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L Thuillier

Publications and source records attributed to L Thuillier.

32 records · Page 2Linked to original sources
PubMed1985

Cytidine deaminase activity of human normal and malignant lymphoid cells.

Cytidine deaminase activity was determined by a radioisotopic assay in extracts of peripheral blood mononuclear cells of normal individuals and of patients with acute lymphoblastic leukaemia. The normal enzyme activity had a broad pH optimum between pH 6.5 and 8.0; apparent Km values for cytidine and deoxycytidine were 3.6 +/- 0.6 mumol/l and 26.5 +/- 3.5 mumol/l, respectively; the activity was resistant to heat inactivation; of the various effectors tested, only uridine, deoxyuridine and tetrahydroxyuridine had inhibitory effects. Cytidine deaminase activity was markedly decreased in lymphoblasts of patients with acute lymphoblastic leukaemia; enzyme activity was related to the percentage of circulating blast cells, and not to the clinical, cytological or immunological characters of the leukaemia.

B-Lymphocytes↗
PubMed1985

Interleukin 2 liberation and absorption capacities of rat T lymphocytes in conditions of severe adenylic nucleotide pool depletion due to adenosine deaminase deficiency.

In rat lymph node lymphocytes stimulated for 24 h by concanavalin A in the presence of 10(-5) M 2'-deoxycoformycin (a potent inhibitor of adenosine deaminase) and 10(-5) M 2'-deoxyadenosine the adenylic nucleotide pool was reduced by 55.5% without modification of either the adenylic energy charge or the ability of the cells to liberate interleukin 2. In the same conditions, the ability of rat spleen cells to bind exogenous interleukin 2 activity was not modified. The proliferative response to concanavalin A stimulation was completely inhibited after a 86-h culture period under adenosine deaminase deficiency conditions. It could not be restored by elimination of 2'-deoxyadenosine after a 20-h pretreatment, when adenylic nucleotide pool depletion was 72.4% whereas the interleukin 2 liberation ability was not suppressed. These results suggest that among the early consequences of adenosine deaminase deficiency conditions, which occur before S phase of the cell cycle, the depletion of adenylic nucleotide pool, rather than the impairment of interleukin 2 liberation and absorption capacities, may account for the inability of the lymphocytes to respond to mitogenic stimulation.

Absorption↗
PubMed1984

Consequences of the salvage of purine compounds on the proliferation of rat T-lymphocytes with normal or inhibited purine de novo synthesis.

We studied the ability of purine compounds to restore the proliferation of concanavalin-A-stimulated rat T-lymphocytes under conditions of purine de novo synthesis inhibition and, on the other hand, the inhibition by purine nucleosides of the response of these cells to a mitogenic stimulation under conditions of normal purine de novo synthesis. The use of 50 microM azaserine, a potent inhibitor of purine de novo synthesis, allowed us to define the physiologically active salvage pathways of purine bases, ribo- and deoxyribonucleosides in concanavalin-A-stimulated rat T-lymphocytes. Except for guanylic compounds, all purines completely restored cell proliferation at a concentration of 50 microM. Guanine, guanosine and 2'-deoxyguanosine at concentrations up to 500 microM did not allow us to restore more than 50% of the cell proliferation. In conditions of normal purine de novo synthesis, the addition of 1000 microM adenine, adenosine, 2'-deoxyadenosine or 100 microM 2'-deoxyguanosine inhibited rat T-lymphocyte proliferation. The differences between the degree of inhibition of cell proliferation could be explained only in part by the differences between the capacities of salvage of these compounds. Furthermore, the fact that 2'-deoxyguanosine toxicity was dependent and 2'-deoxyadenosine toxicity independent on the activation state of the cells provided more evidence that the biochemical mechanisms of inhibition of cell proliferation should be different for these two nucleosides.

AMP Deaminase↗
PubMed1984

Salvage of 5'-deoxy-methylthioadenosine into purines and methionine by lymphoid cells and inhibition of cell proliferation.

5'-Deoxy-5'-methylthioadenosine, a by-product of polyamine metabolism, is a potent inhibitor of cell proliferation. MTA phosphorylase cleaves MTA into adenine and 5'-methylthioribose-1-P. We studied MTA inhibition and salvage into purine compounds and methionine in concanavalin A-stimulated rat T lymphocytes and in Raji cells. When de novo purine synthesis was inhibited by azaserine (20 microM), low concentrations of MTA, (less than or equal to 20 microM), were able to completely restore cell proliferation in both types of cells. When cells were cultured in a methionine-free medium, MTA (15 microM) completely fulfilled the methionine requirement of Raji cells but only 50% of that of rat T lymphocytes. MTA displayed a dose-dependent inhibition of the proliferation of both types of cells, but in the case of MTA salvage into purines or methionine, the curves were shifted to higher MTA concentrations. In vitro studies by Backlund et al. (Backlund, P.S., Chang, C.P. and Smith, R.A. (1982) J. Biol. Chem. 257, 4196-4202) on rat liver homogenates, suggested that the last step of MTA salvage into methionine may be the transamination of 2-keto-4-methylthiobutyrate to methionine. We present evidence that this is a step physiologically efficient in intact cells.

Adenosine↗
PubMed1983

5'-Deoxy-5'-methylthioadenosine inhibition of rat T lymphocyte phosphodiesterase: correlation with inhibition of Con A induced proliferation.

5'-Deoxy-5'-methylthioadenosine inhibits Concanavalin A induced rat T lymphocyte proliferation in a dose dependent manner (50 microM to 1000 microM). The extent of inhibition by MTA of lymphocyte proliferation was greatest when MTA was added to the cells at the same time as Concanavalin A. The determination of cyclic AMP level from 30 min to the 6th hour shows that 5'-Deoxy-5'-methylthioadenosine inhibition is correlated with an elevation of cyclic AMP at this mitogen recognition phase. 5'-Deoxy-5'-methylthioadenosine concentrations that inhibit rat T lymphocyte proliferation also inhibit phosphodiesterase activity. This biochemical mechanism could be specific to 5'-Deoxy-5'-methylthioadenosine inhibition since in an another model of inhibition of rat T lymphocyte proliferation (2'-Deoxyadenosine 10 microM, in adenosine deaminase deficiency conditions: 2'-Deoxycoformycin 10 microM), no significant modification of cyclic AMP level can be demonstrated.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1982

Uridine as the only alternative to pyrimidine de novo synthesis in rat T lymphocytes.

Concanavalin A-induced proliferation of rat T-lymphocytes is completely inhibited by 10(-5) M pyrazofurin, a potent inhibitor of pyrimidine de novo synthesis, as judged by cell viability and [3H]thymidine incorporation. Proliferation is completely restored by 5 X 10(-5) M uridine. Cytidine, deoxycytidine, deoxyuridine and thymidine 10 X 10(-5) M each, fail to re-establish proliferation but produce an isotropic dilution of [3H]thymidine uptake in DNA. Bases (cytosine, uracil and thymine) neither restore proliferation nor induce isotopic dilution. The unexpected inability of cytidine to reverse de novo pyrimidine synthesis inhibition suggests a lack of cytidine deaminase activity in rat T-lymphocytes. This is confirmed by a direct sensitive radioisotopic assay (less than 0.001 nmol X min-1 X 10(-6) cells).

Amides↗
PubMed1982

Isopycnic centrifugation as method for the separation of rat thymocyte subpopulations.

Rat thymocytes were separated into five density layers by isopycnic centrifugation on a discontinuous-density Ficoll gradient and each fraction was tested for metabolic functions at different times during a 96-h culture period in various conditions. Immediately after separation the rate of oxygen consumption per unit of cellular volume is virtually the same in all fractions. However, the capacity to synthesize nucleic acids, proteins, purine and pyrimidine nucleotides by the de novo pathway and the activity of the salvage pathway of purine nucleotides is much greater in the low density fractions (fractions 1 and 2). In in vitro cell culture conditions (1) in the absence of mitogens this metabolic activity (which demonstrates a high spontaneous mitotic capacity of cells in the low-density fractions) decays rapidly to a level which is practically negligible after 24 h of culture; (2) in the presence of mitogens alone: PHA, Con A, PWM or PNA, with or without prior neuraminidase treatment, after 24 h an induction of the synthesis of nucleic acids and protein is observed concomitant to the apparition of blast cells. This blastogenesis peaks at 72 h and occurs in fractions 1 and 2 only; (3) in the presence of Con A and 2-mercaptoethanol the blast transformation corresponding to the response of the thymocytes to TCGF is observed in all of the thymocyte fractions and extends beyond 72 h of culture. It remains much more intense in the low-density fractions 1 and 2.

Animals↗
PubMed1981

Consequences of adenosine deaminase deficiency on thymocyte metabolism.

Using 2'-deoxycoformycin inhibition of adenosine deaminase as a model of adenosine deaminase deficiency, the effects of 10 microM 2'-deoxyadenosine (dAdo) on the metabolism of concanavalin A (Con A)-stimulated rat thymocytes were studied. When dAdo and Con A were added simultaneously, a strong inhibition of the incorporation of [3H]thymidine (84%); [3H]uridine (98%) and L-[3H] leucine (46%) in the acid-insoluble fraction, and of [14C]formate (78%) and H14CO-3 (43%) uptake is observed after 48 h of incubation. When dAdo is added after 12 h of Con A stimulation, no such inhibition is observed, but when added after 24 h of stimulation, there is an enhancement of blastogenesis as measured by nucleic acid, protein, and purine and pyrimidine base synthesis. More detailed studies of thymocytes stimulated by Con A for 0-72 h, followed by short-term incubation periods with dAdo (1-5 h), revealed that thymocyte metabolism becomes progressively less sensitive to dAdo-mediated inhibition during the course of blastogenesis. These results suggest that (a) the inhibition of ribonucleotide reductase is not the only mechanism involved in the inhibition of blastogenesis by dAdo and that (b) such inhibition of thymocyte metabolism is essentially dependent upon the activation state of the cell.

Adenosine↗
PubMed1981

Study of rat lymphocytes by use of peanut agglutinin.

Peanut agglutinin (PNA) was used to identify rat lymphocyte subpopulations. The binding of PNA on the surface of lymphocytes was assessed by fluorescence microscopy using fluoresceinated PNA. It was shown that most of the thymocytes and bone marrow cells have receptors for PNA; in spleen and lymph nodes the percentages of PNA-positive cells are around 40%. PNA was also used as a potentially mitogenic lectin. It was ascertained that lymphocytes incubated with PNA are stimulated only if they are pretreated with neuraminidase, and that only 'immunocompetent' thymocytes exhibit a very weak response. The fact that PNA slightly inhibits the [3H]uridine uptake of resting thymocytes might explain the weak mitogenicity of PNA.

Animals↗
PubMed1978

Purification and kinetic properties of human erythrocyte Mg2+-dependent inorganic pyrophosphatase.

Inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) from human erythrocyte hemolysates has been purified up to 10 000-fold. The purified enzyme is homogenous and has a specific activity of 79.75 mumol PPi hydrolysed.min-1.mg-1 at pH 8 and 37 degrees C. It was confirmed that it is a dimer with a molecular weight of 42 000, composed of two identical protomers. From kinetic studies, it is proposed that human erythrocyte inorganic pyrophosphatase activity depends on free Mg2+ concentration in different ways. This ion constitutes part of the substrate (the Mg.PPi complex; Km = 1.4.10(-4) M) and probably acts as an allosteric activator (kinetic activation constant: KMg2+a = 7.5.10(-4) M). Equilibrium binding studies performed in the absence of PPi showed 4 binding sites for Mg2+, all having the same high affinity (dissociation constant: KMg2+d = 4.10(-6) M). Since the concentration of free Mg2+ in red blood cells is very low and may vary with the oxygenation state, it is likely that in vivo erythrocyte pyrophosphatase activity is regulated.

Enzyme Activation↗