Surface Modification and Electrophoresis of Normal and Transformed BHK21 Cells

1974 ◽  
Vol 14 (1) ◽  
pp. 203-214
Author(s):  
A. L. LATNER ◽  
G. A. TURNER
Keyword(s):  
Electrophoretic Mobility ◽  
Cell Types ◽  
Transformed Cells ◽  
Normal Cells ◽  
Neuraminidase Treatment ◽  
Total Histone ◽  
Electrophoretic Mobilities ◽  
Formaldehyde Treatment ◽  
The Mean ◽  
Number Of Cells

The mean electrophoretic mobilities at pH 7.5 of virus-transformed (Py6) and normal BHK21 cells were very similar, whether they were harvested mechanically or by the use of trypsin. After formaldehyde treatment, there was significantly increased mobility in both cell types; the transformed cells showed significantly the greater change. After neuraminidase treatment, the mean electrophoretic mobility was decreased to the same extent in both types of cell. Treatment with neuraminidase and formaldehyde had no effect on the mean electrophoretic mobility of the normal cells but slowed the transformed variety. The mobility in histone solution had no relationship to histone concentration but was statistically correlated with the amount of histone per cell, calculated from total histone present divided by the total number of cells; a linear relationship being obtained with the normal cells but an initial plateau was demonstrated with the transformed cells. The normal cell line showed a similar plateau after neuraminidase treatment. The possible significance of these results is discussed.

scholarly journals Loss of thrombospondin transcriptional activity in nickel-transformed cells.

1994 ◽  
Vol 14 (1) ◽  
pp. 851-858 ◽  
Author(s):  
K Salnikow ◽  
S Cosentino ◽  
C Klein ◽  
M Costa
Keyword(s):  
Chinese Hamster ◽  
Restriction Enzymes ◽  
Cell Types ◽  
Suppressor Gene ◽  
Transformed Cells ◽  
Normal Cells ◽  
Nickel Compounds ◽  
Embryo Cells ◽  
Rb Gene

mRNA from normal Chinese hamster embryo (CHE) cells was transcribed to cDNA and subtracted with an excess of mRNA from Chinese hamster embryo cells transformed by nickel compounds. Here we report the recovery of a sequence found to be highly homologous to the mouse thrombospondin 1 gene that was obtained by this subtraction procedure. Since thrombospondin is antiangiogenic, cancer cells expressing high levels of thrombospondin cannot grow in vivo because capillaries will not proliferate to cells secreting thrombospondin. To examine expression of thrombospondin, normal CHE cells were stained with monoclonal antibodies to human thrombospondin. The protein was present abundantly in the cytoplasm of normal cells but at greatly reduced levels in Ni-transformed cells. Analysis of mRNA by Northern (RNA) blot revealed transcripts in normal cells but little thrombospondin mRNA in Ni-transformed cells. Loss of thrombospondin mRNA expression was related to Ni treatment rather than transformation, since Ni-resistant cells also exhibited fewer thrombospondin transcripts than did wild-type cells. Digestion of genomic DNA with various combinations of restriction enzymes revealed thrombospondin gene patterns that were identical in both cell types, suggesting that there were no major deletions or rearrangements of the gene in the nickel-transformed cells. The inactivation of the thrombospondin gene was further investigated by analyzing the promoter activity of this gene linked to a chloramphenicol acetyltransferase (CAT) reporter plasmid that was transfected into normal and Ni-transformed cells. The CAT activity in normal cells was significantly higher than in Ni-transformed cells, suggesting that the promoter region of thrombospondin was less efficiently transcribed in Ni-transformed cells. We studied the consequences of enhanced expression of the retinoblastoma (Rb) gene, a known tumor suppressor gene, on CAT transcription driven by the human thrombospondin promoter. Cotransfection of an expression vector containing the mouse Rb gene greatly enhanced the transcription from the thrombospondin promoter such that the expression was higher in normal cells than in transformed cells.

Capillary Electrophoresis Measurements of Electrophoretic Mobility for Colloidal Particles of Biological Interest

1998 ◽  
Vol 64 (7) ◽  
pp. 2572-2577 ◽  
Author(s):  
J. R. Glynn ◽  
B. M. Belongia ◽  
R. G. Arnold ◽  
K. L. Ogden ◽  
J. C. Baygents
Keyword(s):  
Capillary Electrophoresis ◽  
Surface Charge ◽  
Electrophoretic Mobility ◽  
Colloidal Particles ◽  
Bacterial Strains ◽  
Electrophoretic Mobilities ◽  
Biological Interest ◽  
The Mean ◽  
Standard Deviations ◽  
First Time

ABSTRACT The electrophoretic mobilities of three bacterial strains were investigated by capillary electrophoresis (CE) and were compared with results obtained by microelectrophoresis (ME). The CE measurements yielded bimodal electropherograms for two of the strains, thus illustrating for the first time that surface charge variations within a monoclonal population can be probed by CE. Intrapopulation variations were not detected by ME. The mobilities of three chemically distinct types of latex microspheres were also measured. Differences between the mean mobilities obtained by CE and ME were not statistically significant (P ≤ 0.50); the standard deviations of the CE measurements were typically 2 to 10 times smaller than those obtained by comparable ME measurements. The reproducibility of CE permitted batch-to-batch mobility variations to be probed for the bacteria (one of the strains exhibited such variations), and aggregation was evident in one of the latex suspensions. These effects were not measurable with ME.

scholarly journals A new method for the preperative and analytical electrophoresis of cells

2006 ◽  
Vol 11 (4) ◽  
Author(s):  
Anna Wilk ◽  
Kinga Rośkowicz ◽  
Włodzimierz Korohoda
Keyword(s):  
Red Blood Cells ◽  
Surface Properties ◽  
Electrophoretic Mobility ◽  
Blood Cells ◽  
Cell Types ◽  
Porous Matrix ◽  
Simple System ◽  
New Method ◽  
Electrophoretic Mobilities ◽  
Electrophoretic Mobility Of Cells

AbstractIn this paper, a new method is described for the horizontal electrophoresis of cells on a density cushion under near-isopycnic conditions. When cell sedimentation is minimized, the electrophoresis of red blood cells (RBC) used as model cells within an anti-convective porous matrix (with pores over 300 μm in diameter) was capable of separating a mixture of human and chicken RBC according to their electrophoretic mobilities. Samples taken from the separated RBC bands show over 90% purity for each species. The simultaneous electrophoresis of several RBC samples carried out under identical conditions permitted the use of comparative data based on the electrophoretic mobility of cells which differ in their surface properties. We believe that this relatively simple system, in which cell sedimentation and convection are minimized, has the potential to be modified and adapted for the separation of other cell types/organelles.

Electrophoretic variants of alanine aminopeptidase, alkaline phosphatase, and gamma-glutamyltransferase in urine.

1984 ◽  
Vol 30 (6) ◽  
pp. 856-859 ◽  
Author(s):  
K Jung ◽  
U W Wischke
Keyword(s):  
Alkaline Phosphatase ◽  
Electrophoretic Mobility ◽  
Brush Border ◽  
Neuraminidase Treatment ◽  
Electrophoretic Variants ◽  
Brush Border Enzymes ◽  
Gamma Glutamyltransferase ◽  
Electrophoretic Mobilities ◽  
Alanine Aminopeptidase ◽  
Triton X

Abstract The three brush-border enzymes--alanine aminopeptidase (EC 3.4.11.2), alkaline phosphatase (EC 3.1.3.1), and gamma-glutamyltransferase (EC 2.3.2.2)--are present in the urine of healthy persons in two variants, a particulate form and a soluble one. They can be separated by electrophoresis in agarose gel and by ultracentrifugation. The particulate forms exhibit similar electrophoretic mobility, but the soluble forms of these brush-border enzymes differ in their electrophoretic mobilities. The enzyme components of the particulate activity can be mobilized by Triton X-100 and trypsin. The electrophoretic mobility of the soluble forms of alanine aminopeptidase and gamma-glutamyltransferase is slowed by neuraminidase treatment. Both forms of gamma-glutamyltransferase are influenced in their electrophoretic mobility by treatment with n-butanol/diisopropyl ether, showing their lipid dependence. These findings enhance our knowledge of the biochemical nature of brush-border enzymes in urine.

scholarly journals Concomitant loss of cell surface fibronectin and laminin from transformed rat kidney cells

1981 ◽  
Vol 88 (2) ◽  
pp. 352-357 ◽  
Author(s):  
EG Hayman ◽  
E Engvall ◽  
E Ruoslahti
Keyword(s):  
Molecular Weight ◽  
Cell Surface ◽  
Cell Attachment ◽  
Culture Media ◽  
Rat Kidney ◽  
Cell Types ◽  
Transformed Cells ◽  
Kidney Cells ◽  
Normal Cells ◽  
The Matrix

Both fibronectin and laminin were found by immunofluorescence as a matrix at the surface of normal rat kidney cells. These matrices were absent from the surface of virally transformed rat kidney cells. Soluble fibronectin and laminin were detected in the culture media of the transformed as well as the normal cells. Culture supernates of the transformed cells contained even more fibronectin than the supernates of the transformed cells contained even more fibronectin than the supernates of the normal cells while laminin was present in similar amounts in both culture media. This shows that the loss of fibronectin and laminin from the surface of the transformed cells is caused by failure of the cells to deposit these proteins into an insoluble matrix and not caused by inadequate production. Fibronectins isolated from culture media of the normal and transformed cells were similar in SDS polyacrylamide gel electrophresis. Laminin isolated from culture media by affinity chromatography on heparin-Sepharose followed by immunoprecipitation was composed of three main polypeptides, one with a molecular weight of 400,000 and two with a molecular weight close to 200,000 in both cell types. Fibronectins from both cell types were equally active in promoting cell attachment. Rat fibronectin from transformed cells, like normal cells, when applied to culture dishes coated with fibronectin, readily attached and spread on the substratum, requiring approximately the same amount of fibronectin as the normal cells. On the basis of these results it seem that the failure of the transformed cells to incorporate fibronectin into an insoluble cell surface matix is not a consequence of a demonstrable change in the functional characteristics of the fibronectin molecule or in the ability of the cells to interact with fibronectin. It may depend on as yet unidentified interactions of the cell surface. Similar interactions may be needed for the deposition of laminin into the matrix, because laminin was also absent from the surface of transformed cells, despite its being synthesized by these cells.

Loss of thrombospondin transcriptional activity in nickel-transformed cells

1994 ◽  
Vol 14 (1) ◽  
pp. 851-858
Author(s):  
K Salnikow ◽  
S Cosentino ◽  
C Klein ◽  
M Costa
Keyword(s):  
Chinese Hamster ◽  
Restriction Enzymes ◽  
Cell Types ◽  
Suppressor Gene ◽  
Transformed Cells ◽  
Normal Cells ◽  
Nickel Compounds ◽  
Embryo Cells ◽  
Rb Gene

mRNA from normal Chinese hamster embryo (CHE) cells was transcribed to cDNA and subtracted with an excess of mRNA from Chinese hamster embryo cells transformed by nickel compounds. Here we report the recovery of a sequence found to be highly homologous to the mouse thrombospondin 1 gene that was obtained by this subtraction procedure. Since thrombospondin is antiangiogenic, cancer cells expressing high levels of thrombospondin cannot grow in vivo because capillaries will not proliferate to cells secreting thrombospondin. To examine expression of thrombospondin, normal CHE cells were stained with monoclonal antibodies to human thrombospondin. The protein was present abundantly in the cytoplasm of normal cells but at greatly reduced levels in Ni-transformed cells. Analysis of mRNA by Northern (RNA) blot revealed transcripts in normal cells but little thrombospondin mRNA in Ni-transformed cells. Loss of thrombospondin mRNA expression was related to Ni treatment rather than transformation, since Ni-resistant cells also exhibited fewer thrombospondin transcripts than did wild-type cells. Digestion of genomic DNA with various combinations of restriction enzymes revealed thrombospondin gene patterns that were identical in both cell types, suggesting that there were no major deletions or rearrangements of the gene in the nickel-transformed cells. The inactivation of the thrombospondin gene was further investigated by analyzing the promoter activity of this gene linked to a chloramphenicol acetyltransferase (CAT) reporter plasmid that was transfected into normal and Ni-transformed cells. The CAT activity in normal cells was significantly higher than in Ni-transformed cells, suggesting that the promoter region of thrombospondin was less efficiently transcribed in Ni-transformed cells. We studied the consequences of enhanced expression of the retinoblastoma (Rb) gene, a known tumor suppressor gene, on CAT transcription driven by the human thrombospondin promoter. Cotransfection of an expression vector containing the mouse Rb gene greatly enhanced the transcription from the thrombospondin promoter such that the expression was higher in normal cells than in transformed cells.

Differences in Membrane Order between C 3 H 10 T1/2 Cells and Their Transformed Counterparts as Measured by EPR

1992 ◽  
Vol 47 (1-2) ◽  
pp. 148-154 ◽  
Author(s):  
G. F. Grossi ◽  
M. Durante ◽  
M. Napolitano ◽  
A. Lanzone ◽  
P. Riccardi ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Hyperfine Splitting ◽  
Cell Transformation ◽  
Lipid Analysis ◽  
Cell Types ◽  
Transformed Cells ◽  
Fatty Acids Composition ◽  
Normal Cells ◽  
Membrane Order ◽  
Mouse Embryo Fibroblasts

Abstract Membrane order of mouse embryo fibroblasts and their ionizing radiation and chemically transformed counterparts was investigated using EPR spectroscopy after labeling the membrane of the cells with the fatty acid spin label, 5-nitroxy stearic acid. The EPR spectra were recorded at temperatures ranging from 18 to 38 °C for both control and transformed cells. The distance between the outer hyperfine splitting (2 T′||), which is used as an indicator of membrane order, varies in these two cell types. Below 28 °C, 2T′II is higher in transformed fibroblasts than in normal cells, whereas above this temperature membrane order is the same. Lipid analysis as carried out by the measurement of the cholesterol/membrane proteins and sphingomyelin/lecithin ratios, showed no difference in the amounts of the main membrane rigidifiers. These findings suggest that cell transformation of mouse fibroblasts induced by radiation or chemicals may produce alterations in the cell membrane, as evidenced by variations in its order at low temperature. These measured differences are presumably not attributable to its fatty acids composition but to its glycoproteins content, since changes in membrane regidifiers were not observed between normal and transformed cells.

scholarly journals Exosome Traceability and Cell Source Dependence on Composition and Cell-Cell Cross Talk

2021 ◽  
Vol 22 (10) ◽  
pp. 5346
Author(s):  
Rabab N. Hamzah ◽  
Karrer M. Alghazali ◽  
Alexandru S. Biris ◽  
Robert J. Griffin
Keyword(s):  
Donor Cell ◽  
Average Diameter ◽  
Cell Types ◽  
Cell Interaction ◽  
Target Cells ◽  
Remote Communication ◽  
Normal Cells ◽  
Growth Environment ◽  
Cell Components ◽  
The Impact

Exosomes are small vesicles with an average diameter of 100 nm that are produced by many, if not all, cell types. Exosome cargo includes lipids, proteins, and nucleic acids arranged specifically in the endosomes of donor cells. Exosomes can transfer the donor cell components to target cells and can affect cell signaling, proliferation, and differentiation. Important new information about exosomes’ remote communication with other cells is rapidly being accumulated. Recent data indicates that the results of this communication depend on the donor cell type and the environment of the host cell. In the field of cancer research, major questions remain, such as whether tumor cell exosomes are equally taken up by cancer cells and normal cells and whether exosomes secreted by normal cells are specifically taken up by other normal cells or also tumor cells. Furthermore, we do not know how exosome uptake is made selective, how we can trace exosome uptake selectivity, or what the most appropriate methods are to study exosome uptake and selectivity. This review will explain the effect of exosome source and the impact of the donor cell growth environment on tumor and normal cell interaction and communication. The review will also summarize the methods that have been used to label and trace exosomes to date.

Glycosylation of developing human glomeruli: lectin binding sites during cell induction and maturation.

1987 ◽  
Vol 35 (1) ◽  
pp. 33-37 ◽  
Author(s):  
H Holthöfer ◽  
I Virtanen
Keyword(s):  
Binding Sites ◽  
Mesangial Cells ◽  
Ricinus Communis ◽  
Lectin Binding ◽  
Helix Pomatia ◽  
Cell Types ◽  
Basement Membranes ◽  
Phenotypic Change ◽  
Neuraminidase Treatment ◽  
Nephron Development

Expression of cellular glycoconjugates during differentiation of human fetal kidney was studied using fluorochrome-labeled lectins. Each lectin revealed a characteristic binding pattern during the phenotypic change of the nephrogenic mesenchyme and during distinct stages of nephron development. The uninduced mesenchymal cells were positive for Pisum sativum (PSA), Concanavalin A (ConA), Wistaria floribunda (WGA), and Ricinus communis (RCA-I) lectins. However, these lectins failed to react with the uninduced cells of the S-shaped bodies, whereas Maclura pomifera (MPA), Triticum vulgaris (WGA) and, after neuraminidase treatment, Arachis hypogaea (PNA) agglutinins bound intensely to the presumptive podocytes. During later stages of nephrogenesis, MPA positively on the podocytes weakened and could not be observed in adult kidney glomeruli. Binding sites for Helix pomatia (HPA) agglutinin in glomeruli were also expressed only transiently during nephrogenesis. During further development PSA, ConA, WFA, and RCA-I reacted with mesangial cells in addition to the glomerular basement membranes. The segment-specific lectin binding patterns of the tubuli emerged in parallel with the appearance of brush border and Tamm-Horsfall antigens of the proximal and distal tubuli. The results show that nephron site-specific saccharides appear in a developmentally regulated manner and in parallel with morphologic maturation of the nephron. Lectins therefore appear to be useful tools for study of induction and maturation of various nephron cell types.

scholarly journals Impaired clearance of free cystine from lysosome-enriched granular fractions of I-cell-disease fibroblasts

1986 ◽  
Vol 237 (1) ◽  
pp. 9-15 ◽  
Author(s):  
F Tietze ◽  
L H Rome ◽  
J D Butler ◽  
G S Harper ◽  
W A Gahl
Keyword(s):  
Mutant Cell ◽  
Cell Types ◽  
Nephropathic Cystinosis ◽  
Cell Disease ◽  
Rapid Loss ◽  
Normal Cells ◽  
Cultured Fibroblasts ◽  
Carrier Mechanism ◽  
Mucolipidosis Ii

Cultured fibroblasts from patients with I-cell disease (mucolipidosis II) accumulate excessive amounts of free cystine, similarly to cells from patients with nephropathic cystinosis, a disorder of lysosomal cystine transport. To clarify whether the intralysosomal accumulation of cystine in I-cell-disease fibroblasts was due to a defective disposal mechanism, we measured the rates of clearance of free [35S]cystine from intact normal, cystinotic and I-cell-disease fibroblasts. Loss of radioactivity from the two mutant cell types occurred slowly (t 1/2 = 500 min) compared with the rapid loss from normal cells (t 1/2 = 40 min). Lysosome-rich granular fractions isolated from three different cystine-loaded normal, cystinotic and I-cell-disease fibroblast strains were similarly examined for non-radioactive cystine egress. Normal granular fractions lost cystine rapidly (mean t 1/2 = 43 min), whereas cystinotic granular fractions did not lose any cystine (mean t 1/2 = infinity). I-cell-disease granular fractions displayed prolonged half-times for cystine disposal (mean = 108 min), suggesting that I-cell-disease fibroblasts, like cystinotic cells, possess a defective carrier mechanism for cystine transport.

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