针对人SND1基因两个AUG的细胞应激分析

【摘要】目的 针对人SND1基因2个蛋白翻译起始密码子AUG构建真核表达质粒pCMV-N-Flag-SND1-No1/ 2,并分析2个AUG在SND1应激颗粒形成中的作用。方法 以SND1全长转录本为模板,PCR法扩增含BamHⅠ和 EcoRⅠ酶切位点的目的基因SND1-No1/2,双酶切法分别酶切目的基因片段和线性pCMV-N-Flag,以T4-DNA连接酶将两者连接成pCMV-N-Flag-SND1-No1/2重组质粒,然后将构建的重组质粒转染入HeLa细胞内,以Western印迹法检测Flag标签（DYKDDDDK）与SND1-No1/2的融合表达,最后以细胞免疫荧光实验检测在氧化应激状态下Flag SND1-No1/2融合蛋白与内源性SND1应激颗粒的胞内共定位情况。结果以单/双酶切及基因测序法鉴定构建的重组质粒无误,Western印迹结果检测到融合蛋白Flag-SND1-No1/2的表达;细胞免疫荧光结果显示Flag-SND1-No1/2 均可与内源性SND1应激颗粒共定位。结论重组pCMV-N-Flag-SND1-No1/2质粒构建成功,SND1基因第1个 AUG的缺失并不影响SND1应激颗粒的形成。     

P1 Protein of Cocksfoot Mottle Virus is Indispensable for the Systemic Spread of the Virus

Cocksfoot mottle sobemovirus (CfMV) encodes a non-conserved protein P1 from the 5′ ORF1 of genomic RNA. The functions of CfMV P1 are unknown. In the current study we show that P1-deficient CfMV can replicate both in oat leaves and barley suspension culture cells but can not infect oat plants systemically. However, the absence of P1 reduces the efficiency of virus accumulation considerably. The infectivity of the mutant virus restores as a result of the spontaneous transversion. CfMV P1:EGFP shows a very limited cell-to-cell movement in leaf epidermal cells. In Sf9 insect cells CfMV P1 localizes in the fraction of membranes and inclusions but not in soluble cytoplasmic protein fraction.     

Bioequivalence

The bioequivalence of two formulations of the same drug may be determined by evaluating the similarity of their respective plasma concentration curves. The similarity of two plasma concentration functions can be measured by an index called the bioequivalence index. This paper shows how such an index may be defined and calculated.     

冠心病患者大动脉弹性的改变

目的：探讨冠心病患者大动脉弹性特征的变化。方法：对诊断或拟诊为冠心病患者48例，进行冠状动脉造影检查。升主动脉和股动脉压力描计。结果：冠状动脉造影异常患者较正常组脉搏波速度（pulse wave velocity,PWV)，中心动脉反射波增压（central aortic pressure augmentation,AUMG)显著增高（P＜0．01），且随冠脉病变支数增多有上升趋势。结论：冠心病患者大动脉弹性降低。     

Regulation of p21 and p27 expression by the hepatitis B virus X protein and the alternate initiation site X proteins,AUG2 and AUG3

BACKGROUND: The hepatitis B virus X gene has three in-frame start codons encoding the pX, AUG2 and AUG3 proteins. The AUG2 and AUG3 genes are 5'-truncated in respect to the full-length pX gene; however, all three genes terminate at the same stop codon. The activity of pX as an oncogene is well characterized; however, less is known about the AUG2 and AUG3 proteins. METHODS: The effects of pX, AUG2 and AUG3 on p21Cip,1/WAF,1/MDA6 and p27Kip-1 cyclin kinase inhibitor (CKI) protein expression, and the impact they have on proliferation, were investigated in CHO K-1 cells. CHO K-1 cells were chosen because they can be transfected at 100% efficiency. RESULTS: p21- and p27-luciferase reporter expression is modulated by increasing doses of the hepatitis B X proteins. At low concentrations of pX or AUG2, p21- and p27-luciferase activity was increased, and at high concentrations, p21- and p27-luciferase activity was decreased. Expression of the AUG3 gene showed a different profile: it was increasingly stimulatory with dose for both promoters. Western blot analyses demonstrated that p21 and p27 protein levels were modulated as predicted based on data generated in the promoter-luciferase experiments. Tritiated thymidine labeling of DNA showed biphasic kinetics of incorporation in the presence of varying pX and AUG2 concentrations, whereas labeling decreased with AUG3 concentration. The growth inhibitory effect of pX expression was reduced by antisense ablation of either p21 or p27. CONCLUSIONS: The relative expression level of pX, AUG2, and AUG3 impacts on CKI expression and cell proliferation. Our findings may explain why divergent effects of pX expression on growth have been observed by different groups, which may be related to relative pX expression levels.     

Disruption of TWIST1 translation by 5′ UTR variants in Saethre‐Chotzen syndrome

Saethre‐Chotzen syndrome (SCS), one of the most common forms of syndromic craniosynostosis (premature fusion of the cranial sutures), results from haploinsufficiency of TWIST1, caused by deletions of the entire gene or loss‐of‐function variants within the coding region. To determine whether non‐coding variants also contribute to SCS, we screened 14 genetically undiagnosed SCS patients using targeted capture sequencing, and identified novel single nucleotide variants (SNVs) in the 5′ untranslated region (UTR) of TWIST1 in two unrelated SCS cases. We show experimentally that these variants, which create translation start sites in the TWIST1 leader sequence, reduce translation from the main open reading frame (mORF). This is the first demonstration that non‐coding SNVs of TWIST1 can cause SCS, and highlights the importance of screening the 5′ UTR in clinically diagnosed SCS patients without a coding mutation. Similar 5′ UTR variants, particularly of haploinsufficient genes, may represent an under‐ascertained cause of monogenic disease.