To investigate the effect of GFP fused to C terminal of integrin alpha(IIb) on the biosynthesis and expression of alpha(IIb) beta(3) compound, the alpha(IIb) GFP expression plamid, named palpha(IIb) GFP, the cDNA of alpha(IIb) was constructed from p3.1-2b and fused to pEGFP-N1 in frame. When the sequence of palpha(IIb) GFP was confirmed by sequencing it was transferred to Chinese Hamster Ovary (CHO) cells with or without p3.1-3a expressing integrin beta(3). Then the expression of alpha(IIb) GFP fusion protein was confirmed by Western blot and then its subcellular localization was determined with laser confocal scanning microscopy. The results showed that the target gene was cloned into recombinant vector by restriction analysis and sequencing. Overexpression of the fusion protein in the transfected CHO cells was identified with Western blot. Subcellular localization analysis confirmed that alpha(IIb) GFP was expressed in CHO cells and could be transferred from endoplasmic reticulum to Golgi apparatus. It is concluded that the eukaryotic expression plasmid containing alpha(IIb) GFP fusion gene is successfully constructed. GFP fused to the cytoplasmic tail of integrin alpha(IIb) allows the normal expression of alpha(IIb) beta(3) in CHO cells.
Animals ; Blotting, Western ; CHO Cells ; Cricetinae ; Cricetulus ; Endoplasmic Reticulum ; metabolism ; Golgi Apparatus ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; Microscopy, Confocal ; Platelet Glycoprotein GPIIb-IIIa Complex ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection

Objective To study the effects of pressure controlled ventilation(PCV)and volume controlled ventilation(VCV)on diaphragm function and the incidence of postoperative pulmonary complica-tions(PPCs)in patients undergoing laparoscopic surgery.Methods Sixty-six patients underwent laparo-scopic gynecological surgery under general anesthesia,aged 18-64 years,BMI 18-30 kg/m2,ASA physical status Ⅰ or Ⅱ were recruited.The patients were randomly divided into two groups:PCV group(group P)and VCV group(group V),33 cases in each group.All the patients were ventilated in VCV mode after induction.Group P was switched to PCV after pneumoperitoneum and group V maintained VCV until the end of operation after pneumoperitoneum.The diaphragm ultrasonic evaluation indexes including di-aphragmatic excursion(DE),diaphragm contraction velocity(DCV),and diaphragmatic rapid shallow breathing index(D-RSBI)were recorded before anesthesia induction,immediately after extubation,and 30 minutes after extubation.The mechanical ventilation time,artificial pneumoperitoneum time,the time from the end of artificial pneumoperitoneum to extubation,the cumulative dosage of cisatracuriumbesylate,and the patient's observer's assessment alert/sedation(OAA/S)immediately after extubation,the incidence of diaphragm dysfunction immediately after extubation and 30 minutes after extubation,and the cumulative in-cidence of PPCs in 1-3 days after operation.Results Compared with group V,DE in group P was in-creased significantly immediately after extubation(P＜0.05),but there was no significant difference in DE of 30 minutes between the two groups after extubation.Compared with group V,DCV in group P was in-creased significantly immediately after extubation and 30 minutes after extubation(P＜0.05),the inci-dence of PPCs in group P was significantly lower on the 1st day after operation(P＜0.05).There were no significant differences in D-RSBI,time of mechanical ventilation,time of artificial pneumoperitoneum,time from the end of pneumoperitoneum to extubation,cumulative dosage of atracurium besylate,OAA/S score immediately after extubation,and the incidence of diaphragm dysfunction immediately after extubation and 30 minutes after extubation,and the cumulative incidence of PPCs on the 2nd and 3rd day after operation.Conclusion In lower abdominal endoscopic gynecological surgery,compared with volume-controlled venti-lation mode,pressure-controlled ventilation mode dose not reduce the incidence of postoperative diaphragm dysfunction,but dose alleviate the weakening of diaphragm inspiratory force and reduce the incidence of pul-monary complications on the first day after operation.

It has been reported that ODB genes play an important role in homologous recombination-directed DNA repair, suggesting their potential applications in plant breeding. To analyze the expression characteristics of tobacco NtODB gene, the cDNA sequence of NtODB was obtained using in silico cloning technique. The physicochemical properties, signal peptide, and advanced structures of the predicted protein were analyzed using bioinformatics tools. The results showed that the NtODB gene has a 579-bp open reading frame which encodes a protein with 192 amino acid residues. The protein NtODB is predicted to be alkaline and hydrophilic. Real-time quantitative PCR showed that NtODB was constitutively expressed in different tissues. Subcellular localization showed that NtODB was mainly expressed in cell membrane and chloroplast. These results may help us to better understand and elucidate the roles of ODB genes in the homologous recombination-directed DNA repair.
Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; Computational Biology ; Computer Simulation ; DNA, Complementary ; Phylogeny ; Plant Breeding ; Tobacco/genetics*