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Biomedical subjects

Y Beuzard

Publications and source records attributed to Y Beuzard.

At least 73 records · Page 4Linked to original sources
PubMed1990

Entrapment of purified alpha-hemoglobin chains in normal erythrocytes. A model for beta thalassemia.

Altered membrane proteins have been previously described in beta thalassemia and are thought to play an important role in the shortened erythrocyte survival. To investigate the mechanism by which these changes occur, purified heme-containing alpha-hemoglobin chains were entrapped within normal erythrocytes by reversible osmotic lysis. These resealed cells exhibited normal hemoglobin concentration, cell volume, deformability, and no substantial modifications of membrane proteins. Incubation (37 degrees C; up to 20 h) of the alpha-chain-loaded cells resulted in increasing amounts of membrane-associated alpha-chains. This was associated with concurrent decreases in the protein concentrations and reactive thiol groups of spectrin, ankyrin, and actin as determined by gel electrophoresis. The decreases in membrane protein concentration and reactive thiol groups after 20 h of incubation were closely correlated (R2 = 0.947) in the alpha-chain-loaded cells. Indicative of increased oxidant stress within the alpha-chain-loaded erythrocytes, methemoglobin generation was also significantly increased in the alpha-chain-loaded erythrocytes. In addition, entrapment of alpha-chains led to a progressive and significant decrease in erythrocyte deformability. Thus, the entrapment of purified alpha-chains in normal erythrocytes resulted in structural and functional abnormalities very similar to that observed in beta-thalassemic erythrocytes in vivo. The model described provides a means by which the fate of excess alpha-chains, their pathophysiological effects, as well as possible therapeutic approaches to thalassemias can be examined.

Erythrocyte Deformability↗
PubMed1990

Ca2+ permeability in deoxygenated sickle cells.

Deoxygenation of sickle cells is known to increase cation permeabilities (Na+, K+, and Ca2+). The possible mechanisms involved in the increased uptake of Ca2+ were investigated: activation of Ca2+ channels, involvement of the anion channel, and the formation of endocytic vacuoles. The Ca2+ channel blocker nifedipine reduced the deoxy-stimulated Ca2+ uptake by about 30% to 40%. The anion channel inhibitor DIDS (4,4' diisothiocyanate stilbene 2,2' disulfonate) inhibited the deoxy-stimulated Ca2+ uptake by approximately 50%. Maximal possible endocytic uptake, measured by using an impermeant marker ([3H] inuline), accounted for 6% to 9% of the total Ca2+ uptake. These data indicate that the deoxygenation-induced increase in Ca2+ permeability could result from both the activation of a Ca2+ channel and of a transport system for cations involving interactions between polymerized hemoglobin S, band 3 and other membrane components. Endocytosis appears to play only a minor role in the Ca2+ uptake of deoxygenated sickle cells.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1990

Fate of alpha-hemoglobin chains and erythrocyte defects in beta-thalassemia.

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.

Animals↗
PubMed1990

Towards a mouse model for sickle cell disease: HB SAD.

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.

Anemia, Sickle Cell↗
PubMed1989

A study of membrane protein defects and alpha hemoglobin chains of red blood cells in human beta thalassemia.

The soluble pool of alpha hemoglobin chains present in blood or bone marrow cells was measured with a new affinity method using a specific probe, beta A hemoglobin chain labeled with [3H]N-ethylmaleimide. This pool of soluble alpha chains was 0.067 +/- 0.017% of hemoglobin in blood of normal adult, 0.11 +/- 0.03% in heterozygous beta thalassemia and ranged from 0.26 to 1.30% in homozygous beta thalassemia intermedia. This elevated pool of soluble alpha chains observed in human beta thalassemia intermedia decreased 33-fold from a value of 10% of total hemoglobin in bone marrow cells to 0.3% in the most dense red blood cells. The amount of insoluble alpha chains was measured by using the polyacrylamide gel electrophoresis in urea and Triton X-100. In beta thalassemia intermedia the amount of insoluble alpha chains was correlated with the decreased spectrin content of red cell membrane and was associated with a decrease in ankyrin and with other abnormalities of the electrophoretic pattern of membrane proteins. The loss and topology of the reactive thiol groups of membrane proteins was determined by using [3H]N-ethylmaleimide added to membrane ghosts prior to urea and Triton X-100 electrophoresis. Spectrin and ankyrin were the major proteins with the most important decrease of thiol groups.

Erythrocyte Membrane↗
PubMed1989

Alteration in protein kinase C activity and subcellular distribution in sickle erythrocytes.

In agreement with previous data, membrane protein phosphorylation was found to be altered in intact sickle cells (SS) relative to intact normal erythrocytes (AA). Similar changes were observed in their isolated membranes. The involvement of protein kinase C (PKC) in this process was investigated. The membrane PKC content in SS cells, measured by [3H]phorbol ester binding, was about 6-times higher than in AA cells. In addition, the activity of the enzyme, measured by histone phosphorylation was also found to be increased in SS cell membranes but decreased in their cytosol compared to the activity in AA cell membranes and cytosol. The increase in membrane PKC activity was observed mostly in the light fraction of SS cells, fractionated by density gradient, whereas the decrease in cytosolic activity was only observed in the dense fraction. PKC activity, measured in cells from the blood of reticulocyte-rich patients, exhibited an increase in both membranes and cytosol, thus explaining some of the effects observed in the SS cell light fraction, which is enriched in reticulocytes. The increase in PKC activity in the membranes of SS cells is partly explained by their young age but the loss of PKC activity in their cytosol, particularly in that of the dense fraction, seems to be specific to SS erythrocytes. The relative decrease in membrane PKC activity between the dense and the light fractions of SS cells might be related to oxidative inactivation of the enzyme.

Anemia, Sickle Cell↗
PubMed1989

KU 812: a pluripotent human cell line with spontaneous erythroid terminal maturation.

A human leukemic cell line KU 812 was recently established and described as a basophilic cell line. In the present study we show that KU 812 and two of its clones are at least bipotent: in addition to a minor component of basophils, the majority of KU 812 cells belongs to the erythroid cell lineage with a significant percentage (about 15%) of mature hemoglobinized erythroblasts. This terminal differentiation is associated with the synchronized synthesis of the main erythroid proteins, including glycophorins, spectrin beta chain, band 3, and hemoglobin. The predominant hemoglobins are adult, fetal, and Bart's hemoglobin. Adult hemoglobin represented up to 75% of all hemoglobins in the KU 812 F clone in passages containing a high number of mature erythroblasts. Transcripts of all human globin chains were present with ten times less embryonic chain messenger RNA (mRNA) than alpha-, beta- or gamma-chain mRNA. Hemin slightly increased the total hemoglobin production of the cell line, especially gamma-globin chain synthesis, but did not modify the percentage of hemoglobinized cells. Phorbol myristate acetate (PMA) had a complex effect, inducing a proportion of KU 812 cells to adhere to the plastic culture flask. The adherent cell fraction expressed a very low level of specific erythroid proteins, but their ultrastructure was consistent with immature erythroid cells. In contrast, approximately 40% of the nonadherent cells were mature erythroid cells. Cell-sorting experiments showed that this paradoxic effect of PMA is mostly due to cell selection, the more mature cells being unable to adhere. In addition, KU 812 F was found to be sensitive to erythropoietin, which slightly increased its plating efficiency range (from 0% to 50%) in semisolid medium and enhanced hemoglobin accumulation twofold. In binding experiments using 125I erythropoietin, a single class of high-affinity Epo receptors (Kd: 250 pM) was detected by binding with a density of 205 receptors per cell. The KU 812 cell line is therefore a unique model for studying cell commitment toward different hematopoietic lineages and erythroid differentiation.

Antigens, Differentiation↗
PubMed1989

Prenatal diagnosis of haemoglobinopathies by ion exchange HPLC of haemoglobins.

A new method, the cation exchange HPLC of haemoglobins, has been compared to the classical carboxymethyl cellulose (CMC) chromatography of globin chains for the prenatal diagnosis of beta thalassaemia and sickle cell disease. The two methods correlated highly. The HPLC procedure can use two independent and reliable means--optical density at 405 nm and radioactivity to determine the adult Hb/HbF+Fac ratio. The diagnosis is obtained in 15 min by cation exchange HPLC.

Anemia, Sickle Cell↗
PubMed1989

Inhibition of K+ efflux and dehydration of sickle cells by [(dihydroindenyl)oxy]alkanoic acid: an inhibitor of the K+ Cl- cotransport system.

[(Dihydroindenyl)oxy]alkanoic acid (DIOA) was recently introduced as a potent inhibitor of the K+Cl- cotransport system without side effects on other cation transport systems [Garay, R. P., Nazaret, C., Hannaert, P.A. & Cragoe, E. J., Jr. (1988) Mol. Pharmacol. 33, 696-701]. In sickle cells, an abnormal activation of this K+Cl- cotransport system was proposed to be involved in cell K+ loss and dehydration. We found that DIOA inhibited the abnormal sickle cell K+ loss and specifically reduced sickle cell density upon stimulation of the net outward K+Cl- cotransport--i.e., low pH, hypoosmolarity, and activation by N-ethylmaleimide. DIOA opens another therapeutic approach to sickle cell disease by inhibiting cell dehydration, which favors HbS polymerization and reduces erythrocyte deformability.

Anemia, Sickle Cell↗
PubMed1989

Filterability of sickle cells as a function of pO2: role of physico-chemical factors.

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".

Anemia, Sickle Cell↗
PubMed1988

Mouse alpha chains inhibit polymerization of hemoglobin induced by human beta S or beta S Antilles chains.

A murine model of sickle cell disease was tested by studying the polymerization of hybrid hemoglobin tetramers between alpha mouse and human beta S or beta S Antilles chains were prepared from Hb S Antilles, which was a new sickling hemoglobin inducing a sickle cell syndrome more severe than Hb S. The hybrid molecules did not polymerize in solution, indicating that the mouse alpha chains inhibited fiber formation. Consequently, a mouse model for sickle cell disease requires the transfer and expression of both alpha and beta S or beta S Antilles genes.

Amino Acid Sequence↗
PubMed1988

Determination of erythrocyte transit times through micropores. I--Basic operational principles.

To study the transit times of each red blood cell passing through cylindrical micropores and in order to evaluate sub-population of cells with regard to their deformability, we have developed a new system called the cell transit time analyser (CTTA). By using an AC voltage (100 KHz) across a special filter, we measure the electrical conductance change produced by the cells passing through the pores under a known driving pressure. This computer based device provides the distribution of transit times tau for 2000 cells in 1 minute and as a result the mean transit time [tau]. Experiments with red cells were designed to evaluate the flow behavior of both normal cells and cells whose mechanical properties were artificially altered. Cell volume was changed by use of non-isotonic media. Cell shape and cell volume were modified by varying the pH of the suspending buffer. Results of these experiments are: 1) a skew distribution of transit times towards high tau values for both control cells and artificially altered cells is observed: 2) [tau] is minimum for isotonic conditions and increases sharply for either hypotonic or hypertonic media: 3) [tau] is minimum at physiological pH and increases for either acid or alcaline changes of pH.

Blood Flow Velocity↗
PubMed1987

Prenatal diagnosis of beta thalassaemia by reverse phase HPLC.

Reverse phase HPLC of radioactive globin chains has been compared to classical carboxy methyl cellulose chromatography for the prenatal diagnosis of beta thalassaemia. The two methods correlated highly (r = 0.97 p less than 0.0005) and provided an identical diagnosis for 40 fetal blood samples of fetuses homozygous or heterozygous for beta thalassaemia. The HPLC procedure was much faster and required fewer biochemical steps (no globin preparation). It was at least as accurate and more sensitive than the classical chromatography. A single column can be used for 150 analyses and is always ready to be used. Last but not least it is much less expensive than CMC chromatography.

Carboxymethylcellulose Sodium↗
PubMed1987

Ca2+ permeability and cytosolic Ca2+ concentration are not impaired in beta-thalassemic and hemoglobin C erythrocytes.

Total calcium content, determined by atomic absorption spectroscopy, Ca2+ influx, and cytosolic free Ca2+ concentration ( [Ca]i), estimated by a method involving the incorporation of a Ca2+ chelator (Quin 2), were measured in erythrocytes from beta-thalassemic (beta-thal) and hemoglobin C (CC) patients. Elevation of the total calcium content was observed in the cells from all patients, particularly in CC and splenectomized beta-thal. However, [Ca]i was within the normal range (approximately 25 nmol/L) in all the pathologic cells. Ca2+ influx in CC cells and in cells from nonsplenectomized beta-thal patients was also within the same range as that observed in control erythrocytes. In cells from splenectomized beta-thal patients, the kinetic of 45Ca influx was biphasic, indicating the existence of two pools of exchangeable Ca2+. Density fractionation of the cells from one splenectomized beta-thal patient showed that the rapid pool corresponded to the lightest cell fraction, which was also found to have the highest calcium content. The dense cells exhibited a normal Ca2+ influx as well as a smaller increase in total calcium content. It is suggested that, as in sickle cell anemia, the excess of Ca2+ in beta-thal cells is not free in the cytoplasm but trapped within endocytic vacuoles, especially in a population of abnormal cells that are normally removed by the spleen. In CC patients, who have a functional spleen, a different mechanism could be responsible for the calcium retention. In conclusion, the present results demonstrate that in these two cases of hemolytic anemia associated with high calcium content, Ca2+ permeability and the the level of cytosolic Ca2+ are normal.

Adenosine Triphosphate↗
PubMed1986

Contact inhibition within hemoglobin S polymer by thiol reagents.

N-Ethylmaleimide, a thiol reagent, increases the solubility of deoxyhemoglobin S. We investigated which of the two reacted beta 93 cysteine residues of the Hb tetramer was responsible for the inhibition of Hb S polymerization. Accordingly we compared the solubility of equal mixtures of HbA + HbS, HbA NEM + HbS and HbA + HbS NEM. Upon deoxygenation these mixtures contain about 50% a stable and asymmetrical hybrid alpha 2A beta A beta S, alpha 2A beta A,NEM beta S or alpha 2A beta A beta S,NEM respectively and 25% parental molecules as confirmed by ion-exchange HPLC performed in anaerobic conditions. Within the hybrid molecule, beta A or beta A,NEM chain has to be present in the alpha beta dimer located in trans to the dimer which contains the only beta 6 valine residue participating in intermolecular contacts (dimer in cis), while beta S or beta S,NEM must be in cis position in the hybrid molecule. The solubility of mixtures increases 4% for HbA NEM + HbS and 20% for HbA + HbS NEM mixtures compared to HbA + HbS mixture, indicating that the inhibitory effect of N-ethylmaleimide is more effective in cis than in trans position. The absence of a major role played by N-ethylmaleimide located in trans was supported by the solubility study of a mixture of HbS + Hb Créteil beta 89 Ser----Asn. The beta 89 residue in trans next to the cysteine beta 93 modified the T structure similarly to N-ethylmaleimide, and did not affect intermolecular contacts. Crystallographic studies of molecular contacts within deoxyHbS crystals suggest that the cis inhibitory effect of N-ethylmaleimide can be explained by direct inhibition of 'external' contacts between double strands involving the CD corner of the alpha chains.

Centrifugation↗
PubMed1986

Covalent binding of glutathione to hemoglobin. I. Inhibition of hemoglobin S polymerization.

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.

Binding Sites↗
PubMed1986

Covalent binding of glutathione to hemoglobin. II. Functional consequences and structural changes reflected in NMR spectra.

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.

Binding Sites↗