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Biomedical subjects

O Gayet

Publications and source records attributed to O Gayet.

12 recordsLinked to original sources
PubMed2001

Sorting nexin-14, a gene expressed in motoneurons trapped by an in vitro preselection method.

A gene-trap strategy was set up in embryonic stem (ES) cells with the aim of trapping genes expressed in restricted neuronal lineages. The vector used trap genes irrespective of their activity in undifferentiated totipotent ES cells. Clones were subjected individually to differentiation in a system in which ES cells differentiated into neurons. Two ES clones in which the trapped gene was expressed in ES-derived neurons were studied in detail. The corresponding cDNAs were cloned, sequenced, and analysed by in situ hybridisation on wild-type embryo sections. Both genes are expressed in the nervous system. One gene, YR-23, encodes a large intracellular protein of unknown function. The second clone, YR-14, represents a sorting nexin (SNX14) gene whose expression in vivo coincides with that of LIM-homeodomain Islet-1 in several tissues. Sorting nexins are proteins associated with the endoplasmic reticulum (ER) and may play a role in receptor trafficking. Gene trapping followed by screening based on in vitro preselection of differentiated ES recombinant clones, therefore, has the potential to identify integration events in subsets of genes before generation of mouse mutants.

Animals↗
PubMed2001

Juxtaposition of CNR protocadherins and reelin expression in the developing spinal cord.

The CNR (cadherin-related neuronal receptors) family of protocadherins is of great interest because of their potential roles as molecular tags in the formation of specific synaptic connections, and as receptors for reelin, during neuronal migration, and cell body positioning. In order to know more about potential functions of CNRs we have mapped their expression during mouse nervous system development and compared their expression with that of reelin and its intracellular effector Dab1 in several tissues. In spinal cord, CNRs and Dab1 are expressed in motoneurons, while reelin is located in adjacent cells. In the hindbrain, there is a differential expression of CNRs and Dab1 in various motor nuclei. In the retina and olfactory system, we observe CNR and reelin expression but not that of Dab1. These results provide new insights into the potential functions of CNRs and their possible integration in the reelin pathway during development.

Animals↗
PubMed2001

Expression of Deltex1 during mouse embryogenesis: comparison with Notch1, 2 and 3 expression.

The Notch signalling pathway defines a phylogenetically conserved cell-cell communication process that enables cell-fate specification in multicellular organisms. Deltex is a component of the Notch signalling network that physically interacts with the ankyrin repeats of Notch. Here, we report on the expression pattern of the Deltex1 gene during mouse embryonic development and, furthermore, we compare its expression with that of the Notch1, 2 and 3 genes. Complementary and combinatorial expression patterns between Deltex1 and the three Notch genes were observed throughout embryogenesis since Deltex1 expression was related either to cytodifferentiation (i.e. neuronal tissues) or to cell proliferation events (i.e. eye, vascular structures, hematopoiesis).

Animals↗
PubMed1999

Enhanced expression of P-selectin (CD62P) by endothelial cells seeded onto synthetic arterial prostheses (PET, Dacron) is correlated with leukocyte interactions.

This study evaluated the expression by seeded endothelial cells (S-EC) of P-selectin (CD62P/GMP-140/PADGEM), an adhesion molecule implicated in endothelial-leukocyte interactions. Endothelial cells were seeded onto knitted polyethylene terephthalate (PET, Dacron(R)) prostheses and compared with control endothelial cells (C-EC) cultured in flasks to the same stage. Using flow cytometry techniques, we observed that CD62P expression by PET S-EC was significantly increased (p<0.05) compared to C-EC. Moreover, RT PCR techniques showed that the CD62P RNA level was higher on S-ECs compared to C-ECs. Following adhesion assays, increased polymorphonuclear neutrophil (PMN) attachment to the PET-seeded prostheses as compared to control cultures (p<0.001) was observed. PMN adherence was enhanced by TNFalpha activation. PMN adhesion was decreased significantly (p<0.001) after the incubation of resting EC or TNFalpha-activated EC-seeded prostheses with a blocking monoclonal antibody (LYP20) directed against the P-selectin. Such results suggest that: (1) PET prosthetic material may induce the expression of P-selectin by S-EC; (2) seeding conditions provoke an increase in PMN adhesion; (3) increased PMN interactions with seeded PET material is partially dependent upon P-selectin expression by the S-EC.

Animals↗
PubMed1998

Mapping the epitope of a functional P-selectin monoclonal antibody (LYP20) to a short complement-like repeat (SCR 4) domain: use of human-mouse chimaera and homologue-replacement mutagenesis.

P-selectin (CD62P), an adhesion molecule localized in platelet alpha-granules and endothelial cell Weibel-Palade bodies, is rapidly expressed on the surface of activated cells. This adhesion molecule, a member of the selectin family, mediates leucocyte interactions with activated platelets or endothelial cells. The aim of this study was to identify and characterize the epitope of a functional blocking P-selectin monoclonal antibody (mAb), LYP20. LYP20 recognizes human or rat, but not mouse, P-selectin. Human/mouse chimaeras and wild-type constructs, modified by homologue replacement mutagenesis, were expressed in COS cells. Blocking anti-(P-selectin) mAbs (G1, G3 or CLB-thromb/6) were observed, by flow cytometry, to bind to the lectin-like domain. In contrast, LYP20 was found to bind to one of the P-selectin short complement-like repeats (SCR domain 4). Homologue replacement mutagenesis of SCR domain 4 (region delineated by amino acid residues 359-457) identified three amino acids (Cys412-->Ser, Cys416-->Ser or Arg415-->Lys) as being implicated in the LYP20 epitope. Deleting the region bearing the LYP20 epitope, from a wild-type CD62P construct, showed a decrease in polymorphonuclear leucocyte (PMNL) binding to transfected COS cells. In addition, mutation of one of the three amino acids, implicated in the LYP20 epitope, markedly affected PMNL binding to transfected COS cells but did not affect the binding of mAbs G1 and CLB-thromb/6. These results are the first to indicate (1) that a functional blocking anti-P-selectin mAb binds to SCR 4, a site other than the lectin-like/epidermal growth factor-like domain, and (2) that SCR domain 4 has a functional role in P-selectin-leucocyte interactions.

Amino Acid Sequence↗
PubMed1995

The molecular genetic basis of Glanzmann's thrombasthenia in a gypsy population in France: identification of a new mutation on the alpha IIb gene.

Glanzmann's thrombasthenia is a rare inherited bleeding disorder caused by a qualitative or quantitative defect of platelet alpha IIb beta 3. We describe here a new mutation that is the molecular genetic basis of Glanzmann's thrombasthenia in two gypsy families. Our investigation was focused on the alpha IIb gene as a result of biochemical and immunologic analysis of patients' platelets showing undetectable alpha IIb but residual beta 3 levels. The entire alpha IIb cDNA was polymerase chain reaction (PCR) amplified using patients platelet RNA. Sequence analysis showed an 8-bp deletion located at the 3' end of exon 15. This deletion causes a reading-frame shift leading to a premature stop codon and the synthesis of a severely truncated form of alpha IIb. Genomic DNA study showed a G-->A substitution, the Gypsy mutation, at the splice donor site of intron 15. This mutation results in an abnormal splicing occurring at an alternative donor site located 8 bp upstream from the mutation. Based on those results, an allele-specific PCR analysis was developed to allow a rapid identification of the mutation in patients and potential carriers of the gypsy community. This PCR analysis can also be used for genetic counseling and antenatal diagnosis.

Alleles↗
PubMed1994

A new alternative transcript encodes a 60 kDa truncated form of integrin beta 3.

A cDNA for integrin beta 3 isolated from a human erythroleukaemia (HEL) cell library contained a 340 bp insert at position 1281. This mRNA, termed beta 3c, results from the use of a cryptic AG donor splice site in intron 8 of the beta 3 gene, and is different from a previously described alternative beta 3 mRNA. The predicted open reading frame of beta 3C stops at a TAG stop codon 69 bp downstream from position 1281. It starts with the signal peptide and the 404 N-terminal extracellular residues of beta 3, encompassing the ligand binding sites, followed by 23 C-terminal intron-derived residues, corresponding to a truncated form of beta 3 lacking the cysteine-rich, transmembrane and cytoplasmic domains. Expression of beta 3C mRNA was demonstrated in human platelets, megakaryocytes, endothelial cells and HEL cells by reverse transcriptase/PCR. The beta 3C transcript was also demonstrated in the mouse, suggesting its conservation through evolution. Finally, a 60 kDa polypeptide corresponding to the beta 3C alternative transcript was demonstrated in platelets by Western blotting using a polyclonal antibody raised against a synthetic peptide designed from the beta 3C intronic sequence. Taken together, these results suggest a biological role for beta 3C, the first alternative transcript showing an altered extracellular domain of a beta integrin.

Alternative Splicing↗
PubMed1992

Human platelets express the SERCA2-b isoform of Ca(2+)-transport ATPase.

Previous biochemical studies suggested that the human platelet Ca2+ATPase system may be cell-specific. To test this hypothesis, we first undertook the molecular cloning of Ca2+ATPase from human erythroleukaemia (HEL) cells, because this human cell line exhibits megakaryocytic features and expresses a Ca2+ATPase that cross-reacts with platelet Ca(2+)-ATPase. For this cloning, an HEL-cell cDNA library was screened with a rat cardiac Ca2+ATPase cDNA probe. The insert of the longest clone isolated was 3.9 kb and its sequence displayed a 100% identity with that of the non-muscle human Ca2+ATPase 2-b isoform, termed SERCA2-b (sarco-endoplasmic-reticulum Ca2+ATPase). The 3.9 kb cDNA covered a subtotal coding region and part of the 3' non-coding end of the SERCA2-b mRNA. It cross-hybridized with the 4 kb transcript species of cardiac SERCA2-a and with non-muscle SERCA2-b mRNAs, but not with fast-skeletal-muscle SERCA1 mRNA. We next confirmed that SERCA2-b was a component of the platelet Ca2+ATPase system because (1) the platelet clones isolated from a platelet cDNA library exhibited a 100% homology with HEL-cell cDNA; (2) SERCA2-b mRNA was amplified by PCR on total platelet RNA and (3) platelet Ca2+ATPase cross-reacted with a polyclonal SERCA2-b-specific antiserum. Platelets therefore contain a Ca2+ATPase definitely identified as the SERCA2-b isoform of Ca2+ATPase, thus eliminating the possibility that they only contain a single specific Ca2+ATPase.

Animals↗
PubMed1990

Expression of chimeric human transferrin genes in transfected human tumor cell lines.

The iron-binding plasma protein transferrin (TF) is essential for supplying iron to cells and the prevention of iron toxicity. Our laboratory has cloned and characterized the human TF gene. Comparison of promoter regions of TF genes from human, chicken, and mouse reveals a strong nucleotide sequence conservation. This study demonstrates that 5' flanking regions of the TF gene are sufficient for directing expression of a heterologous gene in transgenic mice and transfected cells. For cell-specific expression, more than 150 base pairs appear to be required.

Animals↗
PubMed1988

Cloning of glycoprotein IIIa cDNA from human erythroleukemia cells and localization of the gene to chromosome 17.

Platelet aggregation requires the binding of adhesive proteins such as fibrinogen to the heterodimer of membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa). Human erythroleukemia (HEL) cells synthesize both GPIIb and GPIIIa. Using poly(A+) RNA purified from HEL cells, we constructed a cDNA library in the lambda gt10 phage vector. This library was screened with a 38mer oligonucleotide derived from a platelet GPIIIa peptide, and three overlapping cDNAs were isolated. The three inserts encompassed 3.5 kilobases (kb), including the entire coding region of mature GPIIIa (2,286 basepairs, bp) and 1.3 kb of 3' untranslated sequence. All 222 residues determined directly from platelet GPIIIa tryptic peptides exactly matched the HEL cell-deduced amino acid sequence. The HEL cell sequence matched a previously reported endothelial cell cDNA sequence except for eight nucleotides. Five of these nucleotide differences were silent changes consistent with genetic polymorphisms. The other three differences resulted in changes in the deduced amino acid sequence of GPIIIa; reexamination of the endothelial cell cDNA sequence in these three areas revealed that it is actually identical to the HEL cell sequence. The virtual identity of the endothelial and HEL cell cDNA sequences provides direct evidence that GPIIIa is a subunit common to cell-adhesion receptors present in more than one cell type. We localized the gene for GPIIIa to chromosome 17, the same chromosome to which we had previously mapped the gene for GPIIb.

Amino Acid Sequence↗
PubMedDate not supplied

Characterization of HIV1-PAR, a macrophage-tropic strain: cell tropism, virus/cell entry and nucleotide sequence of the envelope glycoprotein.

The HIV1-PAR strain, isolated from the cerebrospinal fluid of an HIV1-seropositive man suffering from encephalopathy, replicated well in cord blood lymphocytes, poorly in peripheral blood mononuclear cells, and to different levels in blood-derived macrophage (BDM) cultures prepared from different blood donors. In marked contrast to its replication in primocultures, it did not grow in CEM and U937 cell lines. HIV1-PAR production in BDM was inhibited by more than 90% after treatment with OKT4A or 13B8.2 monoclonal antibodies (mAb) binding to adjacent epitopes of the D1 domain of the CD4 molecules. A lower but significant inhibitory effect was observed after BDM treatment with BL4 and OKT4 mAb, directed to the D2 and D3 domain of the CD4 molecule, respectively. The entire HIV1-PAR envelope glycoprotein gene was amplified by polymerase chain reaction and sequenced. The deduced amino acid sequence of HIV1-PAR gp160 revealed the presence of 847 amino acids and 86% homology with the HIV1 LAV virus prototype. An alignment of the amino acid sequence of the envelope glycoprotein of HIV1-PAR and HIV1-LAV showed that the differences were mostly clustered within the five variable regions. Five CD4-binding domains, the gp120/gp41 cleavage site, the putative gp41 fusion domain and 21 out of the 22 cysteine residues were conserved in both isolates. The results further confirm the macrophage-tropic character of the HIV1-PAR virus.

Amino Acid Sequence↗