Cerebral ischemic preconditioning reduces glutamate excitotoxicity by up-regulating the uptake activity of GLT-1 in rats

Our previous study has shown that cerebral ischemic preconditioning (CIP) can up-regulate the expression of glial glutamate transporter-1 (GLT-1) during the induction of brain ischemic tolerance in rats. The present study was undertaken to further explore the uptake activity of GLT-1 in the process by observing the changes in the concentration of extracellular glutamate with cerebral microdialysis and high-performance liquid chromatography. The results showed that a significant pulse of glutamate concentration reached the peak value of sevenfold of the basal level after lethal ischemic insult, which was associated with delayed neuronal death in the CA1 hippocampus. When the rats were pretreated 2 days before the lethal ischemic insult with CIP which protected the pyramidal neurons against delayed neuronal death, the peak value of glutamate concentration decreased to 3.9 fold of the basal level. Furthermore, pre-administration of dihydrokainate, an inhibitor of GLT-1, prevented the protective effect of CIP on ischemia-induced CA1 cell death. At the same time, compared with the CIP + Ischemia group, the peak value of glutamate concentration significantly increased and reached sixfold of the basal level. These results indicate that CIP induced brain ischemic tolerance via up-regulating GLT-1 uptake activity for glutamate and then decreasing the excitotoxicity of glutamate.     

Virus-induced gene silencing for comparative functional studies in Gladiolus hybridus

Key message

Virus-induced gene silencing (VIGS) system could be performed successfully in Gladiolus hybridus with vacuum infiltration of cormels and young plants.

Abstract

Functional analysis of genes in gladiolus has previously been impractical due to the lack of an efficient stable genetic transformation method. However, virus-induced gene silencing (VIGS) is effective in some plants which are difficult to transform through other methods. Although the Tobacco rattle virus (TRV)-based VIGS system has been developed and used for verifying gene functions in diverse plants, an appropriate TRV-VIGS approach for gladiolus has not been established yet. In this report we describe the first use of the TRV-VIGS system for gene silencing in gladiolus. Vacuum infiltration of cormels and young plants with the GhPDS-VIGS vector effectively down-regulated the PHYTOENE DESATURASE ortholog GhPDS gene and also resulted in various degrees of photobleaching in Gladiolus hybridus. The reduction in GhPDS expression was tested after TRV-based vector infection using real-time RT-PCR. In addition, the progress of TRV infection was detected by fluorescence visualization using a pTRV2: CP-GFP vector. In conclusion, the TRV-mediated VIGS described here will be an effective gene function analysis mechanism in gladiolus.     

人羊水祖细胞的分离和基因修饰

探讨从孕中期羊水中分离出人羊水祖细胞的有效方法和FIX基因修饰后的效果,为血友病B的产前治疗提供可行的基础。从镜下分离出呈致密克隆生长的梭形细胞集落,经培养传代后,通过第3代慢病毒载体将hFIX导入该细胞,经酶联免疫反应(ELISA)等方法检测hFIX的表达并检测凝血活性。用这种方法得到的羊水祖细胞呈成纤维细胞样,倍增时间为39.05 h,该细胞在不仅在蛋白水平表达干细胞表面分子SSEA4,TRA1-60,在基因水平还可检测到NANOG,OCT4,SOX2mRNA的表达。羊水祖细胞导入hFIX cDNA后,能合成并分泌hFIX蛋白,传代后48 h在上清液中的浓度为20.37%±2.77%,凝血活性16.42%±1.78%。上清液中的浓度在第4天达到平台期,为50.35%±5.42%,凝血活性可达45.34%±4.67%。ELISA检测显示转染后的羊水细胞表达的hFIX蛋白的水平呈现基本稳定趋势,波动幅度较小;同时检测FIX凝血活性也与蛋白浓度呈正相关。从羊水中可以分离得到具有多能性祖细胞,转染了hFIX的羊水祖细胞在体外能持续合成有凝血功能的hFIX蛋白,为血友病B产前治疗的新方法提供了实验依据。     

产芳香腈水解酶的恶臭假单胞菌Pseudomonas putida CGMCC3830的筛选、鉴定及发酵优化(英文)

近年来微生物腈水解酶水解腈类化合物制备有机酸已逐步受到关注。本研究分离到一株表现出较高腈水解酶活力的细菌菌株,通过形态学、生理生化实验以及16S rRNA基因序列分析将其鉴定为恶臭假单胞菌Pseudomonas putida CGMCC3830。结合单因素及响应面法对该菌株产腈水解酶的发酵条件进行了优化,获得最适培养条件为:甘油13.54 g/L,胰蛋白胨11.59 g/L,酵母粉5.21 g/L,KH2PO4 1 g/L,NaCl 1 g/L,脲1 g/L,初始pH 6.0及培养温度30℃。通过优化,酶活由2.02 U/mL提升至36.12 U/mL。对该菌株底物特异性的考察结果表明,恶臭假单胞菌腈水解酶对芳香族腈类化合物具有较高的水解活力。将其应用于烟酸的生物合成中,2 mg/mL游离细胞能90 min内将20.8 g/L 3-氰基吡啶彻底转化,制备得到相应烟酸。这些结果表明恶臭假单胞菌P.putida CGMCC3830在烟酸的规模化生产中具有一定的应用潜力。     

Engineered Fc based antibody domains and fragments as novel scaffolds

Therapeutic monoclonal antibodies (mAbs) have been successful for the therapy of a number of diseases mostly cancer and immune disorders. However, the vast majority of mAbs approved for clinical use are full size, typically in IgG1 format. These mAbs may exhibit relatively poor tissue penetration and restricted epitope access due to their large size. A promising solution to this fundamental limitation is the engineering of smaller scaffolds based on the IgG1 Fc region. These scaffolds can be used for the generation of libraries of mutants from which high-affinity binders can be selected. Comprised of the CH2 and CH3 domains, the Fc region is important not only for the antibody effector function but also for its long half-life. This review focuses on engineered Fc based antibody fragments and domains including native (dimeric) Fc and monomeric Fc as well as CH2 and monomeric CH3, and their use as novel scaffolds and binders. The Fc based binders are promising candidate therapeutics with optimized half-life, enhanced tissue penetration and access to sterically restricted binding sites resulting in an increased therapeutic efficacy. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.     

Chemical and Genetic Studies on Hybrid of Ligularia subspicata and Ligularia cyathiceps Collected in Yunnan Province of China

Two morphologically ambiguous Ligularia samples (samples A and B), and samples with morphology of Ligularia subspicata (sample C), Ligularia lamarum (sample D), or Ligularia cyathiceps (sample E), were collected at Tianchi Pond, Shangrila County, Yunnan Province, China. Analysis of the nucleotide sequence of the internal transcribed spacers (ITSs) in the nuclear ribosomal RNA gene cluster indicated that not only sample B but also sample D was a hybrid of L. cyathiceps and L. lamarum/L. subspicata. Although the morphology of sample A suggested that it was also a hybrid, the ITS sequence of sample A was that of L. cyathiceps. Twenty compounds were isolated from the five samples, and the structures of two new compounds 7 and 14 were determined. Furanoeremophilanes typical of L. lamarum/L. subspicata were detected not only in samples C and D, but also in samples A and B. These results indicate that the ability to produce root chemicals can spread through introgression.     

Protein extraction from plant tissues for 2DE and its application in proteomic analysis

Plant tissues contain large amounts of secondary compounds that significantly interfere with protein extraction and 2DE analysis. Thus, sample preparation is a crucial step prior to 2DE in plant proteomics. This tutorial highlights the guidelines that need to be followed to perform an adequate total protein extraction before 2DE in plant proteomics. We briefly describe the history, development, and feature of major sample preparation methods for the 2DE analysis of plant tissues, that is, trichloroacetic acid/acetone precipitation and phenol extraction. We introduce the interfering compounds in plant tissues and the general guidelines for tissue disruption, protein precipitation and resolubilization. We describe in details the advantages, limitations, and application of the trichloroacetic acid/acetone precipitation and phenol extraction methods to enable the readers to select the appropriate method for a specific species, tissue, or cell type. The current applications of the sample preparation methods in plant proteomics in the literature are analyzed. A comparative proteomic analysis between male and female plants of Pistacia chinensis is used as an example to represent the sample preparation methodology in 2DE‐based proteomics. Finally, the current limitations and future development of these sample preparation methods are discussed. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP17).