Surface

A specifically marked surface component from 3T3 mouse cells and from SV-40 transformed 3T3 cells has been isolated and partially purified. These components have been shown to be free of RNA, DNA, and lipid. They do contain carbohydrate and peptide, probably as glycoprotein.

Macromolecular Glucosamine-Containing Component of the Surface of Cultivated Mouse Cells

1970 ◽  
Vol 7 (2) ◽  
pp. 337-355
Author(s):  
K. ONODERA ◽  
ROSE SHEININ
Keyword(s):  
Plasma Membrane ◽  
Cell Division ◽  
Cell Surface ◽  
Plating Efficiency ◽  
Transformed Cells ◽  
3T3 Cells ◽  
Vital Stain ◽  
Surface Component ◽  
Mouse Cells ◽  
Macromolecular Component

It has been demonstrated that a glucosamine-containing macromolecular component of the cell surface of 3T3 mouse cells, and SV40-transformed cells, is released from cells by treatment with trypsin under conditions in which the plasma membrane remains functionally intact. This was shown by the fact that the treated cells could be cloned with high plating efficiency and remained impermeable to the vital stain, erythrocin. A method for specifically marking this surface component has been devised based on the finding that in 3T3 cells growing synchronously after subculture by trypsin maximum incorporation of glucosamine into this material occurs 12-13 h thereafter. Of the total radioactive glucosamine incorporated into macro-molecular cell constituents, over 80% was recovered in surface component. Studies on the biosynthesis of surface component revealed that this was periodic during a cycle of cell duplication, with an increased rate of formation immediately after cell division. It was found that the surface component of 3T3 cells differed from that of SV40-transformed cells.

scholarly journals Modulation of the transport of a lysosomal enzyme by PDGF.

1990 ◽  
Vol 110 (2) ◽  
pp. 319-326 ◽  
Author(s):  
E M Prence ◽  
J M Dong ◽  
G G Sahagian
Keyword(s):  
Secretory Pathway ◽  
Cathepsin L ◽  
Control Level ◽  
3T3 Cells ◽  
Lysosomal Protease ◽  
Affinity Ligand ◽  
The Media ◽  
Mouse Cells ◽  
Nih 3T3 ◽  
The Relationship

The major excreted protein (MEP) of transformed mouse fibroblasts is the lysosomal protease, cathepsin L. MEP is also secreted by untransformed mouse cells in response to growth factors and tumor promoters, and is thought to play a role in cell growth and transformation. To determine the relationship between MEP synthesis and MEP secretion, we have examined these events in PDGF-treated NIH 3T3 cells. PDGF enhanced MEP synthesis and caused the diversion of MEP from the lysosomal delivery pathway to a secretory pathway. These two effects were found to be regulated independently at various times after growth factor addition. Short PDGF treatments (0.5 or 1 h) resulted in quantitative secretion of MEP although synthesis was near the control level. High levels of both synthesis and secretion occurred between 2 and 14 h of PDGF treatment. Between 18 and 30 h, the amount of secreted MEP returned to the low control level even though synthesis remained elevated. The secretion was specific for MEP; other lysosomal enzymes were not found in the media from PDGF-treated cells. PDGF-induced secretion of MEP was inhibited 84% by cycloheximide, suggesting that protein synthesis is required to elicit this effect. PDGF also caused a time-dependent increase in mannose 6-phosphate (Man-6-P) receptor-mediated endocytosis. These data support a model in which PDGF alters the distribution of Man-6-P receptors such that the Golgi concentration of receptors becomes limiting, thereby causing the selective secretion of the low affinity ligand, MEP.

Control of proliferin gene expression in serum-stimulated mouse cells

1987 ◽  
Vol 7 (6) ◽  
pp. 2080-2086
Author(s):  
D I Linzer ◽  
E L Wilder
Keyword(s):  
Protein Synthesis ◽  
S Phase ◽  
Mrna Levels ◽  
3T3 Cells ◽  
Serum Stimulation ◽  
Posttranscriptional Processing ◽  
A Cell ◽  
Mouse Cells ◽  
Stimulation Of

The serum-inducible expression of proliferin genes in BALB/c 3T3 cells was found to be dependent on both protein synthesis and an extended presence of serum in the medium. Even though no mature proliferin mRNA was detected in serum-starved cells, transcription of the proliferin genes occurred in these resting-cell cultures, indicating that posttranscriptional events may be important for regulating proliferin mRNA levels. These results suggest that protein synthesis after serum stimulation of quiescent mouse fibroblasts is required for posttranscriptional processing or stabilization of proliferin RNA. Proliferin RNA levels were found to be heterogeneous among serum-stimulated cells analyzed by in situ hybridization. This heterogeneity is probably due to asynchrony in the population and may point to a correlation between the time of proliferin expression and the time of entry of a cell into S phase.

scholarly journals Control of proliferin gene expression in serum-stimulated mouse cells.

1987 ◽  
Vol 7 (6) ◽  
pp. 2080-2086 ◽  
Author(s):  
D I Linzer ◽  
E L Wilder
Keyword(s):  
Protein Synthesis ◽  
S Phase ◽  
Mrna Levels ◽  
3T3 Cells ◽  
Serum Stimulation ◽  
Posttranscriptional Processing ◽  
A Cell ◽  
Mouse Cells ◽  
Stimulation Of

The serum-inducible expression of proliferin genes in BALB/c 3T3 cells was found to be dependent on both protein synthesis and an extended presence of serum in the medium. Even though no mature proliferin mRNA was detected in serum-starved cells, transcription of the proliferin genes occurred in these resting-cell cultures, indicating that posttranscriptional events may be important for regulating proliferin mRNA levels. These results suggest that protein synthesis after serum stimulation of quiescent mouse fibroblasts is required for posttranscriptional processing or stabilization of proliferin RNA. Proliferin RNA levels were found to be heterogeneous among serum-stimulated cells analyzed by in situ hybridization. This heterogeneity is probably due to asynchrony in the population and may point to a correlation between the time of proliferin expression and the time of entry of a cell into S phase.

scholarly journals Bacterial Conjugation in the Cytoplasm of Mouse Cells

2008 ◽  
Vol 76 (11) ◽  
pp. 5110-5119 ◽  
Author(s):  
Yin Mei Lim ◽  
Ad J. C. de Groof ◽  
Mrinal K. Bhattacharjee ◽  
David H. Figurski ◽  
Eric A. Schon
Keyword(s):  
High Frequency ◽  
Mammalian Cells ◽  
Yersinia Pseudotuberculosis ◽  
Shigella Flexneri ◽  
Antibiotic Resistance Genes ◽  
Conjugative Plasmid ◽  
Bacterial Conjugation ◽  
3T3 Cells ◽  
Transfer Genes ◽  
Mouse Cells

ABSTRACT Intracellular pathogenic organisms such as salmonellae and shigellae are able to evade the effects of many antibiotics because the drugs are not able to penetrate the plasma membrane. In addition, these bacteria may be able to transfer genes within cells while protected from the action of drugs. The primary mode by which virulence and antibiotic resistance genes are spread is bacterial conjugation. Salmonellae have been shown to be competent for conjugation in the vacuoles of cultured mammalian cells. We now show that the conjugation machinery is also functional in the mammalian cytosol. Specially constructed Escherichia coli strains expressing Shigella flexneri plasmid and chromosomal virulence factors for escape from vacuoles and synthesizing the invasin protein from Yersinia pseudotuberculosis to enhance cellular entry were able to enter 3T3 cells and escape from the phagocytic vacuole. One bacterial strain (the donor) of each pair to be introduced sequentially into mammalian cells had a conjugative plasmid. We found that this plasmid could be transferred at high frequency. Conjugation in the cytoplasm of cells may well be a general phenomenon.

scholarly journals Nicotine-stimulated proteins in mouse cells are distinct from heat-shock proteins

1984 ◽  
Vol 224 (1) ◽  
pp. 87-92 ◽  
Author(s):  
L A Hunt ◽  
K S Kelley
Keyword(s):  
Heat Shock ◽  
Sodium Dodecyl ◽  
Cellular Proteins ◽  
Mouse Tissue ◽  
3T3 Cells ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Mouse Cells ◽  
Altered Cell ◽  
Shock Proteins

Treatment of mouse tissue-culture cells with nicotine concentrations of 1 mM or less had no significant effects on cell viability, morphology or protein synthesis, but higher concentrations resulted in both altered cell morphology (rounding and vacuolization) and alterations in [3H]leucine-labelled protein profiles on sodium dodecyl sulphate/polyacrylamide gels. The synthesis of a Mr-70 000 protein was increased more than 2-fold relative to that of other major cellular proteins in 3T3 and L929 cells treated with 5 mM-nicotine and in B16 cells treated with 10 mM-nicotine, and this protein appeared to be a soluble cytoplasmic polypeptide. The radiolabelling of several additional polypeptides (Mr 62 000 in 3T3 cells, and Mr 45 000 and 38 000 in B16 cells) was also stimulated by nicotine. The nicotine-enhanced Mr-70 000 protein was distinct, however, from a major cell stress/heat-shock protein whose synthesis was stimulated after incubation of cells at 43.5 degrees C for 20 min.

scholarly journals Conditionally oncogenic forms of the A-Raf and B-Raf protein kinases display different biological and biochemical properties in NIH 3T3 cells.

1995 ◽  
Vol 15 (11) ◽  
pp. 6430-6442 ◽  
Author(s):  
C A Pritchard ◽  
M L Samuels ◽  
E Bosch ◽  
M McMahon
Keyword(s):  
Protein Kinases ◽  
Intracellular Signaling ◽  
Mammalian Cells ◽  
Map Kinases ◽  
Prolonged Exposure ◽  
Feedback Regulation ◽  
Biochemical Properties ◽  
3T3 Cells ◽  
In Cells

The protein kinase domains of mouse A-Raf and B-Raf were expressed as fusion proteins with the hormone binding domain of the human estrogen receptor in mammalian cells. In the absence of estradiol, 3T3 and rat1a cells expressing delta A-Raf:ER and delta B-Raf:ER were nontransformed, but upon the addition of estradiol the cells became oncogenically transformed. Morphological oncogenic transformation was more rapid and distinctive in cells expressing delta B-Raf:ER compared with cells expressing delta A-Raf:ER. Biochemical analysis of cells transformed by delta A-Raf:ER and delta B-Raf:ER revealed several interesting differences. The activation of delta B-Raf:ER consistently led to the rapid and robust activation of both MEK and p42/p44 MAP kinases. By contrast, the activation of delta A-Raf:ER led to a weak activation of MEK and the p42/p44 MAP kinases. The extent of activation of MEK in cells correlated with the ability of the different Raf kinases to phosphorylate and activate MEK1 in vitro. delta B-Raf:ER phosphorylated MEK1 approximately 10 times more efficiently than delta Raf-1:ER and at least 500 times more efficiently than delta A-Raf:ER under the conditions of the immune-complex kinase assays. These results were confirmed with epitope-tagged versions of the Raf kinase domains expressed in insect cells. The activation of all three delta Raf:ER proteins in 3T3 cells led to the hyperphosphorylation of the resident p74raf-1 and mSOS1 proteins, suggesting the possibility of "cross-talk" between the different Raf kinases and feedback regulation of intracellular signaling pathways. The activation of either delta B-Raf:ER or delta Raf-1:ER in quiescent 3T3 cells was insufficient to promote the entry of the cells into DNA synthesis. By contrast, the activation of delta A-Raf:ER in quiescent 3T3 cells was sufficient to promote the entry of the cells into S phase after prolonged exposure to beta-estradiol. The delta Raf:ER system has allowed us to reveal significant differences between the biological and biochemical properties of oncogenic forms of the Raf family of protein kinases. We anticipate that cells expressing these proteins and other estradiol-regulated protein kinases will be useful tools in future attempts to unravel the complex web of interactions involved in intracellular signal transduction pathways.

scholarly journals Biochemical and genetic characterization of three hamster cell mutants resistant to diphtheria toxin

1979 ◽  
Vol 83 (1) ◽  
pp. 116-125 ◽  
Author(s):  
RK Draper ◽  
D Chin ◽  
D Eurey-Owens ◽  
IE Scheffler ◽  
MI Simon
Keyword(s):  
Diphtheria Toxin ◽  
Elongation Factor ◽  
Cross Resistance ◽  
3T3 Cells ◽  
Toxin Resistance ◽  
Gene Coding ◽  
Recessive Phenotype ◽  
Mouse Cells ◽  
Pseudomonas Aeruginosa Exotoxin

We describe here three different hamster cell mutants which are resistant to diphtheria toxin and which provide models for investigating some of the functions required by the toxin inactivates elongation factor 2 (EF-2). Cell-free extracts from mutants Dtx(r)-3 was codominant. The evidence suggests that the codominant phenotype is the result of a mutation in a gene coding for EF-2. The recessive phenotype might arise by alteration of an enzyme which modifies the structure of EF-2 so that it becomes a substrate for reaction with the toxin. Another mutant, Dtx(r)-2, contained EF-2 that was sensitive to the toxin and this phenotype was recessive. Pseudomonas aeruginosa exotoxin is known to inactivate EF-2 as does diphtheria toxin and we tested the mutants for cross-resistance to pseudomonas exotoxin. Dtx(r)-1 and Dtx(r)-3 were cross-resistant while Dtx(r)-2 was not. It is known that diphtheria toxin does not penetrate to the cytoplasm of mouse cells and that these cell have a naturally occurring phenotype of diphtheria toxin resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance of the hybrid cells to diphtheria toxin. Intraspecies hybrids containing the genome of mutants Dtx(r)-1 and Dtx(r)-3 had some resistance while those formed with Dtx(r)-2 were as sensitive as hybrids derived from fusions between wild-type hamster cells and mouse 3T3 cells.

scholarly journals HER4-mediated Biological and Biochemical Properties in NIH 3T3 Cells

1996 ◽  
Vol 271 (9) ◽  
pp. 4813-4818 ◽  
Author(s):  
Bruce D. Cohen ◽  
Janell M. Green ◽  
Linda Foy ◽  
H. Perry Fell
Keyword(s):  
Biochemical Properties ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

scholarly journals Mycoplasma-induced BALB/c 3T3 collagenase is a mammalian enzyme

1983 ◽  
Vol 212 (3) ◽  
pp. 641-647 ◽  
Author(s):  
B Kluve ◽  
W C Merrick ◽  
H Gershman
Keyword(s):  
Type I Collagen ◽  
Type Ii Collagen ◽  
Collagen Molecule ◽  
Relative Molecular Mass ◽  
Type Iii Collagen ◽  
Type I ◽  
Reaction Products ◽  
3T3 Cells ◽  
Type Iv ◽  
Mouse Cells

A collagenase previously reported to accumulate in the medium of cultures of BALB/c 3T3 cells on infection with Mycoplasma orale [Kluve, Merrick, Stanbridge & Gershman (1981) Nature (London) 292, 855-857] was partially purified and characterized. With regard to purification properties, activation, sensitivity to inhibitors and relative molecular mass the enzyme was similar to previously reported vertebrate collagenases, but could not be unequivocally distinguished from bacterial collagenases. With regard to substrate-specificity and reaction products, however, the collagenase was typical of vertebrate collagenases and distinct from bacterial collagenases. Specifically, the enzyme displayed a preference for type III collagen and type I collagen, a somewhat decreased ability to degrade type II collagen, and a very limited ability to degrade type IV collagen. The initial products of the action of the collagenase on type I collagen were characterized as fragments one-quarter and three-quarters of the length of the intact collagen molecule. Because the properties of the collagenase produced by cultures of mycoplasma-infected BALB/c 3T3 cells are those of a mammalian-type (vertebrate-type) enzyme, we have concluded that the collagenase is a product of the mouse (BALB/c 3T3) genome, and is not produced by the mycoplasma. Therefore it appears that infection of BALB/c 3T3 mouse fibroblasts with Mycoplasma orale induces the mouse cells to produce and secrete collagenase.

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