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Past Issue in 2013
Volume: 3, Issue: 12
Biochemistry
Paper Chromatography as Exemplified by Separation of Urocanic Acid and Deaminohistidine
Paper chromatography is an ancient technique to separate low molecular mass compounds based on their distribution between mobile phase (solvent) and stationary phase (cellulose and cellulose-bound water). Paper chromatography has been largely replaced by thin layer chromatography and high performance liquid chromatography as the latter methods have higher resolution capability. Nevertheless due to low cost and availability of great number of protocols for separation of various compounds, paper chromatography is still a powerful analytical tool. In the current protocol this technique is exemplified by separation of urocanic acid and deaminohistidine.
Cell Biology
Calcium Mobilization Assay to Measure the Activity of Gq-coupled Receptors
Calcium mobilization assay is a cell-based second messenger assay to measure the calcium flux associated with Gq-protein coupled receptor activation or inhibition. The method utilizes a calcium sensitive fluorescent dye that is taken up into the cytoplasm of most cells. In some cell lines in which organic-anion transporters are particularly active (e.g. CHO, HeLa), addition of probenecid, an inhibitor of anion transport, is required for retention of this dye in the cells. The dye binds the calcium released from intracellular store and its fluorescence intensity increases. The change in the fluorescence intensity is directly correlated to the amount of intracellular calcium that is released into cytoplasm in response to ligand activation of the receptor of interest. This protocol can be applied to most mammalian cell lines expressing both endogenous and transiently/stably transfected receptors. The method is sensitive enough to be used for low-expressing systems or high throughput screening of target of interest. Note: The method does not differentiate the Ca2+ mobilization induced by Gqα from the Ca2+ mobilization induced by Gβγ.
LC3B Labeling on Terrestrial Isopod Adipocytes
The LC3B protein plays a critical role in autophagy. Normally, this protein resides in the cytosol, but following cleavage and lipidation with phosphatidylethanolamine, LC3B associates with the phagophore. This localization can be used as a general marker for autophagic membranes. To visualize the LC3B, we used the LC3B Antibody Kit for Autophagy (Invitrogen). As this kit was designed to work with cell coming from cell culture it was not possible to perform it on compact tissues such as nerve cord or ovaries. We thus adapted LC3B labeling initial protocol to be able to label autophagic membranes of adipocytes from terrestrial isopods which form a loose tissue that can be more easily penetrated by antibodies. The following protocol permits a visualization of the autophagic vesicles directly in terrestrial isopod cells freshly sampled in animals.
Developmental Biology
Labeling of Precursor Granule Cells in the Cerebellum by ex vivo Electroporation
This protocol will be useful to introduce the genes of interest into the cerebellar granule cells at early stages of development. Since the granule cell precursors are localized in the external granule layer before migration, DNA plasmids can be specifically incorporated into the granule cells by injecting DNA solution into the cerebellar fissures followed by application of electric pulses. This technique can be performed prior to the preparation of either dissociated or organotypic culture, which can be used to study the molecular mechanisms of cell migration, axon elongation and synapstogenesis during development.
Microbiology
Macromolecular Biosynthesis Assay for Evaluation of Influence of an Antimicrobial on the Synthesis of Macromolecules
One of the most compelling approaches in the discovery of novel antimicrobials is screening of natural sources. In our publication we report on the activity of a compound 8-hydroxyserrulat-14-en-19-oic acid (EN4), a diterpene isolated from the Australian plant Eremophila neglecta. We evaluate its applicability for treatment of implant-associated infections. A comprehensive analysis of the mechanism of action of EN4 against staphylococci revealed its membranolytic properties and a general inhibition of macromolecular biosynthesis, which was confirmed in a macromolecular biosynthesis assay and suggested a multitarget activity. The method used to investigate an influence of EN4 on the synthesis of peptidoglycan, RNA, DNA and proteins is based on precipitation of macromolecules with trichloroacetic acid. These macromolecules are synthesised from respective [3H]-labelled precursors. The incorporated radioactivity with and without an antimicrobial is measured and it reflects the mode of action of the tested compound. Antibiotics with known mechanisms of action are used as controls.
EdU labeling of Trypanosome Cells and Their Kinetoplast DNA (kDNA)
Trypanosome mitochondrial genome, known as Kinetoplast DNA (kDNA), is a massive network of interlocked DNA rings. The studies of kDNA replication and architecture are of major significance since kDNA is a valid drug target. However, DNA in procyclic trypanosomes can not be labeled with tracer concentrations of 3[H]-thymidine, possibly because they lack a high-affinity transporter for thymidine. Therefore, BrdU, a thymidine analog, has been used at high concentrations to study kDNA replication. However, the detection of BrdU with anti-BrdU antibody requires harsh conditions such as the acid or heat treatment to seperate double DNA strand, which affects the ability for other antibodies to bind as well as the morphology and ability for dyes that require dsDNA to bind efficiently. Instead, EdU (5-Ethynyl-2′-deoxyuridine), a novel thymidine analog, can be used to study kDNA replication and cell proliferation with a simplified protocol. Detection of EdU is based on a click reaction, which is a copper (I) catalyzed reaction between an azide and an alkyne. This click reaction does not require DNA denaturation and it is multiplex compatible, such as other antibodies and dyes for cell cycle analyses. To visualize trypanosome replicating nuclear DNA and kDNA, EdU is added into the medium of cell culture and incubated for 0.5 h to 3 h and then detected by the following procedures.
IFN-α Inhibition Assay in vitro
During viral infections Interferon-α (IFN-α) is expressed by infected host cells. IFN-α binds to its receptor (IFNAR1/2), which leads to the activation of downstream signaling via JAK-STAT. This signaling cascade results in the expression of several hundred different genes, so called interferon-stimulated gene, which lead to an antiviral state of the infected and the neighboring cells.
Amino Acid Transport Assays in Resting Cells of Lactococcus lactis
Many bacteria are auxotrophic for at least a number of amino acids for which they lack the biosynthetic pathways. The organisms are still able to grow in media containing free amino acids as the sole source of amino acid when transport systems for the free amino acids are present in the cytoplasmic membrane. A range of transport systems for essential as well as non-essential amino acids has been described that use the proton motive force (by proton symport) or ATP hydrolysis as driving force to allow for the accumulation of the amino acid in the cells. The most widely used assay for uptake of amino acids (or any other substrate) is the rapid filtration assay using radiolabelled substrates. Here we describe the assay for uptake in resting cells of Lactococcus lactis (L. lactis) that are energized by glucose.
Measurement of IFN-α Subtype Concentrations (Virus-free, Cell-based Bioassay)
The induction of type I IFN is the immediate host response against viral infections. Type I IFNs belong to a multigene family including up to 14 different IFN-α subtypes and one IFN-β. They are highly conserved and bind the same receptor (IFNAR1/2) with varying affinities, although they differ in their biological activities.
Molecular Biology
Structural Based Strategy for Predicting Transcription Factor Binding Sites
Scanning through genomes for potential transcription factor binding sites (TFBSs) is becoming increasingly important in this post-genomic era. The position weight matrix (PWM) is the standard representation of TFBSs utilized when scanning through sequences for potential binding sites. Many transcription factor (TF) motifs are short and highly degenerate, and methods utilizing PWMs to scan for sites are plagued by false positives. Furthermore, many important TFs do not have well-characterized PWMs, making identification of potential binding sites even more difficult. One approach to the identification of sites for these TFs has been to use the 3D structure of the TF to predict the DNA structure around the TF and then to generate a PWM from the predicted 3D complex structure. However, this approach is dependent on the similarity of the predicted structure to the native structure. We introduce here a novel approach to identify TFBSs utilizing structure information that can be applied to TFs without characterized PWMs, as long as a 3D complex structure (TF/DNA) exists. Our approach utilizes an energy function that is uniquely trained on each structure thus leads to increased prediction accuracy and robustness compared with those using a more general energy function. The software is freely available upon request. Please see reference supplementary material for details.
Plant Science
Hairy Root Transformation in Lotus japonicus
In L. japonicus, hairy root transformation is the very useful technique to generate transformed root systems in a short term. This protocol was previously described (Kumagai and Kouchi, 2003) with some modifications. After the infection of Agrobacterium rhizogenes, L. japonicus develops not only transformed but also untransformed roots. Thus, transgenic roots need to be identified by certain indications. In this protocol, we use the GFP florescent signals as such indication.
Stable Transformation in Lotus japonicus
This is a protocol to produce stable transgenic plants in Lotus japonicus, which is established based on methods previously reported (Handberg and Stougaard, 1992; Stiller et al., 1997; Thkjaer et al., 1998) with some modifications. In this protocol, hygromycin is used to select transgenic plants.
Growth Assay and Detection of TRP and Indole Derivatives in Piriformospora indica Culture Supernatant by LC-MS/MS
The mutualistic root endophyte Piriformospora indica colonizes a wide range of plants and the colonization of root cells by this fungus is very often associated with beneficial effects to its host, such as growth promotion and increased biotic and abiotic stress tolerance. These traits may be based on general mechanisms and signaling pathways common to many different plant species. One such mechanism could be the recruitment of phytohormone pathways by P. indica. It is known, that many mutualistic microorganisms are able to synthesize and secrete phytohormones during the interaction with their host plants. This protocol has been successfully utilized to analyze tryptophan (TRP)-dependent biosynthesis of indole-3-acetic acid (IAA) and its indole derivatives by P. indica as well as their influence on the growth of this fungus (Hilbert et al., 2012).
Indole Derivative Feeding Test and Detection of TRP and Indole derivatives by Thin Layer Chromatography
The mutualistic root endophyte Piriformospora indica colonizes a wide range of plants and the colonization of root cells by this fungus is very often associated with beneficial effects to its host, such as growth promotion and increased biotic and abiotic stress tolerance. These traits could be based on general mechanisms and signaling pathways common to many different plant species. One such mechanism could be the recruitment of phytohormone pathways by P. indica. It is known, that many mutualistic microorganisms are able to synthesize and secrete phytohormones during the interaction with their host plants. This protocol has been successfully utilized to analyze tryptophan (TRP)-dependent biosynthesis of indole-3-acetic acid (IAA) and its indole derivatives by P. indica (Hilbert et al., 2012).
Systems Biology
RNA-Seq Library Generation from Rare Human Cells Isolated by FACS
High throughput RNA Sequencing has revolutionized transcriptome analyses. However, most available protocols require micrograms of RNA rendering this technique not feasible for analyzing small numbers of cells, including precious rare cell types isolated from human tissues or organs. Here, we used an RNA Amplification System and describe a method for preparing RNA sense-strand cDNA libraries compatible with an Illumina sequencing platform starting from limited numbers of human fetal germ cells as well as human embryonic stem cells (hESCs) isolated using Fluorescence Activated Cell Sorting (FACS). With this protocol we generated seven RNA-Seq libraries starting from 4,000 germ cells sorted from fetal ovaries (n = 2) and fetal testes (n = 2) at 16-16.5 weeks of development and 4,000 sorted hESCs (n = 3). We predict that multiplexed libraries can also be generated by replacing the single-plex 3’ adapter used here with a multiplexing compatible 3’ adapter and indexed PCR primers.