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Past Issue in 2013
Volume: 3, Issue: 13
Biochemistry
Analysis of the Incorporation of Carbon Atoms from Radioactive Lactate into Proteins
This method allows to analyze if the carbon atoms of lactate are embedded into proteins. Indeed, mammalian cells express the transporter of monocarboxylic acids (called MCT1) that allows the entry of lactate into the cell. To this end, cells are incubated for 24 h with the culture medium containing lactate uniformly labeled with carbon 14 and then, lactate inside the cell is evaluated by counting the radioactivity by a scintillator.
Cancer Biology
Genomic Signature of Homologous Recombination Deficiency in Breast and Ovarian Cancers
Homologous recombination deficiency, mainly resulted from BRCA1 or BRCA2 inactivation (so called BRCAness), is found in breast and ovarian cancers. Detection of actual inactivation of BRCA1/2 in a tumor is important for patients’ treatment and follow-up as it may help predicting response to DNA damaging agents and give indication Homologous recombination deficiency, mainly resulted from BRCA1 or BRCA2 inactivation (so called BRCAness), is found in breast and ovarian cancers. Detection of actual inactivation of BRCA1/2 in a tumor is important for pat for genetic testing. This protocol describes how to detect impairment of homologous recombination based on the tumor genomic profile measured by SNP-array. The proposed signature of BRCAness is related to the number of large-scale chromosomal breaks in a tumor genome calculated after filtering and smoothing small-scale alterations. The procedure strongly relies on good quality SNP-arrays preprocessed to absolute copy number and allelic content (allele-specific copy number) profiles. This genomic signature of homologous recombination deficiency was shown to be highly reliable in predicting BRCA1/2 inactivation in triple-negative breast carcinoma (97% accuracy; for more details, see Popova et al., 2012) and predictive of survival in ovarian carcinoma (unpublished data). Authors are grateful to Dominique Stoppa-Lyonnet, Anne Vincent-Salomon, Thierry Dubois, and Xavier Sastre-Garau for their contributions. (Patent was deposited: Reference number EP12305648.3, June 7, 2012)
Construction of Human Monocyte Derived Macrophages Armed with Oncolytic Viruses
Macrophages are involved in many key physiological processes and complex responses such as inflammatory, immunological, infectious and neoplastic diseases. The appearance and activation of macrophages are thought to be rapid events in the development of many pathological lesions, including malignant tumours, atherosclerotic plaques, and arthritic joints. This has prompted recent attempts to use macrophages as novel cellular vehicles for gene therapy, in which macrophages are genetically modified ex vivo and then reintroduced into the body with the hope that a proportion will then home to the diseased site. Here, we describe a protocol for preparing monocyte-derived macrophages (MDM) and arming these with oncolytic viruses (OV) as a novel way for delivering anti-cancer therapies. In this approach, proliferation of macrophages co-transduced with a hypoxia-regulated E1A/B construct and an E1A-dependent oncolytic adenovirus, is restricted to prostate tumour cells using prostate-specific promoter elements from the TARP, PSA, and PMSA genes (Muthana et al., 2013; Muthana et al., 2011). When such co-transduced cells reach an area of extreme hypoxia (like that found in tumours), the E1A/B proteins are expressed, thereby activating replication of the adenovirus. The virus is subsequently released by the host macrophage and infects neighboring tumour cells. The virus then infects neighboring cells but only proliferates and is cytotoxic in prostate tumour cells. OV kill cancer cells by a number of mechanisms, including direct lysis, apoptosis, autophagy and shutdown of protein synthesis, as well as the induction of anti-tumoural immunity. Using macrophages to deliver OV ensures that they are protected from the many hazards they face in circulation including neutralizing antibodies, complement activation and non-specific uptake by other tissues such as the liver and spleen.
Cell Biology
Transmission Electron Microscopy (TEM) Protocol: Observation Details within Cells
Transmission electron microscopy is a technique for observing the fine details of organelles in cells or tissues. This protocol is to be used to exam the membrane structure in cells with or without virus infection. Modifications should be made if users want to get images from tissues.
cAMP Accumulation Assays Using the AlphaScreen® Kit (PerkinElmer)
Cyclic adenosine monophosphate (cAMP) is an intracellular signaling messenger derived from the catalytic conversion of ATP, and is a major product of activated Gs protein-coupled receptors. Conversely, formation of cAMP is inhibited by Gi protein-coupled receptors. This protocol has been optimized for the detection of ligand-mediated cAMP accumulation in adherent immortal cell lines expressing Gs-coupled receptors.
Isolation, Culture and Differentiation of Primary Acinar Epithelial Explants from Adult Murine Pancreas
The adult pancreas possesses an intrinsic developmental plasticity whereby acinar cells can convert into ductal structures under some pathological conditions. Acinar tissue can be isolated from murine pancreas and kept in three-dimensional collagen culture. Acinar to ductal metaplasia can be induced in primary acinar epithelial explants by treatment with growth factors. This method can be utilized in ex vivo studies involving pancreatic epithelial differentiation.
MACS Isolation and Culture of Mouse Liver Mesothelial Cells
Mesothelial cells (MCs) form a single squamous epithelial cell layer and cover the surfaces of the internal organs, as well as the walls of cavities. The isolation of MCs is of great importance to study their function and characteristics for the understanding of physiology and pathophysiology of the liver. Glycoprotein M6a (GPM6A) was originally identified as a cell surface protein expressed in neurons and recently its expression was reported in epicardium and liver MCs (Wu et al., 2001; Bochmann et al., 2010; Li et al., 2012). Here we describe a method to isolate MCs from the adult mouse liver with anti-GPM6A antibodies. Under the low glucose and serum concentration, primary MCs grow and form epithelial colonies (Figure 1). Figure 1. Liver MCs 2 days in culture (20x objective)
pERK Detection Assays Using the Surefire AlphaScreen® Kit (TGR Biosciences and PerkinElmer)
Extracellular signal-regulated kinase 1 and 2 (ERK1/2) are serine/threonine protein kinases that are phosphorylated on Thr202/Tyr204 (ERK1) and Thr185/Tyr187 (ERK2). Phosphorylation of ERK1/2 (pERK1/2) arises from multiple stimuli, resulting in physiological responses that include cell growth, proliferation and differentiation. This protocol has been optimized for the detection of ligand-mediated pERK1/2 in adherent immortal cell lines expressing G protein-coupled receptors (GPCRs).
Detection of Released CO2 by Radioactive Lactate
This method allows to evaluate the degradation of lactate during cellular respiration. During this metabolic process, carbon atoms of lactate can be transformed in carbon dioxide. For this purpose, the radioactive lactate is added to the cells and the amount of radioactive carbon dioxide liberated is monitored. The radioactive carbon dioxide generated during cellular respiration is released into the culture medium and it is further converted into gas through the addition of sulfuric acid to culture media. A piece of Whatman paper wet with phenyl-ethylamine-methanol is placed inside the petri dish to trap radioactive carbon dioxide whose production is then evaluated by scintillator counting.
Microbiology
RNA-Affinity Chromatography
RNA-affinity chromatography assays are used to identify proteins binding specific RNA sequences. These proteins represent potential factors contributing to the function of RNA molecules. In our lab, we have used this protocol to identify proteins binding sequence motifs involved in replication and transcription of positive strand RNA viruses. The assay described in this protocol consists on the immobilization of 5’-biotinylated RNA oligonucleotides (30-40 nt) on a streptavidin-conjugated, paramagnetic solid matrix. Then, cytoplasmic protein extracts pre-cleared on the solid matrix to decrease nonspecific binding, were incubated with the immobilized RNA molecules in the presence of a nonspecific competitor. RNA-protein complexes immobilized on the paramagnetic solid matrix were isolated using a magnet and the bound proteins were separated by polyacrylamide gel electrophoresis for proteomic analysis.
Bacterial Conjugation in Rhodobacter capsulatus
Bacterial conjugation of plasmids is the common method of introducing foreign DNA into Rhodobacter capsulatus because transformational systems have not been shown as efficient methods of introducing DNA to R. capsulatus. For R. capsulatus bacterial conjugation using an Escherichia coli donor can be used to introduce replicating vectors, and non-replicating vectors for targeted chromosomal modifications.
Preparation of Pneumococcal Proteins for Western Blot Analysis
This protocol was developed in a study aimed to determine the cellular localization of the lysin of pneumococcal phage SV1 (Frias et al., 2013). We obtained proteins from the surface of Streptococcus pneumoniae by elution with choline or those secreted to the medium. The analysis by Western blot of these fractions allowed us to demonstrate that the phage lysin localizes to the cell wall, associating with choline residues in the teichoic acids. Hence, protein extracts can be used to determine the localization of uncharacterized proteins and can also be useful for other biochemical analyses such as protein identification. This protocol can be easily adapted to different pneumococcal strains and growth conditions and it is well suited to isolate other proteins of interest.
Plant Science
Cellular Extract Preparation for Superoxide Dismutase (SOD) Activity Assay
Superoxide dismutase (SOD) acts as a primary defence against reactive oxygen species (ROS) by converting O2- to O2 and H2O2. Members of this enzyme family include CuZnSOD, MnSOD and FeSOD. Most eukaryotes harbor CuZnSOD and MnSOD, and FeSOD is found in plants and prokaryotes. This protocol is to demonstrate how to prepare the cellular extract for the identification and characterization of SODs in planta.
Fluorescent Dye Based Measurement of Vacuolar pH and K+
Availability of ion specific fluorescent dyes has enabled the possibility to perform in vivo ion specific measurements using live cell imaging in many cellular compartments (Krebs et al., 2010; Bassil et al., 2011; Halperin and Lynch, 2003; Swanson et al., 2011; O'Connor and Silver, 2007). The importance of ion and pH homeostasis of intracellular compartments, including the vacuole, to cell growth is critical and well established (Krebs et al., 2010; Bassil et al., 2011).* Dedicated to Stephen Halperin who tragically and unexpectedly passed away.