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Past Issue in 2015
Volume: 5, Issue: 16
Biochemistry
Sample Preparation of Telomerase Subunits for Crystallization
Telomerase is a large ribonucleoprotein complex that replicates the linear chromosome ends in most eukaryotes. Large-scale preparation of the telomerase core components in vitro has long been a big challenge in this field, hindering the understanding of the catalytic mechanism of telomerase, as well as slowing down the development of telomerase inhibitors for cancer therapy. We have successfully developed a protocol for large-scale preparation of the TRBD-CR4/5 complex of the medaka telomerase in vitro, and used this method to study the high-resolution structure of the TRBD-CR4/5 complex by X-ray crystallography. This procedure may be also adapted to purify other protein-RNA complexes for structural studies.
Cancer Biology
Determining Leukocyte Origins Using Parabiosis in the PyMT Breast Tumor Model
Tumors develop in a complex microenvironment alongside numerous cell types that impact their survival. Immune cells make up a large proportion of these accessory cells and many are known to promote tumor progression. Macrophages, in particular, are associated with poor patient prognosis and are therefore potential candidates for therapeutic targeting in cancer. However, to develop successful strategies to target macrophages, it is important to clarify whether these cells are derived from blood-borne precursors or a tissue-resident population. Parabiosis, or the surgical connection of two mice resulting in a shared blood circulation, allows the distinction between these two cellular sources. Here, we describe the use of parabiosis to define cell ontogeny in a mouse model of breast cancer.
Adoptive Transfer of Myeloid-Derived Suppressor Cells and T Cells in a Prostate Cancer Model
The adoptive transfer of immune cells for cancer, chronic infection, and autoimmunity is an emerging field that has shown promise in recent trials. The transgenic adenocarcinoma mouse prostate (TRAMP) is a classical mouse model of prostate cancer (PCa) and TRAMP cell lines were derived from a TRAMP mouse tumor. TRAMP-C2 is tumorigenic when (subcutaneously) s.c. grafted into syngeneic C57BL/6 host mice (Foster et al., 1997). This protocol will describe the adoptive transfer of purified CD11b+Gr1+ double positive (DP) myeloid-derived suppressor cells (MDSC) and CD3+ T cells in the TRAMP-C2 prostate cancer mouse model in order to establish the intrinsic functionality of these immune cells and to determine their role in tumorigenesis in vivo (Yan et al., 2014).
Cell Biology
Labeling of the Intestinal Lumen of Caenorhabditis elegans by Texas Red-dextran Feeding
In this method, the intestinal lumen of Caenorhabditis elegans (C. elegans) is labeled with a fluorescent fluid-phase marker, Texas Red-dextran. Since dextran conjugates are membrane impermeable, animals fed with it show a red fluorescent signal in the lumen of the intestine. Texas Red-dextran in the lumen is not efficiently endocytosed by intestinal cells and is not effectively transported to the body cavity paracellularly. It is useful to determine whether round-shaped membrane structures are invaginations from the apical membrane or cytoplasmic vesicles. If the barrier function of the intestinal epithelium is impaired, Texas Red-dextran can leak from the intestinal lumen to the body cavity. Therefore, this method can be used to visualize apical membrane morphology in intestinal cells and to investigate the barrier properties of the intestinal epithelium.
Immunology
Separation of Intracellular Vesicles for Immunoassays
The endosome/lysosome systems play important roles in immune cell functions as signaling platforms. Immune cells utilize these endosome/lysosome for signal transduction or intercellular communication to elicit the proper immune responses, regulating the localization or the association of the signaling complexes. Here we introduce the procedures to separate the intracellular vesicles such as endosomes or lysosomes, which could be useful to identify the subcellular localization of the signaling complexes.
Adoptive Transfer of Memory B Cells
The adoptive transfer of antigen-specific B cells into mice that cannot recognize that specific antigen has two main advantages. The first is determining exactly when the B cells were transferred and exposed to antigen. The second is that all B cells that can bind that antigen are the ones that were transferred; no new antigen-specific B cells will emerge from the bone marrow. Thus all B cells that were exposed to the antigen and still alive after at least 4 weeks (8 weeks or more is ideal), are memory B cells. Splenic B cells from B1-8 mice were prepared with an EasySep Mouse B Cell Enrichment Kit according to the manufacturer’s protocol. Single-cell suspensions were transferred intravenously into tail veins of recipient mice. Approximately 1 million NP+ B cells were transferred per mouse. Approximately 12-24 h after transfer, mice were immunized intra-peritoneally with 50 µg of NP-CGG precipitated in alum.
Microbiology
Isolation of Rhizosphere Bacterial Communities from Soil
Rhizosphere bacterial communities have become a major focal point of research in recent years, especially regarding how they affect plants and vice versa (Philippot et al., 2013). Changes in microbial density and diversity within the rhizosphere occur in a spatial temporal manner. The soil zone closest to the plant roots has the most density and diversity of microbes (Clark, 1940). The lack of methods to consistently isolate rhizosphere samples in a spatially defined manner is a major bottleneck in rhizosphere microbiology. We hypothesized that microbes with increasing affinities to and distance from the plant root can be isolated using increasing strengths of physical disruption. Sonication is an excellent choice due to the ability to gently remove rhizosphere soil and bacterial biofilms without damaging plant roots (Doi T et al., 2007; Bulgarelli et al., 2012; Lundberg et al., 2012). In addition, simply increasing the time of sonication can increase the amount of physical force. We used such an approach to consistently isolate microbial communities with different affinities to the soybean roots (White et al., 2014). This article describes the use of successive sonication to isolate distal, middle, and proximal soil from the rhizosphere of soybean roots.
RNase H Polymerase-independent Cleavage Assay for Evaluation of RNase H Activity of Reverse Transcriptase Enzymes
The ribonuclease H (RNase H) polymerase-independent cleavage assay allows detection and quantification of RNase H activity of reverse transcriptase (RT) enzymes with a hybrid substrate formed by a fluorescein labeled RNA annealed with Dabcyl DNA (Figure 1). Here we describe a protocol that we have adapted for HIV-1 RT expressed from a p(His)6-tagged p66/p51 HIV-1HXB2 RT-prot plasmid and for RT of the prototype foamy virus (PFV RT). Figure 1. Scheme of the principle of the experiment. The RNA substrate (blue) labeled with the fluorophore fluorescein (F, yellow) is annealed with complementary DNA strand (green) labeled with a quencher molecule Dabcyl (D, red). Panel A. In the intact substrate the quencher is so close to the fluorophore that it can quench the fluorescence emitted after excitation. Panel B. After the RNA substrate is cut by the RNase H a few ribonucleotides oligo labeled with the fluorescein is free to escape from the quencher, and to release fluorescence after excitation.
Plant Science
Protocol to Treat Seedlings with Brassinazole and Measure Hypocotyl Length in Arabidopsis thaliana
The plant hormones brassinosteroids (BR) promote hypocotyl elongation of Arabidopsis thaliana (A. thaliana) seedlings both under light and dark (etiolated) conditions. A common assay to determine if a mutant or transgenic line is affected in BR biosynthesis or response is a sensitivity assay to brassinazole (BRZ), an inhibitor of P450 cytochromes specific to BR biosynthesis. Here we provide a protocol to compare BRZ sensitivity of different A. thaliana genotypes in terms of hypocotyl elongation (Bernardo-García et al., 2014).
In vitro Colorimetric Method to Measure Plant Glutamate Dehydrogenase Enzyme Activity
Glutamate dehydrogenase (GDH) is an NAD(H) dependent enzyme that catalyzes, in vitro, the reversible amination of glutamate. Here we describe how to determine spectrophotometrically GDH activity monitoring NADH evolution. This protocol is described here for Arabidopsis thaliana (A. thaliana) although it is also valid for other plant species. GDH protein is a hexamer composed, in the case of Arabidopsis, of a combination of GDHα, GDHβ and GDHγ subunits. Every combination of subunits is possible; however, it is still barely known whether different combinations affect the enzymatic properties of the hexamers. In other species, hexamers are a combination of GDHα and GDHβ but it cannot be discarded the existence of other genes since for instance GDHγ subunit in Arabidopsis was described in Fontaine et al. (2012).Glutamate + NAD+ + H+ → 2-Oxoglutarate + NADH + NH4+
High-throughput Quantification of Ammonium Content in Arabidopsis
This protocol is a simple colorimetric assay for internal ammonium quantification in aqueous extracts from plant tissues. The method is based on the phenol hypochlorite assay (Berthelot reaction): NH4+ + hypochlorite + OH- + phenol → indophenol The oxidation of indophenol caused by phenol oxidation is a blue dye that is quantified at 635 nm in a spectrophotometer. Per ammonium molecule one molecule of indophenol is formed. The protocol described here is for Arabidopsis thaliana (A. thaliana) leaves and roots, although it is also valid for other plants species.
Quantification of Sodium Accumulation in Arabidopsis thaliana Using Inductively Coupled Plasma Optical Emission Spectrometery (ICP-OES)
Salt stress is a major issue for plants growing in both natural and agricultural settings (Deinlein et al., 2014). For example, irrigation can lead to the build up of salts in the soil as the irrigation water evaporates, leading to salinization, inhibition of plant growth, reduced productivity and eventually to loss of agriculturally usable land. One key element in trying to understand how salt stress impacts plant growth and development, in defining plant salt sensing and response mechanisms and eventually in the breeding or engineering of plants resistant to this stress is monitoring their salt uptake and redistribution. Methods such as imaging Na-sensitive fluorescent probes (Kader and Lindberg, 2005) and use of Na-ion selective microelectrodes (Shabala et al., 2005) offer the potential to follow Na levels in the plant in a non-destructive manner but are technically demanding and not applicable to field, or even many laboratory, conditions. However, tissue sampling followed by inductively coupled plasma spectroscopy (ICP) represents a simple, quantitative assay to monitor total Na levels in plant samples. ICP analysis is also applicable to plants in any environment where samples can be harvested. The approach uses tissue digestion in acid solutions, followed by injection of the resulting sample into an inductively coupled plasma spectrometer and monitoring the characteristic emitted spectrum from Na. As Na is stable, no complex sample preservation is required. Care needs to be taken with possible Na contamination in standards and samples from the water used for sample preparation and from glassware but otherwise, the approach is simple and robust.
Glucosinolates Determination in Tissues of Horseradish Plant
Glucosinolates (GLS) are secondary metabolites mainly found in plants belonging to the Brassicaceae family, including also horseradish (Armoracia rusticana G. Gaertn., B. Mey. & Scherb), a popular spice with a characteristic pungent flavor due to the abundance of GLS. Such compounds exhibit antibacterial, antifungal, and insecticidal activities, as well as human health properties. Therefore, it is very important to have a full understanding of their levels and profiles in plants. However, the characterization of GLS from horseradish crude extracts is a tough task, due to the complexity of the vegetal matrix and the occurrence of many GLS in trace amounts. Here we describe two alternative effective and rapid methods for GLS characterization in horseradish plants: Liquid chromatography coupled to high resolution mass spectrometry (LC-MS) for determination of intact GLS and HPLC-UV for determination of desulfo-GLS.
Stem Cell
FACS-based Satellite Cell Isolation From Mouse Hind Limb Muscles
Fluorescence Activated Cell Sorting (FACS) is a sensitive and accurate method for purifying satellite cells, or muscle stem cells, from adult mouse skeletal muscle (Liu et al., 2013; Sacco et al., 2008; Tierney et al., 2014). Mechanical and enzymatic digestion of hind limb muscles releases mononuclear muscle cells into suspension. This protocol employs fractionation strategies to deplete cells expressing the cell surface markers CD45, CD31, CD11b and Ly-6A/E-Sca1, both by magnetic separation and FACS-based exclusion, and positively select for cells expressing a7-integrin and CD34. This enables the researcher to successfully enrich satellite cells that uniformly express the paired-box transcription factor Pax7 and are capable of long-term self-renewal, skeletal muscle repair and muscle stem cell pool repopulation.
Systems Biology
Character-State Reconstruction to Infer Ancestral Protein-Protein Interaction Patterns
Protein-protein interactions are at the core of a plethora of developmental, physiological and biochemical processes. Consequently, insights into the origin and evolutionary dynamics of protein-protein interactions may provide information on the constraints and dynamics of specific biomolecular circuits and their impact on the organismal phenotype. This protocol describes how ancestral protein-protein interaction patterns can be inferred using a set of known protein interactions from phylogenetically informative species. Although this protocol focuses on protein-protein interaction data, character-state reconstructions can in general be performed with other kinds of binary data in the same way.