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Methods in Molecular Biology

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https://www.readbyqxmd.com/read/28205182/measurement-of-macrophage-specific-in-vivo-reverse-cholesterol-transport-in-mice
#1
Wendy Jessup, Maaike Kockx, Leonard Kritharides
Reverse cholesterol transport (RCT) is one of the main processes that is thought to protect against cardiovascular disease. RCT constitutes the removal of cholesterol from peripheral sites, its transport through the plasma compartment for delivery to the liver for excretion. Here, we describe an in vivo RCT method that incorporates these steps, measuring movement of cholesterol from macrophages to the plasma, the liver, and finally to the feces in mice.
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28205181/abc-transporter-mediated-sterol-export-from-cells-using-radiolabeled-sterols
#2
Alryel Yang, Ingrid C Gelissen
Cholesterol export from cells to extracellular acceptors represents the first step of the reverse cholesterol transport process and is an essential part of the multifaceted pathway for cells to control their cholesterol levels. Malfunction of this pathway leads to cholesterol accumulation in cells such as macrophages, which can form the basis of conditions like atherosclerosis. A number of ATP-binding cassette (ABC) transporters, namely ABCA1, ABCA7, ABCG1, and ABCG4, play an essential role in this process...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28205180/methods-for-monitoring-abca1-dependent-sterol-release
#3
Yoshio Yamauchi, Shinji Yokoyama, Ta-Yuan Chang
Releasing sterols to the extracellular milieu is an important part of sterol homeostasis in cells and in the body. ATP-binding cassette transporter A1 (ABCA1) plays an essential role in cellular phospholipid and sterol release to lipid-free or lipid-poor apolipoprotein A-I (apoA-I), the major apolipoprotein in high-density lipoprotein (HDL), and constitutes the first step in the formation of nascent HDL. Loss-of-function mutations in the ABCA1 gene lead to a rare disease known as Tangier disease that causes severe deficiency in plasma HDL level...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28205179/measurement-of-rates-of-cholesterol-and-fatty-acid-synthesis-in-vivo-using-tritiated-water
#4
Adam M Lopez, Jen-Chieh Chuang, Stephen D Turley
Every organ in the body is capable of synthesizing cholesterol de novo but at rates that vary with a constellation of factors. A significant proportion of the hydrogen atoms present in cholesterol that is synthesized in the body are derived from water. Thus, although water ordinarily makes up the bulk of body mass, the acute enrichment of the body water pool with a sufficiently large amount of tritiated water over a short interval of time (usually 1 h) yields measurable rates of incorporation of the labeled water into newly generated cholesterol and also fatty acids...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28205178/sterol-analysis-by-quantitative-mass-spectrometry
#5
Andrew M Jenner, Simon H J Brown
Analysis of sterols by mass spectrometry is a fundamental technique allowing for both qualitative and quantitative characterization of sterol molecular lipid species. Lipids are isolated from matrix or matrices by homogenization and solvent extraction, and converted into species amenable for ionization either by derivatization or adduct formation. Chromatogaphy (either gas or liquid phase) can assist with the resolution of sterols. Tandem mass spectrometry allows the precise identification of sterol lipid species, while comparison to internal standards added during extraction enables accurate quantification...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28205177/measuring-activity-of-cholesterol-synthesis-enzymes-using-gas-chromatography-mass-spectrometry
#6
Anika V Prabhu, Winnie Luu, Andrew J Brown
The development of gas chromatography/mass spectrometry (GC/MS) technology has improved the ease and efficiency with which sterols in biological samples can be analyzed. Its advantages include that it needs only a small amount of sample, a short analysis time, and has enhanced specificity over traditional methods. Furthermore, a major benefit is its nonselective properties, which means that a complete scan of the sample will display the relative abundance of every sterol in the sample. This property has made it possible to define the abnormal, but distinctive, sterol profiles in a number of inborn errors of cholesterol synthesis...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28205176/determining-the-topology-of-membrane-bound-proteins-using-pegylation
#7
Vicky Howe, Andrew J Brown
Biochemical methods can help elucidate the membrane topology of hydrophobic membrane proteins where X-ray crystallography is difficult or impractical, providing important structural data. Here, we describe the method of PEGylation, which uses a cysteine-reactive molecule, maleimide polyethylene glycol (mPEG), to determine the cytosolic accessibility of introduced cysteine residues. This accessibility is visualized using Western blotting to detect a band shift that indicates cysteine labeling by mPEG. Using scanning cysteine mutagenesis, followed by PEGylation, one can map the accessibility of the introduced cysteines, hence inferring the membrane topology of the protein...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28205175/membrane-extraction-of-hmg-coa-reductase-as-determined-by-susceptibility-of-lumenal-epitope-to-in-vitro-protease-digestion
#8
Lindsey L Morris, Russell A DeBose-Boyd
Although many aspects of the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway have been elucidated, methods to detect and examine intermediate steps in the process are lacking. Here, we describe the use of a protease protection assay to study the metabolically regulated ERAD substrate HMG CoA reductase. Studies utilizing this assay reveal that ubiquitinated reductase becomes extracted across the ER membrane prior to its cytosolic release and proteasomal degradation through reactions mediated by distinct AAA-ATPases...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28205174/identifying-sterol-response-elements-within-promoters-of-genes
#9
Laura J Sharpe, Andrew J Brown
Cholesterol levels are under tight control within cells. This involves a complex interplay of balancing synthesis, uptake, and export. A major player in the transcriptional regulation of cholesterol levels is sterol regulatory element binding protein (SREBP). SREBP is upregulated in conditions of low cholesterol, and then binds to sterol regulatory elements (SREs) that exist within the promoters of genes involved in cholesterol synthesis and uptake.Here, we describe a method to identify sterol response elements (SREs) using in silico and experimental approaches...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28205173/measurement-of-mitochondrial-cholesterol-import-using-a-mitochondria-targeted-cyp11a1-fusion-construct
#10
Barry E Kennedy, Mark Charman, Barbara Karten
All animal membranes require cholesterol as an essential regulator of biophysical properties and function, but the levels of cholesterol vary widely among different subcellular compartments. Mitochondria, and in particular the inner mitochondrial membrane, have the lowest levels of cholesterol in the cell. Nevertheless, mitochondria need cholesterol for membrane maintenance and biogenesis, as well as oxysterol, steroid, and hepatic bile acid production. Alterations in mitochondrial cholesterol have been associated with a range of pathological conditions, including cancer, hepatosteatosis, cardiac ischemia, Alzheimer's, and Niemann-Pick Type C Disease...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28205172/measurement-of-cholesterol-transfer-from-lysosome-to-peroxisome-using-an-in-vitro-reconstitution-assay
#11
Jie Luo, Ya-Cheng Liao, Jian Xiao, Bao-Liang Song
Low-density lipoproteins (LDLs) are taken up by the cell mainly through receptor-mediated endocytosis. LDL-derived cholesterol leaves lysosome and further transports to downstream organelles for specific cellular needs. We recently report that cholesterol transfers from lysosome to peroxisome through lysosome-peroxisome membrane contact (LPMC). Here, we use iodixanol density gradient centrifugation to isolate lysosomes and peroxisomes separately for the in vitro reconstitution of LPMC. We also apply (3)H-cholesterol-labeled lysosomes and peroxisomes in vitro to measure (3)H-cholesterol transfer through LPMC...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28205171/synthesis-and-live-cell-imaging-of-fluorescent-sterols-for-analysis-of-intracellular-cholesterol-transport
#12
Maciej Modzel, Frederik W Lund, Daniel W├╝stner
Cellular cholesterol homeostasis relies on precise control of the sterol content of organelle membranes. Obtaining insight into cholesterol trafficking pathways and kinetics by live-cell imaging relies on two conditions. First, one needs to develop suitable analogs that resemble cholesterol as closely as possible with respect to their biophysical and biochemical properties. Second, the cholesterol analogs should have good fluorescence properties. This interferes, however, often with the first requirement, such that the imaging instrumentation must be optimized to collect photons from suboptimal fluorophores, but good cholesterol mimics, such as the intrinsically fluorescent sterols, cholestatrienol (CTL) or dehydroergosterol (DHE)...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28205170/transport-assays-for-sterol-binding-proteins-stopped-flow-fluorescence-methods-for-investigating-intracellular-cholesterol-transport-mechanisms-of-npc2-protein
#13
Leslie A McCauliff, Judith Storch
In this chapter we describe the use of stopped flow fluorescence spectroscopy to analyze the kinetic mechanisms of protein mediated cholesterol transfer to, from, and between model membranes. These assays allow for the detection of protein-membrane interactions that may occur during cholesterol transfer by simply modifying donor or acceptor concentrations, membrane composition, or buffer properties, and analyzing resultant transfer rates.
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28205169/quantitative-measurement-of-cholesterol-in-cell-populations-using-flow-cytometry-and-fluorescent-perfringolysin-o
#14
Jian Li, Peter L Lee, Suzanne R Pfeffer
Methods to quantify intracellular cholesterol are valuable for the study of its trafficking and storage in normal cells and in lysosomal storage disorders. Traditionally, cholesterol has been tracked using the small molecule, filipin. Filipin can be difficult to visualize and visualization can be cytotoxic as it requires UV illumination. Here we describe a method to measure cholesterol using a fluorescently labeled, mutant form of Perfringolysin O, a soluble protein toxin that binds cholesterol specifically...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28205168/crispr-cas9-mediated-generation-of-niemann-pick-c1-knockout-cell-line
#15
Ximing Du, Ivan Lukmantara, Hongyuan Yang
Generating a cholesterol storage phenotype of Niemann-Pick Type C (NPC) disease is important for investigating the mechanisms of intracellular cholesterol trafficking, as well as screening drugs for potential treatment of NPC disease. The use of the CRISPR/Cas9 technology to knockout specific genes within the genome of mammals has become routine in the past few years. Here, we describe a protocol for producing a cellular NPC cholesterol storage phenotype in HeLa cells using the CRISPR-Cas9 system to disrupt the NPC1 gene...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28205167/the-use-of-l-sidol-transgenic-mice-as-a-murine-model-to-study-hypercholesterolemia-and-atherosclerosis
#16
Eser J Zerenturk, Anna C Calkin
There are many advantages to the use of mice as a model to study the regulation of cholesterol metabolism. Common models of hypercholesterolemia include low-density lipoprotein receptor deficient (LDLR -/-) mice and apolipoprotein E deficient (ApoE) -/- mice. Herein, we describe the recently generated mouse model, L-sIDOL Tg mice, which express a dominant active form of Inducible Degrader Of the Low-density lipoprotein receptor (IDOL) in a liver-specific manner. This murine model offers significant advantages over previously established models for the study of hypercholesterolemia and atherosclerosis...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28205166/assaying-low-density-lipoprotein-ldl-uptake-into-cells
#17
Anke Loregger, Jessica K Nelson, Noam Zelcer
Determination of LDL particle uptake into cells is a valuable technique in the field of cholesterol metabolism. This allows assessment of LDL uptake capacity in different adherent and non-adherent cells types, as well as the effect of cellular, genetic, or pharmacological perturbations on this process. Here, we detail a general procedure that describes the production of fluorescently-labeled LDL particles and quantitative and non-quantitative assays for determining cellular LDL uptake.
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28205165/manipulating-cholesterol-status-within-cells
#18
Winnie Luu, Ingrid C Gelissen, Andrew J Brown
Cellular cholesterol levels are intricately controlled to maintain homeostasis. Here, we describe ways in which cellular cholesterol status can be manipulated for the study of cholesterol homeostasis, including sterol starvation (by culturing cells in lipoprotein-deficient serum and pretreating/treating with the cholesterol-lowering drug, statin) and sterol enrichment (using cholesterol complexed to cyclodextrin, and low-density lipoprotein). We also describe how to prepare lipoprotein-deficient serum and complex cholesterol to cyclodextrin...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28205164/structural-stringency-of-cholesterol-for-membrane-protein-function-utilizing-stereoisomers-as-novel-tools-a-review
#19
Md Jafurulla, Amitabha Chattopadhyay
Cholesterol is an important lipid in the context of membrane protein function. The function of a number of membrane proteins, including G protein-coupled receptors (GPCRs) and ion channels, has been shown to be dependent on membrane cholesterol. However, the molecular mechanism underlying such regulation is still being explored. In some cases, specific interaction between cholesterol and the protein has been implicated. In other cases, the effect of cholesterol on the membrane properties has been attributed for the regulation of protein function...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28205163/hybrid-in-silico-in-vitro-approaches-for-the-identification-of-functional-cholesterol-binding-domains-in-membrane-proteins
#20
Coralie Di Scala, Jacques Fantini
In eukaryotic cells, cholesterol is an important regulator of a broad range of membrane proteins, including receptors, transporters, and ion channels. Understanding how cholesterol interacts with membrane proteins is a difficult task because structural data of these proteins complexed with cholesterol are scarce. Here, we describe a dual approach based on in silico studies of protein-cholesterol interactions, combined with physico-chemical measurements of protein insertion into cholesterol-containing monolayers...
2017: Methods in Molecular Biology
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