Engineering mouse apolipoprotein A-I into a monomeric, active protein useful for structural determination

Apolipoprotein AI (apoAI), the major protein component of HDL, is one of the best predictors of coronary artery disease (CAD), with high apoAI and HDL levels being correlated with low occurrences of CAD. The primary function of apoAI is to recruit phospholipid and cholesterol for assembly of HDL particles. Like other exchangeable apolipoproteins, lipid-free apoAI forms a mixture of different oligomers even at 1.0 mg/mL. This self-association property of the exchangeable apolipoproteins is closely associated with the lipoprotein-binding activity of this protein family. It is unclear if the self-association property of apolipoprotein is required for its lipoprotein-binding activity. We developed a novel method for engineering an oligomeric protein to a monomeric, biologically active protein. Using this method, we generated a monomeric mouse apoAI mutant that is active. This mutant contains the first 216 residues of mouse apoAI and replaces six hydrophobic residues with either polar or smaller hydrophobic residues at the defined positions (V118A/A119S/L121Q/T191S/T195S/T199S). Cross-linking results show that this mutant is greater than 90% monomeric at 8 mg/mL. CD, DSC, and NMR results indicate that the mutant maintains an identical secondary, tertiary structure and stability as those of the wild-type mouse apoAI. Lipid-binding assays suggest that the mutant shares an equal lipoprotein-binding activity as that of the wild-type apoAI. In addition, both the monomeric mutant and the wild-type protein make nearly identical rHDL particles. With this monomeric mouse apoAI, high-quality NMR data has been collected, allowing for the NMR structural determination of lipid-free apoAI. On the basis of these results, we conclude that this apoAI mutant is a monomeric, active apoAI useful for structural determination.     

N-糖基化位点鉴定方法和非经典N-糖基化序列

蛋白质的N-糖基化修饰是生物体调控蛋白质在组织和细胞中的定位、功能、活性、寿命和多样性的一种普遍的翻译后方式。N-糖基化位点是理解糖链功能的重要前提之一。应用新的糖蛋白、糖肽富集技术和质谱技术,科学家们在不同组织中完成了对N-糖基化位点的鉴定。此外,不同于经典三联子的N-糖基化序列的发现使人们对N-糖基化过程的认识向纵深发展。     

盐池县2000-2012年植被变化及其驱动力

荒漠草原区的植被对防治荒漠化、维护生态屏障具有决定性作用,宁夏盐池县作为其典型代表,近13年的植被变化深受气候变化和人类活动的综合影响。基于MODIS NDVI等数据,运用趋势分析、经验模态分解和空间叠置分析等方法,对盐池县2000—2012年的植被动态变化进行研究,结果表明:(1)2000—2012年盐池县NDVI在0.2—0.4之间呈波动上升趋势,上升幅度为0.078/10 a,上升趋势显著;总体来说,植被稳定性低,年际间波动或转换频繁、幅度大;(2)NDVI的波动分量与残余分量方差贡献率各占50%,且NDVI波动呈减弱趋势。促使NDVI波动的主控因子是年降水量,但其影响在减弱;(3)推动NDVI趋势性上升的主要因素是土地利用方式改善和类型变化,但土地利用方式改善对NDVI的贡献远远大于土地利用类型变化对NDVI的贡献。因此,荒漠草原区的生态改善应以保护为主,辅之以必要的生态重建,走以适度开发带动整体保护的道路。     

CO2浓度升高对西花蓟马和花蓟马成虫体内解毒酶和保护酶活性的影响

【目的】阐明大气CO₂浓度升高对外来入侵昆虫西花蓟马Frankliniella occidentalis及其本地近缘种花蓟马F. intonsa的影响机制。【方法】测定和分析了CO₂人工气候箱内不同CO₂浓度（400 μL/L和800 μL/L）下饲养3代的这两种蓟马体内3种解毒酶［羧酸酯酶（CarE）、乙酰胆碱酯酶（AchE）和微粒体多功能氧化酶(MFO）］和3种保护酶［超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD）］的活性。【结果】西花蓟马成虫体内的CarE, AchE, MFO, CAT和POD酶活性随着CO₂浓度的升高而显著上升（P＜0.05）,其中800 μL/L CO₂浓度下CarE和MFO酶活性分别比400 μL/L CO₂浓度下增加了24.78%和16.05%;800 μL/L CO2浓度下花蓟马成虫体内的CarE, MFO和CAT酶活性显著高于400 μL/L CO₂浓度下花蓟马成虫体内的相应酶活性,而AchE和POD酶活性在两种CO2浓度间差异不显著（P＞0.05）。800 μL/L CO₂浓度下西花蓟马和花蓟马成虫体内的SOD酶活性均显著低于400 μL/L CO₂浓度下的相应蓟马酶活性（P＜0.05）,分别下降了65.22%和42.20%。【结论】CO₂浓度升高是导致两种蓟马成虫体内CarE,MFO和SOD酶活性上升的主要原因,而AchE, CAT和POD酶活性的变化主要受蓟马种类的影响。两种蓟马可能通过改变体内解毒酶或保护酶的活性来适应高浓度CO₂的环境。     

高仿真组织工程神经支架制备工艺的参数优化

目的：对直接影响神经支架微观结构的关键因素进行分析,以确定制备不同孔径仿真支架的制备工艺。方法：用前期开发的神经支架制备工艺,应用不同浓度的醋酸浓度和冷淋速度制备仿真神经支架,以扫描电镜观察神经支架结构特征,以确定醋酸浓度和冷淋速度对神经支架内部结构的影响。结果：醋酸浓度和冷淋速度对神经支架内部结构具有重要影响。醋酸浓度为0mg/ml时,无法制备定向结构的神经支架,当醋酸浓度为1mg/ml、2mg/ml、3mg/ml和4mg/ml时,可制备轴定向仿真支架,并且神经支架的孔径随醋酸浓度增大而增大;当冷淋速度为1×10-5m/s、2×10-5m/s和5×10-5m/s时,所制备的仿真支架内部均呈明显的轴向微管结构,其中冷淋速度为2×10-5m/s时,其轴向微管结构排列最为有序、规律。当速度为1×10-6m/s,2×10-6m/s,5×10-6m/s以及1×10-4m/s时,所制备的材料内部微管结构走向无明显规律。结论：醋酸浓度和冷淋速度是影响神经支架内部结构的两个关键因素,通过改变醋酸浓度和冷淋速度可制备不同孔径的仿真神经支架。     

长吻对鱼粉等六种商品饲料中粗蛋白和能量的消化率

本试验以三氧化二铬（Ｃｒ２Ｏ３）为指示物测定Ⅰ龄长吻对鱼粉、猪血粉、豆饼粉、菜籽饼粉、小麦粉和玉米蛋白粉等六种商品饲料原料粗蛋白和总能量的消化率。试验饲料由参考饲料和试验原料以７０∶３０的比例混合挤压制成颗粒饲料。试验鱼粪便样品用虹吸方式收集,并将测试的消化率结果经过降低１０％的校正。由此所得消化率值更能反应长吻的消化生理状况。另外本文对六种饲料的消化率结果进行了讨论。     

Rapid determination of gatifloxacin in biological samples and pharmaceutical products using europium‐sensitized fluorescence spectrophotometry

A europium‐sensitized fluorescence spectrophotometry method using an anionic surfactant, sodium dodecyl benzene sulphonate (SDBS), was developed for the determination of gatifloxacin (GFLX). The GFLX–Eu³⁺–SDBS system was studied and it was found that SDBS significantly enhanced the fluorescence intensity of the GFLX–Eu³⁺ complex (about 25‐fold). The optimal experimental conditions were determined as follows: excitation and emission wavelengths of 338 and 617 nm, pH 7.5, 3.0 × 10^–6 mol/L europium(III), and 5.0 × 10^–5 mol/L SDBS. The enhanced fluorescence intensity of the system (ΔI_f) showed a good linear relationship with the concentration of GFLX over the range 1.0 × 10^–8–8.0 × 10^–7 mol/L with a correlation coefficient of 0.9990. The detection limit (S:N = 3) was determined as 1.0 × 10^–9 mol/L. This method has been successfully applied for the determination of GFLX in pharmaceuticals and human urine/serum samples. Compared with most other methods reported, the rapid and simple procedure proposed here offered higher sensitivity, wider linear range and good stability. The luminescence mechanism of the system is also discussed in detail. Copyright © 2008 John Wiley & Sons, Ltd.     

益生菌Lactobacillus casei Zhang固态发酵条件的优化

利用一株分离自传统发酵酸马奶中的益生干酪乳杆菌（Lactobacillus casei Zhang）进行固态发酵（Solid State Fermentation,SSF）。以发酵物中的活菌数为主要指标,采用九因素四水平（L32（4^9））的正交试验优化固态发酵培养基,并在优化的培养基基础上研究不同的初始含水量及培养时间对Lactobacillus casei Zhang活菌数的影响。实验结果表明,在固态发酵培养基组成为4g豆粕、5g麸皮、0．6g乳清粉、0．3g葡萄糖、0．3g碳酸钙、0．02g硫酸铵、0．01g硫酸镁,初始含水量为55％的优化条件下,37℃发酵60h,发酵物中Lactobacillus casei Zhang活菌数可达到4．08×10^10CFU／g。     

猪丹毒丝菌天然SpaA和重组SpaA-N免疫保护效果的评价

【目的】本试验以小鼠为动物模型,评估了猪丹毒丝菌重组表面保护性抗原A的N端保护区域(rSpaA-N)和天然SpaA的免疫保护效果。【方法】将猪丹毒丝菌C43311株SpaA-N以可溶形式表达在大肠杆菌BL21中,用GST Bind Resin纯化试剂盒纯化rSpaA-N,采用电洗脱法从猪丹毒丝菌C43311株NaOH提取抗原中纯化天然SpaA,将rSpaA-N、天然SpaA和NaOH提取抗原制成亚单位疫苗,同时设GST及生理盐水对照组,间隔2周分3次皮下免疫小鼠,第3次免疫后2周用100LD50猪丹毒丝菌C43065株进行腹腔攻毒,采用间接ELISA方法检测免疫组小鼠血清的抗体动态变化。【结果】SDS-PAGE结果显示,采用GST Bind Resin纯化试剂盒和电洗脱法纯化得到了66kDa的rSpaA-N和64kDa的天然SpaA,蛋白含量分别为1.34mg/mL和1.26mg/mL,而Western印迹结果表明rSpaA-N和纯化前后的SpaA具有良好的免疫反应性。保护试验结果表明,不同免疫剂量的rSpaA-N组、天然SpaA组和NaOH提取抗原组均能完全保护小鼠受强毒株C43065的致死性攻击,而GST组和生理盐水组小鼠攻毒后全部死亡。ELISA检测结果表明,在不同免疫剂量的rSpaA-N组、天然SpaA组和NaOH提取抗原组小鼠血清中的抗体效价之间无显著差异(P0.05)。【结论】本研究结果表明rSpaA-N具有良好的免疫保护作用,可以作为猪丹毒亚单位疫苗。