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Thymidine transport in phytohaemagglutinin-stimulated pig lymphocytes.

ABSTRACT: Thymidine and uridine transporters in peripheral pig lymphocytes have structural features in common, but are not identical. Accelerated entry of [3H]thymidine begins 12h after the addition of phytohaemagglutinin. The increased thymidine uptake into the cells is characterized by an increase in Vmax. Without alteration of the apparent Km(0.6+/-0.08muM). Thymidine kinase activity is increased 12h after stimulation. Both the increased thymidine uptake and the increased thymidine kinase activity are inhibited in cultures incubated with puromycin: rates of degradation of the two systems are unchanged after phytohaemagglutinin addition, and indicate similar half-lives of about 2h. Thymidine kinase is rate-limiting for thymidine entry up to 18h after phytohaemagglutinin addition; increase in its synthesis is detectable about 6h before net incorporation of thymidine into DNA is significantly promoted.

SUBMITTER: Barlow SD

PROVIDER: S-EPMC1165537 | biostudies-other | 1975 May

REPOSITORIES: biostudies-other

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Deoxycytidine transport and pyrimidine deoxynucleotide metabolism in phytohaemagglutinin-stimulated pig lymphocytes.

Project description:Increased entry of deoxy[3H]cytidine begins at about 12h after addition of phytohaemagglutinin to peripheral pig lymphocyte cultures, and is accompanied by a parallel stimulation of deoxycytidine kinase up to the beginning of DNA synthesis at 24h. The increased deoxycytidine uptake is characterized by an increase in Vmax. without alteration of the apparent Km (0.7 +/- 0.11 muM). Although the entries of both nucleosides are promoted at the same time, the stimulation of deoxycytidine uptake is less than that of thymidine, and the two nucleosides are transported by separate systems. In addition to deoxycytidien kinase, the synthesis of deoxycytidylate deaminase and thymidylate synthetase are stimulated after addition of phytohaemagglutinin, but to a lesser extent than that of thymidine kinase. The importance of the latter enzyme in forming dTMP, and of thymidylate kinase in providing dTTP, is discussed.

| S-EPMC1172720 | biostudies-other

The effect of various concentrations of nucleobases, nucleosides or glutamine on the incorporation of [3H]thymidine into DNA in rat mesenteric-lymph-node lymphocytes stimulated by phytohaemagglutinin.

Project description:1. The rate of [3H]thymidine incorporation into DNA was measured in phytohaemagglutinin-stimulated lymph-node lymphocytes of the rat. 2. Addition of nucleobases or nucleosides to culture medium that already contained 0.2 mM-glutamine had a small stimulatory effect on incorporation. At lower concentrations of glutamine, adenosine (even at 1 microM) caused a marked increase in the rate of incorporation. 3. In the absence of added glutamine, addition of nucleosides or nucleobases markedly increased the rate of incorporation: nucleosides were more effective than nucleobases; and the rate of proliferation in the presence of 10 microM-adenosine plus 10 microM-uridine was similar to that in the presence of optimal concentrations of glutamine. 4. The rate of incorporation was dramatically decreased by an inhibitor of the pathway of pyrimidine nucleotide synthesis de novo. Addition of the pyrimidine nucleosides completely overcame the inhibition; addition of the pyrimidine nucleobases was much less effective. 5. These results indicate that, for proliferation of lymphocytes, glutamine is not essential and can be partially or totally replaced by nucleosides and, to some extent, by nucleobases.

| S-EPMC1131741 | biostudies-other

The effect of glutamine concentration on the activity of carbamoyl-phosphate synthase II and on the incorporation of [3H]thymidine into DNA in rat mesenteric lymphocytes stimulated by phytohaemagglutinin.

Project description:The maximum catalytic activities of carbamoyl-phosphate synthase II, a limiting enzyme for pyrimidine nucleotide synthesis, are very much less than those of glutaminase, a limiting enzyme for glutamine utilization, in lymphocytes and macrophages; and the flux through the pathway for pyrimidine formation de novo is only about 0.4% of the rate of glutamine utilization by lymphocytes. The Km of synthase II for glutamine is about 16 microM and the concentration of glutamine necessary to stimulate lymphocyte proliferation half-maximally is about 21 microM. This agreement suggests that the importance of glutamine for these cells is provision of nitrogen for biosynthesis of pyrimidine nucleotides (and probably purine nucleotides). However, the glutamine concentration necessary for half-maximal stimulation of glutamine utilization (glutaminolysis) by the lymphocytes is 2.5 mM. The fact that the rate of glutamine utilization by lymphocytes is markedly in excess of the rate of the pathway for pyrimidine nucleotide synthesis de novo and that the Km and 'half-maximal concentration' values are so different, suggests that the glutaminolytic pathway is independent of the use of glutamine nitrogen for pyrimidine synthesis.

| S-EPMC1138925 | biostudies-other

Enhanced transport of natural amino acids after activation of pig lymphocytes.

Project description:Na+-dependent uptake of the amino acids L-proline and L-methionine was greatly accelerated when pig lymphocytes were activated with phytohaemagglutinin or other mitogens. The increased influx was apparent after incubation with phytohaemagglutinin for 1 h, and reached a maximum after 24 h. The lymphocytes appear to possess at least three different transport systems for neutral amino acids with properties similar to, but not identical with, those described for other cells. The activity of a system resembling the A system of other cells was increased most dramatically after activation, its activity in unstimulated lymphocytes being extremely low or absent. A second Na+-dependent system, which has properties similar to those of the ASC system in other cells, but with a broader specificity for amino acids, was more active in unstimulated lymphocytes, and uptake by this system was also accelerated after incubation with phytohaemagglutinin. The activity of a third system, very similar to the L system in other cells, was increased to a much smaller extent after lymphocyte activation.

| S-EPMC1161230 | biostudies-other

Early effects of phytohaemagglutinin on glucose metabolism of normal human lymphocytes.

Project description:1. Phytohaemagglutinin induced early changes in the catabolism of glucose by normal human lymphocytes suspended in a bicarbonate buffer. During 4hr. incubation glucose utilization was almost doubled. 2. The rates of several reactions in the metabolism of glucose were estimated. Total pyruvate formation, lactate production and fatty acid synthesis were stimulated to the same degree as was glucose utilization. The pentose cycle and the glycogen synthesis were also stimulated but less than was glucose utilization. The pentose cycle was found to account for 1.4% and 0.9% of the total glucose utilization without and with phytohaemagglutinin respectively. In these cells rates of triose phosphate iso-merization were at least six to seven times the rate of glucose phosphorylation. On an average 55-60% of the total carbon dioxide evolved was derived from decarboxylation of pyruvate, 25-30% from the tricarboxylic acid cycle and about 15% from the pentose cycle. Observed ratios of (14)C specific yields in glycogen from [1-(14)C]- and [6-(14)C]-glucose could possibly be explained by assuming the existence of two separate glucose 6-phosphate pools. 3. During 4hr. incubation in bicarbonate buffer (14)C from [U-(14)C]serine was incorporated into perchloric acid-insoluble material. This incorporation was stimulated by phytohaemagglutinin but was almost completely inhibited by puromycin. Puromycin also abolished phytohaemagglutinin-induced stimulation of glycolysis.

| S-EPMC1187214 | biostudies-other

Components of the cytosolic and released virtosomes from stimulated and non-stimulated human lymphocytes.

Project description:Abstract aimThis work intends to analyse the structure and the composition of virtosomes and their role.BackgroundVirtosomes are newly synthesized DNA-RNA-lipoprotein complexes released from living cells in a regulated and energy-dependent manner.MethodsVirtosome fractions were isolated by ultracentrifugation from human lymphocytes cytoplasm and from culture medium before and after stimulation with phitoemoagglutinin (PHA). The composition in DNA, RNA, protein and lipids was determined. The virtosomes present in the culture medium were put in contact with lymphocytes.ResultsVirtosome fractions released from non-stimulated lymphocytes are shown to reduce replication of stimulated lymphocytes and those from stimulated lymphocytes to increase multiplication of non-stimulated lymphocytes. Biochemical analyses of the virtosomal complexes have shown that those from stimulated lymphocytes have five proteins that are absent from non-stimulated virtosome fractions. A comparison of the cytosolic versus released virtosome fractions from non-stimulated lymphocytes indicated that there is a greater percentage of phospholipids in the released virtosomes with a corresponding decrease in the percentage of protein.ConclusionAlthough there is a presence of cholesterol in the virtosomes, the low levels of phosphatidylcholine and cholesterol, together with the low ratios of cholesterol: phospholipids leads to a confirmation of the apparent lack of a limiting membrane around the virtosomes.General significanceVirtosomes are structural particles formed in the cytoplasm, released from the cells and capable to be transferred in other cells influencing their behaviour.

| S-EPMC5600339 | biostudies-literature

The role of zinc ions in the transformation of lymphocytes by phytohaemagglutinin.

Project description:1. Incorporation of [(3)H]thymidine into DNA was inhibited by EDTA and diethylenetriamine-NNN'N''N''-penta-acetate but not by nitrilotriacetate even when Ca(2+) was present at more than twice the concentration of the chelators. 2. The inhibition caused by EDTA was most effectively reversed by Zn(2+) but also to a lesser extent by Cd(2+). Very little if any activation of the EDTA-inhibited system was obtained with Co(2+), Cu(2+), Fe(3+), Mn(2+) or Ni(2+) added alone. 3. Fe(3+) added to the Zn(2+)-activated lymphocytes in the presence of EDTA markedly increased thymidine incorporation. Addition of Cd(2+) prevented the inhibition of incorporation which occurred at high Zn(2+) concentrations. 4. If EDTA was added more than 15h after phytohaemagglutinin, the inhibition of incorporation was less than that obtained by its addition at zero time. If Zn(2+) was added later than 12h after EDTA and phytohaemagglutinin, the inhibition of incorporation by EDTA was not fully reversed. A study of the time-course of the effects of delayed additions of EDTA and Zn(2+) suggested that, on average, the cells required Zn(2+) between 20 and 30h after phytohaemagglutinin addition to allow the full rate of thymidine incorporation at 37h. 5. The increase in the rate of protein synthesis caused by phytohaemagglutinin was not inhibited by EDTA until about 8h. The further increase after this was totally inhibited by EDTA but this inhibition was fully reversible with Zn(2+). The rate of protein synthesis in EDTA-inhibited cultures at 40h was the same as that at 10h. 6. There was a close similarity between the effects of EDTA on lymphocyte stimulation and those reported by Kay et al. (1969) with low doses of actinomycin D.

| S-EPMC1174308 | biostudies-other

Inhibition of the specific binding of human lactotransferrin to human peripheral-blood phytohaemagglutinin-stimulated lymphocytes by fluorescein labelling and location of the binding site.

Project description:Labelling of human lactotransferrin with fluorescein 5'-isothiocyanate (FITC) in an equimolar ratio inhibits the binding of the protein to phytohaemagglutinin-activated human peripheral-blood lymphocytes. Therefore it can be assumed that FITC reacts at, or near, the receptor-binding site. Three FITC-labelled peptides have been purified from a tryptic digest of the FITC-labelled lactotransferrin. The determination of their amino acid sequence and their localization on the primary structure of the protein permitted the identification of two FITC-accessible areas in the N-terminal lobe and one in the C-terminal lobe. In fact, only 10% of the total FITC was conjugated to one lysine residue (Lys579) of the C-terminal lobe, whereas most (80%) of the FITC was conjugated to three close lysine residues [Lys263 (65% of total fluorescence), Lys280 and Lys282 (15% of total fluorescence)] located in beta-turn structures, of the N-terminal domain I of human lactotransferrin. The results obtained show that the receptor-binding site should be located in the vicinity of the FITC-accessible Lys263, Lys280 and Lys282, and corroborate our preliminary results reporting the involvement of the N-terminal domain I in the binding of human lactotransferrin to mitogen-stimulated lymphocytes [Rochard, Legrand, Mazurier, Montreuil & Spik (1989) FEBS Lett. 255, 201-204]. In any case, FITC labelling is not suitable for studying the binding of lactotransferrin to activated lymphocytes and its use may lead to erroneous interpretations of cell binding experiments.

| S-EPMC1151065 | biostudies-other

Translational activity of messenger ribonucleic acid isolated from unstimulated and phytohaemagglutinin-activated lymphocytes.

Project description:Purified cytoplasmic poly(A)+ RNA isolated from unstimulated pig lymphocytes has the same ability to direct translation in a range of cell-free systems as the corresponding mRNA from 20h phytohaemagglutinin-activated lymphocytes. Additional methylation of the mRNA is not required for maximum protein synthesis in the wheat-germ cell-free system. Misleading results are obtained if the mRNA preparations used are not adequately purified, and a method suitable for routine assessment of the degree of purification achieved is described. Cell-free protein-synthesizing systems from unstimulated lymphocytes translate added lymphocyte mRNA with lower efficiency than do comparable systems from phytohaemagglutinin-activated lymphocytes, whatever the source of the mRNA used.

| S-EPMC1161762 | biostudies-other

Mucociliary Transport in Healthy and Cystic Fibrosis Pig Airways.

Project description:Cystic fibrosis (CF) lung disease is the major cause of morbidity and mortality in people with CF. Abnormal mucociliary transport has been the leading hypothesis for the underlying pathogenesis of CF airway disease. However, this has been difficult to investigate at very early time points. A porcine CF model, which recapitulates many features of CF disease in humans, enables studies to be performed in non-CF and CF pigs on the day that they are born. In newborn CF pigs, we found that under basal conditions, mucociliary transport rates in non-CF and CF pigs are similar. However, after cholinergic stimulation, which stimulates submucosal gland secretion, particles become stuck in the CF airways owing to a failure of mucus strands to release from submucosal glands. In this review, we summarize these recent discoveries and also discuss the morphology, composition, and function of mucins in the porcine lung.

| S-EPMC6322029 | biostudies-literature

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