RESUMEN
Angiosperms exhibit double fertilization, a process in which one of the sperm cells released from the pollen tube fertilizes the egg, while the other sperm cell fertilizes the central cell, giving rise to the embryo and endosperm, respectively. We have previously reported two polar nuclear fusion-defective double knockout mutants of Arabidopsis thaliana immunoglobulin binding protein (BiP), a molecular chaperone of the heat shock protein 70 (Hsp70) localized in the endoplasmic reticulum (ER), (bip1 bip2) and its partner ER-resident J-proteins, ERdj3A and P58IPK (erdj3a p58ipk). These mutants are defective in the fusion of outer nuclear membrane and exhibit characteristic seed developmental defects after fertilization with wild-type pollen, which are accompanied by aberrant endosperm nuclear proliferation. In this study, we used time-lapse live-cell imaging analysis to determine the cause of aberrant endosperm nuclear division in these mutant seeds. We found that the central cell of bip1 bip2 or erdj3a p58ipk double mutant female gametophytes was also defective in sperm nuclear fusion at fertilization. Sperm nuclear fusion was achieved after the onset of the first endosperm nuclear division. However, division of the condensed sperm nucleus resulted in aberrant endosperm nuclear divisions and delayed expression of paternally derived genes. By contrast, the other double knockout mutant, erdj3b p58ipk, which is defective in the fusion of inner membrane of polar nuclei but does not show aberrant endosperm nuclear proliferation, was not defective in sperm nuclear fusion at fertilization. We thus propose that premitotic sperm nuclear fusion in the central cell is critical for normal endosperm nuclear proliferation.
Asunto(s)
Núcleo Celular/metabolismo , Proliferación Celular/fisiología , Endospermo/fisiología , Fertilización/fisiología , Fusión Nuclear , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Retículo Endoplásmico/metabolismo , Endospermo/citología , Endospermo/genética , Fertilización/genética , Técnicas de Inactivación de Genes , Proteínas del Choque Térmico HSP40/genética , Proteínas HSP70 de Choque Térmico , Chaperonas Moleculares/genética , Membrana Nuclear , Óvulo Vegetal/genética , Polen/metabolismo , Tubo Polínico/metabolismoRESUMEN
Using structure based drug design, novel aminobenzisoxazoles as coagulation factor IXa inhibitors were designed and synthesized. Highly selective inhibition of FIXa over FXa was demonstrated. Anticoagulation profile of selected compounds was evaluated by aPTT and PT tests. In vitro ADMET and pharmacokinetic (PK) profiles were also evaluated.
Asunto(s)
Factor IXa/antagonistas & inhibidores , Isoxazoles/química , Animales , Sitios de Unión , Coagulación Sanguínea/efectos de los fármacos , Cristalografía por Rayos X , Perros , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Factor IXa/metabolismo , Semivida , Humanos , Concentración 50 Inhibidora , Isoxazoles/metabolismo , Isoxazoles/farmacología , Simulación de Dinámica Molecular , Tiempo de Tromboplastina Parcial , Estructura Terciaria de Proteína , Tiempo de Protrombina , Ratas , Relación Estructura-ActividadRESUMEN
Immunoglobulin-binding protein (BiP) is a molecular chaperone of the heat shock protein 70 (Hsp70) family. BiP is localized in the endoplasmic reticulum (ER) and plays key roles in protein translocation, protein folding and quality control in the ER. The genomes of flowering plants contain multiple BiP genes. Arabidopsis thaliana has three BiP genes. BIP1 and BIP2 are ubiquitously expressed. BIP3 encodes a less well conserved BiP paralog, and it is expressed only under ER stress conditions in the majority of organs. Here, we report that all BiP genes are expressed and functional in pollen and pollen tubes. Although the bip1 bip2 double mutation does not affect pollen viability, the bip1 bip2 bip3 triple mutation is lethal in pollen. This result indicates that lethality of the bip1 bip2 double mutation is rescued by BiP3 expression. A decrease in the copy number of the ubiquitously expressed BiP genes correlates well with a decrease in pollen tube growth, which leads to reduced fitness of mutant pollen during fertilization. Because an increased protein secretion activity is expected to increase the protein folding demand in the ER, the multiple BiP genes probably cooperate with each other to ensure ER homeostasis in cells with active secretion such as rapidly growing pollen tubes.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/genética , Gametogénesis en la Planta/genética , Genes de Plantas , Polen/fisiología , Proteínas de Arabidopsis/metabolismo , Segregación Cromosómica , Retículo Endoplásmico/metabolismo , Germinación/genética , Mitosis/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutación/genética , Polen/genética , Tubo Polínico/crecimiento & desarrollo , Tubo Polínico/metabolismo , Regiones Promotoras Genéticas/genéticaRESUMEN
This study was conducted to examine the bond strength of rebonded orthodontic brackets after adhesive residuals on the surface of the bracket bases were removed by Er,Cr:YSGG lasers. Seventy-six brackets bonded to premolars with a self-etching primer adhesive system were equally divided into four groups after the first debonding with the bracket bases (Group 1) untreated, and treated by (Group 2) Er,Cr:YSGG laser, (Group 3) sandblaster, and (Group 4) Er,Cr:YSGG laser/sandblaster. The treated brackets were rebonded to the new premolars in the same manner as the first-stage experiment. The shear bond strengths were measured, with the bonding/debonding procedures repeated once after the first debonding, and the bracket/adhesive failure modes were evaluated after each debonding. The treated bracket base surfaces were observed under a scanning electron microscopy (SEM). The mean rebond strengths were significantly lower in group 1 than in other groups, and there were no significant differences between the other groups. The mean initial bond strength was significantly higher than the mean rebond strength in group 1 but there was no significant difference between the two in the other three groups. Failures at the bracket-adhesive interface occurred frequently at second debonding in group 1. Under the SEM, residual adhesive was removed from the bracket bases by Er,Cr:YSGG laser, while adhesive remnant was seen underneath the meshwork of the bracket bases and microroughness appeared on the meshwork after sandblasting. Er,Cr:YSGG laser certainly could serve the purpose of promoting the use of recycled orthodontic brackets.