RESUMEN
Human monoclonal antibodies (HuMAbs) against HR1a from Protobothrops (formerly Trimeresurus) flavoviridis venom were obtained by the fusion of SP2/0-Ag14 myeloma cells and spleen cells from KM mice immunized with purified HR1a. The ability of HuMAbs to neutralize the HR1a was determined by in vitro neutralization assay and by neutralization of the hemorrhagic activity. The initial screening of over 300 hybridoma fusion wells resulted in the establishment of 80 HR1a-reactive hybridomas. Of the reactive clones, HuMAb HR1a-7 and HR1a-18 neutralized both proteolytic and hemorrhagic activity of HR1a. Mapping of epitope recognized by the reactive clones was performed by using an ELISA that measured antibody binding to overlapping peptides (15 amino acid peptide offset frameshifted by three residues) covering the metalloproteinase domain sequence of HR1a. HuMAbs HR1a-7 and HR1a-18 neutralized HR1a by reacting with peptides of EQQRYLNNFRFIELV and IVNTLNETYRYL. The three-dimensional structure of HR1a based on a homology modeling predicted that these two epitopes are surface exposed.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Metaloproteasas/antagonistas & inhibidores , Venenos de Víboras/enzimología , Viperidae , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Mapeo Epitopo , Humanos , Hibridomas , Masculino , Metaloproteasas/química , Metaloproteasas/inmunología , Metaloproteasas/metabolismo , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Pruebas de Neutralización , Conformación Proteica , Venenos de Víboras/antagonistas & inhibidores , Venenos de Víboras/inmunologíaRESUMEN
Neural crest (NC) cells are a migratory, multipotent cell population that arises at the neural plate border, and migrate from the dorsal neural tube to their target tissues, where they differentiate into various cell types. Abnormal development of NC cells can result in severe congenital birth defects. Because only a limited number of cells can be obtained from an embryo, mechanistic studies are difficult to perform with directly isolated NC cells. Protein zero (P0) is expressed by migrating NC cells during the early embryonic period. In the P0-Cre;Z/EG transgenic mouse, transient activation of the P0 promoter induces Cre-mediated recombination, indelibly tagging NC-derived cells with enhanced green fluorescent protein (EGFP). Induced pluripotent stem cell (iPSC) technology offers new opportunities for both mechanistic studies and development of stem cell-based therapies. Here, we report the generation of iPSCs from the P0-Cre;Z/EG mouse. P0-Cre;Z/EG mouse-derived iPSCs (P/G-iPSCs) exhibited pluripotent stem cell properties. In lineage-directed differentiation studies, P/G-iPSCs were efficiently differentiated along the neural lineage while expressing EGFP. These results suggest that P/G-iPSCs are useful to study NC development and NC-associated diseases.
Asunto(s)
Diferenciación Celular , Células Madre Pluripotentes Inducidas/citología , Células Madre Multipotentes/citología , Cresta Neural/citología , Animales , Linaje de la Célula/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Inmunohistoquímica , Células Madre Pluripotentes Inducidas/metabolismo , Operón Lac/genética , Ratones Endogámicos BALB C , Ratones Desnudos , Ratones Transgénicos , Microscopía Confocal , Microscopía Fluorescente , Células Madre Multipotentes/metabolismo , Proteína P0 de la Mielina/genética , Proteína P0 de la Mielina/metabolismo , Cresta Neural/embriología , Cresta Neural/metabolismoRESUMEN
We designed and synthesized N(3)-substituted 5-fluorodeoxyuridines as radiation-activated prodrugs of the antitumor agent, 5-fluorodeoxyuridine (5-FdUrd). A series of 5-FdUrd derivatives possessing a 2-oxoalkyl group at the N(3)-position released 5-FdUrd in good yield via one-electron reduction initiated by hypoxic irradiation. Cytotoxicity of the 5-FdUrd derivative possessing the 2-oxocyclopentyl group (3d) was low, but was enhanced by hypoxic irradiation resulting in 5-FdUrd release.