RESUMEN
The zinc-finger transcriptional repressor Blimp1 (Prdm1) controls gene expression patterns during differentiation of B lymphocytes and regulates epigenetic changes required for specification of primordial germ cells. Blimp1 is dynamically expressed at diverse tissue sites in the developing mouse embryo, but its functional role remains unknown because Blimp1 mutant embryos arrest at E10.5 due to placental insufficiency. To explore Blimp1 activities at later stages in the embryo proper, here we used a conditional inactivation strategy. A Blimp1-Cre transgenic strain was also exploited to generate a fate map of Blimp1-expressing cells. Blimp1 plays essential roles in multipotent progenitor cell populations in the posterior forelimb, caudal pharyngeal arches, secondary heart field and sensory vibrissae and maintains key signalling centres at these diverse tissues sites. Interestingly, embryos carrying a hypomorphic Blimp1gfp reporter allele survive to late gestation and exhibit similar, but less severe developmental abnormalities, whereas transheterozygous Blimp1(gfp/-) embryos with further reduced expression levels, display exacerbated defects. Collectively, the present experiments demonstrate that Blimp1 requirements in diverse cell types are exquisitely dose dependent.
Asunto(s)
Desarrollo Embrionario/genética , Proteínas Represoras/genética , Proteínas Represoras/fisiología , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Animales , Secuencia de Bases , Región Branquial/embriología , Cartilla de ADN/genética , Células Madre Embrionarias/citología , Corazón Fetal/embriología , Miembro Anterior/embriología , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Heterocigoto , Ratones , Ratones Mutantes , Ratones Transgénicos , Células Madre Multipotentes/citología , Especificidad de Órganos , Fenotipo , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Proteínas Recombinantes/genética , Vibrisas/embriologíaRESUMEN
Bone is a highly organized structure comprising a calcified connective tissue matrix formed by mature osteoblasts, which develop from the proliferation and differentiation of osteoprogenitor cells. The osteogenic cell lineage is thought to arise from a population of uncommitted multipotential stromal precursor cells (SPC) which reside close to all bone surfaces, in the bone marrow spaces and the surrounding connective tissue. These SPC also give rise to related cell lineages which form cartilage, smooth muscle, fat, and fibrous tissue. Due to the lack of well defined cell surface markers, little is known of the precise developmentally regulated changes in phenotype which occur during the differentiation and maturation of human osteoprogenitor cells into functional osteoblasts and ultimately, terminally differentiated osteocytes. In order to identify antibody reagents with greater specificity for osteoprogenitors we generated a series of antibodies following immunization with freshly isolated human bone marrow stromal fibroblasts. One such antibody, HOP-26, reacts with a cell surface antigen expressed by SPC and developing bone cells. We now demonstrate that this mAb identifies a member of the tetraspan family of cell surface glycoproteins, namely CD63. Western blot analysis of human bone marrow stromal cells (HBMSC) has revealed that like a well defined CD63 mAb 12F12, HOP-26 interacts with a heavily glycosylated cell surface protein with an apparent molecular weight of 50-60 kD.
Asunto(s)
Antígenos CD/genética , Antígenos de Superficie/genética , Osteoblastos/inmunología , Glicoproteínas de Membrana Plaquetaria/genética , Células Madre/inmunología , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Antígenos CD/metabolismo , Antígenos de Superficie/metabolismo , Secuencia de Bases , Células de la Médula Ósea/inmunología , Células Cultivadas , Clonación Molecular , ADN Complementario/genética , Humanos , Datos de Secuencia Molecular , Glicoproteínas de Membrana Plaquetaria/metabolismo , Homología de Secuencia de Ácido Nucleico , Células del Estroma/inmunología , Tetraspanina 30RESUMEN
OBJECTIVE: To investigate the toxic effect of gentamicin at the high concentrations that can be achieved by local administration in the management of bone infection. DESIGN: Randomized, prospective study in cultured cells, with drug exposure duration of 4 days. SETTING: Cell culture in Dulbecco's modification of Eagle's minimal essential medium with supplements at 37 degrees C in air:CO2 (v:v, 95:5). MATERIALS: Human osteoblastlike cells derived from cancellous bone collected from four adult patients without systemic disease during total hip replacement were cultured in antibiotic-free medium for 4 weeks. INTERVENTION: The cultured cells were exposed to media containing various concentrations of gentamicin (0-1000 microg/mL) for 4 days. MAIN OUTCOME MEASUREMENTS: Alkaline phosphatase activity, total DNA, and 3H-thymidine incorporation. RESULT: Alkaline phosphatase activity was significantly decreased (p < 0.05) in all of the cultures at gentamicin concentrations of 100 microg/mL and above. 3H-thymidine incorporation was also decreased (p < 0.05) in three out of four cultures at 100 microg/mL and above. Total DNA was significantly decreased (p < 0.05) at 700 microg/mL and above. CONCLUSION: Gentamicin, at high concentrations, as achieved following topical application, inhibits cell proliferation in vitro and, therefore, may be detrimental to the repair process in vivo.