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Title: Carbohydrate metabolism of cells transformed by the polyoma virus
Author: Broadfoot, Marcella M.
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1966
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1. Nutritional studies on strain L cells. (a) The amino acids essential for the growth of strain L cells in serum-free medium were found to be: arginine, cystine, glutamine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Giutamic acid, aspartic, acid and plycine were not essential for optimal growth over a four day period. It is suggested that the requirement for proline and serine (which are not present in some other synthetic media) was due to the low cell inoculum used; and the short growth period. Conversion was shown to occur between phenylalanine and tyrosine in vitro but not between cystine and methionine. (b) Interaction was shown to exist between isoleucine, leucine, and valine, and between arginine and lysine. Thus not only the concentre of these amino acids in media are important but also the ratios of their concentrations. (c) From these studies HERT 1 medium was evolved, containing the optimal concentrations of these essential amino acids in a medium based on Waymouth's MB 752/1 medium (Waymouth, 1959). 2. Environmental factors and respiration. The respiration of five cell strains (normal human foreskin fibroblast, HeLa, HLM, Y5 and L5178Y strains) was studied with particular reference to influences of environmental factors on cellular respiration. (a) The respiration of human fibroblast and L5178Y cells declined rapidly after inoculation into new medium. This fall in respiration could be prevented by adding Kreba citric acid cycle intermediates to the medium. Strain L cells have been shown to exhibit similar behaviour (Danes and Paul, 196l). The respiration of HeLa, HLM, mid Y5 cells was not affected by change in medium. (b) In all cells respiration was less in cultures maintained at pH 7.8 than in cultures maintained at 7.4. Respiration was also lower at pH 6.8, with the exception of HLM cells. (c) All cells exhibited a Crabtree effect (inhibition of respiration by glycolysis). (d) Respiration was reduced when cultures were maintained in oxygen tensions above or below that obtained in equilibrium with air. (e) The respiratory rate of all cells was higher in the presence of 1 per cent. CO2 than at lower concentrations. Higher concentrations were slightly inhibitory. Carbohydrate metataolism of BHK 21 strain. Comparisons of carbohydrate metabolism were mad& between clones of BHK21 strains which had been transformed by polyoma virus in vitro and clones which had not been transformed. Since 'transferred' cells produced tumours in vivo, the use of this system enabled comparisons to be made between cells, capable and incapable of producing tumours in vivo, both derived from the same cell. Both cell types were swing at similar rates in a controlled Environment. Six lines of cells were used, of which three were 'normal' and three 'transformed'. (a) Respiration. The factors previously shown to affect the respiration of cultured cells were found to affect respiration of 'normal' and 'transformed' BHK21 ceils in a similar manner. When respiration was measured under standard conditions, there was no significant difference in the respiratory rate of 'normal' and 'transformed' BHK21 cells. (b) Glycolysis. Environmental factors have previously been shown to affect glycolysis (Paul, 1959). Glycolytic rates of BHK21 cells were therefore measured under four sets of standard conditions (pH's 6.8 or 7.4 in an atmosphere of air/5 per cent, CO2 or nitrogen/5 per cent. 602). Under all these environmental conditions, glycolysis was higher in the 'transformed' cells than in the 'normal' calls. (c) Enzymes The activities of several enzymes of carbohydrate metabolism were assayed. No glucose-6-phosphatase activity was demonstrated. The activities of lactic dehydrogenase, malic dehydrogenase, and 6- phosphogluconate dehydrogenase were similar in the 'normal' and 'transformed' cells. Hexokinase activity was higher in the 'transformed' cell than in the 'normal' cell, a finding which correlates with the increased glycolysis in the 'transformed' cell. Isocitric dehydrogenase was also higher in the 'transformed' cell though this result is not parallelled by an increased respiratory rate. Glucose-6-phosphate dehydrogenase activity was lower in the 'transformed' cell than in the 'normal' cell. It is suggested that, since both cell types have similar growth rates, this low activity in the 'transformed' cell must be sufficient to provide pentoses for nucleic acid production during growth. Possible reasons for the differences are discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available