Mechanisms of autoinhibition of IRF-7 and a probable model for inactivation of IRF-7 by Kaposi's sarcoma-associated herpesvirus protein ORF45

IRF-7 is the master regulator of type I interferon-dependent immune responses controlling both innate and adaptive immunity. Given the significance of IRF-7 in the induction of immune responses, many viruses have developed strategies to inhibit its activity to evade or antagonize host antiviral responses. We previously demonstrated that ORF45, a KSHV immediate-early protein as well as a tegument protein of virions, interacts with IRF-7 and inhibits virus-mediated type I interferon induction by blocking IRF-7 phosphorylation and nuclear translocation (Zhu, F. X., King, S. M., Smith, E. J., Levy, D. E., and Yuan, Y. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 5573-5578). In this report, we sought to reveal the mechanism underlying the ORF45-mediated inactivation of IRF-7. We found that ORF45 interacts with the inhibitory domain of IRF-7. The most striking feature in the IRF-7 inhibitory domain is two α-helices H3 and H4 that contain many hydrophobic residues and two β-sheets located between the helices that are also very hydrophobic. These hydrophobic subdomains mediate intramolecular interactions that keep the molecule in a closed (inactive) form. Mutagenesis studies confirm the contribution of the hydrophobic helices and sheets to the autoinhibition of IRF-7 in the absence of viral signal. The binding of ORF45 to the critical domain of IRF-7 leads to a hypothesis that ORF45 may maintain the IRF-7 molecule in the closed form and prevent it from being activated in response to viral infection.     

Cell Population Kinetics of Collagen Scaffolds in Ex Vivo Oral Wound Repair

Biodegradable collagen scaffolds are used clinically for oral soft tissue augmentation to support wound healing. This study sought to provide a novel ex vivo model for analyzing healing kinetics and gene expression of primary human gingival fibroblasts (hGF) within collagen scaffolds. Sponge type and gel type scaffolds with and without platelet-derived growth factor-BB (PDGF) were assessed in an hGF containing matrix. Morphology was evaluated with scanning electron microscopy, and hGF metabolic activity using MTT. We quantitated the population kinetics within the scaffolds based on cell density and distance from the scaffold border of DiI-labled hGFs over a two-week observation period. Gene expression was evaluated with gene array and qPCR. The sponge type scaffolds showed a porous morphology. Absolute cell number and distance was higher in sponge type scaffolds when compared to gel type scaffolds, in particular during the first week of observation. PDGF incorporated scaffolds increased cell numbers, distance, and formazan formation in the MTT assay. Gene expression dynamics revealed the induction of key genes associated with the generation of oral tissue. DKK1, CYR61, CTGF, TGFBR1 levels were increased and integrin ITGA2 levels were decreased in the sponge type scaffolds compared to the gel type scaffold. The results suggest that this novel model of oral wound healing provides insights into population kinetics and gene expression dynamics of biodegradable scaffolds.     

冰核细菌在我国北方玉米上的消长动态规律

研究证明,菠萝欧文氏菌(Erwinia ananas)为我国北方玉米上优势冰核细菌种类,占总体INA细菌95 %以上。采用定量定性和定期取样分离方法,首次研究INA细菌在玉米上的消长动态规律。结果表明:玉米不同生长发育阶段是影响INA细菌在玉米上数量分布和消长动态变化的重要因素,以抽雄至成熟期间分布INA细菌数量最多,高达10 7～10 8CFU/ g,比拔节至抽雄期高出2～3个数量级,比苗期至拔节期高出4～5个数量级;同时还指出,玉米不同播期,对INA细菌数量分布影响显著,差异很大,其中INA细菌分布数量消长变化,以正常播种(1.9×10 7CFU / g) >中期播种(7.9×10 5CFU/ g) >晚期播种(5 .0×10 4 CFU/ g) ;研究指出,处于抽雄至成熟期间的玉米上分布的INA细菌数量最多,因此期间(8月上旬至9月下旬) ,气温逐渐降低,昼夜温差大,田间结露多,加上玉米处于成熟阶段,抗INA细菌能力弱,这些因素有利于低温(5～2 0℃范围内生长)型INA细菌生长繁殖,故使INA细菌分布数量最多     

蛋白激酶D1催化结构域在昆虫细胞/杆状病毒表达系统内的表达、纯化和活性测定

蛋白激酶D(Protein kinase D,PKD)是一种新的丝氨酸/苏氨酸蛋白激酶家族和甘油二酯(Diacylglycerol,DAG)受体,参与细胞内多种生理生化过程。为获得高纯度的PKD1的催化结构域(PKD1-cat)用于晶体学结构的研究,将带有GST标签的PKD1-cat基因克隆到杆状病毒转移载体pFastBac1中,构建了重组质粒。将重组质粒转化到含穿梭载体Bacmid的DH10Bac感受态细胞中,转座后获得了含目的基因GST-PKD1-cat的重组Bacmid。重组Bacmid DNA转染Sf9昆虫细胞后,获得重组杆状病毒并扩毒。将毒种以5 PFU/cell的感染复数感染悬浮培养的T.ni昆虫细胞,SDS-PAGE和Western blotting检测表达产物。结果显示,表达产物在分子量约68 kDa处有一特异条带可与GST单克隆抗体发生反应。经谷胱甘肽琼脂糖凝胶亲和层析纯化和PreScission Protease切除GST标签后,得到了纯度很高的分子量约42 kDa的目的蛋白PKD1-cat。体外PKD激酶活性实验结果显示,随着PKD1-cat浓度的增加,激酶活性增高。这些结果显示截短的重组PKD1-cat有很高的催化活性和纯度,为采用核磁共振或晶体学方法解析PKD1-cat的三维结构奠定了基础。     

Development, validation and implementation of immobilized metal affinity for phosphochemicals (IMAP)-based high-throughput screening assays for low-molecular-weight compound libraries

This protocol describes assay development, validation and implementation of automated immobilized metal affinity for phosphochemicals (IMAP)-based fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET) high-throughput screening (HTS) assays for identification of low-molecular-weight kinase inhibitors. Both procedures are performed in miniaturized kinase reaction volumes and involve the stepwise addition of test or control compounds, enzyme and substrate/ATP. Kinase reactions are stopped by subsequent addition of IMAP-binding buffer. Assay attributes of the IMAP FP and TR-FRET methodologies are described. HTS assays developed using these procedures should result in Z-factors and low assay variability necessary for robust HTS assays. Providing that the required reagents and equipment are available, one scientist should be able to develop a 384-well, miniaturized HTS assay in approximately 6-8 weeks. Specific automated HTS assay conditions will determine the number of assay plates processed in a screening session, but two scientists should expect to process between 100 and 150 assay plates in one 8-h screening day.     

ROC曲线分析在评价入侵物种分布模型中的应用

生态位模型（ecological niche models,ENMs）已广泛应用于物种潜在分布区预测,ENMs的应用也为外来入侵物种的风险分析提供了重要的定量化分析工具,但如何评价不同模型之间的预测效果成了当今研究的热点问题。本文介绍了受试者工作特征（ROC）曲线分析在评价不同生态位模型预测效果中的应用原理和分析方法,并以一种植物病原线虫-相似穿孔线虫（Radopholus similis）为例,应用ROC曲线分析法对其5种模型（BIOCLIM,CLIMEX,DOMAIN,GARP,MAXENT）的预测结果进行了比较分析。5种模型的ROC曲线下面积AUC（Area Under Curve）值分别为0.810,0.758,0.921,0.903和0.950,以MAXENT模型的AUC值最大,表明其预测效果最好;方差分析结果表明,除GARP与DOMAIN模型之间AUC值差异不显著外,其余各模型之间差异显著。     

用天然13C丰度法评估贵州茂兰喀斯特森林区玉米地土壤中有机碳的来源

当森林生态系统转变成农田生态系统时,会把C4植物有机质导入到曾在C3植被下发育的土训中去,使土壤中含有来源不同的土壤有机质,引起碳同位素组成变化。因此,可以利用碳同位素来区分土壤有机质来源,实验结果表明,耕作几十年后原森林土壤有机质的含量仍占有主要地位,来源于原始C3植被的有机碳的比例为66.7％,但容易矿化的、对植物营养有效的有机质含量较低,这与当地的耕作方式有关,需要加强对植物残留物返回土壤工作的管理。     

花青素生物合成关键酶的研究进展

花青素是植物花呈现不同色彩的物质基础,其生物合成途径主要受到查尔酮合成酶(CHS)、查尔酮异构酶(CHI)、黄烷酮3-羟化酶(F3H)、类黄酮3'-羟化酶(F3'H)、类黄酮3’,5’-羟化酶(F3'5'H)、二羟基黄酮醇还原酶(DFR)、花色素苷合成酶(ANS)以及类黄酮3-O-糖基转移酶(UFGT)等关键酶的控制.主要介绍花青苷生物合成途径、关键酶晶体结构及利用基因工程改造花色的研究进展,讨论目前花色改造存在的问题,并对今后的研究前景进行展望.     

The OsmiR396c‐OsGRF4‐OsGIF1 regulatory module determines grain size and yield in rice

Grain weight is the most important component of rice yield and is mainly determined by grain size, which is generally controlled by quantitative trait loci (QTLs). Although numerous QTLs that regulate grain weight have been identified, the genetic network that controls grain size remains unclear. Herein, we report the cloning and functional analysis of a dominant QTL, grain length and width 2 (GLW2), which positively regulates grain weight by simultaneously increasing grain length and width. The GLW2 locus encodes OsGRF4 (growth‐regulating factor 4) and is regulated by the microRNA miR396c in vivo. The mutation in OsGRF4 perturbs the OsmiR396 target regulation of OsGRF4, generating a larger grain size and enhanced grain yield. We also demonstrate that OsGIF1 (GRF‐interacting factors 1) directly interacts with OsGRF4, and increasing its expression improves grain size. Our results suggest that the miR396c‐OsGRF4‐OsGIF1 regulatory module plays an important role in grain size determination and holds implications for rice yield improvement.