Volume: 5 Issue: 17 —BIO-PROTOCOL

Biochemistry

Immunolocalization of Proteins in Corals: the V-type H⁺-ATPase Proton Pump Authors: Katie L. Barott and Martin Tresguerres, date: 09/05/2015, view: 8506, Q&A: 0

Here we describe the immunolocalization of a membrane-bound proton pump, the V-type H⁺-ATPase (VHA), in tissues and isolated cells of scleractinian corals. Immunolocalization of coral proteins requires additional steps not required for various model organisms, such as decalcification of the coral skeleton for immunohistochemistry or ...

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Post-crystallization Improvement of RNA Crystals by Synergistic Ion Exchange and Dehydration Authors: Jinwei Zhang and Adrian R. Ferré-D’Amaré, date: 09/05/2015, view: 6623, Q&A: 0

Compared to the recent dramatic growth in the numbers of genome-wide and functional studies of complex non-coding RNAs, mechanistic and structural analyses have lagged behind. A major technical bottleneck in the structural determination of large RNAs and their complexes is preparation of diffracting crystals. Empirically, a vast majority of such ...

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Cancer Biology

[14C]-Tryptophan Metabolic Tracing in Liver Cancer Cells Authors: Krishna S. Tummala and Nabil Djouder, date: 09/05/2015, view: 7255, Q&A: 1

Nicotinamide adenine dinucleotide (NAD⁺) is a coenzyme for many NAD⁺-consuming proteins with diverse biological functions. Oscillations in NAD⁺ levels may influence several cellular signaling pathways. NAD⁺ synthesis via Preiss-Handler route (salvage reactions) has been extensively reported. However, the ...

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Immunology

Chitin-challenged Mice Model to Study M2 Macrophages Polarization Authors: Lidia Jiménez-García, Sandra Herránz, Alfonso Luque and Sonsoles Hortelano, date: 09/05/2015, view: 7288, Q&A: 1

Chitin is a key component of insects, fungi, and house-dust mites. Chitin has been shown to induce M2-type immune responses in vivo. Intranasal or intraperitoneal (i.p.) administration of chitin particles results in infiltration of eosinophils to the local sites and activation of macrophages with a M2 phenotype. Chitin-challenged mice ...

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Thioglycollate-elicited Peritoneal Macrophages Preparation and Arginase Activity Measurement in IL-4 Stimulated Macrophages Authors: Lidia Jiménez-García, Sandra Herránz, Alfonso Luque and Sonsoles Hortelano, date: 09/05/2015, view: 16990, Q&A: 0

Macrophages are an essential cell population of innate immunity that plays important roles in inflammatory processes. Two main different phenotypes have been described with opposing activities: The classically activated macrophages (M1) and the alternatively activated macrophages (M2). Alternative activation of mouse macrophages can be induced by ...

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Microbiology

Spot Assays for Viability Analysis of Cyanobacteria Authors: Anja Katharina Dörrich and Annegret Wilde, date: 09/05/2015, view: 8999, Q&A: 0

Cyanobacteria are prokaryotic organisms performing oxygenic photosynthesis. The cyanobacterium Synechocystis sp. PCC 6803 is a model organism for the study of photosynthesis, gene regulation and biotechnological applications because it is easy to manipulate genetically. Moreover, this cyanobacterium can grow photoautotrophically ...

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In vitro Real-time Measurement of the Intra-bacterial Redox Potential Authors: Joris van der Heijden and B. Brett Finlay, date: 09/05/2015, view: 7305, Q&A: 0

All bacteria that live in oxygenated environments have to deal with oxidative stress caused by some form of exogenous or endogenous reactive oxygen species (ROS) (Imlay, 2013). Large quantities of ROS damage DNA, lipids and proteins which can eventually lead to bacterial cell death (Imlay, 2013). In contrast, smaller quantities of ROS can play ...

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Determination of Hydroquinone Dioxygenase Activity Authors: Stefano Mancini and Marc Solioz, date: 09/05/2015, view: 6078, Q&A: 0

We recently demonstrated the presence of a quinone detoxification pathway present in Firmicutes. It is based on two enzyme activities, namely a hydroquinone dioxygenase, YaiA, described here, and a hydroquinone reductase, YaiB, described in a separate protocol. In Lactococcus lactis (L. lactis), these enzymes are encoded by the ...

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Determination of Quinone Reductase Activity Authors: Stefano Mancini and Marc Solioz, date: 09/05/2015, view: 6908, Q&A: 0

We recently demonstrated the presence of a quinone detoxification pathway present in Firmicutes. It is based on two enzyme activities, namely a quinone reductase, YaiB, described here, and a hydroquinone dioxygenase, YaiA, described in a separate protocol. In Lactococcus lactis (L. lactis), these enzymes are encoded by the ...

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Electron Paramagnetic Resonance (EPR) Spectroscopy to Detect Reactive Oxygen Species in Staphylococcus aureus Authors: Vinai Chittezham Thomas, Sujata S. Chaudhari, Jocelyn Jones, Matthew C. Zimmerman and Kenneth W. Bayles, date: 09/05/2015, view: 8037, Q&A: 0

Under aerobic conditions, Staphylococcus aureus (S. aureus) primarily metabolizes glucose to acetic acid. Although normally S. aureus is able to re-utilize acetate as a carbon source following glucose exhaustion, significantly high levels of acetate in the culture media may not only be growth inhibitory but also ...

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Plant Science

Expression and Partial Purification of His-tagged Proteins in a Plant System Author: Amancio de Souza, date: 09/05/2015, view: 10737, Q&A: 0

Plant protein expression can be a challenging enterprise in any biochemical or molecular biology research project. Several heterologous systems like bacteria, yeast, insect cells and cell free systems have been used to produce plant proteins for in vitro experiments and structural characterization. However, due to particularities of plant ...

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Cryo-focused Ion Beam Sample Preparation for Imaging Vitreous Cells by Cryo-electron Tomography Authors: Miroslava Schaffer, Benjamin D. Engel, Tim Laugks, Julia Mahamid, Jürgen M. Plitzko and Wolfgang Baumeister, date: 09/05/2015, view: 17590, Q&A: 0

Cryo-electron tomography (CET) is a well-established technique for imaging cellular and molecular structures at sub-nanometer resolution. As the method is limited to samples that are thinner than 500 nm, suitable sample preparation is required to attain CET data from larger cell volumes. Recently, cryo-focused ion beam (cryo-FIB) milling of ...

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Detection of Poly (A) RNA in Mesophyll Cells of Nicotiana benthamiana Using in situ Hybridization Authors: Yuri Mizuno and Daigo Takemoto, date: 09/05/2015, view: 6804, Q&A: 0

Export of transcribed mRNAs from nucleoplasm to cytosol is an essential process for the translation of genes into proteins. This process is tightly regulated by nuclear pores, composed of about 30 nucleoporin proteins (Nups). Whether or not the mRNAs are able to be appropriately exported to cytoplasm is of an importance for understanding the role ...

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A Non-Radioactive Method for Measuring PP2A Activity in Plants Authors: Jian Chen, Guoxin Shen and Hong Zhang, date: 09/05/2015, view: 7130, Q&A: 0

Protein phosphatase 2A (PP2A) is a group of important cellular regulators in eukaryotes that dephosphorylate more than 30% of cellular proteins whose activities are turned on or off by phosphorylation. In plants, PP2A was found to regulate critical components involved in plant growth and development, and in response to biotic and abiotic stresses. ...

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A Bioimaging Pipeline to Show Membrane Trafficking Regulators Localized to the Golgi Apparatus and Other Organelles in Plant Cells Authors: Satoshi Naramoto, Tomoko Dainobu and Marisa S. Otegui, date: 09/05/2015, view: 7714, Q&A: 0

The plant Golgi apparatus is composed of numerous stacks of cisterna, designated as cis, medial, and trans Golgi cisternae; these stacks move within the cytoplasm along the actin cytoskeleton. Cis cisternae receive secretory products from endoplasmic reticulum (ER) and they subsequently progress through the ...

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Past Issues in 2015

Volume: 5 Issue: 17

Biochemistry

Cancer Biology

Immunology

Microbiology

Plant Science