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Oxidized Low-Density Lipoprotein

Oxidized low-density lipoprotein (Ox-LDL) has been studied for over 25 years. Numerous pro- and anti-atherogenic properties have been attributed to Ox-LDL. Yet, Ox-LDL has neither been defined nor characterized, as its components and composition change depending on its source, method of ...

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Lipoxygenase-Catalyzed Phospholipid Peroxidation: Preparation, Purification, and Characterization of Phosphatidylinositol Peroxides

The importance of understanding the mechanisms of modulation of cellular signaling cascades by the peroxidized membrane phospholipids (PLs) is well recognized. The enzyme-catalyzed peroxidation of PLs, as opposed to their oxidation by air and metal catalysis, is well controlled a ...

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Simultaneous Analysis of Multiple Lipid Oxidation Products In Vivo by Liquid Chromatographic-Mass Spectrometry (LC-MS)

Free radical-induced oxidation of polyunsaturated fatty acid (PUFAs) has been linked to a number of human diseases including atherosclerosis and neurodegenerative disorders. Oxidation of PUFAs generates hydroperoxides and cyclic peroxides that are reduced to lipid alcohol, ...

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Oxidative Lipidomics of Apoptosis: Quantitative Assessment of Phospholipid Hydroperoxides in Cells and Tissues

Oxidized phospholipids play essential roles in execution of mitochondrial stage of apoptosis and clearance of apoptotic cells. The identification and quantification of oxidized phospholipids generated during apoptosis can be successfully achieved by oxidative lipido ...

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Analysis of Urinary 8-oxo-7,8-dihydro-2-deoxyguanosine by Liquid ChromatographyTandem Mass Spectrometry

The ability to non-invasively monitor DNA oxidation and its repair has significant utility in large-scale, population-based studies. Such studies could include assessments of the efficacy of antioxidant intervention strategies, pathological roles of DNA oxidation in various ...

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A General Method for Quantifying Sequence Effects on Nucleobase Oxidation in DNA

Oxidative damage to DNA has long been associated with aging and disease, with guanine serving as the primary target for oxidation owing to its low ionization potential. Emerging evidence points to a critical role for sequence context as a determinant of the guanine ionization potential and the ...

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Gene Therapy Techniques for the Delivery of Endothelial Nitric Oxide Synthase to the Lung for Pulmonary Hypertension

Pulmonary hypertension (PH) is a serious, often fatal disease characterized by remodeling of the pulmonary vascular bed, increased pulmonary arterial pressure, and right heart failure. The increased vascular resistance in the pulmonary circulation is due to structural changes a ...

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Redox Homeostasis and Cellular Stress Response in Aging and Neurodegeneration

Decreased expression and/or activity of antioxidant proteins leads to oxidative stress, accelerated aging, and neurodegeneration. While overwhelming levels and uncontrolled/dysregulated actions of reactive oxygen species (ROS) lead to deleterious effects, tighter r ...

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Simultaneous Analysis of Expression of Multiple Redox-Sensitive and Apoptotic Genes in Hypothalamic Neurons Exposed to Cholesterol Secoaldehyde

Oxidative stress and apoptotic cell death are two important processes that occur under several disease states and in conditions of toxicant insult. Traditionally, investigators have chosen a variety of analytical methods to detect and/or quantify oxidative stress and apoptosis. T ...

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Isolation of Inositol 1,4,5-Trisphosphate Receptor-Associating Proteins and Selective Knockdown Using RNA Interference

Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are IP3-gated Ca2+ release channels localized on intracellular Ca2+ stores and play a role in the generation of complex patterns of intracellular Ca2+ signals. We show herein experimental protocols for the identification of asso ...

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Diphosphosinositol Polyphosphates and Energy Metabolism: Assay for ATP/ADP Ratio

Several inositol compounds undergo rapid cycles of phosphorylation and dephosphorylation. These cycles are dependent on ATP and energy metabolism. Therefore, interfering with the cellular energy metabolism can change the concentration of rapidly turning over inositols. Ma ...

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Synthesis and Nonradioactive Micro-analysis of Diphosphoinositol Phosphates by HPLC with Postcolumn Complexometry

A nonradioactive high-performance anion-exchange chromatographic method based on MDD-HPLC (Mayr Biochem. J. 254:585–591, 1988) was developed for the separation of inositol hexakisphosphate (InsP 6, phytic acid) and most isomers of pyrophosphorylated inositol phosphates, s ...

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A Femtomole-Sensitivity Mass Assay for Inositol Hexakisphosphate

Inositol hexakisphosphate (InsP 6) is an important component of cells, and its mass levels are usually assayed by either (a) equilibrium labelling of cell cultures with radiolabelled inositol or (b) by a variety of mass assays of differing sensitivities and ambiguities. Here, we describe a ma ...

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Molecular Manipulation and Analysis of Inositol Phosphate and Pyrophosphate Levels in Mammalian Cells

Lipid-derived inositol phosphates (InsPs) comprise a family of second messengers that arise through the action of six classes of InsP kinases, generally referred to as IPKs. Genetic studies have indicated that InsPs play critical roles in embryonic development, but the mechanisms of ac ...

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HPLC Separation of Inositol Polyphosphates

High performance liquid chromatography (HPLC) is an essential analytical tool in the study of the large number of inositol phosphate isomers. This chapter focuses on the separation of inositol polyphosphates from myo-inositol labeled tissues and cells. We review the different HPLC co ...

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The Role of Inositol and the Principles of Labelling, Extraction, and Analysis of Inositides in Mammalian Cells

Inositides have an important impact on diverse areas of cellular regulation. However, since this area has grown exponentially from the mid 1980s onwards, many workers find themselves relatively new to the field. In this chapter, we establish a broad foundation for the rest of the book by covering ...

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Imaging Phosphoinositide Dynamics in Living Cells

To improve our understanding of the important roles played by inositol lipid derivatives in signalling and other cellular processes, it is crucial to measure phosphoinositide concentration changes in individual cells with high spatial and temporal resolution. A number of protein d ...

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Phosphoinositide Analysis by Liquid ChromatographyMass Spectrometry

The phosphoinositides are a highly dynamic group of molecules implicated in many cellular control processes; however, the analysis of many of these structures has proven very difficult and time-consuming, with limited sensitivity and/or discrimination. Recent developments in L ...

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Measurement of Phosphoinositide 3-Kinase Products in Cultured Mammalian Cells by HPLC

The phosphoinositide 3-kinase (PI3K) family catalyses the addition of a phosphate group to the D-3 position of polyphosphoinositides (PPIn). Since the discovery in the late 80s that phosphatidylinositol is phosphorylated in the D-3 position in eukaryotic cells, there has been an explo ...

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Methods to Assess Changes in the Pattern of Nuclear Phosphoinositides

Phosphatidylinositol (PtdIns) and its phosphorylated derivatives represent less than 5% of total membrane phospholipids in cells. Despite their low abundance, they form a dynamic signalling system that is regulated in response to a variety of extra and intra-cellular cues (Curr Opin ...

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