RUS家族对植物生长发育的调控作用

叶绿体基因组编码许多参与光合作用和其他代谢过程的关键蛋白质,在叶绿体中合成的代谢物对于植物正常的生长发育至关重要。根对紫外线-B辐射敏感[Root-UVB (ultraviolet radiation B)-sensitive, RUS]蛋白属于叶绿体蛋白,由高度保守的DUF647结构域组成,在参与植物形态发生、物质运输和能量代谢等多种生命活动的调控中发挥作用。本文就近年来关于RUS家族在植物的胚胎发育、光形态建成、维生素B6稳态、生长素转运和花药发育等生长发育过程中的相关研究进行回顾和总结,为深入研究其在植物生长发育中的分子调控机制提供了参考。     

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.     

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.     

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.     

微核技术研究进展

微核试验作为检测染色体损伤最常用而有效的细胞遗传学检测方法之一,经济、简便、快速,在敏感性、特异性和准确性方面,与经典的染色体畸变分析方法基本相当。主要综述国内外微核检测技术的最新研究进展,尤其是微核的自动化检测技术。其中流式细胞仪自动化检测和激光扫描细胞仪自动化检测,以及微核试验高内涵筛选方法由于其特有的优势,应用和发展前景广阔。     

中华按蚊CYP6Y亚家族基因的鉴定和生物信息学分析

【目的】鉴定中华按蚊Anopheles sinensis CYP6Y亚家族基因,分析它们的结构和特征,推测其可能的功能。【方法】以冈比亚按蚊An. gambiae CYP6Y1作为询问序列,通过双向Blast方法检索中华按蚊转录组中CYP6Y亚家族基因,并通过生物信息学方法分析基因结构、特征及可能的功能。【结果】从中华按蚊转录组测序数据中鉴定出2条CYP6Y亚家族基因,分别命名为AsCYP6Y1（GenBank登录号：KF709397）和AsCYP6Y2（GenBank登录号：KF709398）。序列分析显示,AsCYP6Y1和AsCYP6Y2全长分别为1 713 bp和1 815 bp,分别编码502和526个氨基酸。基因结构分析显示,该亚家族基因仅含有1个相位1型内含子并与其他P450基因形成保守的共线性分布。蛋白结构分析显示,这2个基因编码的蛋白含P450特有的5个特征序列和6个底物结合位点,且均不存在信号肽,其亚细胞定位为细胞质。3D结构分析显示,AsCYP6Y1有18条α螺旋和13股反向平行的β折叠,AsCYP6Y2有19条α螺旋和11股反向平行的β折叠。通过同样的方法,在达林按蚊An. darlingi中也鉴定出2个CYP6Y亚家族基因。系统进化分析显示,AsCYP6Y1和AsCYP6Y2分别与其他3种按蚊的CYP6Y1和CYP6Y2聚成一支,Bootstrap值均大于90%。替换率分析显示,中华按蚊AsCYP6Y1和AsCYP6Y2与其他3种按蚊同源基因的Ka/Ks均小于1。相对进化速率分析显示,中华按蚊CYP6Y和CYP6M亚家族的相对进化速率均显著快于CYP6P亚家族,而CYP6Y和CYP6M亚家族之间没有显著差异。【结论】在中华按蚊和达林按蚊中存在2个CYP6Y亚家族基因,之前在冈比亚按蚊和不吉按蚊An. funestus中也发现2个CYP6Y亚家族基因,表明CYP6Y亚家族基因可能在按蚊属广泛存在,且可能为按蚊属昆虫所特有。     

河南蚱属二新种记述(直翅目:蚱科)

记述采自河南省蚱科二新种,即鸡公山蚱Tetrix jigongshanensis sp. nov.和突背蚱Tetrix glochinota sp. nov..鸡公山蚱近似于凹额蚱Tetrix cavifrontalis Liang, 1998, 主要区别为:1)头顶前缘平直;2)侧面观,头顶与额面隆起呈钝角形;3)侧面观,额面隆在侧单眼前直,不凹陷;4)侧观,前胸背板上缘呈弧形;5)前胸背板前缘钝角形突出;6)后突到达后足股节中部;7)后翅略不到达后突顶端;8)中足股节明显宽于前翅宽.突背蚱近似于仿蚱Tetrix simulans(B.-Bienko, 1929)及云龙蚱Tetrix yunlongensis Zheng et Mao, 2002, 其与二者的区别为:1)头顶前缘平直;2)侧面观,背板上缘前半部球形突出,后半段平直;3)后突到达后足股节顶端.其区别于前者为:1)侧面观,额面隆起在侧单眼前凹陷;2)中足股节宽于前翅等宽.其区别于后者为头顶宽为一眼宽的1.7倍.模式标本保存于陕西师范大学动物研究所昆虫标本室.