RESUMEN
Roberts syndrome and SC phocomelia (RBS/SC) are genetic autosomal recessive syndromes caused by establishment of cohesion 1 homolog 2 ( ESCO 2) mutation. RBS/SC appear to have a variety of clinical features, even with the same mutation of the ESCO2 gene. Here, we established and genetically characterized a medaka model of RBS/SC by reverse genetics. The RBS/SC model was screened from a mutant medaka library produced by the Targeting Induced Local Lesions in Genomes method. The medaka mutant carrying the homozygous mutation at R80S in the conserved region of ESCO2 exhibited clinical variety (i.e. developmental arrest with craniofacial and chromosomal abnormalities and embryonic lethality) as characterized in RBS/SC. Moreover, widespread apoptosis and downregulation of some gene expression, including notch1a, were detected in the R80S mutant. The R80S mutant is the animal model for RBS/SC and a valuable resource that provides the opportunity to extend knowledge of ESCO2. Downregulation of some gene expression in the R80S mutant is an important clue explaining non-correlation between genotype and phenotype in RBS/SC.
Asunto(s)
Acetiltransferasas/genética , Anomalías Craneofaciales/genética , Modelos Animales de Enfermedad , Ectromelia/genética , Hipertelorismo/genética , Oryzias , Acetiltransferasas/metabolismo , Animales , Apoptosis/genética , Clonación Molecular , Anomalías Craneofaciales/metabolismo , Ectromelia/metabolismo , Genotipo , Hipertelorismo/metabolismo , Oryzias/genética , Oryzias/metabolismo , Fenotipo , Polimorfismo de Nucleótido Simple , Receptor Notch1/biosíntesis , Genética InversaRESUMEN
Ureaplasma spp. is detected in the urogenital tract, including the vagina, cervix, chorioamnion, and placenta. Their colonization is associated with histologic chorioamnionitis (CAM), often observed in placentas from preterm delivery. We isolated Ureaplasma spp. from 63 preterm placentas among 151 specimens, which were delivered at <32 wk of gestation. Of the 63 placentas, 52 (83%) revealed CAM in cultures positive for Ureaplasma spp., however, CAM was observed only in 30% (26/88) of cultures negative for Ureaplasma spp. (p < 0.01). Colonization by Ureaplasma spp. was an independent risk factor for CAM (OR, 11.27; 95% CI, 5.09-24.98). Characteristic neutrophil infiltration was observed in the amnion and subchorion (bistratified pattern) in cultures positive for Ureaplasma spp. FISH analysis of CAM placenta with male infant pregnancy indicated that bistratified infiltrated neutrophils showed the XX karyotype and umbilical vein infiltrated neutrophils showed XY karyotype. The distribution of sulfoglycolipid, the receptor of Ureaplasma spp., was mainly detected in the amnion. Ureaplasmal urease D protein and ureB gene were both detected in the amnion, indicating direct colonization by Ureaplasma spp.
Asunto(s)
Corioamnionitis/microbiología , Placenta/microbiología , Nacimiento Prematuro/microbiología , Infecciones por Ureaplasma/microbiología , Ureaplasma/aislamiento & purificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Estudios de Casos y Controles , Corioamnionitis/genética , Corioamnionitis/inmunología , Cromosomas Humanos X , Cromosomas Humanos Y , Femenino , Edad Gestacional , Glucolípidos/metabolismo , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Modelos Logísticos , Masculino , Infiltración Neutrófila , Oportunidad Relativa , Placenta/inmunología , Reacción en Cadena de la Polimerasa , Embarazo , Nacimiento Prematuro/genética , Nacimiento Prematuro/inmunología , Medición de Riesgo , Factores de Riesgo , Ureaplasma/genética , Ureaplasma/metabolismo , Infecciones por Ureaplasma/complicaciones , Infecciones por Ureaplasma/genética , Infecciones por Ureaplasma/inmunología , Ureasa/genética , Ureasa/metabolismoRESUMEN
Annexin A2, a Ca2+-dependent phospholipid binding protein, is abundantly expressed in various human organs, which exists as either a membrane-associated, cytosolic or soluble form in serum. We constructed expression systems for recombinant human annexin A2 (rhA2) using Pichia pastoris. The systems are designed to secrete rhA2 as either the N- or C-terminally His6-tagged form to facilitate purification. Both types of rhA2 were overexpressed, but in the N-terminal-truncated form as revealed from the results of N-terminal amino acid sequencing and Western blotting. Therefore, further purification of N-terminally His6-tagged rhA2 was not feasible because of the removal of the N-terminal His6-tag sequence. C-terminally His6-tagged rhA2 was expressed as either a glycosylated or a nonglycosylated form, and the nonglycosylated form was purified using the combination of nickel-immobilized affinity, concanavalin A and cation exchanged column chromatographies. The solid-phase binding of rhA2 was examined by enzyme-linked immunosorbent assay (ELISA), which revealed the specific reactivity of rhA2 against an anti-annexin A2 monoclonal antibody. These results suggest that the expression system using P. pastoris is useful for the preparation of rhA2 that is applicable to the ELISA detection of the anti-annexin A2 antibody.