Pierre Cartier

Cellular Immune Deficiency in Two Siblings with Hereditary Orotic Aciduria

HEREDITARY orotic aciduria is a rare inborn error of pyrimidine metabolism that is associated with autosomal recessive inheritance.1 The disease is usually characterized by retarded growth and deve...

Les anomalies de la glycolyse au cours de l'anémie hémolytique par déficit du globule rouge en pyruvate kinase

Abstract In two cases of hemolytic anemia with pyruvate kinase deficiency, we have studied the modifications of glycolysis in red blood cells and the kinetic behaviour of pyruvate kinase. Red cell glycolysis was markedly diminished. This alteration of the metabolic capacity coincides with an accumulation of phosphoenol pyruvate and phospho-glycerates and with a low level of hexose phosphates and triose phosphates. ATP content is very much decreased (35 % of normal values) ; 2,3-diphosphoglycerate is slightly increased. The level of pyridine nucleotides was found to be low, with predominance of reduced forms. These modifications are interpreted in relation to pyruvate kinase deficiency. The kinetic studies of the purified enzyme revealed two forms of pyruvate kinase in normal erythrocytes as well as in pyruvate kinase-deficient cells : — pyruvate kinase-B, with hyperbolic kinetics and K m app. (phosphoenol-pyruvate) = 3.6· 10 −5 M , insensitive to fructose diphosphate. — pyruvate kinase-A, with allosteric kinetics and K m app. (phosphoenol-pyruvate) = 19.3· 10 −5 M . The allosteric form is activated by fructose diphosphate at very low concentrations ( K m app. (fructose diphosphate) = 43·10 −9 M ). In activated A-form, K m (phosphoenolpyruvate) is lowered at the same value as the B-form (= 2.8 · 10 −5 M ). ADP is inhibitor at concentrations above 30 · 10 −5 M , and this action is overcome by fructose diphosphate. ATP inhibits at physiological concentrations. Pyruvate kinase-A and pyruvate kinase-B have identical V max and are interconvertible. The transition factors are discussed. In this study, no differences appeared in the molecular structure of pyruvate kinase in normal and deficient cells.

Enzymatic activities on purine and pyrimidine metabolism in nine mycoplasma species contaminating cell cultures

The study of mycoplasma enzymes involved in nucleotide degradation and base salvage pathways allowed determination of enzyme profiles for some species. The activity of adenosine phosphorylase seems to be the biochemical test of choice to detect mycoplasma contamination in cell cultures. All mycoplasmas tested had a very high level of activity compared to very low activity in human cell cultures, rendering the technic sensitive even at a low degree of contamination.

Les activités purine-phosphoribosyl transférasiques des globules rouges humains technique de dosage

Abstract A rapid and sensitive assay for hypoxanthine phosphoribosyl transferase (IMP: pyrophosphate phosphoribosyltransferase; EC 2.4.2.8) and adenine phosphoribosyl transferase: (AMP: pyrophosphate phosphoribosyltransferase; EC 2.4.2.7) in red blood cells is described. After enzymic conversion of [ 14 C]purines (at 37° for 20 min) to radioactive nucleotides, the reaction was stopped by addition of an excess of EDTA. Radioactive nucleotides were separated using DEAE-cellulose paper and Paladini-Leloir solvent, by descending chromatography. The nucleotide remained adsorbed at the top of the strips and was separated from the purine. Both sides of the paper were counted in a liquid scintillation counter: the percentage of transformation was determined and activity expressed in micromoles/ml red cells/minute. The amount of [ 14 C] purine nucleotide formed was directly proportional to the concentration of the hemolysate, and to the incubation time. The conditions affecting accuracy and reliability have been studied. The reaction rate of guanine phosphoribosyltransferase is 30% higher than with hypoxanthine. This procedure is relatively simple and requires only 0.1 ml of total blood. The phosphoribosyltransferase activity (with hypoxanthine, guanine and adenine) of human red blood cells has been reported. The values observed showed no significant variation with age (within two days and 60 years) and sex.

Measurement of inorganic pyrophosphate in biological fluids and bone tissues

Abstract A simple and rapid technique of measuring pyrophosphates in plasma, urine, and bone tissue is described, using the UDPG-pyrophosphorylase reaction and fluorimetrical determination of NADPH formed in a combined system of phosphorylation and reduction. This very specific method avoids the separation of Pi 2 by column chromatography, and its very great sensitivity enables measurement on a final sample corresponding to 0.2 ml of plasma, with a precision of about 3%. The mean plasma PPi concentration (±SE of mean) was 3.53 ± 0.19 μmoles/liter (SE=0.93), or 0.207 ± 0.006 mg Pi/liter. The normal range for this population (99% confidence limit) is therefore between 1.10 and 5.90 μmoles/liter or 0.068 – 0.366 mg Pi/liter. Analysis of the variation of the duplicate measurements shows a very small divergence of 3.4% or ±0.12 μ m . Normal 24 hr urinary excretion is 53.0 ± 6.8 μmoles with 99% confidence limits of 10.0 and 96.0 μmoles. The average PPi concentration in calvaria of 20 10-day-old rats is 3.685 ± 0.16 nmoles/mg fresh weight.

Metabolism of pyrimidine bases and nucleosides by pyrimidine-nucleoside phosphorylases in cultured human lymphoid cells

The anabolism of pyrimidine ribo- and deoxyribonucleosides from uracil and thymine was investigated in phytohemagglutinin-stimulated human peripheral blood lymphocytes and in a Burkitts lymphoma-derived cell line (Raji). We studied the ability of these cells to synthesize pyrimidine nucleosides by ribo- and deoxyribosyl transfer between pyrimidine bases or nucleosides and the purine nucleosides inosine and deoxyinosine as donors of ribose 1-phosphate and deoxyribose 1-phosphate, respectively: these reactions involve the activities of purine-nucleoside phosphorylase, and of the two pyrimidine-nucleoside phosphorylases (uridine phosphorylase and thymidine phosphorylase). The ability of the cells to synthesize uridine was estimated from their ability to grow on uridine precursors in the presence of an inhibitor of pyrimidine de novo synthesis (pyrazofurin). Their ability to synthesize thymidine and deoxyuridine was estimated from the inhibition of the incorporation of radiolabelled thymidine in cells cultured in the presence of unlabelled precursors. In addition to these studies on intact cells, we determined the activities of purine- and pyrimidine-nucleoside phosphorylases in cell extracts. Our results show that Raji cells efficiently metabolize preformed uridine, deoxyuridine and thymidine, are unable to salvage pyrimidine bases, and possess a low uridine phosphorylase activity and markedly decreased (about 1% of peripheral blood lymphocytes) thymidine phosphorylase activity. Lymphocytes have higher pyrimidine-nucleoside phosphorylases activities, they can synthesize deoxyuridine and thymidine from bases, but at high an non-physiological concentrations of precursors. Neither type of cell is able to salvage uracil into uridine. These results suggest that pyrimidine-nucleoside phosphorylases have a catabolic, rather than an anabolic, role in human lymphoid cells. The facts that, compared to peripheral blood lymphocytes, lymphoblasts possess decreased pyrimidine-nucleoside phosphorylases activities, and, on the other hand, more efficiently salvage pyrimidine nucleosides, are consistent with a greater need of these rapidly proliferating cells for pyrimidine nucleotides.

Inability of immunocompetent thymocytes to produce T-cell growth factor under adenosine deaminase deficiency conditions

Abstract Five density-defined subpopulations of rat thymocytes were separated by isopycnic centrifugation on a discontinuous density Ficoll gradient and compared with respect to their response to Con A stimulation under normal and adenosine deaminase (ADA) deficiency conditions. This study shows that (a) immunocompetent (low-density) thymocytes and splenic T lymphocytes produce T-cell growth factor (TCGF) in response to mitogenic stimulation in normal culture conditions, but are unable to synthesize effective TCGF in the presence of an adenosine deaminase inhibitor and excess substrate (ADA deficiency conditions), and (b) high-density immunoincompetent thymocytes proliferate and differentiate into mature T cells in response to Con A if effective exogenous TCGF is added to the culture medium but are unable to do so under ADA deficiency conditions.

Biochemical study of a case of hemolytic anemia with increased (85-fold) red cell adenosine deaminase☆

An increased red cell adenosine deaminase (ADA) activity (85-fold) was found in a 10-year-old male child suffering from severe hemolytic disease. Evidence is given that the excessive ADA activity in erythrocytes is due to an abnormal amount of a catalytically and immunologically normal enzyme. Metabolic studies with the patients erythrocytes show that the low ATP concentration in these cells (64% of comparably reticulocyte-rich blood) is due both to a diminished synthesis of adenylic nucleotides from adenosine, and to an excessive catabolism of AMP.

Dosage de l'activité adénosine désaminasique dans les érythrocytes et les lymphocytes humains

Abstract A radiochromatographic method is described for measuring enzymatic activity of adenosine deaminase in human erythrocytes and lymphocytes. [8-14C]-adenosine is converted into inosine and hypoxanthine; after Chromatographie separation of the products, the radioactivity is determined. The kinetic properties of the enzyme have been studied. The Km values for the erythrocyte and lymphocyte enzymes are higher as compared with purified deaminase. Optimum conditions for substrate concentration for assay were established. The mean normal activity (± S.E. of mean) is: for erythrocytes, 494 ± 61; nmol min-1 ml-1; for lymphocytes, 147 ± 0.18 nmol min−1 106 cellules. The mean values are higher than that given by other methods working at a lower (non-staurating) substrate concentration.

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 × 10−5 M uridine. Cytidine, deoxycytidine, deoxyuridine and thymidine 10 × 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 (<0.001 nmol.min−1.10−6 cells).