On the reduced intercellular adhesiveness of virally transformed BHK21 cells

1979 ◽  
Vol 35 (1) ◽  
pp. 307-320
Author(s):  
J.G. Edwards ◽  
J.M. Dysart ◽  
D.H. Edgar ◽  
R.T. Robson
Keyword(s):  
Growth Medium ◽  
Caproic Acid ◽  
Transformed Cells ◽  
Baby Hamster Kidney ◽  
Short Term ◽  
Cell Sheets ◽  
Aggregation Assay ◽  
Intracellular Pool ◽  
Rous Sarcoma ◽  
Gyratory Shaker

Baby hamster kidney fibroblasts (BHK21 cells) transformed by polyoma or Rous sarcoma viruses aggregate less than the untransformed parental cells when incubated in growth medium in a gyratory shaker for 18–24 h. This difference can be measured by electronic particle counting, or by filtering aggregated suspensions of 32P-labelled cells through bolting fabric. The aggregation of transformed derivatives is not enhanced by the presence, during aggregation of epsilon-amino caproic acid, an inhibitor of plasmin activation. Some lines of transformed BHK21 cells do not appear less adhesive than untransformed cells in a short-term aggregation assay, and none adheres markedly less well when seeded onto homotypic cell sheets. The decreased aggregation of transformed cells is consistent with suggestions that LETS protein is involved in intercellular adhesion of fibroblasts as well as in attachment of cells to non-cellular substrates. If so, the short-term aggregation of freshly trypsinized cells may depend on secretion of LETS from an intracellular pool.

scholarly journals Tropomyosin isoforms in chicken embryo fibroblasts: purification, characterization, and changes in Rous sarcoma virus-transformed cells.

1985 ◽  
Vol 100 (3) ◽  
pp. 692-703 ◽  
Author(s):  
J J Lin ◽  
D M Helfman ◽  
S H Hughes ◽  
C S Chou
Keyword(s):  
Rous Sarcoma Virus ◽  
Chicken Embryo ◽  
Actin Binding ◽  
Transformed Cells ◽  
Two Dimensional ◽  
Tropomyosin Isoforms ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Seven polypeptides (a, b, c, 1, 2, 3a, and 3b) have been previously identified as tropomyosin isoforms in chicken embryo fibroblasts (CEF) (Lin, J. J.-C., Matsumura, F., and Yamashiro-Matsumura, S., 1984, J. Cell. Biol., 98:116-127). Spots a and c had identical mobility on two-dimensional gels with the slow-migrating and fast-migrating components, respectively, of chicken gizzard tropomyosin. However, the remaining isoforms of CEF tropomyosin were distinct from chicken skeletal and cardiac tropomyosins on two-dimensional gels. The mixture of CEF tropomyosin has been isolated by the combination of Triton/glycerol extraction of monolayer cells, heat treatment, and ammonium sulfate fractionation. The yield of tropomyosin was estimated to be 1.4% of total CEF proteins. The identical set of tropomyosin isoforms could be found in the antitropomyosin immunoprecipitates after the cell-free translation products of total poly(A)+ RNAs isolated from CEF cells. This suggested that at least seven mRNAs coding for these tropomyosin isoforms existed in the cell. Purified tropomyosins (particularly 1, 2, and 3) showed different actin-binding abilities in the presence of 100 mM KCl and no divalent cation. Under this condition, the binding of tropomyosin 3 (3a + 3b) to actin filaments was significantly weaker than that of tropomyosin 1 or 2. CEF tropomyosin 1, and probably 3, could be cross-linked to form homodimers by treatment with 5,5'-dithiobis-(2-nitrobenzoate), whereas tropomyosin a and c formed a heterodimer. These dimer species may reflect the in vivo assembly of tropomyosin isoforms, since dimer formation occurred not only with purified tropomyosin but also with microfilament-associated tropomyosin. The expression of these tropomyosin isoforms in Rous sarcoma virus-transformed CEF cells has also been investigated. In agreement with the previous report by Hendricks and Weintraub (Proc. Natl. Acad. Sci. USA., 78:5633-5637), we found that major tropomyosin 1 was greatly reduced in transformed cells. We have also found that the relative amounts of tropomyosin 3a and 3b were increased in both the total cell lysate and the microfilament fraction of transformed cells. Because of the different actin-binding properties observed for CEF tropomyosins, changes in the expression of these isoforms may, in part, be responsible for the reduction of actin cables and the alteration of cell shape found in transformed cells.

scholarly journals Identification of Phosphotyrosine-Containing Proteins in Untransformed and Rous Sarcoma Virus-Transformed Chicken Embryo Fibroblasts

1982 ◽  
Vol 2 (6) ◽  
pp. 653-665 ◽  
Author(s):  
Ricardo Martinez ◽  
Kenji D. Nakamura ◽  
Michael J. Weber
Keyword(s):  
Protein Kinases ◽  
Rous Sarcoma Virus ◽  
Chicken Embryo ◽  
Cellular Protein ◽  
Transformed Cells ◽  
Phosphoamino Acid ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Phosphorylation on tyrosine residues mediated by pp60srcappears to be a primary biochemical event leading to the establishment of the transformed phenotype in Rous sarcoma virus (RSV)-infected cells. To identify the cellular proteins that undergo tyrosine phosphorylation during transformation, a32P-labeled RSV-transformed chicken embryo cell extract was analyzed by electrophoresis on a polyacrylamide gel. After slicing the gel into approximately 60 slices, phosphoamino acid analyses were carried out on the protein recovered from each gel slice. Phosphotyrosine was found in every gel slice, with two major peaks of this phosphoamino acid aroundMr's of 59 and 36 kilodaltons. When the same analysis was performed with cells infected with a transformation-defectivesrcdeletion mutant of RSV (tdNY101), significant and reproducible peaks of phosphotyrosine were found in only 2 of 60 gel slices. These gel slices corresponded toMr's of 42 and 40 kilodaltons. Identical results were obtained with normal uninfected chicken embryo fibroblasts. We conclude from these observations that pp60srcor the combined action of pp60srcand pp60src-activated cellular protein kinases cause the tyrosine-specific phosphorylation of a very large number of cellular polypeptides in RSV-transformed cells. In addition, untransformed cells appear to possess one or more active tyrosine-specific protein kinases which are responsible for the phosphorylation of a limited number of proteins. These proteins are different from the major phosphotyrosine-containing proteins of the transformed cells.

Detection of phosphotyrosine-containing proteins in polyomavirus middle tumor antigen-transformed cells after treatment with a phosphotyrosine phosphatase inhibitor

1987 ◽  
Vol 7 (2) ◽  
pp. 905-913
Author(s):  
W Yonemoto ◽  
A J Filson ◽  
A E Queral-Lustig ◽  
J Y Wang ◽  
J S Brugge
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tumor Antigen ◽  
Tyrosine Kinases ◽  
Cellular Transformation ◽  
Cellular Protein ◽  
Cellular Proteins ◽  
Transformed Cells ◽  
Major Protein ◽  
Rous Sarcoma ◽  
Phosphotyrosine Phosphatases

Cells transformed with the middle tumor antigen (mT) of polyomavirus were treated with sodium orthovanadate (Na3VO4), an inhibitor of phosphotyrosine phosphatases, to enhance for the detection of cellular proteins which are phosphorylated on tyrosine. Na3VO4 treatment of mT-transformed rat F1-11 cells resulted in a 16-fold elevation in the level of phosphotyrosine associated with total cellular proteins. Parental F1-11 cells displayed only a twofold increase in phosphotyrosine following Na3VO4 treatment. The abundance of phosphotyrosine in Na3VO4-treated mT-transformed F1-11 cells was twofold higher than in untreated Rous sarcoma virus (RSV)-transformed F1-11 cells and 3.5-fold lower than in Na3VO4-treated RSV-transformed F1-11 cells. Tyrosine phosphorylation of many cellular proteins, including p36, the major substrate of the RSV pp60v-src protein, was detected in Na3VO4-treated mT-transformed F1-11 cells at levels comparable to those observed in RSV-transformed cells. Some of the major protein species recognized by antiphosphotyrosine antibodies in Na3VO4-treated mT-transformed cells displayed electrophoretic mobilities similar to those detected in RSV-transformed F1-11 cells. Tyrosine phosphorylation of p36 was also detected in fibroblasts infected with polyomavirus. There was no detectable difference in the kinase activity of pp60c-src:mT extracted from untreated and Na3VO4-treated mT-transformed cells; however, Na3VO4 treatment of F1-11 and mT-transformed F1-11 cells was shown to inhibit the activity of phosphotyrosine phosphatases in a crude assay of total cellular activity with pp60v-src as the substrate. Thus, Na3VO4 treatment may allow the detection of phosphotyrosine-containing proteins in mT-transformed cells by preventing the turnover of phosphate on substrates phosphorylated by activated cellular protein-tyrosine kinases associated with mT. These results suggest that tyrosine phosphorylation of cellular proteins may be involved in the events that are responsible for mT-induced cellular transformation.

Volatile acid production by Clostridium botulinum type F

1970 ◽  
Vol 16 (6) ◽  
pp. 421-425 ◽  
Author(s):  
C. Wayne Moss ◽  
R. T. Howell ◽  
D. C. Farshy ◽  
V. R. Dowell ◽  
J. B. Brooks
Keyword(s):  
Growth Medium ◽  
Butyric Acid ◽  
Acid Production ◽  
Clostridium Botulinum ◽  
Caproic Acid ◽  
Isovaleric Acid ◽  
Volatile Acid ◽  
Fatty Acid Production ◽  
Botulinum Type ◽  
Geographical Locations

The proteolytic activity and volatile fatty acid production of 10 isolates of Clostridium botulinum type F from diverse geographical locations were determined. Two of the 10 strains were non-proteolytic, 3 were slightly proteolytic, and 5 were strongly proteolytic. The non-proteolytic cultures and the slightly proteolytic cultures produced acetic and butyric acid. The strongly proteolytic cultures produced mainly acetic, butyric, isobutyric, and isovaleric acid, and small to trace amounts of propionic, isocaproic, and caproic acid. The relative amounts of the various acids produced were markedly influenced by the growth medium. The addition of glucose to the growth medium caused an increase in the relative amount of butyric acid and a decrease in isobutyric and isovaleric acid.

scholarly journals Expression of human cholesterol 7α-hydroxylase in atherosclerosis-susceptible mice via adenovirus infection

1997 ◽  
Vol 324 (3) ◽  
pp. 863-867 ◽  
Author(s):  
Gina L. MOORE ◽  
Christian A. DREVON ◽  
Dietrich MACHLEDER ◽  
John D. TRAWICK ◽  
Alan McCLELLAND ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Transcriptional Repression ◽  
Adenovirus Vector ◽  
Chow Diet ◽  
Adenovirus Infection ◽  
Hepatic Inflammation ◽  
Short Term ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Virus Promoter

Adenovirus is a vector for the delivery of genes mainly to the liver. Short-term (~3 days) studies using adenovirus transfection have provided valuable insights into how genes can complement normal and pathological phenotypes. When atherosclerosis-susceptible C57BL/6 mice were infected with an adenovirus vector containing the human 7α-hydroxylase cDNA (AV17h1) and fed on a chow diet, human 7α-hydroxylase mRNA and enzyme activity doubled compared with that in mice infected with an adenovirus vector (AV1Null) alone. In AV17h1-infected mice fed on a high fat cholic acid (HFCA) diet, mRNA expression and activity of both the endogenous and adenovirus (human) 7α-hydroxylase were repressed. AV17h1-infected mice fed on a HFCA diet and killed at mid-light had increased 7α-hydroxylase activity and mRNA compared with mice killed at mid-dark. Since expression of AV17h1 is driven by a constitutive Rous sarcoma virus promoter, the repression of human 7α-hydroxylase by the HFCA diet was unexpected. In spite of this post-transcriptional repression by the HFCA diet, AV17h1-infected mice expressed the human 7α-hydroxylase mRNA, causing its enzyme activity to be 3-fold greater than in AV1Null-infected mice. In AV17h1-infected mice, the 7α-hydroxylase enzyme activity varied as a linear function of human mRNA abundance. In conclusion, the accumulation of apolipoprotein B-containing lipoproteins in plasma of C57BL/6 mice fed on the HFCA diet was not reduced by longer-term (2 weeks) 7α-hydroxylase expression, probably because of its diminished expression caused by the diet and hepatic inflammation from the adenovirus infection. These results may suggest that adenovirus is effective in promoting longer-term (2 weeks) expression of 7α-hydroxylase.

Immunological study of a cellular 35K phosphorylated polypeptide detected in Rous sarcoma virus transformed cells

1983 ◽  
Vol 77 (2-4) ◽  
pp. 195-208 ◽  
Author(s):  
S. Y. Lee ◽  
J. Paire ◽  
G. Vernet ◽  
J. M. Biquard ◽  
V. Krsmanovic
Keyword(s):  
Rous Sarcoma Virus ◽  
Transformed Cells ◽  
Immunological Study ◽  
Rous Sarcoma ◽  
Sarcoma Virus
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