往期刊物2016
卷册: 6, 期号: 16
生物化学
Quantification of 2-Hydroxyglutarate Enantiomers by Liquid Chromatography-mass Spectrometry
液相色谱质谱法量化2-羟戊二酸对映体
Two enantiomers of 2-hydroxyglutarate (2HG), L (L2HG) and D (D2HG), are metabolites of unknown function in mammalian cells that were initially associated with separate and rare inborn errors of metabolism resulting in increased urinary excretion of 2HG linked to neurological deficits in children (Chalmers et al., 1980; Duran et al., 1980; Kranendijk et al., 2012). More recently, investigators have shown that D2HG is produced by mutant isocitrate dehydrogenase enzymes associated with a variety of human malignancies, such as acute myeloid leukemia, glioblastoma multiforme, and cholangiocarcinoma (Cairns and Mak, 2013; Dang et al., 2009; Ward et al., 2010). By contrast, we and others have shown that L2HG accumulates in response to cellular reductive stressors like hypoxia, activation of hypoxia inducible factors, and mitochondrial electron transport chain defects (Oldham et al., 2015; Reinecke et al., 2011; Intlekofer et al., 2015; Mullen et al., 2015). Each enantiomer is produced and metabolized in independent biochemical pathways in reactions catalyzed by separate enzymes and utilizing different cofactors with presumably different consequences for cellular metabolism (Kranendijk et al., 2012). Therefore, as research into the roles of D2HG and L2HG in human metabolism continues, it becomes increasingly important for investigators to consider each enantiomer independently (Struys, 2013). Several methods for quantification of biochemically relevant enantiomers in general have been developed and typically include enzymatic assays using enzymes specific for one enantiomeric species or the other, the use of chiral chromatography medium to facilitate chromatographic separation of enantiomers prior to spectroscopy, or the use of chiral derivatization reagents to convert a mixture of enantiomers to diastereomers with differing physical and chemical properties facilitating their chromatographic separation. In this protocol, we report the adaptation of a previously published derivatization method using diacetyl-L-tartaric anhydride (DATAN) for the quantification of 2HG enantiomers (Figure 1) (Oldham et al., 2015; Struys et al., 2004).Figure 1. Reaction scheme for the derivatization protocol
Determination of the H+-ATP Synthase and Hydrolytic Activities
测定 H+-ATP 合成酶的合成和水解活性
The H+-ATP synthase of the inner mitochondrial membrane utilizes the proton gradient generated by the respiratory chain to synthesize ATP. Under depolarizing conditions, it can function in reverse by hydrolyzing ATP to generate a proton gradient. The protocols presented here allow the facile determination of both the synthetic and hydrolytic activities of the H+-ATP synthase in isolated mitochondria and in permeabilized mammalian cells. Since the protocol requires the isolation of polarized and well-coupled mitochondria, first we describe the protocol for mitochondrial isolation from mouse tissues. Second, we describe the protocol for measuring the ATP synthetic activity as end-point and kinetic modes in isolated mitochondria and in permeabilized cells. Finally, we describe the protocol for the determination of the ATP hydrolytic activity of the enzyme in isolated mitochondria.
Determination of VPS34/PIK3C3 Activity in vitro Utilising 32P-γATP
采用 32P-γATP体外测定VPS34/PIK3C3的活性
VPS34 is the only class III phosphatidylinositol-3-kinase (PI3K) in mammalian cells and produces the vast majority of cellular phosphatidylinositol-3-phosphate [PI(3)P]. PI(3)P is a key signalling lipid that plays many membrane trafficking roles in processes such as endocytosis and autophagy. VPS34 is a key cellular regulator, loss of function can have catastrophic effects and is embryonic lethal (Zhou et al., 2011). The levels of cellular PI(3)P can be determined by fluorescent staining techniques and can be used to monitor effects upon VPS34 activity, however it is important to verify that any changes are mediated by VPS34, particularly as alternate pathways of PI(3)P production are possible such as via class II PI3Ks (Devereaux et al., 2013). Assaying VPS34 activity directly in vitro can be a key stage in delineating the action of a particular stimulus.
Purification of Flagellin from Acidovorax avenae and Analysis of Plant Immune Responses Induced by the Purified Flagellin
燕麦食酸菌鞭毛蛋白的纯化及其诱导植物免疫反应的分析
Plants sense potential pathogens by recognizing conserved pathogen-associated molecular patterns (PAMPs) that cause PAMP-triggered immunity (PTI) including the generation of reactive oxygen species, callose deposition, and expression of several PTI-related genes. Acidovorax avenae is a Gam-negative bacterium that causes a seedling disease characterized by the deposition of brown stripes on the sheaths of infected plants. We previously reported that flagellin isolated from the rice avirulent A. avenae N1141 strain induces PTI, while flagellin isolated from the rice virulent A. avenae K1 strain does not induce PTI. To examine the molecular mechanism of specific PTI induction by N1141 flagellin, highly purified flagellin from N1141 or K1 strains is required. Here, we describe a high quality purification method for the A. avenae flagellins and for using it in PTI induction study.
细胞生物学
Determination of Cellular Phosphatidylinositol-3-phosphate (PI3P) Levels Using a Fluorescently Labelled Selective PI3P Binding Domain (PX)
采用荧光标记选择性PI3P结合域(PX)测定细胞PI3P含量
The lipid Phosphatidylinositol-3-phosphate [PtdIns3P or PI(3)P] plays many membrane trafficking roles and is primarily produced by the Class III PI3K, VPS34. Determining the level of cellular PI(3)P however can be complex. Extraction of cellular lipids by methanol/chloroform can struggle to separate and identify distinct phospholipid species. Alternately mass spectrometry may be utilised but this requires significant set up of specialised equipment and time to utilise.Use of a PI(3)P-binding-specific recombinant protein domain is a quick method for ascertaining cellular PI(3)P levels and can also allow visualisation of sub-cellular localisation. The PX domain of p40phox (herein referred to as PX) is very specific for PI(3)P over other phospholipid species (Kanai et al., 2001). However, expressing PX directly in cells can be problematic, as it will act in a dominant negative manner to bind and sequester PI(3)P with greater affinity than endogenous proteins, thus disturbing cellular pathways and the normal balance of PI(3)P levels. Using fluorescently labelled PX following cell fixation is therefore more suitable, as it is able to highlight PI(3)P rich structures without risk of perturbing the system.
免疫学
Chromium-51 (51Cr) Release Assay to Assess Human T Cells for Functional Avidity and Tumor Cell Recognition
铬-51 (51Cr) 释放试验评估T淋巴细胞活性及对肿瘤细胞识别能力
Cytotoxic CD8+ T cells are able to specifically recognize and kill target cells through specific interaction between their T cell receptors (TCRs) and small immunogenic peptides (antigens) presented by major histocompatibility complex (MHC) molecules. The antigen recognition capacity and in vitro lytic activity of antigen-specific cytotoxic T cells can be assessed functionally in the so-called chromium 51 (51Cr) release assay, which was developed almost 50 years ago in our institution (Brunner et al., 1968). Radioactively-labelled cells deficient for endogenous antigen presentation [e.g., transporter for antigen presentation (TAP)-deficient T2 cells] and stably transfected with the MHC of interest (e.g., HLA-A2+) are typically used as targets during this 4h assay. Alternatively, 51Cr-labelled virus-infected or tumor cell lines presenting immunogenic antigens endogenously can serve as target cells (e.g., for the assessment of tumor recognition).In a peptide titration assay (section A), radioactively labelled target cells are pulsed with a serial dilution of the antigenic peptide and incubated at an effector (e.g., a CD8+ T cell clone) to target (51Cr -T2 cells) ratio (E:T) of 10:1 in a 96-well V-bottom plate for 4 h at 37 °C. In a tumor killing assay (section B), cytotoxic CD8+ effector cells are incubated at different ratios with the 51Cr-labelled target cell line (typically at E:T ratios of 30:1, 10:1, 3:1 and 1:1) in the presence or absence of the specific antigenic peptide (1 μM) and incubated for 4 h at 37 °C. At the end of the test, the amount of radioactivity release from the lysed target cells is determined in the supernatant using a liquid scintillation counter. The percentage of specific lysis, as well as the EC50 (i.e., 50% of maximal killing) and EMax values are then calculated, providing quantitative information about the antigen-specific functional avidity (i.e., the relative efficiency of T cell function based on antigen recognition via a defined TCR and maximal killing capacity of the analyzed T cells).
Macrophage Polarization by Tumor-induced MDSCs Assay
利用肿瘤诱导的骨髓源抑制细胞（MDSCs）进行的巨噬细胞极化实验
Myeloid derived suppressor cells (MDSCs) are a subset of granulocytes (immature myeloid cells) that exploit a variety of mechanism to modulate the innate and adaptive immune system. MDSCs are present normally in the body, but their numbers increase during inflammation and in cancer, promoting an immunosuppressive microenvironment. In addition to MDSCs, macrophages also play an important role during cancer development. There are two subsets of tumor associated macrophages (TAMs): M1 and M2. M1 are “anti-tumor” macrophages that are activated by interferon gamma (IFN-γ) and/or Lipopolysaccharide (LPS) and secrete high amount of interleukin 12 (IL-12) thereby inducing a Th1 anti-tumor immune response. M2 or “pro-tumorigenic” macrophages are activated by interleukin 4 (IL-4) and interleukin 10 (IL-10) and secrete large amounts of IL-10, which promotes tumor progression (Gabrilovich et al., 2012).Interaction between MDSCs and macrophages in the tumor microenvironment was shown to enhance immune suppression mediated by these subsets. MDSCs influence TAMs by producing IL-10 that, in turn, induces a down-regulation of IL-12 and polarizes M1 into M2 macrophages. In our study, we use the following protocol to evaluate the ability of tumor induced MDSCs to polarize LPS activated M1 into M2 macrophages (Vences-Catalan et al., 2015). This protocol was adapted from a previous study (Sinha et al., 2007).
Adoptive Transfer of Tumor Expanded Regulatory T Cells (Tregs)
肿瘤扩增调节性T细胞的过继转移
Regulatory T cells (Tregs), a subset of CD4+CD25+ T cells, infiltrate tumors and suppress antitumor activity of effector T and NK cells. Depletion of Tregs by anti CD25+ antibodies has been shown to reduce tumor growth and metastasis (Olkhanud et al., 2009). Conversely, adoptive transfer of Tregs induced immune suppression and promoted tumor growth (Smyth et al., 2006; Janakiram et al., 2015). We have adoptively transferred Tregs to evaluate their immunosuppressive function in vivo. Our study (Vences-Catalan et al., 2015) compared the immunosuppressive efficacy of Tregs derived from tumor-bearing wild type to those of CD81KO mice. The following protocol could be adapted to any other source of Tregs.Lymph node or splenic tumor-induced Tregs are isolated and purified by a two-step procedure using CD4+CD25+ regulatory T cell isolation kit from MACS Miltenyi Biotec. First, CD4+ T cells are enriched by negative selection, followed by positive selection of CD25+ T cells. Tumor-induced purified Tregs (CD3+CD4+CD25+FoxP3+) are then co-injected subcutaneously together with tumor cells into naïve mice (Winn assay) (Winn, 1960). Tregs could also be injected intravenously once or several times, according to the research needs. The effect of the adoptively transferred Tregs on tumor growth is then measured by caliper or by in vivo imaging techniques.
微生物学
A Modified Chromogenic Assay for Determination of the Ratio of Free Intracellular NAD+/NADH in Streptococcus mutans
改进型显色试验测定变异链球菌中的游离胞内NAD+/NADH比率
Nicotinamide adenine dinucleotide is a coenzyme present in all kingdoms of life and exists in two forms: oxidized (NAD+) and reduced (NADH). NAD(H) is involved in a multitude of essential metabolic redox reactions, providing oxidizing or reducing equivalents. The ratio of free intracellular NAD+/NADH is fundamentally important in the maintenance of cellular redox homeostasis (Ying, 2008). Various chromogenic cycling assays have been used to determine the ratio of NAD+/NADH in both bacterial and mammalian cells for more than forty years (Bernofsky and Swan, 1973; Nisselbaum and Green, 1969).Here, we describe in detail an assay to determine the ratio of free intracellular NAD+ to NADH in Streptococcus mutans. This cycling assay is a modified version of the protocol first described by Bernofsky and Swan (Bernofsky and Swan, 1973), using the extraction buffer described by Frezza et al. (2011), followed by the reduced MTT precipitation described by Gibbon and Larher (Gibon and Larher, 1997). As depicted in Figure 1, alcohol dehydrogenase is used to drive a series of redox reactions utilizing exogenously added ethanol and NAD+ from sample extracts as initial substrates, phenazine ethosulfate (PES) as an electron carrier, and thiazolyl blue tetrazolium bromide (MTT) as a terminal electron acceptor. 6 M NaCl is used to stop the reaction. The reduced MTT (formazan dye) is purple in color and can be quantified by measuring absorbance at 570 nm. This protocol is divided into three steps: A. Preparation of cell pellets of S. mutans; B. Preparation of deproteinated cell extracts containing NADtotal or NADH; C. NAD+/NADH cycling assay. This method has proven robust in measuring the NAD+/NADH ratio in S. mutans under a variety of conditions, and should be applicable to other Gram-positive bacteria.Figure 1. Flowchart illustrating protocol Procedure parts B-C
神经科学
A Controlled Cortical Impact Mouse Model for Mild Traumatic Brain Injury
轻度脑外伤模型建立：小鼠脑皮层可控性撞击模型
Traumatic brain injury (TBI) affects millions of people worldwide; however, the immediate impact of TBI and the secondary injury mechanisms are still not fully understood. TBI can cause devastating neuromotor deficits in both acute and chronic stages. Time course studies utilizing animal models of focal TBI have provided essential insight into TBI neuropathology. Here, we describe a surgical technique for creating a mouse model of focal, mild TBI (Dixon et al., 1991; Smith et al., 1995; Bolkvadze and Pitkanen, 2012). Furthermore, we provide protocols for validating TBI models using behavioral tests that examine post-traumatic neuromotor deficits resulting from TBI neuropathology (Fujimoto et al., 2004; Febinger et al., 2015; Smith et al., 1995; Bolkvadze and Pitkanen, 2012).
Primary Explosive Blast-induced Traumatic Brain Injury Model in PC12 Cell Culture
PC12 细胞培养中建立的爆炸冲击诱导的初级脑损伤模型
While it is understood that structural damage occurs at the cellular level from the traumatic brain injury event, the effect on functional activity remains largely unknown. Simplified models such as in vitro models of primary explosive blast are critically needed to deconvolute mechanisms of cellular damage. This protocol details an in vitro indoor experimental system setup (Zander et al., 2015) using real military explosive charges to more accurately represent battlefield blast exposure, and probe the effects of primary explosive blast on dissociated neurons.
植物科学
15N-nitrate Uptake Activity and Root-to-shoot Transport Assay in Rice
水稻吸收和转运N15-硝酸盐的活力实验
15N is a nonradioactive heavy isotope of nitrogen, widely used for biochemical and physiological research in plants. For instance, 15N-KNO3 was used as the nitrogen source in plants in order to investigate nitrate uptake activity and transport from roots to shoots (Lin et al., 2008). Here, we describe a detailed pipeline used for labeling living rice (Oryza sativa) plants with 15N-KNO3 and determination of net nitrate uptake and transport activity, and this protocol was proved to be valid in Arabidopsis and rice (Lin et al., 2008; Hu et al., 2015).
Experimental Pipeline for SNP and SSR Discovery and Genotyping Analysis of Mango (Mangifera indica L.)
芒果SNP和SSR的发现和基因分型分析的实验方法
Establishing a reservoir of polymorphic markers is an important key for marker-assisted breeding. Many crops are still lack of such genomic infrastructure. Single nucleotide polymorphisms (SNPs) and simple sequence repeats (SSRs) are useful as markers because they are widespread over the genome and many technologies were developed for high throughput genotyping. We present here a pipeline for developing a reservoir of SNP and SSR markers for Mangifera indica L. as an example for fruit tree crops having no genomic information available. Our pipeline includes de novo assembly of reference transcriptome with MIRA and CAP3 based on reads produced by 454-GS FLX technology; Polymorphic loci discovery by alignment of Illumina resequencing to the transcriptome reference; Identifying a subset of loci that are polymorphic in the entire germplasm collection for downstream diversity analysis by genotyping with Fluidigm technology.
Determination of Recombinant Mannitol-1-phosphatase Activity from Ectocarpus sp.
水云属中重组甘露醇-1-磷酸酶活性的测定
Brown algae belong to a phylogenetic lineage distantly related to green plants and animals, and are found predominantly, but not exclusively, in the intertidal zone, a harsh and frequently changing environment. Because of their unique evolutionary history and of their habitat, brown algae feature several peculiarities in their metabolism. One of these is the mannitol cycle, which plays a central role in their physiology, as mannitol acts as carbon storage, osmoprotectant, and antioxidant. This polyol is derived directly from the photoassimilate fructose-6-phosphate via the action of a mannitol-1-phosphate dehydrogenase (M1PDH, EC 1.1.1.17) and a mannitol-1-phosphatase (M1Pase, EC 3.1.3.22). This protocol describes the biochemical characterization of a recombinant M1Pase of Ectocarpus sp. The M1Pase enzyme catalyzes the conversion of mannitol-1-phosphate to mannitol (Figure 1).Figure 1. Reaction catalyzed by a mannitol-1-phosphatase
干细胞
Bone Marrow-derived Endothelial Progenitor Cell Intercellular Adhesion Assay
骨髓源内皮祖细胞胞间粘附试验
Cell-cell adhesion ensures tight contacts between neighbouring cells, which is necessary for cell segregation, as well as for the morphological and functional differentiation of different tissues. Evidently there are cell-cell recognition systems that make cells of the same type preferentially adherent to one another. Homotypic cell adhesion is particularly important in mediating a range of physiological processes such as cell survival, migration and remodeling of vessels. Thus in the present study we selected two populations of endothelial progenitor cells which are from the same donor to investigate the possible effect of a small molecule compound Icariin on homotypic cell adhesion. Many angiogenic factors can destabilize the organization of intercellular junctions, causing endothelial barrier opening. In the present study, we observed that Icariin treatment reduced the level of VE-cadherin expression in EPCs indicating a decrease in cell-cell adhesion-a proof of the pro-angiogenic effect of Icariin. In summary, the observed loss of homotypic adhesion of EPCs may contribute to the enhanced angiogenic effect exerted by Icariin.