RESUMO
Exosomes are vesicles formed in the endosomal compartment and released in the extracellular medium during reticulocyte maturation into erythrocytes. They have a clearing function because of their enrichment with some proteins known to decrease or disappear from the cell surface during maturation, e.g. acetylcholinesterase and transferrin receptor. We show here that integrin alpha4beta1, present on the surface of erythroid precursors but absent from the mature red cell membrane, is at least partly cleared from the reticulocyte plasma membrane by the exosomal pathway. Using flow cytometry, we found that the alpha4 subunit disappears from the reticulocyte surface during in vitro maturation. Two different monoclonal antibodies (B-5G10 and HP 2/1) were used to demonstrate the presence of the alpha4 chain on the exosome surface. Moreover, membrane acetylcholinesterase and lumenal peroxidase-like (i.e. hemoglobin) enzymatic activities were assayed to demonstrate exosome binding to plates coated with increasing fibronectin (FN) concentrations. This interaction was dependent on divalent cations (MnCl2 > MgCl2 > CaCl2). Similarly, vesicles bound to plates coated with the chymotryptic 40 K fragment (FN-40) containing the heparin-binding region of FN. This binding was inhibited by exosome preincubation with fibronectin CS1 peptide and with a monoclonal antibody (HP 2/1) against the integrin alpha4-chain, confirming an alpha4beta1-induced interaction. The importance of the exosome clearance function is highlighted here, since the presence of VLA-4 on reticulocytes often leads to blood circulation complications in some diseases. Moreover, the presence of alpha4beta1 on the exosome surface, by allowing binding to endothelial cells through vascular cell adhesion molecule 1 (VCAM-1), might confer another physiological function to the secreted vesicles.
Assuntos
Fibronectinas/metabolismo , Integrinas/metabolismo , Organelas/metabolismo , Receptores de Retorno de Linfócitos/metabolismo , Reticulócitos/fisiologia , Animais , Anticorpos/metabolismo , Anticorpos/farmacologia , Sítios de Ligação , Cátions/metabolismo , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Regulação para Baixo , Membrana Eritrocítica/metabolismo , Humanos , Integrina alfa4beta1 , Integrinas/imunologia , Membranas Intracelulares/metabolismo , Fragmentos de Peptídeos/metabolismo , Ratos , Receptores de Retorno de Linfócitos/imunologiaRESUMO
PURPOSE: To characterize the effect on mRNA splicing of a yet undescribed mutation located in intron 13 splice-donor sequence (IVS13 + 3A --> C) in the Rab-Escort-protein 1 gene of a patient with choroideremia. METHODS: The base substitution was firstly detected by the Single Strand conformation analysis from genomic DNA. A REP-1 cDNA region encompassing exons 10-14 was then specifically amplified from lymphocytes-derived mRNA. RESULTS: We could demonstrate that this substitution affects REP-1 RNA processing. The patient revealed only one aberrantly spliced mRNA lacking exon 13 and no normal transcript. CONCLUSION: The skipping of exon 13 results in the creation of a stop codon at the misspliced junction. This is the first case of nucleotide substitution at the +3 position of a splice donor site so far described in choroideremia.
Assuntos
Alquil e Aril Transferases , Proteínas de Transporte/metabolismo , Coroideremia/genética , DNA Recombinante , Proteínas rab de Ligação ao GTP , Proteínas Adaptadoras de Transdução de Sinal , Adulto , Sequência de Bases , Coroideremia/patologia , Sequência Consenso , Éxons/genética , Angiofluoresceinografia , Humanos , Masculino , Mutação , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , RNA Mensageiro/genética , Transcrição GênicaRESUMO
By using the single strand conformational analysis to search for point mutations in the choroideremia gene, we have identified an intronic polymorphism within the intron 2 of the CHM gene. We have studied the frequency of this polymorphism in the population from South of France.
Assuntos
Coroideremia/genética , Éxons/genética , Poli T/genética , Splicing de RNA/genética , Coroideremia/diagnóstico , Feminino , França , Frequência do Gene/genética , Humanos , Íntrons , Masculino , Mutação Puntual/genética , Polimorfismo GenéticoRESUMO
In spite of recent developments there is still considerable conflict over the definitions of extended role and competency. This article reviews the legal and professional support systems underpinning the adoption by nurses of duties traditionally considered to be doctors' tasks.
