α-酮戊二酸依赖型双加氧酶催化特性及反应耦联辅因子对其催化羟基化反应的影响

【目的】以重组大肠杆菌表达的枯草芽孢杆菌(Bacillus subtilis)L-异亮氨酸双加氧酶(L-isoleucine dioxygenase,IDO)为研究对象,考察其催化L-异亮氨酸(L-Ile)羟基化反应的影响因素,构建IDO催化合成羟基氨基酸的反应体系。【方法】通过Ni-NTA亲和层析法从重组大肠杆菌(Escherichia coli)BL21/p ET28a-ido中纯化获得重组IDO,以L-Ile为底物,考察重组IDO催化羟基化反应的影响因素,并进一步针对耦联反应优化α-酮戊二酸(α-KG)在重组IDO酶促转化体系中的添加浓度。【结果】基于重组IDO催化L-Ile羟基化的活性测定,计算该酶Km为0.247 mmol/L,kcat为1.260 s-1,kcat/Km为5.101 L/(mmol·s),与其他同源酶动力学参数比较分析表明,重组IDO的底物亲和性及催化效率较高。重组IDO催化反应的最适温度为20°C、最适p H为7.0;在35°C以下较为稳定;反应体系中Fe2+最适浓度为1 mmol/L。重组IDO可催化不同L-氨基酸反应,对L-异亮氨酸、L-正亮氨酸、L-甲硫氨酸的活性较高。通过优化α-KG浓度,反应体系中添加30 mmol/Lα-KG时,可将底物浓度提高至70 mmol/L,产物4-羟基异亮氨酸(4-HIL)的摩尔产率达66.20%,表明α-KG作为反应耦联辅因子,其浓度对重组IDO催化L-Ile羟基化具有显著影响。【结论】重组IDO的底物亲和性、催化效率、最适催化条件、稳定性等基本性质有利于催化L-Ile羟基化反应。在其催化反应体系中,α-KG作为反应耦联辅因子,对酶促转化效果影响显著。研究结果为4-HIL及其他羟基氨基酸的酶促转化提供了研究基础。     

Comparing the folding landscapes of evolutionarily divergent procaspase-3

All caspases evolved from a common ancestor and subsequently developed into two general classes, inflammatory or apoptotic caspases. The caspase-hemoglobinase fold has been conserved throughout nearly one billion years of evolution and is utilized for both the monomeric and dimeric subfamilies of apoptotic caspases, called initiator and effector caspases, respectively. We compared the folding and assembly of procaspase-3b from zebrafish to that of human effector procaspases in order to examine the conservation of the folding landscape. Urea-induced equilibrium folding/unfolding of procaspase-3b showed a minimum three-state folding pathway, where the native dimer isomerizes to a partially folded dimeric intermediate, which then unfolds. A partially folded monomeric intermediate observed in the folding landscape of human procaspase-3 is not well-populated in zebrafish procaspase-3b. By comparing effector caspases from different species, we show that the effector procaspase dimer undergoes a pH-dependent conformational change, and that the conformational species in the folding landscape exhibit similar free energies. Together, the data show that the landscape for the caspase-hemoglobinase fold is conserved, yet it provides flexibility for species-specific stabilization or destabilization of folding intermediates resulting in changes in stability. The common pH-dependent conformational change in the native dimer, which yields an enzymatically inactive species, may provide an additional, albeit reversible, mechanism for controlling caspase activity in the cell.     

鄱阳湖浮游甲壳动物群落结构特征

鄱阳湖是中国第一大淡水湖,具有"丰水为湖,枯水为河"的独特特点。为探讨鄱阳湖浮游甲壳动物群落结构及其时空分布的特征,于2009年全年采集其不同季节、不同水位期的浮游甲壳动物样品进行定量分析。结果显示,鄱阳湖浮游甲壳动物群落结构总体与河流浮游甲壳群落具有相似性。无节幼体、象鼻溞、剑水蚤等河流优势类群在鄱阳湖浮游甲壳动物中占优势;而哲水蚤和溞属仅在低水位季节占优势,枝角类丰度仅在高温、高水位、流速缓的季节高过桡足类。丰水期浮游甲壳动物平均丰度和生物量远远高于枯水期,可达枯水期的50倍,差异极其显著(P0.01)。温度和水位变化引起的环境因子改变是导致鄱阳湖浮游甲壳动物发生季节演替的主要原因;而营养盐对浮游甲壳动物的影响并不显著。空间上浮游甲壳动物群落构成明显不同,年均丰度最高和最低的点均出现在河口地区。因此:对于换水周期短,水交换速率快的水体,应该充分考虑水文条件对生物的影响。     

Cross-seeding of WT amyloid-β with Arctic but not Italian familial mutants accelerates fibril formation in Alzheimer's disease

Alzheimer’s disease (AD) involves the neurotoxic self-assembly of a 40 and 42 residue peptide, Amyloid-β (Aβ). Inherited early-onset AD can be caused by single point mutations within the Aβ sequence, including Arctic (E22G) and Italian (E22K) familial mutants. These mutations are heterozygous, resulting in an equal proportion of the WT and mutant Aβ isoform expression. It is therefore important to understand how these mixtures of Aβ isoforms interact with each other and influence the kinetics and morphology of their assembly into oligomers and fibrils. Using small amounts of nucleating fibril seeds, here, we systematically monitored the kinetics of fibril formation, comparing self-seeding with cross-seeding behavior of a range of isoform mixtures of Aβ42 and Aβ40. We confirm that Aβ40(WT) does not readily cross-seed Aβ42(WT) fibril formation. In contrast, fibril formation of Aβ40(Arctic) is hugely accelerated by Aβ42(WT) fibrils, causing an eight-fold reduction in the lag-time to fibrillization. We propose that cross-seeding between the more abundant Aβ40(Arctic) and Aβ42(WT) may be important for driving early-onset AD and will propagate fibril morphology as indicated by fibril twist periodicity. This kinetic behavior is not emulated by the Italian mutant, where minimal cross-seeding is observed. In addition, we studied the cross-seeding behavior of a C-terminal-amidated Aβ42 analog to probe the coulombic charge interplay between Glu22/Asp23/Lys28 and the C-terminal carboxylate. Overall, these studies highlight the role of cross-seeding between WT and mutant Aβ40/42 isoforms, which can impact the rate and structure of fibril assembly.     

青藏高原1979-2007年间的积雪变化

利用雪深被动微波遥感数据产品,对青藏高原1979—2007年积雪深度、积雪日数的分布变化及其趋势进行了分析。结果表明:青藏高原积雪日数、积雪深度和海拔三者之间在空间上具有显著正相关;青藏高原积雪在1988年发生突变,该年前后积雪分布有显著不同,这与20世纪80年代中后期青藏高原由暖干时期进入暖湿时期有关;将青藏高原按夏季水汽来源不同将其分为南北两部分,发现29年来北部积雪日数变化与全国积雪变化相反呈极显著增加趋势(R2=0.39,P0.01),以1.40 d/a的趋势增加,主要原因是西北部地区冬季积雪日数增加;南部积雪深度与全国积雪变化一致呈极显著减少趋势(R2=0.24,P0.01),以-0.04 cm/a的趋势减少,主要原因是东南部春、夏、秋三季积雪深度减少。     

HIV gag基因修饰在不同载体系统中对免疫效果的影响

目的:了解gag基因修饰前后在不同载体系统中的表达水平差异对免疫效果的影响,为确定HIV疫苗中能诱发较高水平细胞免疫的Gag靶抗原奠定实验基础。方法:将含有优化前后gag基因的HIV-1 DNA(pVRC)疫苗和重组痘苗病毒(rVV)载体疫苗单独或联合免疫BALB/c小鼠,利用IFN-γ酶联免疫斑点法和胞内细胞因子染色检测各组的细胞免疫效果,ELISA检测体液免疫水平,分别比较基因优化前后及在不同载体内的Gag诱发的免疫效果。结果:DNA疫苗中gag基因修饰后细胞免疫反应由472提高至925 SFC/106MNC,抗体滴度随免疫次数增加而提高,基因修饰后第二针的抗体水平由104.2提高至105.3,三针后则没有差别;而以rVV为载体的疫苗基因修饰前后细胞免疫反应(~320 SFC/106MNC)和抗体水平(~104.4)均没有差异。2种疫苗联合免疫均可显著提高Gag修饰前后在小鼠体内的免疫效果,基因修饰后细胞免疫反应由1700提高至2100 SFC/106MNC,抗体水平则没有差别。结论:gag基因修饰明显提高常规DNA疫苗免疫效果,并可进一步提高联合免疫效果,但对rVV疫苗单独免疫效果无明显影响。     

地毯草黄单胞菌双组分系统VgrS-VgrR基因敲除及表型筛选

【目的】研究地毯草黄单胞菌双组分系统VgrS-VgrR与致病性的关系,为木薯细菌性病害的高效防控提供分子生物学证据。【方法】采用同源重组方法构建vgrS和vgrR的插入失活突变体,用可移动的cosmid载体p HM1构建互补菌株。检测突变体的致病性、细菌游动性、胞外酶、胞外多糖的变化,观察细菌对H_2O_2和金属离子胁迫的反应。【结果】相比野生型菌株,vgrS和vgrR突变体接种寄主植物木薯后致病力显著降低,突变体的游动性减少、蛋白酶活性减弱、H_2O_2耐受性降低,在高浓度金属离子Fe²⁺、Fe³⁺、Cu²⁺、Ni²⁺、Zn²⁺、Co²⁺的胁迫条件下菌体生长显著减弱。然而,vgrS和vgrR突变体的胞外多糖含量显著升高,分别是野生型的2.14和1.89倍。【结论】阐明了VgrS-VgrR系统在细菌致病过程中发挥的重要作用,为鉴定VgrS-VgrR调控机制提供线索,为药物筛选提供靶向目标。     

The chemiluminescence immunoassay for aflatoxin B1 based on functionalized magnetic nanoparticles with two strategies of antigen probe immobilization

A rapid and sensitive chemiluminescence immunoassay (CLIA) based on magnetic nanoparticles (MNPs) was developed to detect aflatoxin B1 (AFB1), which is a potent carcinogen in nature. We prepared monodisperse MNPs (300 nm in diameter) according to the solvothermal synthesis reaction and the MNPs were coated with silica by the Stöber method. Triethox was used as a one‐step carboxylation reagent, and 3‐aminopropyltriethoxysilane (APTES) an amination reagent, to modify the MNPs. We prepared two types of solid phase antigens using the above synthesized functionalized MNPs coupled with the later prepared AFB1‐oxime active ester and the purchased BSA–AFB1 respectively. 2′,6′‐dimethylcarbonylphenyl‐10‐sulfopropylacridinium‐9‐carboxylate 4′‐N‐hydroxysuccinimide (4′‐NHS) ester (NSP–DMAE–NHS), as a novel luminescent reagent, was used to label anti‐AFB1 antibodies. The two CLIA calibration curves based on the two types of solid phase antigens were obtained and compared. The acquired limit of detection (LOD) was about 0.001 ng/mL for the two functionalized MNPs‐based immunoassays, and the half maximal inhibitory concentration (IC₅₀) was 0.51 ng/mL for the MNPs–AFB1‐based method and 0.72 ng/mL for the MNPs–BSA–AFB1‐based method.