琼脂糖和聚丙烯酰胺凝胶电泳技术检测小麦基因组DNA RAPD扩增产物的方法学比较

通过20%(wv)的琼脂糖凝胶和5%(wv)的聚丙烯酰胺凝胶电泳对小麦白粉病抗、感特性品种基因组DNA的RAPD检测结果表明:5%聚丙烯酰胺凝胶对线性DNA分子(01～20kb)和长度相差100bp以下的DNA分子的分离较20%的琼脂糖凝胶电泳效果好。因此,我们研究出了一项利用聚丙烯酰胺凝胶电泳检测小麦白粉病抗、感特性的新技术,在工作中建立了一种适合于检测小麦基因组DNA结构差异的电泳方法。该方法主要包括:(1)丙烯酰胺和亚甲基双丙烯酰胺的新配比;(2)分离DNA片段的最佳凝胶浓度;(3)电泳条件;(4)脱色、漂洗、银染、显色过程。实验发现,该技术对于小麦白粉病抗、感特性检测中的小片段和长度相差100bp以下的线性DNAPCR扩增结果的分辨效果较好。应用该技术在抗感品种间已经发现了DNA水平上的差异。     

O‐GlcNAcylation of protein kinase A catalytic subunits enhances its activity: a mechanism linked to learning and memory deficits in Alzheimer's disease

Alzheimer's disease (AD) is characterized clinically by memory loss and cognitive decline. Protein kinase A (PKA)‐CREB signaling plays a critical role in learning and memory. It is known that glucose uptake and O‐GlcNAcylation are reduced in AD brain. In this study, we found that PKA catalytic subunits (PKAcs) were posttranslationally modified by O‐linked N‐acetylglucosamine (O‐GlcNAc). O‐GlcNAcylation regulated the subcellular location of PKAcα and PKAcβ and enhanced their kinase activity. Upregulation of O‐GlcNAcylation in metabolically active rat brain slices by O‐(2‐acetamido‐2‐deoxy‐d ‐glucopyranosylidenamino) N‐phenylcarbamate (PUGNAc), an inhibitor of N‐acetylglucosaminidase, increased the phosphorylation of tau at the PKA site, Ser214, but not at the non‐PKA site, Thr205. In contrast, in rat and mouse brains, downregulation of O‐GlcNAcylation caused decreases in the phosphorylation of CREB at Ser133 and of tau at Ser214, but not at Thr205. Reduction in O‐GlcNAcylation through intracerebroventricular injection of 6‐diazo‐5‐oxo‐l ‐norleucine (DON), the inhibitor of glutamine fructose‐6‐phosphate amidotransferase, suppressed PKA‐CREB signaling and impaired learning and memory in mice. These results indicate that in addition to cAMP and phosphorylation, O‐GlcNAcylation is a novel mechanism that regulates PKA‐CREB signaling. Downregulation of O‐GlcNAcylation suppresses PKA‐CREB signaling and consequently causes learning and memory deficits in AD.     

婴儿泪在室内生长的光照范围研究

研究了婴儿泪适宜生长的光照范围问题。通过测定试验房间光照的分布图 ,选择试验房间内具有代表性的光区进行栽培试验。实验环境温度为 15～ 2 5℃ ,湿度为 33%～ 6 9%。经栽培 4 0d后 ,用LI 6 4 0 0测定其不同光强下的光合特性 ,计算光补偿点、光饱和点、最大净光合速率。同时测定不同试验区植株的枝长与叶片数量的生长量。结果表明 :婴儿泪在 >8 5μmol·m-2 ·s-1～ <2 99 0 μmol·m-2 ·s-1(4 2 5～ 14 95 0lx ,RH33%～ 6 9% ,温度 15～ 2 5℃ )的光照条件下能够生长     

条件性缺氧诱导因子-1α RNAi转基因小鼠模型的建立

缺氧诱导因子1a (hypoxia inducible factor-1 a, HIF-1 a)是细胞在缺氧等条件下稳定表达的具有转录活性的蛋白,通过与多种靶基因调控区的缺氧反应元件(hypoxia response element, HRE)结合, 调控靶基因表达, 使机体对缺氧、缺血等病理生理过程产生适应性反应。为从整体动物水平研究HIF-1 a的作用, 需要建立HIF-1 a相关遗传修饰小鼠。分别针对HIF-1 a mRNA序列的两个靶位点合成两对互补的寡核苷酸链, 构建可诱导的RNA干扰真核表达载体HIF-AB和HIF-CD。分别将CRE重组酶真核表达载体CRE-ERT2与HIF-AB或HIF-CD转染入RAW264.7细胞, 筛选得到稳定表达CRE-ERT2与HIF-AB, 或CRE-ERT2与HIF-CD的稳定细胞系。在用4-HT诱导去除上述细胞系中HIF-AB或HIF-CD所含的Neo基因后, 用CoCl2诱导HIF-1 a表达, 采用半定量RT-PCR检测HIF-AB或HIF-CD对HIF-1 a 基因表达的影响。结果发现干扰载体(HIF-AB和HIF-CD)对HIF-1 a mRNA序列的沉默效果分别为85%和72%。选择干扰效率较高的表达载体HIF-AB经显微注射获得HIF-1 a基因敲低小鼠模型, 经PCR以及测序验证获得2个转基因阳性小鼠(Founders, G0代)。G0代雄鼠与FVB/N雌鼠交配后获得2只F1代(first filial generation)转基因阳性小鼠, 经与EIIA-Cre转基因小鼠交配, 得到EIIA-Cre; HIFRNAiflox/+小鼠, RT-PCR结果显示, EIIA-Cre; HIFRNAiflox/+小鼠肝、肺、肾等组织的HIF-1 a mRNA水平明显降低, 分别约为正常对照的44%、38.2%和23.5%。该小鼠模型的建立为进一步研究HIF-1 a的功能及作用机制提供了新的手段。     

弗氏志贺菌磷酸化蛋白的检测

目的:检测弗氏志贺菌全菌蛋白中被磷酸化修饰的蛋白。方法:制取弗氏2a志贺菌2457T野生株全菌磷酸化蛋白样品时加入磷酸化酶抑制剂,随后对样品进行双向电泳,以抗磷酸丝氨酸/苏氨酸/酪氨酸抗体为免疫探针,通过Western印迹找到被磷酸化的蛋白,并进行胶内酶解及MALDI-TOF质谱分析。结果与结论:共检测到13个磷酸化蛋白,其中9个为代谢途径中的酶。     

甘露糖受体的结构特征和免疫功能

甘露糖受体可以通过其胞外3个结合区域使组分内化。它可以介导糖蛋白递呈和蛋白糖基化。甘露糖在维持内环境稳定方面发挥作用,同时还有识别病原体和抗原递呈功能。     

5’核苷酸酶碱性磷酸酶双重染色法在毛细淋巴管研究中的应用

本文应用5’-N-ALP双重染色法观察了裸鼠皮肤及人胃癌组织内淋巴管的形态和细微分布.在光镜下毛细淋巴管、淋巴管呈5’-N强阳性反应,管壁显示明显的棕色或深棕色,而毛细血管、血管的ALP呈强阳性,管壁呈明显的蓝色.据此可用组化方法将毛细淋巴管和毛细血管区别开来.本法能显示呈褐色的毛细淋巴管,特别是呈实性条索状的毛细淋巴管,因而双重染色比HE染色更能客观、准确地显示毛细淋巴管的分布.     

Therapeutic potential of α,β‐thujone through metabolic reprogramming and caspase‐dependent apoptosis in ovarian cancer cells

The therapeutic potential of α,β‐thujone, a functional compound found in many medicinal plants of the Cupressaceae, Asteraceae, and Lamiaceae families, has been demonstrated, including in inflammation and cancers. However, its pharmacological functions and mechanisms of action in ovarian cancer remain unclear. We investigated the anticancer properties of α,β‐thujone in ES2 and OV90 human ovarian cancer cells and its effect on sensitization to cisplatin. α,β‐thujone inhibited cancer cell proliferation and induced cell death through caspase‐dependent intrinsic apoptotic pathways. Moreover, α,β‐thujone‐mediated endoplasmic reticulum stress was associated with the loss of mitochondrial functions and altered metabolic landscape of ovarian cancer cells. α,β‐Thujone attenuated blood vessel formation in transgenic zebrafish, implying it has significant antiangiogenic potential. In addition, α,β‐thujone sensitized ovarian cancer cells to cisplatin, causing synergistic pharmacological effects. Collectively, our results suggest that α,β‐thujone has therapeutic potential in human ovarian cancer and functions via regulating multiple intracellular stress‐associated metabolic reprogramming and caspase‐dependent apoptotic pathways.     

Li–Ti Cation Mixing Enhanced Structural and Performance Stability of Li‐Rich Layered Oxide

Li‐rich layered metal oxides are one type of the most promising cathode materials in lithium‐ion batteries but suffer from severe voltage decay during cycling because of the continuous transition metal (TM) migration into the Li layers. A Li‐rich layered metal oxide Li_1.2Ti_0.26Ni_0.18Co_0.18Mn_0.18O₂ (LTR) is hereby designed, in which some of the Ti⁴⁺ cations are intrinsically present in the Li layers. The native Li–Ti cation mixing structure enhances the tolerance for structural distortion and inhibits the migration of the TM ions in the TMO₂ slabs during (de)lithiation. Consequently, LTR exhibits a remarkable cycling stability of 97% capacity retention after 182 cycles, and the average discharge potential drops only 90 mV in 100 cycles. In‐depth studies by electron energy loss spectroscopy and aberration‐corrected scanning transmission electron microscopy demonstrate the Li–Ti mixing structure. The charge compensation mechanism is uncovered with X‐ray absorption spectroscopy and explained with the density function theory calculations. These results show the superiority of introducing transition metal ions into the Li layers in reinforcing the structural stability of the Li‐rich layered metal oxides. These findings shed light on a possible path to the development of Li‐rich materials with better potential retention and a longer lifespan.     

Acceleration of cyanobacterial dominance in north temperate‐subarctic lakes during the Anthropocene

Increases in atmospheric temperature and nutrients from land are thought to be promoting the expansion of harmful cyanobacteria in lakes worldwide, yet to date there has been no quantitative synthesis of long‐term trends. To test whether cyanobacteria have increased in abundance over the past ~ 200 years and evaluate the relative influence of potential causal mechanisms, we synthesised 108 highly resolved sedimentary time series and 18 decadal‐scale monitoring records from north temperate‐subarctic lakes. We demonstrate that: (1) cyanobacteria have increased significantly since c. 1800 ce , (2) they have increased disproportionately relative to other phytoplankton, and (3) cyanobacteria increased more rapidly post c. 1945 ce . Variation among lakes in the rates of increase was explained best by nutrient concentration (phosphorus and nitrogen), and temperature was of secondary importance. Although cyanobacterial biomass has declined in some managed lakes with reduced nutrient influx, the larger spatio‐temporal scale of sedimentary records show continued increases in cyanobacteria throughout the north temperate‐subarctic regions.