ApoE4 increases glycogen synthase kinase 3β expression and Tau phosphorylation in U87 cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To explore the relations among apolipoprotein E4, Tau protein and glycogen synthase kinase 3β (GSK-3β).</p><p><b>METHODS</b>U87 cells were transfected with pIRES-EGFP (control) or the recombinant plasmids ApoE4/pIRES-EGFP or ApoE3/pIRES-EGFP, and the expression levels of p-Tau/Tau and GSK-3β in the cells were examined with Western blotting. To further confirm the effect of ApoE on GSK-3β and p-Tau expressions, a short interfering RNA (siRNA) targeting ApoE (ApoE-siRNA) was transfected into U87 cells via Lipofectamine 2000 and the protein expressions were examined 24 h later.</p><p><b>RESULTS</b>Compared with those in the control group, the expressions levels of both GSK-3β and p-Tau/Tau increased significantly in the cells transfected with ApoE4 and ApoE3 plasmids (P<0.01), and the ApoE4 plasmid produced a more potent effect than the ApoE3 plasmid on the protein expressions (P<0.01). ApoE knockdown resulted in significantly reduced expressions of GSK-3β (P<0.001) and p-Tau (P<0.01) in the cells.</p><p><b>CONCLUSION</b>ApoE4 can enhance Tau phosphorylation though upregulating GSK-3β, which sheds light on a new role of ApoE4 in Alzheimer's disease.</p>

Construction and identification of a RNA interference plasmid for rat BNIP3 gene / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a RNA interfering plasmid targeting rat Bcl-2/E1B-19K-interacting protein 3 (BNIP3) and assess its effect on exogenous BNIP3 expression in HEK293 cells.</p><p><b>METHODS</b>The miRNA sequences were designed using Invitrogen BLOCK-iT RNAi Designer and synthesized into double-strand oligonucleotides, which were cloned into the pcDNATM6.2-GW/EmGFP-miR vector, followed by transformation of the product into competent Top10 E. coli cells. After expansion of the transformed bacteria, the plasmid was extracted and sequenced before its co-transfection with pEGFP-C3- rBNIP3 plasmid into HEK293 cells. The interference effect of the constructed plasmid on BNIP3 mRNA and protein expression were detected by real-time PCR and Western blotting.</p><p><b>RESULTS</b>The sequencing result indicated that the interfering plasmid targeting rat BNIP3 was constructed correctly. After transfection into HEK293 cells, the interfering plasmid significantly inhibited exogenous BNIP3 mRNA and protein expressions.</p><p><b>CONCLUSION</b>The RNA interfering plasmid targeting rat BNIP3 has been constructed successfully, which provides a useful tool for studying the function of BNIP3.</p>

Expression and identification of the extracellular domain of human epidermal growth factor receptor type III variant / 南方医科大学学报

<p><b>OBJECTIVE</b>To clone the extracellular domain (ECD) of the type III variant of human epidermal growth factor receptor (EGFRvIII) and construct the recombinant expression plasmid.</p><p><b>METHODS</b>A DNA fragment (vIII ECD) encoding the extracellular domain of human EGFRvIII was obtained by PCR, and its T-A was cloned and sequenced. The DNA fragment was then ligated into the GST fusion expression vector to construct the recombination plasmid. After identification with restriction digestion and DNA sequencing, the recombinant plasmid was transformed into E. coli BL21 (DE3) for expression of the recombinant protein. The target protein was identified by SDS-PAGE and Western blotting.</p><p><b>RESULTS</b>The results of restriction digestion and DNA sequencing confirmed the successful construction of the recombinant plasmid. SDS-PAGE showed that the fusion protein was expressed as inclusion bodies in E. coli BL21 (DE3), and the amount of the fusion protein expressed in the bacteria, after induction for 4 h, accounted for up to 15% of the total bacterial proteins. Western blotting demonstrated that the fusion protein could be recognized by the specific anti-EGFR antibody.</p><p><b>CONCLUSION</b>We have successfully constructed the recombinant expression vector of vIII ECD and induced the expression of the fusion protein, which may facilitate functional and immunological studies of EGFRvIII.</p>

Advances in anti-tumor therapy targeted type Ⅲ variant of the epidermal growth factor receptor mutation / 国外医学(肿瘤学分册)

The epidermal growth factor receptor (EGFR) is overexpressed in many epithelial cancers,and is commonly caused by EGFR gene amplification and gene mutations. The most frequently occurring variant,the type III mutation (EGFRvIII) ,is characterized by an inframe deletion of exons 2-7 of the coding sequence. It is expressed only in tumors and not found in normal tissues, and therefore represents an attractive therapeutic target. The tumor therapy methods targeted for EGFRvIII include immu-notherapy, ribozyme, RNA interference, etc.

Screening of peptide inhibitors of acetylcholinesterase from 12-mer random phage display peptide library / 南方医科大学学报

<p><b>OBJECTIVE</b>To screen the peptide inhibitor of acetylcholinesterase (AChE) from 12-mer random phage display peptide library.</p><p><b>METHODS</b>Human AChE was used as the target to screen its binding peptides from 12-mer random phage display peptide library. The positive phage clones were isolated after three rounds of biopanning followed then by sequence analysis and their activity evaluation.</p><p><b>RESULTS</b>Six positive phage clones binding to human AChE were obtained, and 4 of them sharing the conservative sequence W(S/P)HY inhibited the enzyme activity of AChE.</p><p><b>CONCLUSION</b>Acquisition of AChE inhibitor from phage display library provides clues for designing peptide inhibitors of AChE.</p>

Isolation, expression analysis of a chilling induced cDNA from rice root with differential display: an evidence role for caffeine-sensitive calcium signal / 生物工程学报

Chilling-sensitive rice varieties acquire chilling tolerance when their roots are exposed to water stress for short time. Caffeine-sensitive calcium signal was involved in this procedure. By using total RNA differential display, a chilling induced cDNA(ICT: induction of chilling treatment) was isolated from roots of chilling-sensitive rice variety. It was determined that it is a novel cDNA by homology searching. The transcript level of ict mRNA is up-regulated under chilling stress, it is decreased to low level when the samples were transferred to standard culture conditions. Pre-treated with mannitol for two hours is beneficial to inducing ICT level of expression. This chilling induction was inhibited by caffeine, suggesting that it may play a putative role in signal transduction of caffeine-sensitive calcium.

Congenital cystic adenomatoid malformation of lung in adult / 中华放射学杂志

Objective To investigate of the radiological manifestations in congenital cystic adenomatoid malformation (CCAM) of lung in adult and to improve the diagnostic accuracy of CCAM of lung in adult.Methods Five cases with pathologically proved CCAM of lung in adult were retrospectively analyzed.Chest X-ray was available in 5 cases and chest CT was performed in 2 cases.Results On plain chest radiography,thin wall air cystic lesions with air-fluid level were detected in 3 cases.Honeycomb like small cystic lesion was detected in 1 case.Multiple round cystic lesions were found in 1 case.CT scan of the chest demonstrated a round thin wall air cystic lesion in the lateral segment of right lung's middle lobe,and a thin wall air cystic lesion with the wall merged into the shadow of chest wall in the left apicoposterior segment in one case.Small cystic lesions just like honeycomb were found in bilateral basal segments of the inferior lobes,with a 0.8—1.0 cm sized round mass revealed in the right lung′s inferior lobe basal segment, and the mass was spiculated in another case.Conclusion The imaging signs of CCAM of lung in adult is cyst or cyst-solid and at the risk of developing carcinoma.