10 results on '"Kieffer N"'
Search Results
2. A naturally occurring Arg -> Gln substitution at position 93 of platelet beta 3 integrin abolishes anti-HPA-1a binding
- Author
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Watkins, NA, Schaffner-Reckinger, E, Allen, D, Metcalfe, P, Murphy, M, Kieffer, N, and Ouwehand, WH
- Published
- 2000
3. A three amino acid deletion in glycoprotein IIIa is responsible for type I Glanzmann's thrombasthenia: importance of residues Ile325Pro326Gly327 for beta3 integrin subunit association
- Author
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Mc, Morel-Kopp, Kaplan C, valerie proulle, Jallu V, Melchior C, Peyruchaud O, Mh, Aurousseau, and Kieffer N
- Subjects
Male ,Proline ,Glycine ,CHO Cells ,Platelet Membrane Glycoproteins ,Transfection ,Polymerase Chain Reaction ,Consanguinity ,Antigens, CD ,Cricetinae ,Animals ,Humans ,Amino Acid Sequence ,Isoleucine ,Polymorphism, Single-Stranded Conformational ,DNA Primers ,Sequence Deletion ,Base Sequence ,Integrin beta3 ,Infant ,Recombinant Proteins ,Pedigree ,Algeria ,Female ,France ,Thrombasthenia - Abstract
Glanzmann's thrombasthenia (GT) is a recessive autosomal bleeding disorder characterized by abnormal platelet aggregation due to a qualitative or quantitative defect of the glycoprotein (GP) IIb-IIIa complex (integrin alphaIIb beta3). We describe a new mutation in the GPIIIa gene responsible for type I GT in a consanguineous Algerian family. A discordance between phenotyping and genotyping of the GPIIIa-related HPA-1 platelet alloantigen system in three family members heterozygous for the disease suggested a genetic defect in the GPIIIa gene and a normal GPIIb gene. Sequence analysis of amplified genomic DNA fragments showed a 6-bp deletion in exon 7 of the GPIIIa gene resulting in the amino acid deletion/substitution (Ile325pro326Gly327 --Met) and creating a new BspHI restriction site. Expression of the mutated integrin beta3 subunit cDNA in Chinese hamster ovary cells showed that the cDNA gene was transcribed into a full-length beta3 protein with an apparent molecular weight identical to wild-type beta3 and accumulated as a single-chain molecule in the cell cytoplasm. The absence of heterodimeric complex formation of the mutant beta3 protein with endogenous alpha v was shown by immunoprecipitation experiments, intracellular immunofluorescent labeling, and a semiquantitative enzyme-linked immunosorbent assay using the alpha vbeta3 complex-specific monoclonal antibodies LM609 and 23C6. Substitution of the methionine residue by a proline, present at position 326 of wild-type beta3, did not restore the ability of the recombinant mutant beta3 protein to associate with alpha v , suggesting that the Ile-Pro-Gly motif is located in a beta3 domain important for integrin subunit interaction. The association of a BspHI restriction site with this newly identified mutation has allowed allele-specific restriction analysis of Algerian GT individuals and the identification of two new unrelated type I patients exhibiting the same mutation, suggesting that the described mutation might be significant in this population and that BspHI restriction analysis will provide a useful screening assay for antenatal diagnosis and genetic counselling.
- Published
- 1997
4. Serine 752 in the cytoplasmic domain of the beta 3 integrin subunit is not required for alpha v beta 3 postreceptor signaling events
- Author
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Guinet Jm, Bron N, Gouon, Kieffer N, Melchior C, and Michels S
- Subjects
Alpha-v beta-3 ,Proline ,Fibrinogen receptor ,Recombinant Fusion Proteins ,Integrin ,Molecular Sequence Data ,Alpha (ethology) ,CHO Cells ,Platelet Glycoprotein GPIIb-IIIa Complex ,Platelet Membrane Glycoproteins ,Transfection ,chemistry.chemical_compound ,Antigens, CD ,Cricetinae ,von Willebrand Factor ,Cell Adhesion ,Serine ,Animals ,Humans ,Point Mutation ,Receptors, Vitronectin ,Integrin Alpha-IIb/Beta-3 ,Amino Acid Sequence ,Vitronectin ,Beta (finance) ,Cytoskeleton ,Extracellular Matrix Proteins ,Alanine ,biology ,Genetic Complementation Test ,Integrin beta3 ,Antibodies, Monoclonal ,Fibrinogen ,General Medicine ,Molecular biology ,chemistry ,biology.protein ,Oligopeptides ,Sequence Alignment ,ATP synthase alpha/beta subunits ,Protein Binding ,Signal Transduction ,Thrombasthenia - Abstract
A naturally occurring point mutation (Ser752Pro substitution) in the beta subunit cytoplasmic domain of the platelet fibrinogen receptor GPIIb-IIIa (integrin alpha IIb beta 3), causing Glanzmann's thrombasthenia, has been shown to abrogate bidirectional transmembrane signaling of GPIIb-IIIa when expressed in heterologous cells (Chen YP, 1994, Blood 84, 1857-1865). As the vitronectin receptor alpha v beta 3 constitutively mediates cell attachment to RGD containing extracellular matrix proteins, the purpose of this study was to explore the regulatory role of Ser752 in alpha v beta 3 vitronectin receptor function, by cotransfecting recombinant human alpha v cDNA together with human beta 3 mutant cDNA into Chinese hamster ovary (CHO) cells. CHO cells expressing wild type human alpha v beta 3 acquired the ability to attach and spread on fibrinogen and von Willebrand factor, in contrast to non transfected CHO cells that only bound to vitronectin and fibronectin. Overexpression of a truncated recombinant beta 3 subunit (beta 3 delta 744) generated alpha v (hamster) beta 3 (human) chimers that mediated attachment but lost the ability to promote cell spreading on vitronectin, von Willebrand factor and fibrinogen, and to concentrate in focal contact sites, demonstrating a negative effect of beta 3 delta 744 on alpha v beta 3 dependent postreceptor occupancy events. Transfection of beta 3Ser752Pro reproduced the same negative effect as beta 3 delta 744, whereas beta 3Ser752Ala restored normal receptor function by allowing pronounced attachment and spreading on fibrinogen and von Willebrand factor. Our results provide evidence that (1) the C-terminal cytoplasmic domain of beta 3 (amino acids 744-762) is essential for alpha v beta 3 integrin postreceptor occupancy events; (2) within this domain, the Ser752Pro mutation affects alpha v beta 3 postreceptor occupancy events by preventing cell spreading and focal contact localization; (3) the defective receptor function of the vitronectin receptor alpha v beta 3 is due to the presence of Pro752, rather than the absence of Ser752, as a Ser to Ala substitution at position 752 restores normal beta 3 integrin cell spreading and adhesive plaque formation.
- Published
- 1996
5. Modulation of the activity and assessment of the receptor selectivity in a series of new RGD-containing peptides
- Author
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Angela D. Morris, Kieffer N, Christophe Thurieau, S. Simonet, Tony Verbeuren, and Jean-Luc Fauchère
- Subjects
Integrins ,Alpha-v beta-3 ,Integrin ,Molecular Sequence Data ,Platelet Membrane Glycoproteins ,Receptors, Cytoadhesin ,Biochemistry ,Cell Line ,chemistry.chemical_compound ,Structure-Activity Relationship ,Dogs ,Cell surface receptor ,Peptide synthesis ,Cell Adhesion ,Animals ,Humans ,Receptors, Vitronectin ,Amino Acid Sequence ,Cell adhesion ,biology ,Lysine ,Cell Adhesion Inhibition ,chemistry ,biology.protein ,Platelet aggregation inhibitor ,Vitronectin ,Oligopeptides ,Platelet Aggregation Inhibitors - Abstract
We have investigated the structure-activity relationship of a series of new synthetic RGD analogs and their potential use as specific platelet aggregation inhibitors. Twelve short linear peptides showed high potency to inhibit aggregation in ADP-stimulated dog platelets. In order to assess the selectivity of these analogs towards platelet integrin GPIIb-IIIa, a new cell adhesion inhibition system was devised which was able to discriminate between the two closely related beta 3-integrins of the vasculature, GPIIb-IIIa (alpha IIb beta 3), present in platelets, and the vitronectin receptor (alpha v beta 3), expressed in endothelial cells and platelets. As reported for other peptides by Scarborough et al. (1993, J. Biol. Chem. 268, 1066), the analogs containing lysine instead of arginine in position 1 showed increased selectivity towards GPIIb-IIIa. One of them, in which the piperidine carboxylic group was attached to the N-terminus of KGDW, not only strongly inhibited platelet aggregation, but also selectively abolished cell adhesion mediated by GPIIb-IIIa without effect on the vitronectin receptor.
- Published
- 1993
6. The human platelet membrane glycoprotein complex GP IIb-IIIa expresses antigenic sites not exposed on the dissociated glycoproteins
- Author
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Jp, Rosa, Kieffer N, Didry D, Dominique Pidard, Tj, Kunicki, and At, Nurden
- Subjects
Blood Platelets ,Counterimmunoelectrophoresis ,Immunology ,Antibodies, Monoclonal ,Membrane Proteins ,Cell Biology ,Hematology ,Platelet Membrane Glycoproteins ,Biochemistry ,Polyethylene Glycols ,Epitopes ,Animals ,Humans ,Binding Sites, Antibody ,Rabbits ,Glycoproteins - Abstract
A number of recent reports have described murine monoclonal antibodies that react specifically with the complex formed by human platelet membrane glycoproteins (GP) IIb and IIIa. We show that the IgG L, a previously described human alloantibody isolated from a polytransfused thrombasthenia patient, has similar properties. When used in non- precipitating amounts in crossed immunoelectrophoresis (CIE), 125I-IgG L bound strongly to the IIb-IIIa complex. However, after dissociation of the complex with EDTA, only a weak binding to GP IIb and no binding to GP IIIa was detected. In further studies, increased amounts of IgG L were interacted with 125I-labeled membrane glycoproteins in (a) CIE and (b) classical indirect immunoprecipitation experiments. Although the antibody was able to quantitatively precipitate the IIb-IIIa complex from Triton X-100-soluble extracts of platelet membranes, no precipitation of GP IIb or GP IIIa was observed after divalent cation chelation. Addition of EDTA to immunoprecipitates containing GP IIb- IIIa resulted in dissociation and partial release of both glycoproteins. The interaction of the IgG L with electrophoretically separated GP IIb and GP IIIa was studied using a Western blot procedure in the presence of Ca2+, Mg2+, or EDTA. The presence of divalent cations did not increase the reactivity of the antibody with the individual glycoproteins. Overall, our results show that acquired antibodies to IIb-IIIa, such as the IgG L, may predominantly react with complex-dependent determinants.
- Published
- 1984
7. Evaluation of Response to Selection and Inbreeding in a Closed Line of Hereford Cattle
- Author
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Brinks, J. S., Clark, R. T., and Kieffer, N. M.
- Subjects
Livestock Production/Industries, Production Economics - Published
- 1965
8. Expression of platelet glycoproteins by erythroid blasts in four cases of trisomy 21
- Author
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Debili N, Kieffer N, Mt, Mitjavila, Jean-Luc Villeval, Guichard J, Teillet F, Henri A, Kj, Clemetson, Vainchenker W, and Breton-Gorius J
- Subjects
Erythroblasts ,Antibodies, Monoclonal ,Humans ,Leukemia, Erythroblastic, Acute ,Platelet Membrane Glycoproteins ,Down Syndrome ,Blotting, Northern ,Immunohistochemistry - Abstract
In four patients with trisomy 21 (three constitutional, one acquired) with a morphological undifferentiated leukemia, diagnosis of erythroid leukemia was established by both immunophenotyping and ultrastructural studies. Indeed, a majority of blasts from three patients expressed several erythroid markers such as carbonic anhydrase 1, spectrin beta chain, and glycophorin A. In addition, band 3 and hemoglobin were immunologically detected in a fraction of the blast cells from two cases. At ultrastructural level, a majority or all blast cells exhibited erythroid differentiation features such as theta granules and ferritin molecules. However, platelet glycoproteins GP Ib, GP IIb, and GP IIIa were also immunologically detected in a fraction (from 14-82%) of the blasts. Since the ultrastructural study indicated that some promegakaryoblasts were also present in three patients, double labeling between erythroid markers (glycophorin A or carbonic anhydrase I) and platelet glycoprotein (Ib or IIIa) was performed and showed a clear overlap between the two kinds of markers. A similar approach was performed at ultrastructural level and indicated that blast cells with ultrastructural erythroid features of differentiation may have three distinct phenotypes, i.e., presence of glycophorin A without platelet glycoproteins or, conversely, the presence of platelet glycoproteins without glycophorin A and coexpression of glycophorin A and platelet glycoproteins. Expression of glycophorin A correlated directly with the differentiation level of the erythroid blasts, whereas platelet glycoproteins were essentially expressed in the more primitive leukemic erythroid cells. The GP Ib synthesized by these blasts was subsequently studied. The GP Ib alpha mRNA analyzed by Northern blot from these erythroid cells was identical in size with that from megakaryocytic cells as was the molecular weight of the GP Ib molecule from both after immunoprecipitation by a monoclonal antibody. Therefore, "in vivo" erythroid leukemic cells may express the main platelet glycoproteins including GP Ib.
9. Limits of phenotypic markers for the diagnosis of megakaryoblastic leukemia
- Author
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Breton-Gorius J, Jean-Luc Villeval, Kieffer N, Mt, Mitjavila, Guichard J, and Vainchenker W
- Subjects
Blood Platelets ,Phenotype ,Leukemia, Megakaryoblastic, Acute ,Ferritins ,Biomarkers, Tumor ,Fluorescent Antibody Technique ,Humans ,Platelet Membrane Glycoproteins ,Megakaryocytes ,Antibodies - Abstract
Diagnosis of megakaryoblastic and early erythroid leukemia requires the use of differentiation markers that in most cases permit their precise diagnosis. In some cases, their use can be misleading. Here we report and discuss some examples. A platelet peroxidase (PPO) activity is detected in most cases of early erythroid leukemias as well as in the CFU-E-like cells of normal marrow, thus providing evidence that PPO activity must be studied along with other (immunologic or ultrastructural) markers to permit a reliable diagnosis of megakaryoblastic leukemia. Ferritin molecules an erythroid marker, could be detected as a cluster at ultrastructural level in leukemic platelets and in micromegakaryocytes of one patient. However, in blasts of the erythroid lineage, ferritin molecules are also either dispersed in the cytoplasm or localized in theta granules. Immunologic markers have also their own limit. Indeed, in one patient, GB IIb and IIIa were detected on erythroid blasts, resulting in a phenotype very similar to HEL cells. Carbonic anhydrase (CA) I, an early erythroid marker, was detected in the platelets of four leukemic patients and was present along with an increased expression of CA II. This study emphasizes the fact that precise diagnosis of leukemia cannot be performed with a single marker of differentiation, but requires the simultaneous use of several lineage restricted markers.
10. New hematopoietic differentiation antigens detected by anti-K562 monoclonal antibodies
- Author
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Farace F, Mt, Mitjavila, Betaieb A, Mc, Dokhelar, Joëlle Wiels, Finale Y, Kieffer N, Breton-Gorius J, Vainchenker W, and Tursz T
- Subjects
Molecular Weight ,Mice ,Mice, Inbred BALB C ,Immunization, Passive ,Animals ,Antibodies, Monoclonal ,Tetradecanoylphorbol Acetate ,Female ,Fluorometry ,Hematopoietic Stem Cells ,Antigens, Differentiation ,Cell Line - Abstract
Following immunizations of BALB/c mice with K562 cells, we have obtained seven original monoclonal antibodies (MoAbs): (a) One MoAb, GA3, defines an antigen essentially restricted to the red cell series. This antigen is expressed on immature erythroblasts but is not detectable on the surface of early and late erythroid progenitors. GA3 MoAb immunoprecipitates a Mr 105,000 glycoprotein on K562 cells. (b) Two MoAbs, 14B6 and 12B1, react with cells of the monocytic series. MoAb 14B6, which also faintly stains platelets, is reactive with immature myeloid cells and the majority of hematopoietic progenitors. The 14B6 antigen has been immunoprecipitated from 12-O-tetradecanoylphorbol-13-acetate treated K562 cells as a Mr 130,000-100,000 protein. Antigen 12B1 is expressed only on cultured monocyte/macrophages and is restricted to a subpopulation of monocytes and to follicular dendritic cells. It is not detected on hematopoietic progenitors. Immunoprecipitation experiments performed on 12-O-tetradecanoylphorbol-13-acetate treated K562 cells revealed a glycoprotein with a molecular weight of 93,000-86,000. (c) Two anti-K562 MoAbs, CF4 and HE10, recognize a myeloid differentiation antigen expressed from the granulomonocytic colony forming unit stage to polymorphonuclear neutrophils. These MoAbs detect an apparently original glycolipid moiety distinct from LeX. (d) Two MoAbs recognize antigens expressed on the granulomonocytic series. 2E1 recognizes the monocyte low affinity Fc receptor (Mr 40,000) and defines a new cluster of myeloid differentiation (CDw32). The antigen is expressed on a small portion of immature hematopoietic progenitors. 8F5 identifies a Mr 95,000 protein which is also present on plasma cells. In some experiments, it is detected on erythroid colony forming unit analysis. Immunizations with K562 cells thus resulted in the production of antibodies recognizing antigens of the monocytic, granulocytic, as well as erythroid series. However, three of them are also detected on hematopoietic progenitors.
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