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C Vasseur

Publications and source records attributed to C Vasseur.

At least 55 records · Page 3Linked to original sources
PubMed1992

Two new human hemoglobin variants caused by unusual mutational events: Hb Zaïre contains a five residue repetition within the alpha-chain and Hb Duino has two residues substituted in the beta-chain.

With rare exceptions, the more than 600 human hemoglobin variants described are caused by a single point mutation. Other abnormal features, such as unequal crossing-over, frameshift mutagenesis or double mutations in the same polypeptide chain, have seldom been encountered. We report two new variants caused by such rare mutational events. Hb Zaïre [alpha 116(GH4)-His-Leu-Pro-Ala-Glu-117 (GH5)] is the second example in which a short amino acid sequence is inserted within the alpha-chain. This abnormal hemoglobin results from a tandem repetition of 5 amino-acid residues, from sequence 112 through 116, at the end of the GH corner. Hb Duino is an unstable hemoglobin. It presents within the same beta-chain, the association of two rare point mutations; these substitutions are those found in Hb Newcastle [beta 92(F8)His----Pro] and in Hb Camperdown [beta 104(G6)Arg----Ser]. Family studies demonstrated that the Hb Newcastle abnormality was a de novo mutation of a gene already carrying the Hb Camperdown substitution.

Adult↗
PubMed1992

Decreased G3PDH binding to erythrocyte membranes in sickle cell disease.

Several membrane abnormalities have been described in red cells from patients with sickle cell disease, responsible for chronic hemolytic anemia. We describe here a 35-50% inhibition of the binding of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) to the membrane of sickle red cells. Varying the phosphorylation state of the membrane proteins did not change their affinity for the enzyme. Protein band 3 and the cytoplasmic domain of this protein isolated from sickle red cells showed normal interaction with the enzyme. The inhibition observed with intact membranes is not due to short term oxidation of membrane proteins, as various procedures inducing acute oxidative stress in normal membranes did not reproduce the inhibition of G3PDH binding. We conclude that the alteration of the binding of G3PDH to the membrane of sickle erythrocytes is probably related to long term processes involving cycles of HbS polymer formation.

Adult↗
PubMed1991

Human erythrocyte band 3 polymorphism (band 3 Memphis): characterization of the structural modification (Lys 56----Glu) by protein chemistry methods.

Band 3 variants occur rather frequently in different populations. Based on sodium dodecyl sulfate (SDS)-polyacrylamide electrophoretic properties, a widespread polymorphism (band 3 Memphis) has been previously described. It corresponds to a protein that has been hypothesized to be elongated in its N-terminal cytoplasmic domain. Band 3 from a heterozygote subject for this polymorphism and that displays a normal reactivity towards stilbene disulfonates has been isolated and its primary structure determined by protein chemistry. Reverse-phase high performance liquid chromatography tryptic peptide mapping showed, as the only difference with controls, that the enzymatic cleavage between the two N-terminal peptides did not occur, yielding a 69 residue-long fragment. Further cleavages of this peptide (cyanogen bromide, V8 protease), amino acid composition, and sequence analyses demonstrated that the lysine at position 56 was replaced by a glutamic acid. Thus, surprisingly, a single amino acid change is responsible for the large difference in the electrophoretic behavior. This result suggests that single amino acid substitutions may similarly be involved in the structural modification of several other protein variants, described as elongated or shortened based only on SDS-polyacrylamide electrophoresis studies. When deletions/insertions were confirmed by sequence analysis, their extent was often different from that expected from electrophoresis.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1991

Three regions of erythrocyte band 3 protein are phosphorylated on tyrosines: characterization of the phosphorylation sites by solid phase sequencing combined with capillary electrophoresis.

The major part of band 3 phosphorylation was recently shown to concern the first tryptic peptide of the protein (Yannoukakos et al. (1991) Biochim. Biophys. Acta 1061, 253-266). Tyrosine 8 is the prevalent site of phosphorylation, but other phosphorylated regions were found which could not be analyzed with certainty. Direct characterization of the phosphorylated residues in all these phosphorylated fragments was made possible due to recent advances in protein chemistry techniques, such as solid phase sequence analysis and capillary electrophoresis. The present report establishes that band 3 phosphorylation occurs predominantly on tyrosines: besides tyrosine 8 already known in the N-terminal region, two other tyrosines are demonstrated to be targets for the tyrosine kinase, tyrosine 359 and tyrosine 904. These residues lie in regions of band 3 exposed to the cytoplasm, the junction of the cytoplasmic and the membrane-spanning domains, and the C-terminal end of the protein which is also cytosolic, respectively.

Amino Acid Sequence↗
PubMed1991

Phosphorylation sites in human erythrocyte band 3 protein.

The human red cell anion-exchanger, band 3 protein, is one of the main phosphorylated proteins of the erythrocyte membrane. Previous studies from this laboratory have shown that ATP-depletion of the red blood cell decreased the anion-exchange rate, suggesting that band 3 protein phosphorylation could be involved in the regulation of anion transport function (Bursaux et al. (1984) Biochim. Biophys. Acta 777, 253-260). Phosphorylation occurs mainly on the cytoplasmic domain of the protein and the major site of phosphorylation was assigned to tyrosine-8 (Dekowski et al. (1983) J. Biol. Chem. 258, 2750-2753). This site being very far from the integral, anion-exchanger domain, the aim of the present study was to determine whether phosphorylation sites exist in the integral domain. The phosphorylation reaction was carried out on isolated membranes in the presence of [gamma-32P]ATP and phosphorylated band 3 protein was then isolated. Both the cytoplasmic and the membrane spanning domains were purified. The predominant phosphorylation sites were found on the cytoplasmic domain. RP-HPLC analyses of the tryptic peptides of whole band 3 protein, and of the isolated cytoplasmic and membrane-spanning domains allowed for the precise localization of the phosphorylated residues. 80% of the label was found in the N-terminal tryptic peptide (T-1), (residues 1-56). In this region, all the residues susceptible to phosphorylation were labeled but in varying proportion. Under our conditions, the most active membrane kinase was a tyrosine kinase, activated preferentially by Mn2+ but also by Mg2+. Tyrosine-8 was the main phosphate acceptor residue (50-70%) of the protein, tyrosine-21 and tyrosine-46 residues were also phosphorylated but to a much lesser extent. The main targets of membrane casein kinase, preferentially activated by Mg2+, were serine-29, serine-50, and threonine(s)-39, -42, -44, -48, -49, -54 residue(s) located in the T-1 peptide. A tyrosine phosphatase activity was copurified with whole band 3 protein which dephosphorylates specifically P-Tyr-8, indicating a highly exchangeable phosphate. The membrane-spanning fragment was only faintly labeled.

Adenosine Triphosphate↗
PubMed1991

Hemoglobin Redondo [beta 92(F8) His----Asn]: an unstable hemoglobin variant associated with heme loss which occurs in two forms.

Hb Redondo [beta 92(F8) His----Asn] illustrates how post-translational structural modifications may modify the phenotypic expression of an unstable hemoglobin. This variant was found in a Portuguese patient suffering from a chronic hemolytic anemia. The electrophoretic pattern demonstrated that it occurred in two forms, both being semi-hemoglobins: the fastest one migrating like HbS and the other like HbA2; after a few days of storage at 4 degrees C the intensity of the slowest Hb fraction decreased while that of the other increased proportionally. In both cases, the RP-HPLC analysis of the tryptic digest of the aminoethylated beta chains demonstrated the presence of an abnormal beta T10 peptide carrying a His----Asx substitution. Microsequence study of these two peptides demonstrated that the slowest abnormal Hb fraction had a beta 92 His----Asn substitution and the fastest a His----Asp at the same site. All these results suggest that the beta 92 His----Asn variant loses readily its heme group and that a deamidation occurs rapidly in vitro, yielding a beta 92 Asp semi-hemoglobin. The oxygen affinity of the patient's red blood cells was increased, leading to a stimulation of erythropoiesis and to a macrocytic hemolytic disease.

Amino Acid Sequence↗
PubMed1991

Cleavage by protease from Staphylococcus aureus V8: an improvement in the sequence analysis of human hemoglobin variants.

Protease from Staphylococcus aureus V8 cleaves either at glutamic residues or at both aspartic and glutamic residues, depending on the experimental conditions. In structural analyses of human hemoglobin variants, the specificity of this enzyme is of considerable interest to localize substitutions occurring in medium or large size peptides as it cleaves in smaller fragments which may be unambiguously characterized. It may also recognize the replacement of an acidic residue by the corresponding amide, or vice versa, avoiding protein sequence analysis. The various aspects of the use of protease V8 are illustrated by the study of four alpha chain hemoglobin variants concerning peptides alpha T-9 and alpha T-12b.

Amino Acid Sequence↗
PubMed1989

Primary structure of the cytoplasmic domain of human erythrocyte protein band 3. Comparison with its sequence in the mouse.

We report here the peptide profile of the human cytoplasmic domain of band 3 protein (CDB-3). The peptide alignment was designed allowing for maximal homology with the murine protein whose sequence was deduced from cDNA analysis by Kopito and Lodish (Kopito, R.R., Anderson, M. and Lodish, H.F. (1987) J. Biol. Chem. 262, 8035-8040). In the human protein, part of the amino acid sequence has been determined by Kaul et al. (Kaul, R.K., Murthy, P.S.N., Reddy, A.G., Steck. T.L. and Kohler, H. (1983) J. Biol. Chem. 258, 7981-7990). We have sequenced most of the fragment not described by these author. The homology with the murine protein is high (90%), except in a few peptides where it is only 50%. The actual miniaturization of the techniques allows for the determination of a clear peptide profile of human CDB-3 starting from 10 ml blood samples. Our characterization of the peptide profile of membrane proteins is the first step towards the identification of genetic mutations, which have to be looked for in hemolytic anemia when the presence of an abnormal membrane protein is suspected.

Amino Acid Sequence↗
PubMed1988

Inhibition of membrane erythrocyte (Ca2+ + Mg2+)-ATPase by hemin.

Red blood cell lysis is a common symptom following severe or prolonged oxidative stress. Oxidative processes occur commonly in sickle cells, probably mediated through denatured hemoglobin and the accumulation of ferric hemes in the membranes. Calmodulin-stimulated (Ca2+ + Mg2+)-ATPase from sickle red cell membranes is partially inactivated (Leclerc et al. (1987) Biochim. Biophys. Acta 897, 33-40). In this study (Ca2+ + Mg2+)-ATPase activity from normal adult erythrocyte membranes was measured in the presence of hemin. We report a time- and concentration-dependent inhibition of the activity of the enzyme by hemin due to a decrease in the maximum velocity. Only a mild inhibitory effect was observed in the presence of iron-free protoporphyrin IX, indicating the catalytic influence of the iron. Experiments carried out with hemin (ferric iron) liganded with imidazole or with reduced protoheme (ferrous iron) liganded with carbon monoxide, demonstrated that the inhibition requires that hemin be capable of binding additional ligands. The inhibition was not influenced by the absence of oxygen but was prevented by addition of bovine serum albumin. Addition of butylated hydroxytoluene, a protective agent of lipid peroxidation, failed to prevent the inhibition of calmodulin-stimulated (Ca2+ + Mg2+)-ATPase. As dithiothreitol partially restores the enzyme activity, we postulated that hemin interacts with the thiol groups of the enzyme.

Ca(2+) Mg(2+)-ATPase↗
PubMed1988

Automated purification of human protein band 3, the major integral protein of the erythrocyte membrane.

Human erythrocyte protein band 3 was purified from a Triton X-100 extract of white ghosts. This purification, including an ion-exchange chromatography and a group-affinity chromatography, was automated. The apparatus was assembled from commercially available elements and allowed for the recovery of 2 to 3 mg pure band 3 in 2 hr. The purification could be repeated several times a day. The advantages of automation are discussed.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1987

Cation dependence of the phosphorylation of specific residues in red cell membrane protein band 3.

Phosphorylation of anion channel protein (ACP), the major component of erythrocyte protein band 3, was achieved in red cell ghosts in buffers containing vanadate (an inhibitor of phosphatases) and Mg2+ or Mn2+, known specific activators of the various kinases present in the red cell membrane. The anion channel protein was isolated to purity and the phosphorylated aminoacids were determined. The present results show that the phosphorylation of anion channel protein in its membraneous environment leads to an equal phosphorylation of tyrosine and serine plus threonine in the presence of Mg2+. In contrast, phosphotyrosine represents 80% of the total when Mn2+ is the activator.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1984

Evidence for the presence of tropomyosin in the cytoskeletons of ADP- and thrombin-stimulated blood platelets.

Stimulation of porcine platelets with ADP or thrombin and subsequent analyses of their cytoskeletons by SDS-polyacrylamide gel electrophoresis have shown the presence of a 30.5-kDa polypeptide in the cytoskeletons of activated as well as aggregated platelets. This polypeptide comigrates with pure porcine platelet tropomyosin in SDS gels, their mobilities being similarly and markedly decreased in the presence of 6 M urea. One-dimensional peptide mapping after limited proteolysis by Staphylococcus aureus protease gives the same pattern for pure tropomyosin and the 30.5-kDa polypeptide. This latter may thus be identified as the porcine platelet tropomyosin subunit, the role of which may not be solely structural.

Adenosine Diphosphate↗
PubMed1983

Intracardiac electrophysiological study of S-2395 in intact and chemically sympathectomized dogs.

S-2395 is a new, long-acting, non-selective beta-receptor blocking agent without apparent intrinsic cardiodepressive action and no receptor selectivity. Its electrophysiological actions were studied in intact and chemically sympathectomized dogs. In normal dogs, S-2395 very slightly increased atrial monophasic action potential (MAPa) duration, atrioventricular functional (FRP) and ventricular effective (ERP) refractory periods. The onset of the atrial supernormal conduction phenomenon (SNCP) was delayed and the ventricular SNCP was abolished. These results are consistent with the action of low concentrations of beta-blocking agents. In contrast S-2395 reduced the ventricular MAPv and increased the ERP/MAPv ratio of sympathectomized animals. Such modifications are usually seen with concentrations higher than necessary for simple beta-receptor blockade. In conclusion S-2395, like several others beta-blockers, had minor effects on the classical electrophysiological parameters of normal dogs. However, it suppressed ventricular SNCP and had a more pronounced action in sympathectomized dogs who are known to have higher levels of circulating catecholamines and who present post-synaptic supersensitivity. The SNCP has been linked with re-entrant arrhythmias. Considering that beta-blockers prevent arrhythmias specially in hyperadrenergic states, the suppression of the ventricular SNCP by S-2395 could thus be the mechanism by which this drug and possibly other beta-blockers might exert their antiarrhythmic action.

Adrenergic beta-Antagonists↗
PubMed1976

The genesis of bowel sounds: influence of viscus and gastrointestinal content.

This study was undertaken to try to solve the controversy about the influence of gastrointestinal contents on the genesis of bowel sounds, and to probe the respective importance of the various abdominal viscera. Eleven healthy volunteers were intubated by mouth with a multiple-lumen tube. Bowel sounds were recorded for 10 min when the tube was in the stomach, the upper jejunum, and the cecum, while it was left intact in situ, or perfused with isotonic saline (15 ml per min), or with an equal (7.5 ml per min of each) mixture of isotonic saline and air. Using a previously developed method, a computer analysis was made of the recording without any human intervention during the treatment of data. An analysis of variance demonstrated that the effect of perfusion varied according to site, with 46% of counted sounds while the tube was in the stomach, 32% in the jejunum, and 22% in the colon (P less than 0.05). There were two types of sounds: some exceeded in amplitude a preset threshold, and thus were picked up by the computer, but their average absolute value for 20 msec remained inferior to another preset threshold. Their number was kept in memory (NS--sounds having an amplitude exceeding a threshold S1, expressed in number per 10 min). A second type of sounds also exceeded the present threshold but their average absolute value for 20 msec also exceeded another preset threshold. Their number (NE--sounds having an amplitude exceeding the thershold S1 but having also a 20-msec average amplitude above another threshold S2, expressed in number per 10 min) was also memorized. The latter group was composed of two types of sounds: some had a limited spectrum of low frequency (100 Hz) and were of high amplitude and short (congruent to 5 msec) duration (NE1); some others had a higher and more dispersed frequency centered around 300 Hz (NE2). Fifty per cent of high energy (NE) sounds appeared while the tube was in the stomach, 30% in the colon, and 20% in the jejunum (P less than 0.005). Short and high amplitude sounds (NE1) were counted more often (43%) when it was in the colon than in the stomach (38%) and the jejunum (19%) (P less than 0.025), and this was confirmed (P less than 0.005) by a study of the ratio of NE1/NE. On the contrary, higher frequency sounds (NE2) were present more often when the tube was in the stomach (59%) than in the jejunum (24%) and in the colon (17%) (P less than 0.005). There was no influence of the presence of the unperfused tube on the genesis of bowel sounds in different sites (P greater than 0.05). In the stomach and the colon perfusion of the air/saline mixture increased the number of sounds (P less than 0.025) and all types of sounds in the stomach (P less than 0.025), whereas in the jejunum it was the perfusion of saline which increased them (P less than 0.025). It is concluded that the stomach is the most active site of production of bowel sounds, followed by the colon and then the small bowel, that sounds differ in different sites, and that all this is influenced by viscus content.

Cecum↗