Springer protocols feed by Biochemistry 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Chiral Phase-HPLC Separation of Hydroperoxyoctadecenoic Acids and Their Biosynthesis by Fatty Acid Dioxygenases
Fatty acid oxygenases are often characterized by steric analysis of their hydroxy or hydroperoxy metabolites. Chiral phase-HPLC (CP-HPLC) can be used to separate enantiomeric hydroperoxyoctadecenoic acids. This method is based on analysis of seven octadecenoic fatty acids with double bonds at positions 6Z to 13Z, which were oxidized to hydroperoxides by photooxidation. A stationary phase, Reprosil Chiral NR, was found to resolve these hydroperoxy fatty acids with 1-hydroperoxy-2-propene and with 3-hydroperoxy-1-propene elements so that the S hydroperoxy fatty acids consistently eluted before the R stereoisomers. The chiral...
Source: Springer protocols feed by Biochemistry - October 20, 2014 Category: Biochemistry Source Type: news
Quantification of DNA Repair Capacity Towards Oxidatively Damaged DNA in Subcellular and Cellular Systems by a Nonradioactive Cleavage Assay
The identification of appropriate biomarkers for oxidative stress is one major aim in molecular epidemiology. Besides the quantification of specific DNA lesions such as of 8-oxoguanine (8oxoG), another approach consists in the assessment of the repair capacity towards 8oxoG, mediated predominantly by the human 8-oxoguanine DNA glycosylase 1 (hOGG1); further processing of base excision repair involves AP endonuclease 1 (APE1). Thus, during the last few years the so-called cleavage assays have been described, investigating the incision capacity of cell extracts towards 32P-labelled and 8oxoG damaged oligonucleotides. Here, w...
Source: Springer protocols feed by Biochemistry - October 20, 2014 Category: Biochemistry Source Type: news
Nitric Oxide Availability as a Marker of Oxidative Stress
Nitric oxide (NO) is widely considered one of the most important molecules produced in the human body, acting as a necessary regulator in a vast array of vital physiological functions, namely, blood pressure, immune response, and neural communication. Healthy endothelium is defined by the ability to produce adequate levels of NO. Reactive oxygen species (ROS) play a major role in NO-based cell signaling. ROS can affect NO availability both from production to post-production scavenging and lead to a myriad of vascular disorders due to compromised NO functionality. In 2004, it was identified in animal models that oxidative s...
Source: Springer protocols feed by Biochemistry - October 20, 2014 Category: Biochemistry Source Type: news
A Novel Gas Chromatographic Method for Determination of Malondialdehyde from Oxidized DNA
Malondialdehyde (MA) is known to form from various lipids upon oxidation as one of secondary oxidation products. Determination of MA formed from lipid peroxidation has been used to examine occurrence of oxidative damages associated with many diseases, such as cancer, Alzheimer’s, arthritis, inflammation, diabetes, atherosclerosis, and AIDS as well as aging. Analysis of MA is, however, extremely difficult because it is highly reactive and readily polymerized and forming adducts with biological substances such as proteins, phospholipids, and DNA (Shibamoto, J Pharm Biomed Anal 41:12–25, 2002). Gas chromatographic...
Source: Springer protocols feed by Biochemistry - October 20, 2014 Category: Biochemistry Source Type: news
Analysis of Relationship Between Oxidized Phospholipid Structure and Interaction with the Class B Scavenger Receptors
We describe here the design and synthesis of a series of model oxidized phospholipids having various functional groups at sn-1, sn-2, and sn-3 positions. Synthetic methodologies and experimental details for the preparation of specific examples of model oxidized phospholipids are presented. The correlation between their structure and their ability to serve as ligands for CD36 and SR-BI was determined using competitive binding assay on cells overexpressing scavenger receptors, direct binding assay to scavenger receptors expressed as GST-fusion proteins, and cholesterol ester synthesis assay using mouse peritoneal macrophages...
Source: Springer protocols feed by Biochemistry - October 20, 2014 Category: Biochemistry Source Type: news
Specific Imaging and Tracking of Mitochondria in Live Cells by a Photostable AIE Luminogen
Tracking the dynamics of mitochondrial morphology has attracted much research interest because of its involvement in early stage apoptosis and degenerative conditions. To follow this process, highly specific and photostable fluorescent probes are in demand. Commercially available mitochondria trackers, however, suffer from poor photostability. To overcome this limitation, we have designed and synthesized a fluorescent agent, tetraphenylethene-triphenylphosphonium (TPE-TPP), for mitochondrial imaging. Inherent from the mitochondrial-targeting ability of TPP groups and the aggregation-induced emission (AIE) characteristics o...
Source: Springer protocols feed by Biochemistry - October 20, 2014 Category: Biochemistry Source Type: news
Derivatization and Detection of Small Aliphatic and Lipid-Bound Carbonylated Lipid Peroxidation Products by ESI-MS
Double bonds in polyunsaturated fatty acids (PUFA) and lipids are one of the major targets of reactive oxygen species (ROS). The resulting lipid peroxidation products (LPP) represent a group of chemically diverse compounds formed by several consecutive oxidative reactions. Oxidative cleavage leads to the formation of small aliphatic and lipid-bound aldehydes and ketones (oxoLPPs). These strong electrophiles can readily react with nucleophilic substrates, for example, side chains in proteins which can alter structure, function, and cellular distribution of the modified proteins. Despite growing interest in the field of oxid...
Source: Springer protocols feed by Biochemistry - October 20, 2014 Category: Biochemistry Source Type: news
Using Nanosensors for In Situ Monitoring and Measurement of Nitric Oxide and Peroxynitrite in a Single Cell
The cytotoxic peroxynitrite (ONOO−) is an oxidation product of the cytoprotective nitric oxide (NO). Our studies support the hypothesis that the concentration ratio of NO and ONOO−, [NO]/[ONOO−] can be a marker of nitroxidative imbalance, which subsequently correlates well with endothelial dysfunction and dysfunction of the cardiovascular system. Nanosensors, described here, have been used for simultaneous monitoring and measurement of NO and ONOO− release from a single endothelial cell. These nanosensors, with a diameter of 200–300 nm, can be positioned accurately in close proximity of 5&ndas...
Source: Springer protocols feed by Biochemistry - October 20, 2014 Category: Biochemistry Source Type: news
Electrochemical Detection of Glutathione S-Transferase: An Important Enzyme in the Cell Protective Mechanism Against Oxidative Stress
Oxidative stress arises when the antioxidant capacity of cells to clean the excess production of reactive oxygen species (ROS) decreases. Several human diseases seem to be related with an increment in the oxidative stress. In this regard, GSH present in the cells works by neutralizing ROS and other xenobiotics through the glutathione S-transferase (GST) enzyme. Thus, the level of expression of GST is an important factor in determining the sensitivity of cells to toxic chemicals or xenobiotic compounds. Therefore, the detection of GST levels is fundamental in the clinical diagnosis of ROS-related diseases. Here, we describe...
Source: Springer protocols feed by Biochemistry - October 20, 2014 Category: Biochemistry Source Type: news
Measuring Riboswitch Activity In Vitro and in Artificial Cells with Purified Transcription–Translation Machinery
We present a simple method to measure the real-time activity of riboswitches with purified components in vitro and inside of artificial cells. Typically, riboswitch activity is measured in vivo by exploiting β-galactosidase encoding constructs with a putative riboswitch sequence in the untranslated region. Additional in vitro characterization often makes use of in-line probing to explore conformational changes induced by ligand binding to the mRNA or analyses of transcript lengths in the presence and absence of ligand. However, riboswitches ultimately control protein levels and often times require accessory factors. T...
Source: Springer protocols feed by Biochemistry - March 28, 2014 Category: Biochemistry Source Type: news
Nucleotide Kinase-Based Selection System for Genetic Switches
Ever-increasing repertories of RNA-based switching devices are enabling synthetic biologists to construct compact, self-standing, and easy-to-integrate regulatory circuits. However, it is rather rare that the existing RNA-based expression controllers happen to have the exact specification needed for particular applications from the beginning. Evolutionary design of is powerful strategy for quickly tuning functions/specification of genetic switches. Presented here are the steps required for rapid and efficient enrichment of genetic switches with desired specification using recently developed nucleoside kinase-based dual sel...
Source: Springer protocols feed by Biochemistry - March 28, 2014 Category: Biochemistry Source Type: news
Rational Design of Artificial ON-Riboswitches
Riboswitches are composed of two regions: one for binding to the ligand (the aptamer domain) and the other for regulating the expression of the gene (the expression platform). In most riboswitches (both natural and artificial), a part of the aptamer domain required for ligand binding is directly involved in the regulation of expression, so that it is difficult to design other ligand-responsive riboswitches based on these riboswitches even by using artificial aptamers obtained through in vitro selection. This chapter describes a method for rationally constructing a foundational ON-riboswitch, which is easily available for t...
Source: Springer protocols feed by Biochemistry - March 28, 2014 Category: Biochemistry Source Type: news
Engineering Protein-Responsive mRNA Switch in Mammalian Cells
Engineering of translation provides an alternative regulatory layer for controlling transgene expression in addition to transcriptional regulation. Synthetic mRNA switches that modulate translation of a target gene of interest in response to an intracellular protein could be a key regulator to construct a genetic circuit. Insertion of a protein binding RNA sequence in the 5′ UTR of mRNA would allow for the generation of a protein-responsive RNA switch. Here we describe the design principle of the switch and methods for tuning and analyzing its translational activity in mammalian cells. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - March 28, 2014 Category: Biochemistry Source Type: news
Guanine-Tethered Antisense Oligonucleotides as Synthetic Riboregulators
Regulation of gene expression by short oligonucleotides (antisense oligonucleotides), which can modulate RNA structures and inhibit subsequent associations with the translation machinery, is a potential approach for gene therapy. This chapter describes an alternative antisense strategy using guanine-tethered antisense oligonucleotides (G-ASs) to introduce a DNA–RNA heteroquadruplex structure at a designated sequence on RNA targets. The feasibility of using G-ASs to modulate RNA conformation may allow control of RNA function by inducing biologically important quadruplex structures. This approach to manipulate quadrupl...
Source: Springer protocols feed by Biochemistry - March 28, 2014 Category: Biochemistry Source Type: news
In Vitro Selection of Allosteric Ribozymes that Sense the Bacterial Second Messenger c-di-GMP
Recently, a number of study have shown the ligand-dependent allosteric ribozymes can be harnessed as biosensors, high-throughput screening, and agents for the control of gene expression in vivo, called artificial riboswitches. In this chapter, we describe how in vitro selection can be used to create an allosteric ribozyme that senses bacterial second messenger cyclic-di-GMP (c-di-GMP). A hammerhead ribozyme was joined to a natural c-di-GMP class I riboswitch aptamer via communication modules. Both c-di-GMP-activating and -inhibiting ribozyme can be obtained by this approach. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - March 28, 2014 Category: Biochemistry Source Type: news
