64 results on '"Garel MC"'
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2. Isoelectric focusing of human hemoglobin: its application to screening, to the characterization of 70 variants, and to the study of modified fractions of normal hemoglobins
- Author
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Basset, P, Beuzard, Y, Garel, MC, and Rosa, J
- Abstract
Isoelectric focusing on slabs of acrylamide gel was adapted for the screening of abnormal hemoglobins, the characterization of 70 human variants, and the study of minor fractions of normal hemoglobin. The screening method was as fast and inexpensive as conventional techniques, allowed the simultaneous analysis of some 50 samples of whole blood, and yielded resolution superior to that obtained by other methods with hemolysates. Among the 70 variants, 31 mutants could not be separated from HbS by cellulose acetate electrophoresis. The characterization technique of electrofocusing allowed us to distinguish between most variants. Only one mutant, Hb Galveston, could be confused with HbS. Hb Koln, the most frequent unstable mutant, exhibited a special pattern. HbA1C was separated from HbA. Preliminary results indicate that quantitation of HbA1C by gel scanning is feasible.
- Published
- 1978
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3. Enhanced cardiac function in transgenic mice expressing a Ca(2+)-stimulated adenylyl cyclase
- Author
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Jacques Hanoune, Marie-Claude Garel, Nicole Defer, Giovanni Esposito, Larissa Lipskaia, Iman Hajar, Howard A. Rockman, Lipskaia, L, Defer, N, Esposito, Giovanni, Hajar, I, Garel, Mc, Rockman, Ha, and Hanoune, J.
- Subjects
Cardiac function curve ,Genetically modified mouse ,medicine.medical_specialty ,Physiology ,Systole ,Transgene ,Restriction Mapping ,Endogeny ,Mice, Transgenic ,Biology ,Adenylyl cyclase ,Contractility ,chemistry.chemical_compound ,Mice ,In vivo ,Diastole ,Heart Rate ,Internal medicine ,Receptors, Adrenergic, beta ,medicine ,Animals ,Humans ,Receptor ,Myocardium ,Cell Membrane ,Hemodynamics ,Isoproterenol ,Heart ,Cyclic AMP-Dependent Protein Kinases ,Myocardial Contraction ,Kinetics ,Endocrinology ,chemistry ,Echocardiography ,Guanosine 5'-O-(3-Thiotriphosphate) ,Calcium ,Cardiology and Cardiovascular Medicine ,Adenylyl Cyclases - Abstract
Abstract —The predominant functional adenylyl cyclases normally expressed in cardiac tissue and coupled to β-adrenergic receptors are inhibited by micromolar Ca 2+ concentration. To modify the overall balance of activities, we have generated transgenic mice expressing the Ca 2+ -stimulatable adenylyl cyclase type 8 (AC8) specifically in the heart. AC activity is increased by at least 7-fold in heart membranes from transgenic animals and is stimulated by Ca 2+ in the same range of concentration that inhibits the endogenous activity. Moreover, the in vivo basal protein kinase A activity was augmented 4-fold. Overexpression of AC8 in the heart has no detrimental consequences on global cardiac function. Basal heart rate and contractile function, measured by noninvasive echocardiography, were unchanged. In contrast, on release of parasympathetic tone, the intrinsic contractility is heightened and unresponsive to further β-adrenergic receptor stimulation. AC8 transgenic mice thus represent an original model to investigate the relative influence of Ca 2+ and cAMP on cardiac function within a phenotype of enhanced cardiac contractility and relaxation.
- Published
- 2000
4. The cancer chemopreventive agent resveratrol induces tensin, a cell-matrix adhesion protein with signaling and antitumor activities.
- Author
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Rodrigue CM, Porteu F, Navarro N, Bruyneel E, Bracke M, Romeo PH, Gespach C, and Garel MC
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- Actins metabolism, Cell Adhesion, Cell Adhesion Molecules metabolism, Cell Line, Tumor, Cell Movement, Cycloheximide pharmacology, Cytoplasm metabolism, Cytoskeleton metabolism, DNA, Complementary metabolism, Dose-Response Relationship, Drug, Flow Cytometry, Humans, Immunoblotting, K562 Cells, Neoplasm Invasiveness, Phosphatidylinositol 3-Kinases metabolism, Protein Synthesis Inhibitors pharmacology, RNA, Messenger metabolism, Resveratrol, Reverse Transcriptase Polymerase Chain Reaction, Signal Transduction, Tensins, Time Factors, Anticarcinogenic Agents pharmacology, Antineoplastic Agents, Phytogenic pharmacology, Enzyme Inhibitors pharmacology, Gene Expression Regulation, Neoplastic, Microfilament Proteins biosynthesis, Neoplasms prevention & control, Stilbenes pharmacology
- Abstract
During a search to identify resveratrol (3,5,4'-trihydroxy-trans-stilbene, RV) target genes in the human erythroleukemic K562 cell line, we show here that the tensin gene and protein levels are remarkably induced by this dietary polyphenol. Tensin, a cell-matrix adhesion protein binding the integrins and cytoskeletal actin filaments also interacts with PI3-kinase and JNK signaling pathways. Tensin induction by RV is associated with increased K562 cell adhesion to fibronectin, cell spreading and actin polymerization. The same responses were observed in the tensin-deficient MCF7 human breast cancer cell line. In K562 and MCF7 cells treated by RV, tensin was found in punctate and intracytoplasmic areas. In MCF7 epithelial cells, induction of tensin is not exclusively associated with plasma membrane-bound vinculin, suggesting a dual localization of tensin in both focal and fibrillar adhesions. Pharmacological blockade of PI3-kinase and Rho GTPases/Rho-kinase resulted in selective depletion of focal adhesions, disorganization of tensin localization and disruption of stress fibers. RV increased cell motility and attachment to fibronectin in MCF7 cells submitted to mechanical laminar flow stress, and abrogated estrogen-induced MCF7 cancer cell invasion. Our data support the conclusion that induction of tensin by RV contributes to the chemopreventive and anti-invasive activity of this natural dietary compound in tensin-negative and -deficient leukemic cells or epithelioid cancers.
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- 2005
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5. Resveratrol, a natural dietary phytoalexin, possesses similar properties to hydroxyurea towards erythroid differentiation.
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Rodrigue CM, Arous N, Bachir D, Smith-Ravin J, Romeo PH, Galacteros F, and Garel MC
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- Anemia, Sickle Cell blood, Anemia, Sickle Cell drug therapy, Blotting, Western methods, Cell Differentiation drug effects, Cell Division drug effects, Cell Line, Cells, Cultured, Dose-Response Relationship, Drug, Enzyme Inhibitors therapeutic use, Erythroid Precursor Cells drug effects, Erythroid Precursor Cells metabolism, Fetal Hemoglobin biosynthesis, Gene Expression drug effects, Granulocyte-Macrophage Colony-Stimulating Factor pharmacology, Hemoglobins biosynthesis, Humans, Hydroxyurea therapeutic use, Models, Biological, Proto-Oncogene Proteins p21(ras) genetics, Resveratrol, Reverse Transcriptase Polymerase Chain Reaction, Ribonucleotide Reductases antagonists & inhibitors, Stem Cells drug effects, Stem Cells metabolism, Time Factors, Antioxidants pharmacology, Leukemia, Erythroblastic, Acute drug therapy, Stilbenes pharmacology
- Abstract
Resveratrol, a natural dietary polyphenol, has been postulated to be implicated in the cardioprotective effect of red wine and the low incidence of breast and prostate cancers among vegetarians and Orientals respectively. This compound inhibits ribonucleotide reductase as does hydroxyurea, the first therapeutic agent used in the treatment of sickle cell disease. Using the human erythroleukaemic K562 cell line as an in vitro model, we show here that 50 micromol/l of resveratrol induced a higher haemoglobin production (sevenfold) in K562 cells than 500 micromol/l of hydroxyurea (3.5-fold). This erythroid differentiation was linked to a dose- and time-dependent inhibition of cell proliferation associated with an equivalent increased expression of p21 mRNA, but with a higher increased level of p21 protein (sixfold) for cells treated with resveratrol than for those treated with hydroxyurea (1.5-fold). We also show that 50 micromol/l of resveratrol and 25 micromol/l of hydroxyurea induced variable but similar enhancements of fetal haemoglobin synthesis in cultured erythroid progenitors for the majority of the sickle cell patients studied. These inductions were linked to, but not correlated with, a variable decrease in erythroid burst-forming unit clone number. Taken together, these results show that resveratrol merits further investigations in sickle cell disease therapy.
- Published
- 2001
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6. Enhanced cardiac function in transgenic mice expressing a Ca(2+)-stimulated adenylyl cyclase.
- Author
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Lipskaia L, Defer N, Esposito G, Hajar I, Garel MC, Rockman HA, and Hanoune J
- Subjects
- Adenylyl Cyclases genetics, Animals, Cell Membrane enzymology, Cyclic AMP-Dependent Protein Kinases metabolism, Diastole, Echocardiography, Guanosine 5'-O-(3-Thiotriphosphate) pharmacology, Heart Rate, Humans, Isoproterenol pharmacology, Kinetics, Mice, Mice, Transgenic, Myocardial Contraction, Receptors, Adrenergic, beta metabolism, Restriction Mapping, Systole, Adenylyl Cyclases metabolism, Calcium physiology, Heart physiology, Hemodynamics, Myocardium enzymology
- Abstract
The predominant functional adenylyl cyclases normally expressed in cardiac tissue and coupled to beta-adrenergic receptors are inhibited by micromolar Ca(2+) concentration. To modify the overall balance of activities, we have generated transgenic mice expressing the Ca(2+)-stimulatable adenylyl cyclase type 8 (AC8) specifically in the heart. AC activity is increased by at least 7-fold in heart membranes from transgenic animals and is stimulated by Ca(2+) in the same range of concentration that inhibits the endogenous activity. Moreover, the in vivo basal protein kinase A activity was augmented 4-fold. Overexpression of AC8 in the heart has no detrimental consequences on global cardiac function. Basal heart rate and contractile function, measured by noninvasive echocardiography, were unchanged. In contrast, on release of parasympathetic tone, the intrinsic contractility is heightened and unresponsive to further beta-adrenergic receptor stimulation. AC8 transgenic mice thus represent an original model to investigate the relative influence of Ca(2+) and cAMP on cardiac function within a phenotype of enhanced cardiac contractility and relaxation.
- Published
- 2000
- Full Text
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7. Critical role of human bisphosphoglycerate mutase Cys22 in the phosphatase activator-binding site.
- Author
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Ravel P, Craescu CT, Arous N, Rosa J, and Garel MC
- Subjects
- Binding Sites, Bisphosphoglycerate Mutase metabolism, Circular Dichroism, Escherichia coli, Glycolates metabolism, Humans, Phosphoric Monoester Hydrolases metabolism, Phosphorylation, Structure-Activity Relationship, Substrate Specificity, Bisphosphoglycerate Mutase chemistry, Cysteine, Glycolates chemistry, Phosphoric Monoester Hydrolases chemistry
- Abstract
The enzymatic activities catalyzed by bisphosphoglycerate mutase (BPGM, EC 5.4.2.4) have been shown to occur at a unique active site, with distinct binding sites for diphosphoglycerates and monophosphoglycerates. The physiological phosphatase activator (2-phosphoglycolate) binds to BPGM at an undetermined site. BPGM variants were constructed by site-directed mutagenesis of three amino acid residues in the active site to identify residues specifically involved in the binding of the monophosphoglycerates and 2-phosphoglycolate. Substitution of Cys22 by functionally conservative residues, Thr or Ser, caused a great decrease in 2-phosphoglycolate-stimulated phosphatase activity and in the Ka value of the activator, whereas it caused no change in other catalytic activities or in the Km values of 2,3-diphosphoglycerate (2,3-DPG) and glycerate 3-phosphate (3-PG, EC 1.1.1.12), indicating that Cys22 is specifically involved either directly or indirectly in 2-phosphoglycolate binding. Kinetic experiments showed that the Ka of the cofactor and the Km of 3-PG were affected by the substitution of Ser23 indicating that this residue is necessary for the fixation of both 3-PG and 2-phosphoglycolate. The R89K variant has previously been shown to have a modified Km value for monophosphoglycerates, however, its affinity for 2-phosphoglycolate is unaltered, suggesting that Arg89 is specifically involved in monophosphoglycerates binding. CD spectroscopic studies of substrates and cofactor binding showed that 2,3-DPG induced structural modifications of normal and mutated enzymes which could be due to protein phosphorylation. Addition of 2-phosphoglycolate to phosphorylated proteins with normal affinity for the cofactor produced spectra with the same characteristics as unphosphorylated species. In summary, monophosphoglycerates and 2-phosphoglycolate have partially distinct binding sites in human BPGM. The specific implication of the Cys22 residue in 2-phosphoglycolate binding is of great significance in the design of analogs of therapeutic benefit.
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- 1997
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8. New procedures to measure synthase and phosphatase activities of bisphosphoglycerate mutase. Interest for development of therapeutic drugs.
- Author
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Ravel P, Garel MC, and Toullec D
- Subjects
- Binding, Competitive, Chromatography, Ion Exchange methods, Electrophoresis, Paper methods, Glyceric Acids metabolism, Glycolates metabolism, Hemoglobins metabolism, In Vitro Techniques, Spectrophotometry methods, Bisphosphoglycerate Mutase metabolism, Clinical Enzyme Tests methods, Phosphoric Monoester Hydrolases metabolism
- Abstract
In red blood cells, a modulation of the level of the allosteric effector of hemoglobin, 2,3-diphosphoglycerate (2,3-DPG) would have implications in the treatment of ischemia and sickle cell anemia. Its concentration is determined by the relative activities of the synthase and phosphatase reactions of the multifunctional bisphosphoglycerate mutase (BPGM). In this report we develop first a more direct synthase assay which uses glyceraldehyde phosphate to suppress the aldolase and triose phosphate isomerase reactions. Secondly we propose a radioactive phosphatase assay coupled to chromatographic separation and identification of the reaction products by paper electrophoresis. Such identification of these products allow us to show that the multifunctional BPGM expresses its mutase instead of its phosphatase activity in conditions of competition between the 3-phosphoglycerate and the 2-phosphoglycolate activator in the phosphatase reaction. These two more precise procedures could be used to study the effects of substrate and cofactor analogues regarding potential therapeutic approaches and could be used for clinical analyses to detect deficiency of BPGM.
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- 1997
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9. A recombinant bisphosphoglycerate mutase variant with acid phosphatase homology degrades 2,3-diphosphoglycerate.
- Author
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Garel MC, Arous N, Calvin MC, Craescu CT, Rosa J, and Rosa R
- Subjects
- 2,3-Diphosphoglycerate, Amino Acid Sequence, Base Sequence, Binding Sites, Catalysis, DNA Primers chemistry, Diphosphoglyceric Acids metabolism, Glycine chemistry, Histidine chemistry, Humans, Molecular Sequence Data, Mutagenesis, Site-Directed, Structure-Activity Relationship, Substrate Specificity, Bisphosphoglycerate Mutase metabolism, Diphosphoglyceric Acids chemistry
- Abstract
To date no definite and undisputed treatment has been found for sickle cell anemia, which is characterized by polymerization of a deoxygenated hemoglobin mutant (HbS) giving rise to deformed erythrocytes and vasoocclusive complications. Since the erythrocyte glycerate 2,3-bisphosphate (2,3-DPG) has been shown to facilitate this polymerization, one therapeutic approach would be to decrease the intraerythrocytic level of 2,3-DPG by increasing the phosphatase activity of the bisphosphoglycerate mutase (BPGM; 3-phospho-D-glycerate 1,2-phosphomutase, EC 5.4.2.4). For this purpose, we have investigated the role of Gly-13, which is located in the active site sequence Arg9-His10-Gly11-Glu12-Gly13 in human BPGM. This sequence is similar to the Arg-His-Gly-Xaa-Arg* sequence of the distantly related acid phosphatases, which catalyze as BPGM similar phosphoryl transfers but to a greater extent. We hypothesized that the conserved Arg* residue in acid phosphatase sequences facilitates the phosphoryl transfer. Consequently, in human BPGM, we replaced by site-directed mutagenesis the corresponding amino acid residue Gly13 with an Arg or a Lys. In another experiment, we replaced Gly13 with Ser, the amino acid present at the corresponding position of the homologous yeast phosphoglycerate mutase (D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1). Mutation of Gly13 to Ser did not modify the synthase activity, whereas the mutase and the phosphatase were 2-fold increased or decreased, respectively. However, replacing Gly13 with Arg enhanced phosphatase activity 28.6-fold, whereas synthase and mutase activities were 10-fold decreased. The presence of a Lys in position 13 gave rise to a smaller increase in phosphatase activity (6.5-fold) but an identical decrease in synthase and mutase activities. Taken together these results support the hypothesis that a positively charged amino acid residue in position 13, especially Arg, greatly activates the phosphoryl transfer to water. These results also provide elements for locating the conserved Arg* residue in the active site of acid phosphatases and facilitating the phosphoryl transfer. The implications for genetic therapy of sickle cell disease are discussed.
- Published
- 1994
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10. Reactivity of 42 disulfides with thiol group of human haemoglobin and human serum albumin.
- Author
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Mahieu JP, Gosselet NM, Sebille B, Garel MC, and Beuzard Y
- Subjects
- Chromatography, Ion Exchange, Disulfides chemistry, Drug Design, Humans, Kinetics, Molecular Structure, Structure-Activity Relationship, Antisickling Agents chemistry, Cysteine metabolism, Disulfides metabolism, Hemoglobins metabolism, Serum Albumin metabolism
- Abstract
The reactivities of disulfides of different compound families towards thiol groups of human haemoglobin and human serum albumin were determined at physiological pH 7.4 by anion-exchange liquid chromatography. The apparent second-order kinetic rate constants, K1, were calculated for the reaction of these disulfides with each protein. The results show that the studied heterocyclic disulfides are the most reactive compounds with both proteins. The lipophilic properties of these disulfides were evaluated by reversed-phase high performance liquid chromatography, using the percentage of acetonitrile (PAC) required for eluting each compound of the chromatographic column in a water-acetonitrile gradient. The structure-reactivity correlations between log K1 and log PAC are stated for each protein and compared. They fit a parabolic curve which permits one to define a lipophilic domain corresponding to a quantitative reaction of disulfides towards these proteins. The studied disulfides present a similar optimum of reactivity for both proteins.
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- 1993
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11. Amino acid residues involved in the catalytic site of human erythrocyte bisphosphoglycerate mutase. Functional consequences of substitutions of His10, His187 and Arg89.
- Author
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Garel MC, Lemarchandel V, Calvin MC, Arous N, Craescu CT, Prehu MO, Rosa J, and Rosa R
- Subjects
- Base Sequence, Binding Sites, Bisphosphoglycerate Mutase chemistry, Bisphosphoglycerate Mutase genetics, Catalysis, Enzyme Stability, Hot Temperature, Humans, Molecular Sequence Data, Mutagenesis, Site-Directed, Oligodeoxyribonucleotides, Protein Structure, Tertiary, Arginine metabolism, Bisphosphoglycerate Mutase metabolism, Erythrocytes enzymology, Histidine metabolism
- Abstract
Human bisphosphoglycerate mutase (GriP2 mutase) is a trifunctional enzyme which synthesizes and degrades GriP2 in red cells. Among the amino acid residues involved in its active site there are two conserved histidine residues, His10 which is phosphorylated during the catalytic process and His187 for which only speculative data have been made about the potential role during the reactions. Another amino acid residue, Arg89, had not been described as part of this active site but we have recently shown that a natural mutant Arg89-->Cys was highly thermolabile and showed severe perturbations of its enzymatic properties. To understand better the exact role of these residues, replacements of His10 by Gly (H10G) or Asp (H10D), His187 by Asn (H187N), Tyr (H187Y) or Asp (H187D) and Arg89 by Cys (R89C), Ser (R89S), Gly (R89G) or Lys (R89K) were performed by site-directed mutagenesis. The results obtained in this report show that replacement of the His10 residue completely abolished the enzymatic activities. Concerning the His187 residue, our results afford arguments that it plays an essential role in the three catalytic activities. Indeed all these activities are abolished in the two H187Y and H187D variants, whereas they are detectable though strongly diminished, for the H187N variant. In addition mutations at His187 could be distinguishable from those at His10 since the former resulted in a thermolabile enzyme, whereas no significant change in heat stability was observed for the latter. It is noteworthy that the H187N variant is protected against thermal instability by glycerate 2,3-bisphosphate (GriP2). Concerning the Arg89 mutants, R89C, R89S and R89G, the three variants showed characteristics identical to those found in the natural R89C mutant, i.e. loss of 99% of synthase activity, consistent decrease of mutase and 2-phosphoglycolate-stimulated phosphatase activities whereas the unstimulated phosphatase activity was normal. Moreover these mutants were unstable at 55 degrees C but GriP2 was able to protect them against thermal instability. In contrast, the R89K mutant was stable at 55 degrees C. Its synthase and unstimulated phosphatase activities were normal but its mutase and 2-phosphoglycolate-stimulated phosphatase activities were decreased. In addition, Km values for monophosphoglycerates were increased (3.2-fold) in the synthase but normal in mutase activities, whereas Km values for GriP2 were normal in mutase and phosphatase activities.(ABSTRACT TRUNCATED AT 400 WORDS)
- Published
- 1993
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12. Towards a transgenic mouse model of sickle cell disease: hemoglobin SAD.
- Author
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Trudel M, Saadane N, Garel MC, Bardakdjian-Michau J, Blouquit Y, Guerquin-Kern JL, Rouyer-Fessard P, Vidaud D, Pachnis A, and Roméo PH
- Subjects
- Anemia, Sickle Cell mortality, Animals, Chromatography, High Pressure Liquid, DNA genetics, Electrophoresis, Polyacrylamide Gel, Erythrocyte Indices, Globins genetics, Hemoglobin, Sickle genetics, Isoelectric Focusing, Mice, Mice, Transgenic, Oxygen metabolism, Peptide Mapping, Phenotype, Polymerase Chain Reaction, Trypsin, Anemia, Sickle Cell physiopathology, Disease Models, Animal, Hemoglobin, Sickle metabolism
- Abstract
In order to obtain a transgenic mouse model of sickle cell disease, we have synthesized a novel human beta-globin gene, beta SAD, designed to increase the polymerization of the transgenic human hemoglobin S (Hb S) in vivo. beta SAD (beta S-Antilles-D Punjab) includes the beta 6Val substitution of the beta S chain, as well as two other mutations, Antilles (beta 23Ile) and D Punjab (beta 121Gln) each of which promotes the polymerization of Hb S in human. The beta SAD gene and the human alpha 2-globin gene, each linked to the beta-globin locus control region (LCR) were co-introduced into the mouse germ line. In one of the five transgenic lines obtained, SAD-1, red blood cells contained 19% human Hb SAD (alpha 2 human 1 beta 2SAD) and mouse-human hybrids in addition to mouse hemoglobin. Adult SAD-1 transgenic mice were not anemic but had some abnormal features of erythrocytes and slightly enlarged spleens. Their erythrocytes displayed sickling upon deoxygenation in vitro. SAD-1 neonates were anemic and many did not survive. In order to generate adult mice with a more severe sickle cell syndrome, crosses between the SAD progeny and homozygous for beta-thalassemic mice were performed. Hemoglobin SAD was increased to 26% in beta-thal/SAD-1 mice which exhibited: (i) abnormal erythrocytes with regard to shape and density; (ii) an enlarged spleen and a high reticulocyte count indicating an increased erythropoiesis; (iii) mortality upon hypoxia; (iv) polymerization of hemolysate similar to that obtained in human homozygous sickle cell disease; and (v) anemia and mortality during development.
- Published
- 1991
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13. Crystallization and preliminary X-ray diffraction studies of the human erythrocyte bisphosphoglycerate mutase.
- Author
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Cherfils J, Rosa R, Garel MC, Calvin MC, Rosa J, and Janin J
- Subjects
- Bisphosphoglycerate Mutase genetics, Bisphosphoglycerate Mutase metabolism, Cloning, Molecular, Crystallization, Escherichia coli genetics, Gene Expression, Humans, X-Ray Diffraction, Bisphosphoglycerate Mutase chemistry, Erythrocytes enzymology
- Abstract
Bisphosphoglycerate mutase (EC 2.7.5.4) catalyzes the synthesis and breakdown of 2,3-diphosphoglycerate in red cells. The human enzyme, cloned and expressed in Escherichia coli has been crystallized in the rhombohedral space group R32 with a = b = c = 100.4 A and alpha = beta = gamma = 81.2 degrees. The asymmetric unit contains either a dimeric enzyme molecule, or a monomer.
- Published
- 1991
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14. Changes of polymerization and conformation of hemoglobin S induced by thiol reagents.
- Author
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Garel MC, Caburi-Martin J, Domenget C, Kister J, Craescu CT, Poyart C, and Beuzard Y
- Subjects
- 2,2'-Dipyridyl pharmacology, Adult, Anemia, Sickle Cell blood, Humans, Kinetics, Macromolecular Substances, Magnetic Resonance Spectroscopy, Oxyhemoglobins metabolism, Protein Conformation, Reference Values, 2,2'-Dipyridyl analogs & derivatives, Disulfides pharmacology, Hemoglobin A metabolism, Hemoglobin, Sickle metabolism, Hydroxymercuribenzoates pharmacology, Sulfhydryl Reagents pharmacology
- Abstract
Thiol reagents, covalently bound to cysteine beta 93, either inhibit or facilitate the polymerization process of hemoglobin S. The progelling effect of parahydroxymercurybenzoate or 2,2'-dithiodipyridine contrasted with the increased oxygen affinity and the destabilization of the T state of Hb shown by functional and NMR studies. Thiol reagents increased the oxygen affinity of Hb from 30 to 1000%. Such variability was also observed in the reduction (up to 50%) of the alkaline Bohr effect. We show that the antigelling or progelling activity of thiol reagents does not depend solely on the concentration of molecules present in the deoxy T state but that specific effects of the reagent affects molecular interactions of the hemoglobin S polymerization process.
- Published
- 1990
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15. Isolation and characterization of the gene encoding the muscle-specific isozyme of human phosphoglycerate mutase.
- Author
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Castella-Escola J, Ojcius DM, LeBoulch P, Joulin V, Blouquit Y, Garel MC, Valentin C, Rosa R, Climent-Romeo F, and Cohen-Solal M
- Subjects
- Amino Acid Sequence, Base Sequence, Biological Evolution, Bisphosphoglycerate Mutase metabolism, Cloning, Molecular, Consensus Sequence, Cosmids, Erythrocytes enzymology, Exons, Genes, Humans, Introns, Isoenzymes metabolism, Molecular Sequence Data, Poly A, Restriction Mapping, Sequence Homology, Nucleic Acid, Bisphosphoglycerate Mutase genetics, Isoenzymes genetics, Muscles enzymology
- Abstract
The human muscle-specific phosphoglycerate mutase encoding gene (PGAM-M) has been cloned from a genomic cosmid library and sequenced. The sequence corresponding to the coding region was evaluated and revised by sequencing of the protein itself, fully confirming our results. The amino acid sequence of the M isozyme presented a 80.6% homology with the B isozyme (non-muscle-specific isozyme), a value higher than previously reported. The PGAM-M gene is composed of three exons, which consist of 454, 180 and 202 bp, respectively, and are separated by two introns of 103 bp and approx. 5.6 kb, respectively. Comparison of the structure of the human PGAM-M gene with that coding for human bisphosphoglycerate mutase, an erythroid-specific enzyme belonging to the same multifunctional enzyme family, revealed that the location of the second intron is similar in each gene and corresponds to a tertiary subdomain in the spatial structure of the protein. The transcription start point (tsp) in the PGAM-M gene was identified by both primer extension and S1 nuclease-protection experiments. A TATA-box-like element was observed 29 bp upstream from the tsp; the sequence ATTGG, inverse/complementary to CCAAT-box, was found 40 bp upstream from the supposed TATA box. No muscle-specific consensus sequences could be detected in the 5'-untranslated region. Only one polyadenylation AATAAA signal was observed in the short 3'-untranslated region (43 bp long). Finally, only one copy of this gene is present in the human genome instead of the several copies found for the PGAM-B gene, suggesting the possible evolutionary origin of the muscle subunit in a modified copy of the PGAM-B gene.
- Published
- 1990
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16. Human bisphosphoglycerate mutase expressed in E coli: purification, characterization and structure studies.
- Author
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Calvin MC, Blouquit Y, Garel MC, Prehu MO, Cohen-Solal M, Rosa J, and Rosa R
- Subjects
- Amino Acid Sequence, Chromatography, Cloning, Molecular, Electrophoresis, Polyacrylamide Gel, Escherichia coli enzymology, Humans, Immunodiffusion, Kinetics, Molecular Sequence Data, Bisphosphoglycerate Mutase genetics, Escherichia coli genetics
- Abstract
Bisphosphoglycerate mutase (EC 5.4.2.4.) is an erythrocyte-specific enzyme whose main function is to synthesize 2,3-diphosphoglycerate (glycerate-2,3-P2) an effector of the delivery of O2 in the tissues. In addition to its main synthase activity the enzyme displays phosphatase and mutase activities both involving 2,3-diphosphoglycerate in their reaction. Using a prokaryotic expression system, we have developed a recombinant system producing human bisphosphoglycerate mutase in E coli. The expressed enzyme has been extracted and purified to homogeneity by 2 chromatographic steps. Purity of this enzyme was checked with sodium dodecyl sulfate polyacrylamide gel and Cellogel electrophoresis and structural studies. The bisphosphoglycerate mutase expressed in E coli was found to be very similar to that of human erythrocytes and showed identical trifunctionality, thermostability, immunological and kinetics' properties. However, the absence of a blocking agent on the N-terminus results in a slight difference of the electrophoretic mobility of the enzyme expressed in E coli compared to that of the erythrocyte.
- Published
- 1990
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17. Fate of alpha-hemoglobin chains and erythrocyte defects in beta-thalassemia.
- Author
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Rouyer-Fessard P, Scott MD, Leroy-Viard K, Garel MC, Bachir D, Galacteros F, and Beuzard Y
- Subjects
- Animals, Erythrocyte Deformability, Erythrocyte Membrane metabolism, Humans, Macromolecular Substances, Mice, Spectrin metabolism, Erythrocytes metabolism, Hemoglobins metabolism, Thalassemia blood
- Abstract
The fate of alpha-hemoglobin chains and the cause of membrane protein defects in thalassemic erythrocytes have been studied in: (1) human beta-thalassemia syndromes, (2) mouse beta-thalassemia, and (3) normal human erythrocytes loaded with purified alpha-hemoglobin chains. The similarity and differences observed in these three systems underline the importance of insoluble alpha chains and the direct relationship between the amount of these chains and the membrane protein defects. Indeed, in addition to the alpha/non-alpha ratio of globin chain synthesis, the proteolysis and instability of alpha chains are major factors in modulating the cellular defects.
- Published
- 1990
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18. Towards a mouse model for sickle cell disease: HB SAD.
- Author
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Trudel M, Garel MC, Saadane N, Rouyer-Fessard P, Vidaud D, Costantini F, and Beuzard Y
- Subjects
- Animals, Animals, Newborn blood, Disease Models, Animal, Globins genetics, Hypoxia complications, Mice, Mice, Transgenic genetics, Protein Engineering, Recombinant Proteins, Thromboembolism etiology, Anemia, Sickle Cell, Hemoglobin, Sickle genetics, Mice, Transgenic blood
- Abstract
Very recently a high expression of human hemoglobin S, which causes sickle cell disease, has been obtained in transgenic mice. We have constructed a modified beta S gene, beta SAD which carries two additional mutations in order to induce polymerization of transgenic hemoglobin when diluted by endogenous mouse Hb. The transgenic SAD mice are not anemic but exhibit a low percentage of irreversible sickle cells. Sickling is induced by deoxygenation of erythrocytes in vitro. In addition, the anemia of neonates and the low incidence of SAD animals in the progeny suggest a deleterious effect of SAD Hb during development. Finally, hypoxia induces a high mortality in SAD adults suggesting the induction of vaso-occlusive events.
- Published
- 1990
19. Natural and artificial mutants of the human 2,3-bisphosphoglycerate as a tool for the evaluation of structure-function relationships.
- Author
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Garel MC, Lemarchandel V, Prehu MO, Calvin MC, Arous N, Rosa R, Rosa J, and Cohen-Solal M
- Subjects
- 2,3-Diphosphoglycerate, Amino Acid Sequence, Base Sequence, Bisphosphoglycerate Mutase biosynthesis, Cloning, Molecular, DNA genetics, Escherichia coli genetics, Gene Expression, Humans, Molecular Sequence Data, Plasmids, Structure-Activity Relationship, Bisphosphoglycerate Mutase genetics, Diphosphoglyceric Acids metabolism, Mutation, Phosphotransferases genetics
- Abstract
2,3-bisphosphoglycerate mutase is a multifunctional enzyme which catalyses in red blood cells the synthesis and the degradation of 2,3-bisphosphoglycerate, the allosteric effector of hemoglobin. In order to study the structure-function relationships in BPGM, an expression vector was constructed which yielded an active protein, but with a modified electrophoretic mobility, due to a non-blocked N-terminal residue. Using site directed mutagenesis, mutants were produced with shortened chains. Results indicated the importance of residues 252-256 for the function. A natural deficient mutant with the substitution 89 Arg----Cys was described. Artificial mutant with the same substitution reproduced the same defect, as well as mutants Arg----Gly and Arg----Ser, indicating the key role of Arg 89 in the enzymatic mechanism.
- Published
- 1990
20. [Evaluation of three commercial kits for the estimation of total glycolysed hemoglobin: Hb Al (author's transl)].
- Author
-
Rosa J, Senan C, Blouquit Y, Garel MC, and Molko F
- Subjects
- Adolescent, Adult, Blood Specimen Collection, Chromatography, Edetic Acid, Female, Heparin, Humans, Male, Middle Aged, Temperature, Diabetes Mellitus blood, Glycosides analysis, Hemoglobin A analogs & derivatives, Hemoglobin A analysis, Reagent Kits, Diagnostic
- Abstract
It has been demonstrated by numerous workers that variations in levels of glycolysed hemoglobins Hb Al (Alb, Alb, Alc) are an excellent means of control of the quality of diabetes control. The estimation of the main glycolysed hemoglobin (Hb Alc) is very difficult, and various chromatographic methods have been developed to permit rapid estimation of all these glycolysed hemoglobins. Our work consisted of testing the three microcolumn kits on the french market: Isolab, Helena and Biorad. The great thermo-dependency of the first two kits did not permit us to obtain results in correlation with those of our reference technic derived from Trivelli's method. On the other hand, the levels of Hb Al obtained with the Biorad kit were perfectly correlated with the levels of Hb Alc. The average normal of Hb Al was 6,45 +/- 0,66 p. cent, CV 10,2 p. cent; the average value of Hblc was 5,4 +/- 0,4 p. cent. CV 7,4 p. cent. The average levels of Hb A obtained in diabetic subjects was 12 p. cent, the levels of Hblc being 10 p. cent. Certain restrictions in the use of these microcolumns were demonstrated: presence of Hb F or abnormal Hb, hyperlipemia and presence of chylomicrons.
- Published
- 1980
21. Beta-chain contact sites in the haemoglobin S polymer.
- Author
-
Nagel RL, Johnson J, Bookchin RM, Garel MC, Rosa J, Schiliro G, Wajcman H, Labie D, Moo-Penn W, and Castro O
- Subjects
- Binding Sites, Crystallization, Hemoglobin, Sickle genetics, Humans, Mutation, Protein Binding, Hemoglobins, Abnormal genetics
- Abstract
The amino acid residues involved in the areas of contact that stabilise the haemoglobin S polymer fibre seem to be the same ones that stabilise the basic unit of the deoxyhaemoglobin S crystal: the Wishner-Love double strand. The haemoglobin S fibre is probably formed by a unique packing of these double strands.
- Published
- 1980
- Full Text
- View/download PDF
22. Effects of the alpha 20 mutation on the polymerization of Hb S.
- Author
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Rhoda MD, Blouquit Y, Caburi-Martin J, Monplaisir N, Galacteros F, Garel MC, and Rosa J
- Subjects
- Humans, Kinetics, Mutation, Oxyhemoglobins, Polymers, Protein Conformation, Solubility, Structure-Activity Relationship, Globins genetics, Hemoglobin, Sickle genetics
- Abstract
The contribution of the alpha 20 residues in intermolecular contacts present in hemoglobin S fibers was investigated with mixtures of Hb Le Lamentin alpha 2(20)His----Gln beta 2A and of hemoglobin S alpha 2A beta 2(6)Glu----Val and with artificial hybrids alpha 2(20)His----Gln beta 2(6)Glu----Val. This study showed an increased solubility and delay time of polymerization of Hb S in solution only when the mutation at the alpha 20 residue is cis to the beta 6 Val contact. No modification of the polymerization process occurs when the mutation is trans to this beta 6 Val contact. This result is in agreement with the crystal model of Wishner and Love, who showed that one of the two alpha 20 residues of the Hb S tetramer was involved in an axial contact between hemoglobin S molecules in the crystals of Hb S ( Wishner , B.C., Ward, K.B., Lattman , E.E. and Love, W.E. (1975) J. Mol. Biol. 98, 179-194). The present observation is a new illustration of the validity of the crystal model for the structure of the fibers based on pairs of double filaments.
- Published
- 1984
- Full Text
- View/download PDF
23. Hemoglobin Dakar = Hb Grady: demonstration by a new approach to the analysis of the tryptic core region of the alpha chain and oxygen equilibrium properties.
- Author
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Garel MC, Goossens M, Oudart JL, Blouquit Y, Thillet J, and Rosa J
- Subjects
- Amino Acid Sequence, Amino Acids analysis, Blood Protein Electrophoresis, Electrophoresis, Cellulose Acetate, Globins, Hemoglobin A, Humans, Kinetics, Peptide Fragments analysis, Protein Binding, Protein Conformation, Protein Denaturation, Thermolysin, Trypsin, Hemoglobins, Abnormal, Oxygen blood
- Abstract
Preliminary studies have suggested that in Hb Dakar, histidine alpha112 was substituted by a glutamine. A re-investigation on this hemoglobin is presented in this report. A structural study has been performed using a new approach to analyse the tryptic core region of the human hemoglobin alpha chain. After tryptic digestion of the aminoethylated alpha chain, a secondary digestion of the tryptic core was carried out with chymotrypsin and with another protease, thermolysin. Analyses of the chymotryptic and thermolytic peptides indicated that the structure of Hb Dakar was identical to that of Hb Grady previously described by Huisman et al. who showed the insertion of three amino acid residues in position alpha115 or alpha118. The insertion, which was localized near two residues involved in the alpha1beta1 contact, did not produce a dissociation into dimers. Functional studies demonstrated a a slightly increased oxygen affinity, a lowered cooperativity and a normal Bohr effect. The low amount of the abnormal hemoglobin (8%) may in part be explained by a slight instability of the molecule.
- Published
- 1976
- Full Text
- View/download PDF
24. Chromosomal assignment of the human 2,3-bisphosphoglycerate mutase gene (BPGM) to region 7q34----7q22.
- Author
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Barichard F, Joulin V, Henry I, Garel MC, Valentin C, Rosa R, Cohen-Solal M, and Junien C
- Subjects
- Chromosome Banding, DNA genetics, Humans, Karyotyping, Nucleic Acid Hybridization, Bisphosphoglycerate Mutase genetics, Chromosome Mapping, Chromosomes, Human, Pair 7, Phosphotransferases genetics
- Abstract
A 1.1-kb cDNA clone for human 2,3-bisphosphoglycerate mutase (BPGM) (EC 2.7.5.4) was used to map the structural gene to metaphase chromosomes. In situ hybridization experiments localized the human BPGM gene to chromosome 7 and, more precisely, to region 7q34----7q22.
- Published
- 1987
- Full Text
- View/download PDF
25. Isolation and characterization of the human 2,3-bisphosphoglycerate mutase gene.
- Author
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Joulin V, Garel MC, Le Boulch P, Valentin C, Rosa R, Rosa J, and Cohen-Solal M
- Subjects
- Amino Acid Sequence, Base Sequence, Bisphosphoglycerate Mutase isolation & purification, Blotting, Southern, DNA analysis, Humans, Molecular Sequence Data, Bisphosphoglycerate Mutase genetics, Phosphotransferases genetics
- Abstract
The human 2,3-bisphosphoglycerate mutase gene was isolated from genomic libraries and analyzed by Southern blots and DNA sequencing. The transcription initiation site was localized by primer extension as well as by S1 protection of the mRNA. The gene extends over 22 kilobase pairs; it is composed of two introns (8.8 and 11.5 kilobase pairs long) and three exons (84, 662, and 965 base pairs long). The second exon correlates with a functional subdomain of the protein, as shown by comparison with the yeast phosphoglycerate mutase structure. The sequence TAGAAAA was found 30 bases upstream from the transcription initiation site and could be analogous to the TATA box. A sequence homologous to the CCAAT box was found twice, at positions -75 and -178. There is no GC-rich sequence or GC box in the 5'-flanking region of the gene. Northern blot analysis indicates that the 2,3-bisphosphoglycerate mutase mRNA is detected mainly in erythroid tissues and cell lines, although it is also present in low amounts in a nonerythroid tissue. A comparison of the 5'-upstream sequences with other promoters active only in erythroid cells did not reveal any common signal that could be responsible for the "erythroid promoter."
- Published
- 1988
26. Kinetics of polymerization of hemoglobin S modified by thiol reagents and by oxidation.
- Author
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Domenget C, Garel MC, Rhoda MD, Caburi-Martin J, Galacteros F, and Beuzard Y
- Subjects
- Aniline Compounds, Dithionite, Ethylmaleimide pharmacology, Humans, Iodoacetamide pharmacology, Isoelectric Focusing, Kinetics, Methemoglobin, Oxidation-Reduction, Polymers, Sulfhydryl Reagents, Hemoglobin, Sickle metabolism, Sulfhydryl Compounds pharmacology
- Abstract
The effects of four thiol reagents on the kinetics of polymerization of hemoglobin S have been studied in high phosphate buffer (1.8 M), in the presence (3 mM) or absence of sodium dithionite, depending on the reduction of mixed disulfides of Hb in the presence of this reducing agent. The effect of oxidized forms (methemoglobin) of HbS on the kinetics of aggregation of deoxyHbS was also studied because of the presence of 33% metHbS when HbS was modified by 4-aminophenyl disulfide. In the presence of sodium dithionite, the delay times prior to polymerization of deoxyHbS modified by N-ethylmaleimide, iodoacetamide and 4-aminophenyl disulfide were, respectively, 1.5-, 1.35- and 1.15-times longer than that of native deoxyHbS. The results indicate that the radicals bound to the cysteine beta 93 residue inhibit the contacts in the polymer formation to various extents but do not modify the size of the nuclei.
- Published
- 1985
- Full Text
- View/download PDF
27. Covalent binding of glutathione to hemoglobin. I. Inhibition of hemoglobin S polymerization.
- Author
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Garel MC, Domenget C, Caburi-Martin J, Prehu C, Galacteros F, and Beuzard Y
- Subjects
- Binding Sites, Dithiothreitol pharmacology, Humans, Kinetics, Macromolecular Substances, Protein Binding, Cysteine, Glutathione blood, Hemoglobin, Sickle metabolism, Hemoglobins metabolism
- Abstract
Thiol reagents react with cysteine beta 93 of hemoglobin and as a result increase the oxygen affinity of hemoglobin. In the present studies we have used a thiol-disulfide exchange between mixed disulfides of hemoglobin and reduced glutathione to attach intracellular glutathione to hemoglobin and to study its antisickling properties. The rates of production of glutathionyl hemoglobin (G-Hb) depend on the structure of the thiol reagent linked to cysteine beta 93. Up to 25% G-Hb can be produced in normal and sickle red cells because of the high intracellular concentration of reduced glutathione. This high level of G-Hb in normal cells increases the oxygen affinity by about 35% and reduces heme-heme interactions. In sickle cells the increased oxygen affinity is associated with an inhibition of sickling of about 70% at 21 mm Hg. Inhibition of polymerization of deoxy HbS is also due to a direct inhibition of intermolecular contacts in the fibers as demonstrated by the increased solubility and the increased delay time of G-HbS compared to deoxy HbS.
- Published
- 1986
28. Inhibition of erythrocyte sickling by thiol reagents.
- Author
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Garel MC, Domenget C, Galacteros F, Martin-Caburi J, and Beuzard Y
- Subjects
- Erythrocytes physiology, Humans, Kinetics, Methemoglobin metabolism, Oxygen blood, Oxyhemoglobins metabolism, Reference Values, Anemia, Sickle Cell blood, Antisickling Agents, Erythrocytes drug effects, Sulfhydryl Reagents pharmacology
- Abstract
The antisickling effects of eight thiol reagents that cross the red cell membrane and then react with the cysteine beta 93, the only accessible thiol group of hemoglobin, have been investigated at various pO2 values. In spite of completely reacted hemoglobins, the potent antisickling effect varied from one compound to the other and was partially related to the extent of the increased oxygen affinity of intact sickle cells induced by these compounds. The formation of methemoglobin upon the incubation of red blood cells with some disulfides had only a small effect on the sickling process.
- Published
- 1984
29. Hemoglobin J Cairo: beta 65 (E9) Lys leads to Gln, A new hemoglobin variant discovered in an Egyptian family.
- Author
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Garel MC, Hassan W, Coquelet MT, Goossens M, Rosa J, and Arous N
- Subjects
- Adult, Amino Acid Sequence, Amino Acids analysis, Anemia blood, Egypt, Female, Glutamine, Humans, Lysine, Macromolecular Substances, Male, Oxyhemoglobins, Pedigree, Peptide Fragments analysis, Protein Binding, Hemoglobins, Abnormal
- Abstract
The present report describes clinical, hematological and biochemical studies of a 27-year old Egyptian woman in whom a fast moving Hb variant was found. The abnormal Hb constituted 48% of the total erythrocyte Hb of the propositus and her father. Structural studies demonstrated that in the abnormal Hb lysine beta 65 is replaced by glutamine. The new Hb mutant is designated hemoglobin J Cairo beta 65 (E9) Lys leads to Gln. This substitution results in only a moderate decrease in cooperativity. No evidence of Hb instability was found. A slight anemic state has been observed in the propositus since she reached adolescence.
- Published
- 1976
- Full Text
- View/download PDF
30. Haemoglobin Strasbourg alpha2beta2 23 (B5) Val replaced by Asp: revised structure anf functional properties.
- Author
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Cohen-Solal M, North ML, Thillet J, Albrecht-Ellmer K, Garel MC, Blouquit Y, Rosa J, and Valentin C
- Subjects
- Amino Acid Sequence, Diphosphoglyceric Acids blood, Hydrogen-Ion Concentration, Oxyhemoglobins metabolism, Structure-Activity Relationship, Hemoglobins, Abnormal genetics, Hemoglobins, Abnormal isolation & purification
- Published
- 1978
- Full Text
- View/download PDF
31. [An acquired S hemoglobin in the adult].
- Author
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Gibaud A, Braconnier F, Garel MC, Cohen-Solal M, Gibaud H, and Rosa J
- Subjects
- Abortion, Spontaneous etiology, Adult, Amino Acid Sequence, Anemia, Sickle Cell blood, Blood Protein Electrophoresis, Chromatography, Gel, Female, Hemoglobinopathies genetics, Humans, Male, Pregnancy, Protein Denaturation, Hemoglobin, Sickle analysis, Hemoglobinopathies etiology, Hemoglobins, Abnormal analysis, Transfusion Reaction
- Published
- 1974
32. [Use of a new rheometer for the study of the filtrability of a suspension of sickled red cells as a function of PO2].
- Author
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Martin-Caburi J, Hermann T, Garel MC, Domenget C, Galacteros F, Healy JC, Hanss M, and Beuzard Y
- Subjects
- Filtration, Humans, Methods, Oxygen blood, Partial Pressure, Rheology, Anemia, Sickle Cell blood, Erythrocytes physiology
- Abstract
The filtration time of a small volume (0.1 ml) of red cell suspension from normal (AA), heterozygous (AS) and homozygous (SS) subjects for sickle cell disease was investigated as a function of PO2 The curve of filtration time of AS and SS red cell suspensions was biphasic. At high values of PO2, the progressive reduction of filtrability of sickle cell suspensions with decreasing PO2 occurred without new change in morphology of most of the cells. In contrast, at lower PO2 the apparent filtrability was improved and the cells were sickled." However the red blood cells were retained by the filter and the "solvent" filtrability was improved because rigid and highly deformed sickled cells did not clogged completely the pores of the filter. This study allowed to distinguish a new concept of apparent filtrability for red blood cells in sickle cell disease.
- Published
- 1982
33. One- and two-dimensional NMR investigations of the heme pocket in free alpha(CO) chains from human hemoglobin.
- Author
-
Schaeffer C, Craescu CT, Mispelter J, Garel MC, Rosa J, and Lhoste JM
- Subjects
- Amino Acids analysis, Humans, Hydrogen-Ion Concentration, Magnetic Resonance Spectroscopy, Oxyhemoglobins analysis, Structure-Activity Relationship, Heme analysis, Hemoglobins analysis, Myoglobin analysis
- Abstract
Two-dimensional nuclear magnetic resonance techniques were used to assign resonances corresponding to heme pocket residues of the isolated alpha(CO) subunits of the human adult hemoglobin (HbA). The assignment procedure was based on the partial identification of the amino acid spin system from the J-correlated (COSY) spectrum and on the nuclear Overhauser effect connectivities (from NOSEY spectra) with the heme substituents. We present here partial assignments corresponding to five amino acid residues: Leu86, Leu-91, Val-93, Leu-101 and Leu-136. Starting from the known crystallographic structure of the alpha subunit in the hemoglobin tetramer, we applied a dipolar model to compute the ring-current shift of the protons from fifteen amino acid residues in the heme pocket. Comparison of the predicted and observed chemical shifts suggests that there is a very close similarity between the heme pocket tertiary structure of the alpha(CO) subunits in crystals of HbA(CO) and of the free alpha(CO) chains. The one-dimensional NMR spectra were used to monitor the pH-induced structural changes, the effects of chemical modification and of ligand substitution. Upon increasing the pH from 5.6 to 9.0 the structure of the heme environment appears to be invariant with the exception of some residues in the CD corner. The structure is also largely conserved when p-chloromercuribenzoate is bound to Cys-104. In contrast, the substitution of CO by O2 as ligand induces many large changes in the heme cavity which can be partially characterized by NMR spectroscopy.
- Published
- 1988
- Full Text
- View/download PDF
34. Human bisphosphoglycerate mutase. Expression in Escherichia coli and use of site-directed mutagenesis in the evaluation of the role of the carboxyl-terminal region in the enzymatic mechanism.
- Author
-
Garel MC, Joulin V, Le Boulch P, Calvin MC, Préhu MO, Arous N, Longin R, Rosa R, Rosa J, and Cohen-Solal M
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Bisphosphoglycerate Mutase blood, Bisphosphoglycerate Mutase metabolism, Cloning, Molecular, Erythrocytes enzymology, Escherichia coli enzymology, Escherichia coli growth & development, Genetic Vectors, Humans, Kinetics, Molecular Sequence Data, Oligonucleotide Probes chemical synthesis, Plasmids, Recombinant Proteins metabolism, Restriction Mapping, Sequence Homology, Nucleic Acid, Bisphosphoglycerate Mutase genetics, Escherichia coli genetics, Mutation, Phosphotransferases genetics
- Abstract
Bisphosphoglycerate mutase is an erythrocyte-specific enzyme whose main function is to synthesize 2,3-diphosphoglycerate, the allosteric effector of hemoglobin. In addition to its main 2,3-diphosphoglycerate synthase activity, the enzyme displays phosphatase and mutase activities both involving 2,3-diphosphoglycerate in their reaction. The three activities have been demonstrated to be catalysed at a unique active site. To study the structure of such an active site we have developed a recombinant system producing mutants of human bisphosphoglycerate mutase in Escherichia coli, by site-directed mutagenesis. For this purpose the human bisphosphoglycerate mutase cDNA that we had previously cloned has been used to construct a procaryotic high level expression vector bearing the "tac" promoter. Human bisphosphoglycerate mutase produced in E. coli, a species which does not normally synthesize this enzyme, represented 8% of the total soluble bacterial protein and displayed the three catalytic activities (synthase, mutase, and phosphatase) characteristic of the enzyme. Since it has been suggested that the carboxyl-terminal region may be implicated in the catalytic activity of the enzyme, three variants deleted in this part of the protein were produced. Our results indicate that a minimal deletion of 7 amino acid residues in the carboxyl-terminal portion of the human bisphosphoglycerate mutase completely abolished the three catalytic activities of the enzyme. In contrast, the effects of the deletion of the last two lysine residues were limited to a 38% reduction in the synthase activity. These results show that the carboxyl-terminal amino acid residues are either directly or indirectly implicated in the three catalytic functions of the human bisphosphoglycerate mutase, and that the two terminal lysine residues are not essential for the major part of the enzymatic mechanism of the enzyme.
- Published
- 1989
35. Sickle cell hemoglobin fiber formation strongly inhibited by the Stanleyville II mutation (alpha 78 Asn leads to Lys).
- Author
-
Rhoda MD, Martin J, Blouquit Y, Garel MC, Edelstein SJ, and Rosa J
- Subjects
- Chromatography, DEAE-Cellulose, Electrophoresis, Cellulose Acetate, Heterozygote, Humans, Microscopy, Electron, Cytoskeleton ultrastructure, Erythrocytes ultrastructure, Hemoglobin, Sickle isolation & purification, Hemoglobins, Abnormal isolation & purification
- Abstract
A double mutant hemoglobin possessing both the Hb S (beta 6 Glu leads to Val) and the Hb Stanleyville II (alpha 78 Asn leads to Lys) mutations has been purified from the blood of a donor heterozygous for both of the mutations. The purification required two chromatography steps, with the second permitting resolution of the double mutant from Hb A2 remaining after the first step. Measurement of the competence for fiber formation by the double mutant hemoglobin was carried out by the centrifugation of gels to obtain Csat. The double mutant was found to have a greatly elevated Csat, 26.4 gm/dl, compared to 15.2 gm/dl for the Hb S control. The concentration of the pellet of the centrifuged gel for the double mutant was in the range 48-50 gm/dl, suggesting that no major rearrangement in the structure of the fibers had been induced by the Stanleyville II mutation. Electron microscopic observations on the fibers of the double mutant confirmed that a normal appearance was maintained.
- Published
- 1983
- Full Text
- View/download PDF
36. Covalent binding of glutathione to hemoglobin. II. Functional consequences and structural changes reflected in NMR spectra.
- Author
-
Craescu CT, Poyart C, Schaeffer C, Garel MC, Kister J, and Beuzard Y
- Subjects
- Binding Sites, Disulfides metabolism, Humans, Hydrogen-Ion Concentration, Magnetic Resonance Spectroscopy methods, Oxyhemoglobins metabolism, Protein Binding, Protein Conformation, Glutathione blood, Hemoglobin A metabolism, Hemoglobin, Sickle metabolism
- Abstract
Binding of glutathione by disulfide linkage to Cys-beta 93 of hemoglobin tetramers within sickle cells increases the oxygen affinity and significantly inhibits sickling at low partial oxygen pressure (Garel, M-C., Domenget, C., Caburi-Martin, J., Prehu, C., Galacteros, F., and Beuzard, Y. (1986) J. Biol. Chem. 261, 14704-14709). This article reports a characterization of the oxygen-binding properties of glutathionyl hemoglobin (G-Hb) in solution in the presence or absence of allosteric effectors. The studies reveal a nearly 6-fold increase in oxygen affinity compared to native HbA and a Hill coefficient at half-saturation (n50) of 1.50 compared to n50 of approximately 2.9 for HbA. The oxygen Bohr effect measured in the alkaline pH range is reduced by 38%. Addition of 2,3-diphosphoglycerate decreases the oxygen affinity of G-Hb and HbA to a similar extent and increases the Bohr effect, indicating that the binding sites for organic phosphates are not perturbed in G-Hb. The rate of autooxidation of G-HbO2 is slower than of HbAO2. Oxidation by ferricyanide of G-HbCO is also reduced and is biphasic, demonstrating a heterogeneous susceptibility of the hemes in G-Hb. Flash photolysis experiments indicate that the tetramer-dimer dissociation constant is 1 order of magnitude greater for G-HbCO than for HbACO. High resolution NMR spectra at 400 MHz show that in G-Hb: the tertiary structure of the beta heme pocket is significantly perturbed, particularly in the F helix and the EF corner; the formation of the salt bridge between His-beta 146 and Asp-beta 94, a feature of the deoxy state, is precluded; and a deoxy interchain (alpha 1 beta 2) contact between Asp beta 2 99 and Tyr alpha 1 42 is appreciably destabilized. The NMR data provide a structural basis for interpreting the high oxygen affinity, reduced cooperativity, and diminished polymerization of G-HbS.
- Published
- 1986
37. A method for isolation of abnormal haemoglobins with high oxygen affinity due to a frozen quaternary r-structure: application to Hb Creteil alpha 2 A beta 2 (F5) 89 ASN.
- Author
-
Garel MC, Cohen-Solal M, Blouquit Y, and Rosa J
- Subjects
- Amino Acids analysis, Asparagine, Binding Sites, Cellulose, Chromatography, Gel, Chromatography, Ion Exchange, Cystamine, Electrophoresis, Female, France, Humans, Iodoacetates, Methods, Oxyhemoglobins isolation & purification, Pedigree, Peptide Fragments analysis, Protein Binding, Protein Conformation, Serine, Trypsin, Urea, Hemoglobins, Abnormal isolation & purification, Oxygen blood
- Published
- 1974
- Full Text
- View/download PDF
38. Hb Strasbourg alpha2beta2 20 (B2) Val leads to Asp: a variant at the same locus as Hb Olympia beta 20 Val leads to Met.
- Author
-
Garel MC, Blouquit Y, Arous N, and Rosa J
- Subjects
- Adolescent, Amino Acid Sequence, Amino Acids analysis, Aspartic Acid, Blood Protein Electrophoresis, Electrophoresis, Cellulose Acetate, Female, Genetic Variation, Humans, Methionine, Peptide Fragments analysis, Valine, Hemoglobins, Abnormal
- Published
- 1976
- Full Text
- View/download PDF
39. Binding of 21 thiol reagents to human hemoglobin in solution and in intact cells.
- Author
-
Garel MC, Beuzard Y, Thillet J, Domenget C, Martin J, Galacteros F, and Rosa J
- Subjects
- Adult, Disulfides blood, Humans, In Vitro Techniques, Kinetics, Oxidation-Reduction, Protein Binding, Erythrocytes metabolism, Hemoglobins metabolism, Sulfhydryl Reagents blood
- Abstract
The reactivity of the cysteine-beta 93 residue of human hemoglobin was investigated in order to define the optimal structure of potential antisickling agents. The properties of 21 thiol reagents were compared with regard to (a) their binding rate to hemoglobin in solution and within intact cells; (b) the modification of the oxygen dissociation curve of intact cells and (c) the effect on methemoglobin formation in solution or within intact cells. The results show the very different behaviors of these reagents.
- Published
- 1982
- Full Text
- View/download PDF
40. Hemoglobin Dakar.
- Author
-
Garel MC and Rosa J
- Subjects
- Africa, Western, Humans, Hemoglobins, Abnormal
- Published
- 1976
41. Molecular cloning of the human 2,3-bisphosphoglycerate mutase cDNA and revised amino acid sequence.
- Author
-
Cohen-Solal M, Joulin V, Romeo PH, Rosa R, Valentin C, Garel MC, and Rosa J
- Subjects
- Amino Acid Sequence, Bisphosphoglycerate Mutase blood, Cloning, Molecular, Humans, RNA, Messenger blood, RNA, Messenger genetics, Reticulocytes enzymology, Bisphosphoglycerate Mutase genetics, DNA genetics, Phosphotransferases genetics
- Abstract
The human erythrocyte 2,3-bisphosphoglycerate mutase (BPGM) is a multifunctional enzyme which controls the metabolism of 2,3-diphosphoglycerate (DPG), the main allosteric effector of haemoglobin. Several cDNA banks were constructed from reticulocyte mRNA either by conventional cloning methods in plasmid pBR322 and screening with specific mixed oligonucleotide probes, or in the expression vector lambda gt 11. The largest cDNA isolated was 1673 bases, and encodes for a protein of 258 amino acids; it contains a large 3' untranslated region (785 bases). It is slightly smaller than the size of the intact mRNA estimated by Northern blot (1800 bases). Our sequence data indicate differences with the previously published amino acid sequence involving 21% of the residues. They were entirely confirmed by the amino acid composition of the tryptic peptides derived from purified BPGM. The revised amino acid sequence of the human BPGM is presented.
- Published
- 1987
42. Interaction of hemoglobin Siriraj with hemoglobin S: a mild sickle cell syndrome.
- Author
-
Rhoda MD, Arous N, Garel MC, Mazarin M, Monplaisir N, Braconnier F, Rosa J, Cohen-Solal M, and Galacteros F
- Subjects
- Amino Acid Sequence, Genetic Carrier Screening, Genetic Variation, Hemoglobin A metabolism, Hemoglobins, Abnormal genetics, Hemolysis, Humans, Kinetics, Male, Middle Aged, Syndrome, Anemia, Sickle Cell blood, Hemoglobin, Sickle metabolism, Hemoglobins, Abnormal metabolism
- Abstract
Hb Siriraj is a beta chain variant in which beta 7 (A4) Glu is replaced by a lysine. It has been encountered in association with Hb S in a black man from Martinique. Some properties of Hb Siriraj are compared, particularly, with Hb C [alpha 2 beta 26(A3)Glu----Lys], and a study of its in vitro interaction with Hb S is discussed.
- Published
- 1986
- Full Text
- View/download PDF
43. Functional studies of Hb Malmö beta97 (FG4) His leads to Gln.
- Author
-
Thillet J, Garel MC, Blouquit Y, Basset P, Dreyfus B, and Rosa J
- Subjects
- Allosteric Regulation, Diphosphoglyceric Acids blood, Hydrogen-Ion Concentration, Oxidation-Reduction, Oxygen blood, Oxyhemoglobins metabolism, Structure-Activity Relationship, Hemoglobins, Abnormal physiology
- Published
- 1977
- Full Text
- View/download PDF
44. Hemoglobin C Ziguinchor alphaA2 beta62 (A3) Glu leads to Val beta58 (E2) Pro leads to Arg: the second sickling variant with amino acid substitutions in 2 residues of the beta polypeptide chain.
- Author
-
Goossens M, Garel MC, Auvinet J, Basset O, Ferreira Gomes P, Rosa J, and Arous N
- Subjects
- Adult, Amino Acid Sequence, Anemia, Sickle Cell blood, Anemia, Sickle Cell genetics, Genes, Humans, Isoelectric Point, Male, Peptides analysis, Senegal, Hemoglobin C isolation & purification, Hemoglobin, Sickle isolation & purification, Hemoglobins, Abnormal analysis
- Published
- 1975
- Full Text
- View/download PDF
45. Molecular cloning and sequencing of the human erythrocyte 2,3-bisphosphoglycerate mutase cDNA: revised amino acid sequence.
- Author
-
Joulin V, Peduzzi J, Roméo PH, Rosa R, Valentin C, Dubart A, Lapeyre B, Blouquit Y, Garel MC, and Goossens M
- Subjects
- Amino Acid Sequence, Base Sequence, Bisphosphoglycerate Mutase genetics, Bisphosphoglycerate Mutase isolation & purification, DNA Restriction Enzymes, Humans, Molecular Weight, Plasmids, Bisphosphoglycerate Mutase blood, Cloning, Molecular, DNA metabolism, Erythrocytes enzymology, Phosphotransferases blood
- Abstract
The human erythrocyte 2,3-bisphosphoglycerate mutase (BPGM) is a multifunctional enzyme which controls the metabolism of 2,3-diphosphoglycerate, the main allosteric effector of haemoglobin. Several cDNA banks were constructed from reticulocyte mRNA, either by conventional cloning methods in pBR322 and screening with specific mixed oligonucleotide probes, or in the expression vector lambda gt 11. The largest cDNA isolated contained 1673 bases [plus the poly(A) tail], which is slightly smaller than the size of the intact mRNA as estimated by Northern blot analysis (approximately 1800 bases). This cDNA encodes for a protein of 258 residues; the protein yielded 34 tryptic peptides which were subsequently isolated by h.p.l.c. Our nucleotide sequence data were entirely confirmed by the amino acid composition of these tryptic peptides and reveal several major differences from the published sequence; the revised amino acid sequence of human BPGM is presented. These findings represent the first step in the study of the expression and regulation of this enzyme as a specific marker of the erythroid cell line.
- Published
- 1986
- Full Text
- View/download PDF
46. Filterability of sickle cells as a function of pO2: role of physico-chemical factors.
- Author
-
Kraiem A, Craescu CT, Galacteros F, Martin-Caburi J, Domenget C, Garel MC, and Beuzard Y
- Subjects
- Cell Separation, Filtration, Hematocrit, Humans, Hydrogen-Ion Concentration, Osmolar Concentration, Partial Pressure, Temperature, Anemia, Sickle Cell blood, Erythrocyte Deformability physiology, Erythrocytes, Abnormal physiology, Oxygen blood
- Abstract
A rigidity index (RI) related to red blood cell deformability was measured by using the hemorheometre. The RI for 13 patients homozygous for sickle cell disease was 109 +/- 44 at 37 degrees C and at atmospheric pO2. The filtration time curve as a function of pO2 is biphasic for sickle cell suspensions. The pO2 at which filtration time is maximum, pO2max., correlated with the rigidity index measured at atmospheric pO2. This pO2max. value was very sensitive to small changes in physico-chemical parameters such as osmolality, pH, temperature, hematocrit, and cell density. Conditions which reduced the Hb S polymerization induced a leftward shift of pO2max.. The experimental curves are in agreement with theoretical models based on the presence of two abnormal cell types: filtrable "slow cells" and infiltrable "sickled cells".
- Published
- 1989
- Full Text
- View/download PDF
47. Molecular cloning and nucleotide sequence of murine 2,3-bisphosphoglycerate mutase cDNA.
- Author
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Le Boulch P, Joulin V, Garel MC, Rosa J, and Cohen-Solal M
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Binding Sites, Globins genetics, Humans, Mice, Molecular Sequence Data, Nucleic Acid Hybridization, Phylogeny, Rabbits, Structure-Activity Relationship, Bisphosphoglycerate Mutase genetics, Cloning, Molecular, DNA genetics, Phosphotransferases genetics
- Abstract
Cloning and sequencing of a murine cDNA with the entire coding region of 2,3-bisphosphoglycerate mutase is reported, as a prerequisite for further expression studies of this erythroid specific enzyme in Friend mouse erythroleukemia cells. A comparison between species of the deduced amino acid sequences of these proteins shows 20 substitutions between mouse and human and 21 between mouse and rabbit: none of these substitutions are in positions assumed to be in the active site. Amino acid alignment with the other related enzymes, the phosphoglycerate mutases, in combination with crystallographic data from yeast phosphoglycerate mutase, gives some insight into the structure/function correlation for this protein family. Amino acid residues which are most likely critical for either 2,3-bisphosphoglycerate mutase or phosphoglycerate mutase function are pointed out. Concerning the phylogenetic analysis, phosphoglycerate mutases B and M from mammalians appear to have diverged with the yeast enzyme from a common ancestor, before the emergence of the 2,3-bisphosphoglycerate mutases.
- Published
- 1988
- Full Text
- View/download PDF
48. Oxidation properties of two hemoglobin variants with their mutation localized in the heme pocket: Hb Castilla beta 32 (B14) Leu replaced by Arg and Hb Toulouse beta 66 (E10) Lys replaced by Glu, and abnormal functional properties of Hb Castilla.
- Author
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Thillet J, Garel MC, Bierme R, and Rosa J
- Subjects
- Amino Acids analysis, Erythrocytes metabolism, Hemoglobin A metabolism, Hemoglobins, Abnormal physiology, Humans, Isoelectric Focusing, Kinetics, Mutation, Oxidation-Reduction, Protein Conformation, Spectrophotometry, Hemoglobins, Abnormal genetics
- Published
- 1980
- Full Text
- View/download PDF
49. Hemoglobin Castilla beta 32 (B14) Leu leads to Arg; a new unstable variant producing severe hemolytic disease.
- Author
-
Garel MC, Blouquit Y, Rosa J, Arous N, and Romero Garcia C
- Subjects
- Adult, Amino Acid Sequence, Anemia, Hemolytic blood, Anemia, Hemolytic genetics, Anemia, Hemolytic pathology, Female, Heinz Bodies pathology, Humans, Peptides analysis, Protein Conformation, Spain, Structure-Activity Relationship, Hemoglobins, Abnormal analysis
- Published
- 1975
- Full Text
- View/download PDF
50. [Clinical and biochemical studies of a new case of Hb J Baltimore (alpha 2A beta2 16 Gly yields Asp) isolated in France (author's transl)].
- Author
-
Mauran A, Manesse B, Cohen-Solal M, Garel MC, Thillet J, Blouquit Y, Caburi J, Vergne H, and Rosa J
- Subjects
- Amino Acid Sequence, Blood Protein Electrophoresis, Chromatography, Chromatography, Gas, Chromatography, Thin Layer, France, Hematocrit, Hemoglobins, Humans, Methods, Mutation, Oxygen Consumption, Peptide Chain Termination, Translational, Peptide Fragments isolation & purification, Reticulocytes, Staining and Labeling, Trypsin, Hemoglobinopathies epidemiology, Hemoglobins, Abnormal analysis
- Published
- 1974
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