The Effects of Adapting Human Diploid Cells to Grow in Glutamic Acid Media on Cell Morphology, Growth and Metabolism

1973 ◽  
Vol 12 (2) ◽  
pp. 617-629 ◽  
Author(s):  
J. B. GRIFFITHS
Keyword(s):  
Glutamic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Amino Acid Uptake ◽  
Transformed Cells ◽  
Acid Uptake ◽  
Acid Media ◽  
Inhibition Of Growth ◽  
Dependent Inhibition ◽  
Human Diploid Cells ◽  
Diploid Cells

Two lines of human diploid cells, the W1-38 and MRC-5, were adapted to utilize glutamic acid in place of glutamine. This adaptation resulted in (a) more cells per unit culture area, (b) an alteration in cell size and protein content, (c) a morphological change of the cells from fibroblastic to epithelial-like, and (d) increased metabolic activity. Changes in the agglutinability of adapted cells by 3 lectins together with the results from polyacrylamide gel electrophoresis could be interpreted as a change in plasma membrane structure. Comparative studies of unadapted, adapted and transformed cells showed that adaptation to glutamic acid produced cells with a metabolism and amino acid uptake similar to those of transformed cells. These changes were reversible and were not accompanied by any apparent karyological change. The significance of these results for the study of density-dependent inhibition of growth is discussed.

The uptake of amino acids by mouse cells (strain LS) during growth in batch culture and chemostat culture: the influence of cell growth rate

1967 ◽  
Vol 168 (1013) ◽  
pp. 421-438 ◽  
Keyword(s):  
Amino Acids ◽  
Growth Rate ◽  
Amino Acid ◽  
Cell Growth ◽  
Glutamic Acid ◽  
Batch Culture ◽  
Amino Acid Uptake ◽  
Essential Amino Acids ◽  
Growth Yields ◽  
Acid Uptake

The uptake of thirteen essential amino acids by mouse LS cells in suspension culture was determined by bacteriological assay methods. Chemostat continuous-flow cultures were used to determine the effect of different cell growth rates on the quantitative amino acid requirements for growth. The growth yields of the cells ( Y = g cell dry weight produced/g amino acid utilized) were calculated for each of the essential amino acids. A mixture of the non-essential amino acids, serine, alanine and glycine increased the cell yield from the essential amino acids. The growth yields from nearly all the essential amino acids in batch culture were increased when glutamic acid was substituted for the glutamine in the medium. The growth yields from the amino acids in batch culture were much less at the beginning than at the end of the culture. The highest efficiencies of conversion of amino acids to cell material were obtained by chemostat culture. When glutamic acid largely replaced the glutamine in the medium the conversion of amino acid nitrogen to cell nitrogen was 100 % efficient (that is, the theoretical yield was obtained) at the optimum growth rate (cell doubling time, 43 h). The maximum population density a given amino acid mixture will support can be calculated from the data. It is concluded that in several routinely used tissue culture media the cell growth is limited by the amino acid supply. In batch culture glutamine was wasted by (1) its spontaneous decomposition to pyrrolidone carboxylic acid and ammonia, and (2) its enzymic breakdown to glutamic acid and ammonia, but also glutamine was used less efficiently than glutamic acid. Study of the influence of cell growth rate on amino acid uptake rates per unit mass of cells indicated that a marked change in amino acid metabolism occurred at a specific growth rate of 0.4 day -1 (cell doubling time, 43 h). With decrease in specific growth rate below 0.4 day -1 there was a marked stimulation of amino acid uptake rate per cell and essential amino acids were consumed increasingly for functions other than synthesis of cell material.

Liver uptake of amino acids and carbohydrates during a single circulatory passage

1975 ◽  
Vol 228 (4) ◽  
pp. 1155-1161 ◽  
Author(s):  
WM Pardridge ◽  
LS Jefferson
Keyword(s):  
Amino Acids ◽  
Glutamic Acid ◽  
Aspartic Acid ◽  
Amino Acid Uptake ◽  
Ringer Solution ◽  
Transport Systems ◽  
Test Substance ◽  
Acid Uptake ◽  
Liver Uptake ◽  
Rapid Injection

The uptake of 14C-labeled amino acids and carbohydrates by liver following rapid injection into the portal vein was measured relative to a simultaneously injected highly diffusible reference, tritium-labeled water, (3HOH). A 0.25-ml bolus of buffered Ringer solution containing 1-2 muCi of the 14C-labeled test substance and 3-6 muCi of 3HOH was administered by rabid portal injection in anesthetized rats. Circulation was terminated after a single passage of the bolus through the hepatic microvasculature and the tissue was immediately macerated, solubilized, and subjected to liquid scintillation counting. Liver uptake indices (LUI) were calculated from the ratio of 14C to 3H in liver tissue relative to the same ratio in the injection mixture. LUI's of five carbohydrates were measured: sucrose (24.3 percent), inulin (27.7 percent), D-mannitol (80.2 percent), D-glucose (96.8 percent) and L-glucose (26.7 percent). The LUI for cholic acid was 127.1 percent. Among 18 amino acids tested, the LUI's were the highest for the acidic ones (L-aspartic acid, 100.0 percent and L-glutamic acid, 86.4 percent) and lowest for the basic ones (L-arginine, 37.4 percent and L-lysine, 31.4 percent). Stereospecificity for glucose and alanine uptake, saturation kinetics for glutamic acid (Km equal to 4.8 mM) and aspartic acid (Km equal to 2.7 mM), and cross-inhibition among uptake of the acidic amino acids were observed. These findings confirmed the applicability of a technique which was originally developed for studies of amino acid uptake in brain to characterization of transport systems in liver.

Suppression of the transformed phenotype in somatic cell hybrids

1978 ◽  
Vol 33 (1) ◽  
pp. 171-190
Author(s):  
C.J. Marshall ◽  
H. Dave
Keyword(s):  
Somatic Cell ◽  
Tumour Cells ◽  
Transformed Cells ◽  
Somatic Cell Hybrids ◽  
Cell Hybrids ◽  
Recessive Mutations ◽  
Inhibition Of Growth ◽  
Dependent Inhibition ◽  
Hybrid Lines

Somatic cell hybrids between mouse mammary tumour cells (TA3B) and diploid rat embryo fibroblasts (REF) or between TA3B and Syrian hamster sarcoma cells (BI) were examined for the in vitro characteristics of transformed cells as soon as possible after cell fusion. Unlike the parental tumour cells as three of four TA3B X REF and five BI X TA3B independent hybrid lines had low colony-forming efficiencies in agar, exhibited density-dependent inhibition of growth and did not form colonies on confluent monolayers of 3T3 cells, demonstrating that the transformed phenotype was suppressed in these hybrids. In addition tests of some of the hybrid lines for tumour production in nude mice showed that this was also suppressed. Suppression was more stable in the TA3B X REF than in the BI X TA3B hybrids, variants of the BI X TA3B hybrids with the properties of transformed cells could be readily isolated by subculturing cells that had grown in agar. Tumour growth selected for hybrids with the characteristics of transformed cells, and derivatives of the hybrids selected to show the transformed phenotype readily produced tumours. These correlations suggest that the transformed phenotype and malignancy may be under the same control in these cells. The phenomenon of suppression may be explained by the hypothesis that neoplastic transformation results from recessive mutations in genes which control the normal phenotype. On this model the finding of suppression in hybrids between two different tumour lines is interpreted as complementation and indicates that the mutations are not the same in all cell lines.

The Quantitative Utilization of Amino Acids and Glucose and Contact Inhibition of Growth in Cultures of the Human Diploid Cell, WI-38

1970 ◽  
Vol 6 (3) ◽  
pp. 739-749
Author(s):  
J. B. GRIFFITHS
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Basal Medium ◽  
Diploid Cell ◽  
Contact Inhibition ◽  
Growth Media ◽  
Acid Uptake ◽  
Human Diploid Cell ◽  
Inhibition Of Growth ◽  
Diploid Cells

There are many reports in the literature showing that contact inhibition of growth is affected by the culture medium. A quantitative study of amino acid and glucose uptake by the human diploid cell line, WI-38 was carried out to determine more precisely what effect nutritional factors have on contact inhibition of growth. Eagle's minimal essential medium (MEM) was found to support higher cell yields than Eagle's basal medium (BME) and for growth to continue beyond 96 h a medium change was essential. However, analysis of the used growth media showed that neither amino acids nor glucose were fully depleted after 96 h. The rate of glucose utilization was in the range 65-100µg/mg dry wt./h and this agreed very closely with the results of other authors. The pattern of amino acid uptake also closely resembled that for other cell lines except that the utilization of cystine was higher. The nutritional requirements were further studied as the results from the medium analyses failed to explain the growth-promoting activity of MEM. Daily medium changes greatly increased cell yields even though the medium nutrients were not exhausted. This effect was dependent upon fresh medium being used and the only medium component found to be of importance was the amino acid complement. These results are discussed in relation to the low saturation density of diploid cells in culture and a possible explanation is proposed in terms of differences in the cell membrane between normal and altered cells.

scholarly journals Differential effect of clofibrate on inflammation-induced alterations in plasma proteins in the rat

1979 ◽  
Vol 178 (3) ◽  
pp. 633-641 ◽  
Author(s):  
Michael C. Powanda ◽  
Fred B. Abeles ◽  
Karen A. Bostian ◽  
John P. Fowler ◽  
Edward C. Hauer
Keyword(s):  
Amino Acid ◽  
Acute Phase ◽  
Hepatic Metabolism ◽  
Amino Acid Uptake ◽  
Selective Inhibition ◽  
Acid Uptake ◽  
Initial Stimulus ◽  
Dependent Inhibition ◽  
Plasma Copper ◽  
Dose Dependent

Daily intramuscular injections of clofibrate begun 6h before the initiation of inflammation induced by the subcutaneous injection of turpentine exerted a differential, dose-dependent inhibition of the anticipated acute-phase globulin response. Specifically, clofibrate at 140mg/kg muted the increase in α2-macrofoetoprotein, but did not affect that of seromucoid or haptoglobin and only transiently inhibited the rise in copper and the rebound in transferrin. A higher dose, 280mg/kg, markedly suppressed α2-macrofoetoprotein appearance and the rebound in transferrin, somewhat inhibited the increase in seromucoid and haptoglobin and only transiently affected the rise in plasma copper; 420mg of clofibrate/kg very nearly abolished the appearance of α2-macrofoetoprotein, markedly suppressed the transferrin rebound and the increases in seromucoid and haptoglobin and again only transiently affected the increase in copper. Clofibrate did not diminish the localized inflammatory response, did not cause microscopically detectable liver damage and did not prevent the hypozincaemia, hypoalbuminaemia and enhanced amino acid uptake by liver usually associated with inflammation. Thus it is unlikely that clofibrate exerted its dose-dependent selective inhibition by muting the initial stimulus or by impairing hepatic metabolism. This seemingly selective action of clofibrate on plasma-protein alterations during inflammation may provide a means of elucidating the function of individual acute-phase globulin during disease. Clofibrate of itself, apart from inflammation, produced decreases in plasma zinc, copper, transferrin and seromucoid and an increase in hepatic amino acid uptake that were to some extent dependent on the dose of the drug.

Effect of 3-O-substituted analogues of D-glucose and D-allose on amino acid uptake by Penicillium sp.

1985 ◽  
Vol 31 (6) ◽  
pp. 513-518 ◽  
Author(s):  
Robert G. Brown ◽  
John M. Embil
Keyword(s):  
Growth Rate ◽  
Amino Acid ◽  
Glutamic Acid ◽  
Extracellular Enzymes ◽  
Amino Acid Uptake ◽  
Acid Uptake ◽  
Production Time ◽  
Penicillium Species ◽  
Penicillium Sp ◽  
Effect On Growth

3-O-Methyl-D-glucose and 3-O-ethyl-D-allose cause an increase in growth rate of two dextranase-producing Penicillium species which is concomitant with an increase in amino acid uptake. In contrast, 3-O-ethyl-D-glucose and 3-O-methyl-D-allose have no effect on growth and inhibit uptake of L-glutamic acid. These results indicate that considerable specificity is involved in the response of Penicillium sp. to O-alkylated sugars and some O-alkylated sugars may reduce production time of extracellular enzymes.

Studies on Contact Inhibition of Growth in the Mouse Fibroblast, 3T3

1973 ◽  
Vol 12 (3) ◽  
pp. 861-873
Author(s):  
A. I. MEISLER
Keyword(s):  
Amino Acids ◽  
Growth Rate ◽  
Amino Acid ◽  
Amino Acid Uptake ◽  
Mouse Fibroblast ◽  
Acid Uptake ◽  
Initial Growth ◽  
Sephadex Column ◽  
Initial Growth Rate ◽  
Inhibition Of Growth

The effect of amino acid concentration on the initial growth rate of the contact-inhibited mouse fibroblasts 3T3 and its SV40-transformed derivative 3T3T has been studied. In medium in which the serum had been passed through a Sephadex column, and the concentration of amino acids lowered 1000-fold, one acid at a time, the growth rate constants were computed. For most of the 16 amino acids studied, the initial growth rate of 3T3 decreased more profoundly than that of 3T3T. The presence in serum of a factor which stimulates amino acid uptake and growth rate of 3T3T is described.

THE METABOLISM OF ANIMAL TISSUES CULTIVATED IN VITRO: III. AMINO ACID METABOLISM OF STRAIN L CELLS IN COMPLETELY SYNTHETIC MEDIA

1958 ◽  
Vol 36 (1) ◽  
pp. 771-782 ◽  
Author(s):  
Arthur E. Pasieka ◽  
Helen J. Morton ◽  
Joseph F. Morgan
Keyword(s):  
Amino Acid ◽  
Glutamic Acid ◽  
Culture Medium ◽  
Synthetic Medium ◽  
Amino Acid Content ◽  
Amino Acid Uptake ◽  
Acid Uptake ◽  
L Cells ◽  
Synthetic Media

Strain L cells, of mouse fibroblastic origin, have been cultivated in vitro in completely synthetic medium M 150 and in various modifications of this medium. The amino acid changes in the nutrient medium during cell cultivation have been studied by paper chromatography. A characteristic pattern of amino acid uptake and accumulation in the medium has been found. No change in the alanine concentration was observed but omission of alanine from the culture medium resulted in its accumulation in appreciable amounts. Omission of glutamic acid did not alter the pattern of amino acid changes by the cells. Omission of glutamine increased the uptake of amino acids and prevented amino acid accumulation. Omission of both glutamic acid and glutamine resulted in a virtual cessation of amino acid changes in the culture medium. Strain L cells decreased the adenine content of the medium and produced small amounts of hypoxanthine. These changes were not affected by alterations in the amino acid content of the medium. Omission of glutamic acid and glutamine from the culture medium did not cause an appreciable decrease in cell population or apparent degeneration of the cultures over a 30-day period.

Influx of Glycylsarcosine and l-Lysyl-l-Lysine into Hamster Jejunum in Vitro

1980 ◽  
Vol 58 (3) ◽  
pp. 221-225 ◽  
Author(s):  
E. Taylor ◽  
D. Burston ◽  
D. M. Matthews
Keyword(s):  
Amino Acid ◽  
Glutamic Acid ◽  
Amino Acid Uptake ◽  
The Other ◽  
Amino Acid Side Chain ◽  
Acid Uptake ◽  
High Concentration ◽  
Simple Diffusion ◽  
Wide Range

1. This paper reports an investigation of whether the dipeptides glycylsarcosine and l-lysyl-l-lysine share a single mediated transport mechanism into hamster jejunum, or whether one of these peptides is taken up in part by a mediated mechanism unavailable to the other. The investigation, using rings of everted jejunum in vitro, was carried out at pH 5 in order to reduce brush border and/or intramedium hydrolysis of lysyl-lysine. 2. The kinetics of uptake of each peptide was studied over a wide range of concentrations. Estimates of the simple diffusion component in uptake of each peptide were made by the method of self-inhibition of transport as previously described. After correction for simple diffusion, uptake of each peptide conformed to Michaelis-Menten kinetics, and values for Kt and Vmax. were obtained. 3. It was found that each peptide was capable, at infinitely high concentration, of complete inhibition of mediated uptake of the other. The inhibitory effect was competitive. We concluded that glycylsarcosine and lysyl-lysine were taken up by a common mediated mechanism (or possibly mechanisms), neither peptide being taken up by a mediated mechanism unavailable to the other. 4. A previous paper showed that l-glutamyl-l-glutamic acid and glycylsarcosine were taken up by a common mediated mechanism, and this paper shows that l-lysyl-l-lysine and glycylsarcosine are taken up by a common mediated mechanism. It is therefore postulated that the neutral dipeptide glycylsarcosine, the acidic dipeptide glutamyl-glutamic acid and the basic dipeptide lysyl-lysine all share a common mediated mechanism for uptake. This suggests that peptide uptake differs from amino acid uptake in that it is indifferent to the net charge on the amino acid side chain(s).

THE METABOLISM OF ANIMAL TISSUES CULTIVATED IN VITRO: III. AMINO ACID METABOLISM OF STRAIN L CELLS IN COMPLETELY SYNTHETIC MEDIA

1958 ◽  
Vol 36 (7) ◽  
pp. 771-782 ◽  
Author(s):  
Arthur E. Pasieka ◽  
Helen J. Morton ◽  
Joseph F. Morgan
Keyword(s):  
Amino Acid ◽  
Glutamic Acid ◽  
Culture Medium ◽  
Synthetic Medium ◽  
Amino Acid Content ◽  
Amino Acid Uptake ◽  
Acid Uptake ◽  
L Cells ◽  
Synthetic Media

Strain L cells, of mouse fibroblastic origin, have been cultivated in vitro in completely synthetic medium M 150 and in various modifications of this medium. The amino acid changes in the nutrient medium during cell cultivation have been studied by paper chromatography. A characteristic pattern of amino acid uptake and accumulation in the medium has been found. No change in the alanine concentration was observed but omission of alanine from the culture medium resulted in its accumulation in appreciable amounts. Omission of glutamic acid did not alter the pattern of amino acid changes by the cells. Omission of glutamine increased the uptake of amino acids and prevented amino acid accumulation. Omission of both glutamic acid and glutamine resulted in a virtual cessation of amino acid changes in the culture medium. Strain L cells decreased the adenine content of the medium and produced small amounts of hypoxanthine. These changes were not affected by alterations in the amino acid content of the medium. Omission of glutamic acid and glutamine from the culture medium did not cause an appreciable decrease in cell population or apparent degeneration of the cultures over a 30-day period.

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