MedWorm.com provides a medical RSS filtering service. Over 6000 RSS medical sources are combined and output via different filters. This feed contains the latest items from the 'Springer protocols feed by Molecular Medicine' source. Thu, 09 Feb 2012 16:56:54 +0100 http://www.medworm.com/index.php?rid=5568946&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-523-7_25 Over the past decade, an immense amount of biomedical data have become available in the public domain due to the development of ever-more efficient screening tools such as expression microarrays. To fully leverage this important new resource, it has become imperative to develop new methodologies for mining and visualizing data to make inferences beyond the scope of the original experiments. This need motivated the development of a new freely available web-based application called StarNet ( http://vanburenlab.medicine.tamhsc.edu/starnet2.html ). Here we describe the use of StarNet, which functions primarily as a query tool that draws correlation networks centered about a gene of interest. To support inferences and the development of new hypotheses using the result... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5568946 Sun, 01 Jan 2012 05:00:00 +0100 5568946 http://www.medworm.com/index.php?rid=5568945&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-523-7_24 DNA microarray methodologies have proven to be an indispensable tool for genome-wide transcriptional profiling of organs, tissues, and cells. Here, we present a protocol for the optimized isolation and preparation of RNA from rat microvessels (including arteries, veins, and lymphatics) for subsequent use in two-color microarray analysis. The investigation of wide-ranging vessel sizes from all three vessel lineages necessitates an RNA isolation strategy that can effectively isolate high-quality RNA from varying and often very small quantities (<1 mg) of fibrous vessel tissue. Additionally, the lack of sample biomass necessitates the use of amplification strategies to generate enough RNA for use in microarray analysis. While the methods presented here were developed for use with two-color... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5568945 Sun, 01 Jan 2012 05:00:00 +0100 5568945 http://www.medworm.com/index.php?rid=5568944&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-523-7_23 Murine models are currently the preferred approach for studying the molecular mechanisms of cardiac dysfunction resulting from changes in gene expression. Transgenic and gene-targeting methods can be used to generate mice with altered cardiac size and function, and as a result, in vivo techniques are indispensible in evaluating cardiac phenotype. Traditionally, the pathologic assessment of sacrificed hearts was used to study cardiac pathophysiology in small animals. Below we describe the use of ultrasound biomicroscopy-Doppler analysis to temporally assess cardiac function in mouse embryos. Methods are described for obtaining 2D, pulsed-wave Doppler, and M-mode imaging using standard clinical cardiac ultrasound imaging planes. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5568944 Sun, 01 Jan 2012 05:00:00 +0100 5568944 http://www.medworm.com/index.php?rid=5568943&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-523-7_22 The atomic force microscope (AFM) is a state-of-the-art tool that can analyze and characterize samples on a scale from angstroms to 100 μm by physical interaction between AFM cantilever tip and sample surface. AFM imaging has been used incrementally over last decade in living cells in cardiovascular research. Beyond its high resolution 3D imaging, AFM allows the quantitative assessments on the structure and function of the underlying cytoskeleton and cell organelles, binding probability, adhesion forces, and micromechanical properties of the cell by “sensing” the cell surface with mechanical sharp cantilever tip. AFM measurements have enhanced our understanding of cell mechanics in normal physiological and pathological states. (Source: Springer protocols feed by Molecular Me... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5568943 Sun, 01 Jan 2012 05:00:00 +0100 5568943 http://www.medworm.com/index.php?rid=5568942&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-523-7_21 Cardiovascular development is a precisely coordinated process at multilevels. It involves cross-talking among numerous signaling transduction pathways to ensure proper cell polarity, migration, proliferation, differentiation, and programmed death. Here, genome-wide RNA interference screens in Drosophila cells are introduced as novel approaches to discover potential regulators, with special emphases on (1) cell growth and viability, and (2) receptor tyrosine kinase and extracellular-signal-regulated kinase signaling pathway. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5568942 Sun, 01 Jan 2012 05:00:00 +0100 5568942 http://www.medworm.com/index.php?rid=5568941&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-523-7_20 Neonatal rat ventricular myocytes (NRVM) and fibroblasts (FBs) serve as in vitro models for studying fundamental mechanisms underlying cardiac pathologies, as well as identifying potential therapeutic targets. Both cell types are relatively easy to culture as monolayers and can be manipulated using molecular and pharmacological tools. Because NRVM cease to proliferate after birth, and FBs undergo phenotypic changes and senescence after a few passages in tissue culture, primary cultures of both cell types are required for experiments. Below we describe methods that provide good cell yield and viability of primary cultures of NRVM and FBs from 0 to 3-day-old neonatal rat pups. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5568941 Sun, 01 Jan 2012 05:00:00 +0100 5568941 http://www.medworm.com/index.php?rid=5568940&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-523-7_19 The authors describe technical details of experimental protocol of gene transfection in isolated rat mesenteric lymphatic vessels (MLVs). Authors also refer to the recent publication in Microcirculation, which provides wide set of experimental evidences obtained from confocal microscopic imaging and isolated vessels functional tests, which confirmed a successful achievement of the following goals. (1) Optimization of the experimental conditions to maintain the isolated “normal” rat mesenteric vessels in culture for sufficiently long periods of time to permit effective knockdown or overexpression of selected proteins/genes. (2) Development of the effective transfection protocols for lymphatic muscle and/or endothelial cells in intact isolated rat MLVs without nonspecific impairm... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5568940 Sun, 01 Jan 2012 05:00:00 +0100 5568940 http://www.medworm.com/index.php?rid=5568939&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-523-7_18 Apoptosis is the process of programmed cell death that has been identified in the development of heart. It is likely to be regulated by survival and death signals that are also present in many other tissues. To understand cardiomyocyte apoptosis in normal and abnormal development of heart, the cell death detection techniques were applied to various researches. These methods include morphological, histological, or molecular assays based on recent advances in our understanding of the molecular mechanism of cell death, including: (1) terminal deoxynucleotidyl transferase mediated dUTP nick end-labeling of fragmented nuclei, (2) cardiovascular molecular imaging of apoptosis using Annexin V, and (3) immunohistochemical detection of activated caspases. (Source: Springer protocols feed by Molecul... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5568939 Sun, 01 Jan 2012 05:00:00 +0100 5568939 http://www.medworm.com/index.php?rid=5568938&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-523-7_17 Through directed differentiation of embryonic stem cells, it has been demonstrated that mesodermal lineages in the mammalian heart (smooth muscle, endothelial, and cardiac) develop from a common, multipotent cardiovascular precursor (Dev Biol 265:262–275, 2004; Cell 127:1137–1150, 2006; Dev Cell 11:723–732, 2006). Identification of cardiovascular precursor cells at various stages of lineage commitment has been determined by expression of multiple markers, including the stem cell factor receptor c-kit. Utilizing a bacterial artificial chromosome (BAC) transgenic mouse model in which EGFP expression is placed under control of the c-kit promoter (c-kitBAC-EGFP), work from our laboratory indicates that c-kit expression identifies a multipotent cardiovascular precursor cell po... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5568938 Sun, 01 Jan 2012 05:00:00 +0100 5568938 http://www.medworm.com/index.php?rid=5568937&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-523-7_16 Vascular smooth muscle cells (VSMCs) are the cellular components of the normal blood vessel wall that provides structural integrity and regulates the diameter by contracting and relaxing dynamically in response to vasoactive stimuli. The differentiated state of the VSMC is characterized by specific contractile proteins, ion channels, and cell surface receptors that regulate the contractile process and are thus termed contractile cells. In addition to these normal functions, in response to injury or during development, VSMCs are responsible for the synthesis of extracellular matrix proteins, become migratory and proliferate. This phenotype has been termed synthetic cells. To better understand the mechanisms regulating these and other processes, scientists have depended on cultured cells tha... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5568937 Sun, 01 Jan 2012 05:00:00 +0100 5568937 http://www.medworm.com/index.php?rid=5568936&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-523-7_15 Epicardium is the outer cell layer of the heart. Its integrity and function are essential for normal heart development. To study the role of epicardium in both fetal and adult hearts, it is desirable to isolate and culture pure populations of these cells. Here we describe methods with Cre-loxP technology to lineage tag epicardial cells (EpiCs) and epicardium-derived cells (EPDCs), dissociate and isolate them by flow-activated cytometry sorting (FACS), and characterize them by quantitative PCR and immunostaining. This platform allows further characterization and manipulation of EpiCs and EPDCs for expression studies and functional assays. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5568936 Sun, 01 Jan 2012 05:00:00 +0100 5568936 http://www.medworm.com/index.php?rid=5568935&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-523-7_14 The formation of blood vessel networks is a fundamental event in vertebrate embryo development. Angiogenesis and vasculogenesis are the essential processes in vascular formation. Endothelial cells play a key role during angiogenesis and vasculogenesis, and cultured vascular endothelial cells provide an indispensable model for exploring the molecular mechanisms of angiogenesis and vasculogenesis. In this chapter, we described a protocol using PECAM-1-coated Dynabeads for the isolation of vascular endothelial cells from mouse heart and lung. This method will provide up to 107 endothelial cells with high purity (>85%). The isolated endothelial cells retain their in vivo characteristics, such as the expression of the cell surface markers PECAM-1 and ICAM-2. (Source: Springer protocols feed ... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5568935 Sun, 01 Jan 2012 05:00:00 +0100 5568935 http://www.medworm.com/index.php?rid=5568934&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-523-7_13 The heart is the first organ to form and become functional in a developing embryo and its proper function is critical for most, if not all, subsequent stages of an animal’s development. The formation of the heart relies heavily upon the rapid proliferation of cardiomyocytes at a specific stage during early development, and interfering with the ability of these cells to grow during this time frame results in heart defects that include thin ventricle walls, as well as malformed ventricular septums. Thus, since cardiomyocyte proliferation represents a key step in early cardiac development and identifying the mechanisms that underlie cardiomyocyte proliferation has become an important area of study, techniques to identify and study proliferating cardiomyocytes in vivo are needed. Immunof... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5568934 Sun, 01 Jan 2012 05:00:00 +0100 5568934 http://www.medworm.com/index.php?rid=5568933&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-523-7_12 It is important to identify the mechanisms regulating cardiovascular development. However, complex genetic tools are often required, including transgenic animals that express the lacZ transgene encoding the β-galactosidase enzyme under the control of a specific promoter or following recombination with the Cre recombinase. The latter can be useful for identifying specific cell populations of the developing cardiovascular system, including neural crest cells. The tracking of these cells can help clarify their fate in mutant embryos and elucidate the etiology of some congenital cardiovascular birth defects. This chapter highlights the methods used to stain embryonic tissues in whole mount or sections to detect the expression of the lacZ transgene with a focus on tracking cardiac neural c... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5568933 Sun, 01 Jan 2012 05:00:00 +0100 5568933 http://www.medworm.com/index.php?rid=5568932&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-523-7_11 Formation of a fully functional four-chambered heart involves an intricate and complex series of events that includes precise spatial–temporal regulation of cell specification, proliferation, and migration. The formation of the ventricular septum during mid-gestation ensures the unidirectional flow of blood, and is necessary for postnatal viability. Notably, a majority of all congenital malformations of the cardiovascular system in humans involve septal abnormalities which afflict 1 out of 100 newborn children in the United States. Thus, a clear understanding of the precise mechanisms involved in this morphogenetic event will undoubtedly reveal important therapeutic targets. The final step in valvuloseptal morphogenesis occurs, in part, by directed movement of flanking myocytes into ... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5568932 Sun, 01 Jan 2012 05:00:00 +0100 5568932 http://www.medworm.com/index.php?rid=5568931&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-523-7_10 Malformations of heart valves are one of the most common serious congenital defects. Heart valves are developed from endocardial cushions of the heart. The endocardial cushion in early heart development consists of two cell layers: an outer myocardial cell layer and an inner endocardial cell layer with abundant extracellular matrix (cardiac jelly) in between. Endocardial cells of the cushion, triggered by signals from myocardial cells, delaminate from the surface of the endocardial cushion and undergo transdifferentiation into mesenchymal cells. This process of endocardial-to-mesenchymal transformation (EMT) begins in the atrioventricular canal at embryonic day 9 (E9) and in the cardiac outflow tract at E10 of mouse development. Once formed by the EMT, the mesenchymal cells invade the card... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5568931 Sun, 01 Jan 2012 05:00:00 +0100 5568931 http://www.medworm.com/index.php?rid=5568930&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-523-7_9 Assays of angiogenesis in vitro provide insights into vascular development and are useful in studies of agents that modulate blood vessel formation. This chapter describes techniques to induce angiogenesis-like sprouting from aortic and microvascular explants cultured in 3-dimensional native collagen gels. The chapter focuses on explants derived from mice and use of a miniaturized format that permits efficient utilization of reagents, ease of processing, rapid analysis, and conventional imaging. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5568930 Sun, 01 Jan 2012 05:00:00 +0100 5568930 http://www.medworm.com/index.php?rid=5568929&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-523-7_8 The establishment of a blood vessel network is fundamental to embryonic development and plays a critical role in many diseases including coronary heart disease and cancer. Vascular endothelial cells are central players in blood vessel formation and line the inside of the entire blood vessel system. PECAM-1 is expressed in all types of endothelial cells and is therefore a useful marker for the detection of blood vessels. In this manuscript, we describe PECAM-1 staining in whole-mount and sectioned tissues in mouse embryos. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5568929 Sun, 01 Jan 2012 05:00:00 +0100 5568929 http://www.medworm.com/index.php?rid=5568928&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-523-7_7 We introduce a whole-mount immunohistochemistry method for analyzing intricate vascular network formation in mouse embryonic tissues. Laser scanning confocal microscopy with multiple labeling allows for robust imaging of blood and lymphatic vessel branching morphogenesis with excellent resolution. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5568928 Sun, 01 Jan 2012 05:00:00 +0100 5568928 http://www.medworm.com/index.php?rid=5568927&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-523-7_6 Visualizing the circulatory pattern in developing embryos becomes an essential technique for the field of cardiovascular biology. In the zebrafish model system, there are currently several techniques available to visualize the circulatory pattern. Microangiography is a simple technique in which a fluorescent dye is injected directly into the Sinus Venosus and/or the Posterior Cardinal Vein, allowing for the rapid labeling and easy detection of patent vessels. Here, we compare microangiography to other vascular labeling techniques, describe the benefits and potential applications of microangiography, and give step by step instructions for microangiography. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5568927 Sun, 01 Jan 2012 05:00:00 +0100 5568927 http://www.medworm.com/index.php?rid=5568926&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-523-7_5 The chick embryo chorioallantoic membrane (CAM) is an extraembryonic membrane which serves as a gas exchange surface and its function is supported by a dense capillary network. Because of its extensive vascularization and easy accessibility, the CAM has been broadly used to study the morphofunctional aspects of the angiogenesis process in vivo and to investigate the efficacy and mechanisms of action of proangiogenic and antiangiogenic natural and synthetic molecules. The CAM has long been a favored system for the study of tumor angiogenesis and metastasis, because at this stage the chick immunocompetence system is not fully developed and the conditions for rejection have not been established. The CAM may also be used to verify the ability to inhibit the growth of capillaries by implanting ... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5568926 Sun, 01 Jan 2012 05:00:00 +0100 5568926 http://www.medworm.com/index.php?rid=5568925&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-523-7_4 The study of gene function in developmental biology has been significantly furthered by advances in antisense technology made in the early 2000s. This was achieved, in particular, by the introduction of morpholino (MO) oligonucleotides. The introduction of antisense MO oligonucleotides into cells enables researchers to readily reduce the levels of their protein of interest without investing huge financial or temporal resources, in both in vivo and in vitro model systems. Historically, the African clawed frog Xenopus has been used to study vertebrate embryological development, due to its ability to produce vast numbers of offspring that develop rapidly, in synchrony, and can be cultured in buffers with ease. The developmental progress of Xenopus embryos has been extensively characterized an... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5568925 Sun, 01 Jan 2012 05:00:00 +0100 5568925 http://www.medworm.com/index.php?rid=5568924&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-523-7_3 Formation of the cardiac outflow tract and great arteries involves complex morphogenetic processes, whose abnormities result in several clinically important diseases. Studies of these developmental processes are therefore important for understanding congenital vascular defects. However, the three-dimensional structure of arteries makes it challenging to analyze the pattern of vasculature using conventional histological approaches. Here we describe a vascular casting method to visualize the branching and connections of great arteries in developing embryos as well as in adult mice. This technique can be used to study the development of cardiac outflow tract, semilunar valves, and great arteries as demonstrated previously (Circ Res, 2008; Development 135: 3577–3586, 2008). (Source: Spri... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5568924 Sun, 01 Jan 2012 05:00:00 +0100 5568924 http://www.medworm.com/index.php?rid=5568923&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-523-7_2 Zebrafish embryos have been extensively used to study heart development and cardiac function, mainly due to the unique embryology and genetics of this model organism. Since most human heart disease occurs during adulthood, adult zebrafish models of heart disease are being created to dissect mechanisms of the disease and discover novel therapies. However, due to its small heart size, the use of cardiac functional assays in the adult zebrafish has been limited. To address this bottleneck, the transparent fish line casper;Tg(cmlc2:nuDsRed) that has a red fluorescent heart can be used to document beating hearts in vivo and to quantify cardiac functions in adult zebrafish. Here, we describe our methods for quantifying shortening fraction and heart rate in embryonic zebrafish, as well as in the ... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5568923 Sun, 01 Jan 2012 05:00:00 +0100 5568923 http://www.medworm.com/index.php?rid=5568922&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-523-7_1 Congenital heart defects occur in approximately 1% of newborns and are a major cause of morbidity and mortality in infants and children. Many adult cardiac diseases also have developmental basis, such as heart valve malformations, among others. Therefore, dissecting the developmental and molecular mechanisms underlying such defects in embryos is of great importance in prevention and developing therapeutics for heart diseases that manifest in infants or later in adults. Whole embryo culture is a valuable tool to study cardiac development in midgestation embryos, in which ventricular chambers are specified and expand, and the myocardium and endocardium interact to form various cardiac structures including heart valves and trabecular myocardium (Cell 118: 649–663, 2004; Dev Cell 14: 298... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5568922 Sun, 01 Jan 2012 05:00:00 +0100 5568922 http://www.medworm.com/index.php?rid=5168921&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-243-4_1 Androgens and the androgen receptor (AR) are indispensable for expression of the male phenotype. The two most important androgens are testosterone and 5α-dihydrotestosterone. The elucidation of the mechanism of androgen action has a long history starting in the 19th century with the classical experiments by Brown-Séquard. In the 1960s the steroid hormone receptor concept was established and the AR was identified as a protein entity with a high affinity and specificity for testosterone and 5α-dihydrotestosterone. In addition, the enzyme 5α-reductase type 2 was discovered and found to catalyze the conversion of testosterone to the more active metabolite 5α-dihydrotestosterone. In the second half of the 1980s, the cDNA cloning of all steroid hormone receptors, i... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5168921 Sun, 28 Aug 2011 01:40:54 +0100 5168921 http://www.medworm.com/index.php?rid=5168920&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-243-4_2 Androgens are critical for normal prostate development and function, as well as prostate cancer initiation and progression. Androgens function mainly by regulating target gene expression through the androgen receptor (AR). Many studies have shown that androgen-AR signaling exerts actions on key events during prostate carcinogenesis. In this review, androgen action in distinct aspects of prostate carcinogenesis, including (i) cell proliferation, (ii) cell apoptosis, and (iii) prostate cancer metastasis will be discussed. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5168920 Sun, 28 Aug 2011 01:40:54 +0100 5168920 http://www.medworm.com/index.php?rid=5168919&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-243-4_3 Prostate cancer that recurs after androgen deprivation therapy is the second leading cause of cancer-related death in North American men. Clinical and experimental evidences indicate that the development of recurrent prostate cancer is dependent on re-activation of the androgen receptor signaling pathway. Androgen is required for androgen receptor translocation to the nucleus, interaction with androgen response elements, expression of target genes, and prostate cancer cell proliferation. The intra-tissue and serum testosterone and dihydrotestosterone levels are important biomarkers to monitor androgen deprivation therapy efficacy in prostate cancer and recurrent prostate cancer. We have measured testosterone and dihydrotestosterone in procured recurrent prostate cancer specimens using liqu... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5168919 Sun, 28 Aug 2011 01:40:54 +0100 5168919 http://www.medworm.com/index.php?rid=5168918&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-243-4_4 Evaluating endocrine activities of environmental chemicals or screening for new small molecule modulators of the androgen receptor (AR) transcription activity requires standardized and reliable assay procedures. Scintillation proximity assays (SPA) are sensitive and reliable techniques that are suitable for ligand competition binding assays. We have utilized a radiolabeled ligand competition binding assay for the androgen receptor (AR) that can be carried out in a 384-well format. This standardized, highly reproducible and low-cost assay has been automated for high-throughput screening (HTS) purposes. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5168918 Sun, 28 Aug 2011 01:40:54 +0100 5168918 http://www.medworm.com/index.php?rid=5168917&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-243-4_5 Androgen receptor (AR) acts as a ligand-regulated transcription factor that conveys the message of both natural and synthetic androgens directly to the level of gene programs. Reporter gene assays provide a convenient and quantitative way to measure the transcriptional activity of AR and the functionality of its binding sites (AREs) in DNA. Many reporter genes and different transfection methods can be used for this purpose. In this chapter, we describe the use of firefly luciferase gene-based reporters and transfection protocols for the measurement of AR activity in heterologous COS-1 cells cotransfected with an AR expression vector and in VCaP prostate cancer cells expressing endogenous AR. We also discuss the suitability of different reporter constructs and transfection methods for diffe... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5168917 Sun, 28 Aug 2011 01:40:54 +0100 5168917 http://www.medworm.com/index.php?rid=5168916&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-243-4_10 The androgen receptor (AR) is the central component of a dynamic conformational and interaction cascade initiated by androgenic hormones. AR function can be modified by cellular inputs not examined in test tube studies of AR action. Thus, there is a need to measure AR conformation and biochemistry directly within the cell where the intracellular locations, levels and availability of the hormone, AR, AR-interacting factors, DNA-binding sites, enzymes that modify those components of AR action, and factors that compete for the formation of functional AR–cofactor complexes may affect AR action. The dynamic nature of the AR functional cycle itself may introduce temporal fluctuations in factor status and location to affect AR output in the intact cell. This chapter focuses on the method of... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5168916 Sun, 28 Aug 2011 01:40:54 +0100 5168916 http://www.medworm.com/index.php?rid=5168915&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-243-4_6 We describe here the methods most fit to describe an ARE: the electrophoretic mobility shift and the transactivation assays. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5168915 Sun, 28 Aug 2011 01:40:54 +0100 5168915 http://www.medworm.com/index.php?rid=5168914&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-243-4_7 The androgen receptor (AR) plays a pivotal role in the progression of prostate cancer from the androgen-dependent to the castration-resistant state, making it a potential target for therapy. In this chapter, we describe the preparation and use of sublines of LNCaP and C4-2 human prostate cancer cells which have been engineered to stably express a doxycycline (DOX)-inducible AR shRNA in order to study the in vitro and in vivo effects of AR knockdown. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5168914 Sun, 28 Aug 2011 01:40:54 +0100 5168914 http://www.medworm.com/index.php?rid=5168913&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-243-4_8 High-affinity binding of testosterone or dihydrotestosterone to the androgen receptor (AR) triggers the androgen-dependent AR NH2- and carboxyl-terminal (N/C) interaction between the AR NH2-terminal FXXLF motif and the activation function 2 (AF2) hydrophobic binding surface in the ligand-binding domain. The functional importance of the AR N/C interaction is supported by naturally occurring loss-of-function AR AF2 mutations where AR retains high-affinity androgen binding but is defective in AR FXXLF motif binding. Ligands with agonist activity in vivo such as testosterone, dihydrotestosterone, and the synthetic anabolic steroids induce the AR N/C interaction and increase AR transcriptional activity in part by slowing the dissociation rate of bound ligand and stabilizing AR against degradati... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5168913 Sun, 28 Aug 2011 01:40:52 +0100 5168913 http://www.medworm.com/index.php?rid=5168912&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-243-4_13 The recruitment of co-repressors to the androgen receptor is an important mechanism for reducing androgen-mediated gene activation. Importantly, co-repressors play a major role in the treatment of hormone-dependent growing tissue, such as prostate cancer and breast cancer. In line with this, co-repressor dysfunction seems to be a major player for development of castration-resistant prostate cancer or therapy-resistant breast cancer. The molecular basis of hormone therapy by particular antihormones (antagonists) for the androgen receptor (AR) is mediated by enhanced recruitment and activity of co-repressors that cause repression of AR target genes that regulate proliferation and alteration of cancer cells. Therefore co-repressor recruitment is a crucial molecular mechanism of gene repressio... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5168912 Sun, 28 Aug 2011 01:40:52 +0100 5168912 http://www.medworm.com/index.php?rid=5168911&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-243-4_12 Androgen receptor (AR) is a ligand-controlled transcription factor that is deregulated and therefore targeted in prostate cancer. In addition to androgens, AR is regulated by post-translational modifications (PTMs). SUMOylation, conjugation of small ubiquitin-related modifier (SUMO) protein 1, 2, or 3, is a bulky PTM regulating several important physiological processes. We have shown that AR is modified by SUMO-1 at two conserved lysine residues in its N-terminal domain. This agonist-enhanced modification represses the transcriptional activity of the receptor in a reversible and target gene-selective fashion. Acceptor sites for SUMOs are also found in several other nuclear receptors. Since the cellular steady-state level of SUMO modifications of most substrates, including AR, is very ... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5168911 Sun, 28 Aug 2011 01:40:52 +0100 5168911 http://www.medworm.com/index.php?rid=5168910&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-243-4_11 Acetylation is an essential post-translational modification in which an acetyl group is covalently conjugated to a protein substrate. Histone acetylation was first proposed nearly half a century ago by Dr. Vincent Allfrey. Subsequent studies have shown that acetylated core histones are often associated with transcriptionally active chromatin. Acetylation at lysine residues of histone tails neutralizes the positive charge, which decreases the binding ability to DNA and increases the accessibility of transcription factors and co-activators to the chromatin template. In addition to histones, a number of non-histone substrates are acetylated. Acetylation of non-histone proteins governs biological processes, including cellular proliferation and survival, transcriptional activity, and intracellu... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5168910 Sun, 28 Aug 2011 01:40:52 +0100 5168910 http://www.medworm.com/index.php?rid=5168909&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-243-4_18 Systems-level approaches have emerged that rely on analytical, microscopy-based technology for the discovery of novel drug targets and the mechanisms driving AR signaling, transcriptional activity, and ligand independence. Single cell behavior can be quantified by high-throughput microscopy methods through analysis of endogenous protein levels and localization or creation of biosensor cell lines that can simultaneously detect both acute and latent responses to known and unknown androgenic stimuli. The cell imaging and analytical protocols can be automated to discover agonist/antagonist response windows for nuclear translocation, reporter gene activity, nuclear export, and subnuclear transcription events, facilitating access to a multiplex model system that is inherently unavailable through... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5168909 Sun, 28 Aug 2011 01:40:52 +0100 5168909 http://www.medworm.com/index.php?rid=5168908&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-243-4_17 Understanding the biology of prostate cancer and the roles of androgen receptor in prostate cancer progression is essential to the development of novel therapeutic strategies to effectively attack and eradicate this disease. Preclinical, in vivo, studies are critical to further evaluate potential clinical relevance of in vitro findings. Ideally, in vivo studies should employ models that mimic characteristics of prostate cancer from early diagnosis through the period of castration-resistant metastases. In this chapter we describe methodologies used to grow human prostate cancer xenografts in mice. In this setting, roles of androgen receptor signaling in prostate cancer progression and efficacy of novel treatment modalities, including those affecting androgen receptor signaling, can be inves... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5168908 Sun, 28 Aug 2011 01:40:52 +0100 5168908 http://www.medworm.com/index.php?rid=5168907&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-243-4_16 Androgen acting through the androgen receptor (AR) is known to be essential for male sexual differentiation and development. Using Cre-lox technology, we have generated the floxed AR mice, which have been bred with general or tissue-specific Cre expressing transgenic mice to knock out the AR gene in specific target cells. Our findings indicated that AR is required for sexual development and that loss of AR can have significant effects on many aspects of physiological functions and disease progression, such as immune function, metabolism, and tumorigenesis. Furthermore, our strategy can generate AR knockout (ARKO) in female mice, which allows researchers to study the AR function in the female. In brief, our floxed AR mouse model provides a powerful tool to study in vivo AR functions in sele... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5168907 Sun, 28 Aug 2011 01:40:52 +0100 5168907 http://www.medworm.com/index.php?rid=5168906&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-243-4_15 Chromatin immunoprecipitation (ChIP) is an invaluable tool in the study of transcriptional regulation. ChIP methods require both a priori knowledge of the transcriptional regulators which are important for a given biological system and high-quality specific antibodies for these targets. The androgen receptor (AR) is known to play essential roles in male sexual development, in prostate cancer and in the function of many other AR-expressing cell types (e.g. neurons and myocytes). As a ligand-activated transcription factor the AR also represents an endogenous, inducible system to study transcriptional biology. Therefore, ChIP studies of the AR can make use of treatment contrast experiments to define its transcriptional targets. To date several studies have mapped AR binding sites using ChIP i... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5168906 Sun, 28 Aug 2011 01:40:52 +0100 5168906 http://www.medworm.com/index.php?rid=5168905&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-243-4_14 The human androgen receptor (AR) is expressed in nearly all prostate cancers (PCa) and is known to participate in tumor progression through the expression of genes involved in the proliferation and differentiation of PCa. It is suggested that different types of ligands induce a distinct AR conformation that would lead to a specific set of interacting partners for the AR, such as coactivators (CoA) and corepressors (CoR), heat shock proteins (HSP), remodeling factors, kinases, phosphatases, and transcription factors resulting in various degrees of AR activity and stability. The natural ligand of the AR, dihydrotestosterone (DHT), induces a transcriptionally active conformation of the AR while the steroidal antiandrogen cyproterone acetate (CPA) and the nonsteroidal compounds hydroxyflutamid... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5168905 Sun, 28 Aug 2011 01:40:52 +0100 5168905 http://www.medworm.com/index.php?rid=5168904&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-243-4_19 Fusion between androgen-regulated TMPRSS2 and ETS transcription factor gene ERG is the most frequent genetic alteration that occurs in 40–70% of prostate cancers. Not only ERG but also other ETS transcription factor genes are involved in gene fusions. ETV1, ETV4, and ETV5 have all several fusion partners. One common feature shared by the majority of these partners is androgen-regulated expression. Despite its high frequency, the biological and molecular effects of ETS gene fusion in prostate cancer development and progression are unknown. In this chapter quantitative polymerase chain reaction (Q-PCR) is used for detection and further studying the incidence and properties of these fusion transcripts. The focus is on the expression of TMPRSS2–ERG transcripts in clinical prostate ... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5168904 Sun, 28 Aug 2011 01:40:52 +0100 5168904 http://www.medworm.com/index.php?rid=5168903&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-243-4_20 The balance between proliferation and cell death is often disrupted in cancer leading to tumor growth. In prostate cancer, these events are regulated, at least in part, through androgen signaling. Prostate cancer is dependent on androgens for growth in the initial stages where apoptosis is simultaneously inhibited. Androgen signaling remains important in later stages of prostate cancer as well. Here, we provide methods to study apoptosis in prostate cancer cells and its regulation by androgens. In prostate cancer cells grown in vitro, apoptosis can be induced by different stimuli, such as the endoplasmic reticulum Ca2+ ATPase inhibitor Thapsigargin (TG) through the intrinsic apoptosis pathway, or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) plus the inhibition of PI3K, t... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5168903 Sun, 28 Aug 2011 01:40:52 +0100 5168903 http://www.medworm.com/index.php?rid=5168902&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-243-4_22 Increased metabolic activity is a hallmark of proliferating cancer cells. One common deregulated metabolic pathway in prostate cancer is de novo lipogenesis which is highly increased in prostate cancer and is linked to poor prognosis and metastasis. Male sex hormones play an essential role in prostate cancer growth and have been shown to increase the expression and activity of several lipogenic factors, such as fatty acid synthase (FASN) and sterol regulatory element-binding proteins (SREBPs), leading to accumulation of neutral lipids in prostate cancer cells. These factors are being evaluated as potential prognostic markers and therapeutic targets in prostate cancer. Here we describe methods to directly detect and quantify accumulation of neutral lipids and assess concomitant changes in l... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5168902 Sun, 28 Aug 2011 01:40:52 +0100 5168902 http://www.medworm.com/index.php?rid=5168901&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-243-4_21 Much evidence indicates that, with few exceptions, non-genomic actions of steroids are mediated by receptors universally known as nuclear receptors. Steroid receptors do not exhibit intrinsic tyrosine kinase activity. Nevertheless, they stimulate different signaling pathways in cytoplasm of target cells, including those dependent on Src, a cytoplasmic tyrosine kinase. Steroid-induced Src activation regulates cell cycle progression, survival, migration, and associated processes, such as cell growth and differentiation. Androgen stimulation of human prostate cancer-derived LNCaP cells triggers cell cycle progression and proliferation. The key event in this process is the association of androgen receptor (AR) with Src. This association triggers activation of the Src/Ras/Erk pathway and finall... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5168901 Sun, 28 Aug 2011 01:40:51 +0100 5168901 http://www.medworm.com/index.php?rid=5168900&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-243-4_9 Androgen receptor (AR) is a ligand-dependent transcription factor that belongs to the nuclear receptor superfamily. In the presence of its specific ligands, AR translocates into the nucleus, interacts with chromatin at hormone response elements (HREs) and recruits a variety of coregulators and basal transcription factors to regulate transcription. Using green fluorescent protein (GFP) labelling and the tandem gene array system of mouse mammary tumour virus (MMTV), the interaction of AR with HREs can be visualized and studied in live cells. The MMTV array in nuclei can be specifically detected by DNA fluorescence in situ hybridization (DNA FISH) and thereby specific binding of GFP-AR to the array can be confirmed in the presence of specific ligands. The transcriptional activity of GFP-AR at... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=5168900 Sun, 28 Aug 2011 01:40:51 +0100 5168900 http://www.medworm.com/index.php?rid=4794237&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-120-8_12 To understand the links between CFTR mutations and the development of cystic fibrosis (CF) phenotypes, it is imperative to study the transcriptome in affected cell types. Microarray expression profiling provides a platform to study global gene expression in detail. This approach may provide the necessary information to segregate phenotypic characteristics of CF, differentiate between genetic or environmental factors, and assess the advent and progression of disease phenotypes. Moreover, if a “CF signature” of genes with altered expression is defined, this can be used to monitor effectiveness of treatment. We provide here detailed protocols and tips for collecting and preserving tissues and cells, and preparing total RNA. We also outline novel strategies for experimental design ... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4794237 Sat, 07 May 2011 09:02:41 +0100 4794237 http://www.medworm.com/index.php?rid=4794236&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-120-8_11 Cystic fibrosis (CF) is a disease that manifests itself in the context of cell, tissue, and organismal (patho)physiology. While a strong focus on the cystic fibrosis transmembrane conductance regulator (CFTR) since its discovery in 1989 has dominated the field with a wealth of experiments that have provided substantial insight into protein function and structure, a largely untapped area of high relevance to both our basic understanding of CFTR function and its role in clinical disease is the realization that CFTR operates in the context of a cellular network. This is a composite of protein–protein interactions and specific cellular and subcellular environments that balance ion conductance at the cell surface with trafficking through the exocytic and endocytic pathways to promote tiss... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4794236 Sat, 07 May 2011 09:02:41 +0100 4794236 http://www.medworm.com/index.php?rid=4794235&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-120-8_9 Many bacteria can be detected in CF sputum, pathogenic and commensal. Modified Koch’s criteria for identification of established and emerging CF pathogens are therefore described. Methods are described to isolate bacteria and to detect bacterial biofilms in sputum or lung tissue from CF patients by means of conventional culturing and staining techniques and by the PNA FISH technique. Additionally, the confocal scanning laser microscopy technique is described for studying biofilms in vitro in a flow cell system. The recA-gene PCR and the RFLP-based identification methods are described for identification of isolates from the Burkholderia complex to the species level. DNA typing by PFGE, which can be used for any bacterial pathogen, is described as it is employed for Pseudomonas aerugin... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4794235 Sat, 07 May 2011 09:02:41 +0100 4794235 http://www.medworm.com/index.php?rid=4794234&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-120-8_8 We describe methods for the general quantification of heavily glycosylated proteins as the mucins using dot/slot blot. We also describe the separation of the mucins by gel electrophoresis and the identification with specific antibodies on Western blot and by proteomics. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4794234 Sat, 07 May 2011 09:02:41 +0100 4794234 http://www.medworm.com/index.php?rid=4794233&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-120-8_7 The airways are continuously challenged by a variety of stimuli including bacteria, viruses, allergens, and inflammatory factors that act as agonists for G protein-coupled receptors (GPCR). Intracellular calcium (Ca2+ i) mobilization in airway epithelia in response to extracellular stimuli regulates key airway innate defense functions, e.g., Ca2+-activated Cl− secretion, ciliary beating, mucin secretion, and inflammatory responses. Because Ca2+ i mobilization in response to luminal stimuli is larger in CF vs. normal human airway epithelia, alterations in Ca2+ i signals have been associated with the pathogenesis of CF airway disease. Hence, assessment of Ca2+ i signaling has become an important area of CF research. This chapter will focus on measurement... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4794233 Sat, 07 May 2011 09:02:41 +0100 4794233 http://www.medworm.com/index.php?rid=4794232&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-120-8_6 Human airways are kept sterile by a mucosal innate defense system that includes mucus secretion. Mucus is secreted in healthy upper airways primarily by submucosal glands and consists of defense molecules mixed with mucins, electrolytes, and water and is also a major component of sputum. Mucus traps pathogens and mechanically removes them via mucociliary clearance while inhibiting their growth via molecular (e.g., lysozyme) and cellular (e.g., neutrophils, macrophages) defenses. Fluid secretion rates of single glands in response to various mediators can be measured by trapping the primary gland mucus secretions in an oil layer, where they form spherical bubbles that can be optically measured at any desired interval to provide detailed temporal analysis of secretion rates. The composition a... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4794232 Sat, 07 May 2011 09:02:40 +0100 4794232 http://www.medworm.com/index.php?rid=4794231&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-120-8_5 The healthy human respiratory tract is lined with a pseudostratified epithelia composed of ∼80% ciliated cells and ∼20% goblet cells. These cells produce and are bathed by a layer of airway surface liquid (ASL), which plays a critical role in lung defense by helping to maintain the sterility of the lung. This layer is composed of two phases: the mucus layer which functions to trap particulates, bacteria, and viruses, and the underlying periciliary liquid layer (PCL), which provides hydration, enabling mucus transport and clearance. This chapter describes the methods used to measure the structure and height of the ASL by XZ confocal microscopy and mucus transport rates using epifluorescent microscopy in live airway cultures. Furthermore, we also demonstrate that these methods are al... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4794231 Sat, 07 May 2011 09:02:40 +0100 4794231 http://www.medworm.com/index.php?rid=4794230&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-120-8_4 Cystic fibrosis is characterized by excessive pulmonary inflammation, which presents early in life and becomes self-sustaining, eventually leading to the destruction of the lung. Treating inflammation is one of the most pressing needs in CF therapy and has been shown to slow lung function deterioration. However, it remains unclear whether excessive inflammation is a direct result of CFTR dysfunction, and thus innate, or develops in response to early stimulation of inflammatory pathways. Here, we will discuss clinically relevant studies and the methods employed by them. We will focus on investigations in cell and animal models as well as patients. Our discussion will describe the character of pulmonary inflammation in CF and present potential therapeutic approaches that can ameliorate exces... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4794230 Sat, 07 May 2011 09:02:40 +0100 4794230 http://www.medworm.com/index.php?rid=4794229&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-120-8_3 Cystic fibrosis (CF) is a lethal genetic disorder, characterized by both clinical and genetic complexities, and arises as a result of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The gene encodes a Cl− channel belonging to the ABC (ATP Binding Cassette) superfamily of transporters. The members of this superfamily use ATP hydrolysis to fulfill their function as active transporters. So far, CFTR is the only member of this family to function as a cAMP-activated Cl− channel. Intense research following the cloning of the CFTR gene has extended the role of the CFTR beyond that of a Cl− channel. One of the best recognized, yet still controversial, functions of the CFTR is its ability to modulate the functioning of other transporters. The modu... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4794229 Sat, 07 May 2011 09:02:40 +0100 4794229 http://www.medworm.com/index.php?rid=4794228&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-120-8_2 CFTR functions as a chloride channel at the apical membrane of airway, gastrointestinal, and other epithelial cells. Immunofluorescence microscopy is commonly used to assess the subcellular localization and relative abundance of CFTR. Visualization of heterologously overexpressed CFTR is typically unproblematic and straightforward, whereas detection of small quantities of endogenous CFTR in tissues can be challenging and requires highly specific antibodies and optimized staining protocols. CFTR tagged by green fluorescent protein can be employed to study trafficking in live cells. Tagging of CFTR with an extracellular epitope permits detection exclusively at the cell surface and subsequent chasing allows visualization of endocytic trafficking. (Source: Springer protocols feed by Molecular ... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4794228 Sat, 07 May 2011 09:02:40 +0100 4794228 http://www.medworm.com/index.php?rid=4794227&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-120-8_1 Mutation of the CFTR chloride channel was identified as the genetic basis of cystic fibrosis over 20 years ago; however, correlation of the pathophysiological changes occurring in CF lung disease with the mutation of a chloride channel is ongoing. The failure of innate lung defense in CF, and the subsequent cyclical microbial colonization of airways, explains the gross anatomical changes that occur in CF pathophysiology. However, ongoing research is focused on how the lack of the CFTR channel explains the failure of innate lung defense. Hydration status of the mucus blanket is key to understanding this link, and this series of chapters details the recent progress that has been made in understanding the interplay between ion transport activity and innate lung defense, and the initiation of ... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4794227 Sat, 07 May 2011 09:02:40 +0100 4794227 http://www.medworm.com/index.php?rid=4794226&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-120-8_10 One of the main functions of the airway mucosa is to maintain a mechanical barrier at the air–surface interface and to protect the respiratory tract from external injuries. Differentiation of human airway epithelial cells (hAECs) to polarized airway mucosa can be reproduced in vitro by culturing the cells on microporous membrane at the air–liquid interface. Here, we describe approaches to study differentiation as well as repair of the hAECs by using a commercially available airway cell culture model called MucilAir™. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4794226 Sat, 07 May 2011 09:02:40 +0100 4794226 http://www.medworm.com/index.php?rid=4794225&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-120-8_13 Differential proteomics represents an enticing strategy to unmask the proteins involved in CF pathogenesis and to discover potential therapeutic targets and/or markers of disease progression. Quantitative proteomics is possible nowadays owing to the recent progress in protein labelling and/or in label-free approaches, combined to sensitive detection by mass spectrometry (MS). In this chapter, we present one strategy to perform differential quantitative proteomic studies on different cellular compartments of proliferating cell lines expressing wild-type (WT) CFTR and F508del-CFTR using stable isotope labelling in cell culture (SILAC). (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4794225 Sat, 07 May 2011 09:02:39 +0100 4794225 http://www.medworm.com/index.php?rid=4794224&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-120-8_14 The cystic fibrosis transmembrane conductance regulator (CFTR) does not function in isolation, but rather in a complex network of protein–protein interactions that dictate the physiology of a healthy cell and tissue and, when defective, the pathophysiology characteristic of cystic fibrosis (CF) disease. To begin to address the organization and operation of the extensive cystic fibrosis protein network dictated by simultaneous and sequential interactions, it will be necessary to understand the global protein environment (the proteome) in which CFTR functions in the cell and the local network that dictates CFTR folding, trafficking, and function at the cell surface. Emerging mass spectrometry (MS) technologies and methodologies offer an unprecedented opportunity to fully characterize b... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4794224 Sat, 07 May 2011 09:02:39 +0100 4794224 http://www.medworm.com/index.php?rid=4794223&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-120-8_15 As several genomes have been sequenced, post-genomic approaches like transcriptomics and proteomics, identifying gene products differentially expressed in association with a given pathology, have held promise both of understanding the pathways associated with the respective disease and as a fast track to therapy. Notwithstanding, these approaches cannot distinguish genes and proteins with mere secondary pathological association from those primarily involved in the basic defect(s). New global strategies and tools identifying gene products responsible for the basic cellular defect(s) in CF pathophysiology currently being performed are presented here. These include high-content screens to determine proteins affecting function and trafficking of CFTR and ENaC. (Source: Springer protocols feed ... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4794223 Sat, 07 May 2011 09:02:39 +0100 4794223 http://www.medworm.com/index.php?rid=4794222&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-120-8_16 Lipid analysis has been a crucial source of information in cystic fibrosis (CF). New methodologies for qualitative and quantitative lipidomics allow evaluation of a large number of samples, of special interest in patient screening for diagnostic and prognostic biological markers, as well as in cell physiology. In this chapter, two new complementary approaches are described: matrix-assisted laser desorption coupled to time of flight (MALDI-TOF-ClinProTools™) and liquid chromatography coupled to ion trap mass spectrometry (LC-MS n ). MALDI-TOF-ClinProTools™ offers a large unbiased screening for the discovery of potential lipid alterations in diseased patients. LC-MS n represents a state-of-the-art lipidomic tool for the identification and quanti... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4794222 Sat, 07 May 2011 09:02:39 +0100 4794222 http://www.medworm.com/index.php?rid=4794221&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-120-8_17 This section of Cystic Fibrosis Protocols and Diagnosis focuses on resources available to facilitate the activities of the research community in the field of cystic fibrosis (CF). An overview of the protocols and resources described in subsequent chapters of this book section is provided, as well as how they can accelerate research in this area. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4794221 Sat, 07 May 2011 09:02:39 +0100 4794221 http://www.medworm.com/index.php?rid=4794220&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-120-8_19 Animal models of human diseases are critical for dissecting mechanisms of pathophysiology and developing therapies. In the context of cystic fibrosis (CF), mouse models have been the dominant species by which to study CF disease processes in vivo for the past two decades. Although much has been learned through these CF mouse models, limitations in the ability of this species to recapitulate spontaneous lung disease and several other organ abnormalities seen in CF humans have created a need for additional species on which to study CF. To this end, pig and ferret CF models have been generated by somatic cell nuclear transfer and are currently being characterized. These new larger animal models have phenotypes that appear to closely resemble human CF disease seen in newborns, and efforts to c... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4794220 Sat, 07 May 2011 09:02:39 +0100 4794220 http://www.medworm.com/index.php?rid=4794219&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-120-8_18 When primary human airway epithelial (hAE) cells are grown in vitro on porous supports at an air–liquid interface (ALI), they recapitulate in vivo morphology and key physiologic processes. These cultures are useful for studying respiratory tract biology and diseases and for testing new cystic fibrosis (CF) therapies. This chapter gives protocols enabling creation of well-differentiated primary CF and non-CF airway epithelial cell cultures with non-proprietary reagents. We also discuss the production of retroviral and lentiviral vectors, the derivation of hAE cell lines, reporter gene assays, and the evolving science of gene overexpression and knockdown in ALI hAE cultures. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4794219 Sat, 07 May 2011 09:02:39 +0100 4794219 http://www.medworm.com/index.php?rid=4794218&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-120-8_20 The CFTR Folding Consortium (CFC) was formed in 2004 under the auspices of the Cystic Fibrosis Foundation and its drug discovery and development affiliate, CFF Therapeutics. A primary goal of the CFC is the development and distribution of reagents and assay methods designed to better understand the mechanistic basis of mutant CFTR misfolding and to identify targets whose manipulation may correct CFTR folding defects. As such, reagents available from the CFC primarily target wild-type CFTR NBD1 and its common variant, F508del, and they include antibodies, cell lines, constructs, and proteins. These reagents are summarized here, and two protocols are described for the detection of cell surface CFTR: (a) an assay of the density of expressed HA-tagged CFTR by ELISA and (b) the generation and u... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4794218 Sat, 07 May 2011 09:02:39 +0100 4794218 http://www.medworm.com/index.php?rid=4794217&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-120-8_21 Over 1600 novel sequence variants in the CFTR gene have been reported to the CF Mutation Database (http://www.genet.sickkids.on.ca/cftr/Home.html). While about 25 mutations are well characterized by clinical studies and functional assays, the disease liability of most of the remaining mutations is either unclear or unknown. This gap in knowledge has implications for diagnosis, therapy selection, and counseling for patients and families carrying an uncharacterized CFTR mutation. This chapter will describe a critical approach to assessing the disease implications of CFTR mutations utilizing clinical data, literature review, functional testing, and bioinformatic in silico methods. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4794217 Sat, 07 May 2011 09:02:39 +0100 4794217 http://www.medworm.com/index.php?rid=4682895&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-055-3_9 Over the past decade, utilization of microarray technology has flourished in biomedical research. It has evolved rapidly into a revolutionary tool that offers deeper insight into the molecular basis associated with complex diseases, especially in the field of cancer. Specifically, array-based Comparative Genomic Hybridization (aCGH) permits the detection of genome-wide copy number alterations with high resolution. Microarray application to DNA extracted from formalin-fixed paraffin-embedded tissue (FFPE), in particular, poses a challenge due to the partially degraded nature and compromised quality of the DNA. This chapter gives a description of the several CGH-microarray platforms currently available and offers practical steps that guide you through optimal handling and superior aCGH data ... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4682895 Thu, 07 Apr 2011 15:29:03 +0100 4682895 http://www.medworm.com/index.php?rid=4682894&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-055-3_8 Tissue heterogeneity is a common source of unsuccessful experiments. Laser capture microdissection is a tool to prepare homogeneous tissue and cell areas as starting material for reliable and reproducible results as it allows the defined investigation of spatially different tissue areas. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4682894 Thu, 07 Apr 2011 15:29:03 +0100 4682894 http://www.medworm.com/index.php?rid=4682893&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-055-3_7 Laser capture microdissection (LCM) enables researchers to combine structure identification by ­microscopy with structure investigation by modern molecular techniques. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4682893 Thu, 07 Apr 2011 15:29:03 +0100 4682893 http://www.medworm.com/index.php?rid=4682892&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-055-3_6 The HER2/neu gene (also known as ERBB2) is located on chromosome 17 (q11.2–q12) and encodes a glycoprotein known to be a member of the epidermal growth factor receptor family. Clinically, the determination of its amplification status is of utmost importance, as 10–35% of invasive human breast carcinomas come along with HER2/neu overexpression, and treatment has to be adjusted accordingly. Here a method to analyze HER2 FISH samples with digital microscopy, using the slide scanning ­platform Metafer PV (MetaSystems, Altlussheim, Germany), is presented. Metafer PV is a system for the automation of HER2/neu FISH assay analysis of samples hybridized with the Abbott™ PathVysion® probe kit. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4682892 Thu, 07 Apr 2011 15:29:03 +0100 4682892 http://www.medworm.com/index.php?rid=4682891&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-055-3_5 We describe a protocol for CISH in which a microwave-induced target retrieval step is introduced as a replacement for heat pretreatment. The same procedure is performed following enzyme digestion to produce consistent signals in amplified and nonamplified cells that are both larger in size and numbers when compared with those produced by the conventional protocol. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4682891 Thu, 07 Apr 2011 15:29:03 +0100 4682891 http://www.medworm.com/index.php?rid=4682890&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-055-3_4 Multiple immunofluorescent labeling of formalin-fixed paraffin-embedded (FFPE) tissue is not a routinely used method. At least in part, this is due to the perception that the innate autofluorescence of the FFPE material forbids the use of immunofluorescent labeling. As a result, immunohistochemical (immunoperoxidase) staining of FFPE material or cryosectioning methods is used instead. In this chapter, we describe a robust optimized method for high-resolution immunofluorescence labeling of FFPE tissue that involves the combination of antigen retrieval, indirect immunofluorescence, and confocal laser scanning microscopy. Once such samples have been prepared and imaged by confocal microscopy, they can be stored at −20°C for extensive periods (>250 days) and reexamined with minima... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4682890 Thu, 07 Apr 2011 15:29:03 +0100 4682890 http://www.medworm.com/index.php?rid=4682889&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-055-3_3 The rapid acceptance of immunohistology as an invaluable adjunct to morphologic diagnosis has been possible because of the development of new and more sensitive antibodies and detection systems that allow its application to formalin-fixed, paraffin-embedded tissue (FFPT). More importantly, antigen-retrieval techniques have resulted in some degree of consistency allowing immunohistology to be used reliably as a diagnostic tool. The advent of prognostic and predictive biomarkers, and the desire for individualized therapy has resulted in mounting pressure to employ the immunohistological assay in a quantitative manner. While it was not a major issue when the technique was employed in a qualitative manner, the numerous variables in the preanalytical and analytical phases of the test procedure ... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4682889 Thu, 07 Apr 2011 15:29:02 +0100 4682889 http://www.medworm.com/index.php?rid=4682888&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-055-3_2 Tissue microarrays (TMAs) are produced by taking small punches from a series of paraffin-embedded (donor) tissue blocks and transferring these tissue cores into a positionally encoded array in a recipient paraffin block. Though TMAs are not used for clinical diagnosis, they have several advantages over using conventional whole histological sections for research. Tissue from multiple patients or blocks can be examined on the same slide, and only a very small amount of reagent is required to stain or label an entire array. Multiple sections (100–300) can be cut from a single array block, allowing for hundreds of analyses per microarray. These advantages allow the use of TMAs in high-throughput procedures, such as screening antibodies for diagnostics and validating prognostic markers th... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4682888 Thu, 07 Apr 2011 15:29:02 +0100 4682888 http://www.medworm.com/index.php?rid=4682887&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-055-3_1 Formalin-fixed, paraffin-embedded (FFPE) archival tissues and their associated diagnostic records represent an invaluable source of information on diseases where the patient outcomes are already known. Older archives contain many unique FFPE tissue specimens that would be impossible to replicate today due to changes in medical practice and technology. Unfortunately, there is no single regulatory or bioethical standard that covers research with FFPE tissue specimens. This makes it difficult for researchers to prepare protocols involving FFPE tissues and equally difficult for Institutional Review Boards to evaluate them. In this review, focused on US regulatory policy, the application of the Common Rule and the Privacy Rule of the Health Insurance Portability and Accountability Act to resear... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4682887 Thu, 07 Apr 2011 15:29:02 +0100 4682887 http://www.medworm.com/index.php?rid=4682886&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-055-3_19 Molecular analysis of tissue lesions is increasingly used in laboratories to identify new prognostic and therapeutic markers. Formalin has long been the tissue fixative of choice in the laboratories of pathology, as it preserves tissue morphology allowing accurate histological diagnosis. However, formalin is highly toxic and alters nucleic acids and protein integrity, so that new fixatives are critically needed that would allow both morphological and molecular analysis on the same tissue specimen. Recently, we found RCL2®-CS100, a noncross-linking fixative, to display interesting performances regarding tissue morphology and DNA, RNA, and protein quality. We adapted RCL2 tissue fixation protocol so it could be used on a routine and automated laboratory basis, still preserving its good p... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4682886 Thu, 07 Apr 2011 15:29:02 +0100 4682886 http://www.medworm.com/index.php?rid=4682885&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-055-3_18 Formalin-fixed, paraffin-embedded (FFPE) tissue archives and their associated diagnostic records represent an invaluable source of proteomic information on diseases where the patient outcomes are already known. Over the last few years, advances in methodology have made it possible to recover peptides from FFPE tissues that yield a reasonable representation of the proteins recovered from identical fresh or frozen specimens. These new methods, based largely upon heat-induced antigen retrieval techniques borrowed from immunohistochemistry, have developed sufficiently to allow at least a qualitative analysis of the proteome of FFPE archival tissues. This chapter describes the approaches for performing proteomic analysis on FFPE tissues by liquid chromatography and mass spectrometry. (Source: S... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4682885 Thu, 07 Apr 2011 15:29:02 +0100 4682885 http://www.medworm.com/index.php?rid=4682884&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-055-3_17 Gene expression profiling of RNA isolated from formalin fixed, paraffin-embedded (FFPE) tissue samples has been historically challenging. Yet FFPE samples are sought-after because of the in-depth retrospective records typically associated with them rendering these samples a valuable resource for translational medicine studies. Extensive degradation, chemical modifications, and cross-linking have made it difficult to isolate RNA of sufficient quality required for large-scale gene expression profiling studies. NuGEN Technologies’ WT-Ovation™ FFPE System linearly amplifies RNA from FFPE samples through a robust and simple whole-transcriptome approach using as little as 50 ng total RNA isolated from FFPE samples. The amplified material may be labeled with validated kits and/or prot... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4682884 Thu, 07 Apr 2011 15:29:02 +0100 4682884 http://www.medworm.com/index.php?rid=4682883&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-055-3_16 In this study, we prepared miRNA preserving total RNA from matched pairs of FFPE and respective fresh-frozen clinical samples, and used that in microarray experiments to compare miRNA expression profiles between FFPE and fresh-frozen tissue from the same tissue samples. We demonstrate that miRNA expression profile from FFPE tissues closely resembles that from fresh tissues. These results underscore the suitability of FFPE tissues as appropriate resources for miRNA expression analyses. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4682883 Thu, 07 Apr 2011 15:29:02 +0100 4682883 http://www.medworm.com/index.php?rid=4682882&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-055-3_15 A molecular test providing clear identification of individuals at highest risk for developing metastatic disease from among early stage breast cancer patients has proven to be of great benefit in breast cancer treatment planning and therapeutic management. Patients with high risk of disease recurrence can also get an estimate of the magnitude of benefit to be gained by adding chemotherapy to surgery and hormonal therapy. Developing this clinical test was made possible by the availability of technologies capable of identifying molecular biomarkers from the gene expression profiles of preserved surgical specimens. Molecular tests such as the Oncotype DX® breast cancer test are proving to be more effective tools for individualized patient stratification and treatment planning than traditi... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4682882 Thu, 07 Apr 2011 15:29:02 +0100 4682882 http://www.medworm.com/index.php?rid=4682881&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-055-3_14 Although RNA is isolated from archival fixed tissues routinely for reverse transcription polymerase chain reaction (RT-PCR) and microarray analyses to identify biomarkers of cancer prognosis and therapeutic response prediction, the sensitivity of these molecular profiling methods to variability in pathology tissue processing has not been described in depth. As increasing numbers of expression analysis studies using fixed archival tumor specimens are reported, it is important to examine how dependent these results are on tissue-processing methods. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4682881 Thu, 07 Apr 2011 15:29:02 +0100 4682881 http://www.medworm.com/index.php?rid=4682880&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-055-3_13 We describe in this chapter a detailed protocol for MethyLight and its use in the determination of CpG Island Methylator Phenotype status in FFPE colorectal cancer samples. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4682880 Thu, 07 Apr 2011 15:29:02 +0100 4682880 http://www.medworm.com/index.php?rid=4682879&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-055-3_12 Gene promoter hypermethylation is recognised as an important mechanism by which genes may be silenced both physiologically and in disease states. This mechanism of gene silencing has been shown to play a role in many common human tumours. A number of methods are available for the detection of promoter hypermethylation, including the methylation-specific polymerase chain reaction (PCR), bisulphite sequencing, and pyrosequencing. Pyrosequencing is a reproducible method for obtaining data on the methylation status of DNA. It also has the advantage of providing quantitative data regarding the amount of methylation present in multiple CpGs in a given sample. The technique is based on the bisulphite conversion of unmethylated cytosine to uracil and subsequent amplification by PCR. The technique ... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4682879 Thu, 07 Apr 2011 15:29:02 +0100 4682879 http://www.medworm.com/index.php?rid=4682878&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-055-3_11 Whole genome amplification systems were developed to meet the increasing research demands on DNA resources and to avoid DNA shortage. The technology enables amplification of nanogram amounts of DNA into microgram quantities and is increasingly used in the amplification of DNA from multiple origins such as blood, fresh frozen tissue, formalin-fixed paraffin-embedded tissues, saliva, buccal swabs, bacteria, and plant and animal sources. This chapter focuses on the use of GenomePlex® tissue Whole Genome Amplification Kit, to amplify DNA directly from archived tissue. In addition, this chapter documents our unique experience with the utilization of GenomePlex® amplified DNA using several molecular techniques including metaphase Comparative Genomic Hybridization, array Comparative Genom... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4682878 Thu, 07 Apr 2011 15:29:02 +0100 4682878 http://www.medworm.com/index.php?rid=4682877&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-61779-055-3_10 High-density oligonucleotide microarrays are commonly used for GWAS studies as well as for tumor genome alteration identifications. The recent Affymetrix Genome-Wide SNP 6.0 microarray generation has two major advantages: (1) showing high genome coverage and (2) starting with very small amount of DNA material. The hybridization protocol needs to be standardized and highly reproducible, as DNA is first digested by restriction enzymes and then PCR-amplified to reduce genome complexity. Especially the restriction digestion step is highly sensitive to degradation of the initial material. The stronger the sample is degraded, the lower the number of restriction sites still present in the genome, and hence the less-efficient amplification step. (Source: Springer protocols feed by Molecular Medici... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4682877 Thu, 07 Apr 2011 15:29:02 +0100 4682877 http://www.medworm.com/index.php?rid=4682901&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-803-4_4 The clinical effects of heparin are meritorious and heparin remains the anticoagulant of choice for most clinical needs. However, as with any drug, adverse effects exist. Heparin-induced thrombocytopenia (HIT) is an important adverse effect of heparin associated with amputation and death due to thrombosis. Although the diagnosis and treatment of HIT can be difficult and complex, it is critical that patients with HIT be identified as soon as possible to initiate early treatment to avoid thrombosis. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4682901 Sun, 29 Aug 2010 23:00:00 +0100 4682901 http://www.medworm.com/index.php?rid=4682900&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-803-4_5 Currently, there are several lines of evidence supporting the interplay between coagulation and inflammation in the propagation of various disease processes, including venous thromboembolism (VTE) and inflammatory diseases. Major advances in the development of oral anticoagulants have resulted in considerable progress toward the goal of safe and effective oral anticoagulants that do not require frequent monitoring or dose adjustment and have minimal food/drug interactions. Indirect inhibitors such as low-molecular-weight heparin (LMWH) and the pentasaccharide fondaparinux represent improvements over traditional drugs such as unfractionated heparin for acute treatment of VTE, constituting a more targeted anticoagulant approach with predictable pharmacokinetic profiles and no requirement for... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4682900 Sun, 29 Aug 2010 23:00:00 +0100 4682900 http://www.medworm.com/index.php?rid=4682899&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-803-4_7 Antiplatelet drugs represent a key class of drugs that are of proven value in arterial thromboembolic disorders. There is a need for effective, safe antiplatelet agents or their combinations to provide predictable therapeutic benefit, dosage flexibility, and unique pharmacologic profiles, such as rapid onset in acute thrombotic states, as well as sustained antiplatelet effects in chronic platelet-activating states (e.g., post-stent placement). Aspirin, clopidogrel, or their combination have shown improved clinical outcomes in certain unique settings, and the search for additional antiplatelet agents is ongoing. Current studies suggest that combination antiplatelet therapy with existing agents is best considered a use-adapted strategy, with the greatest clinical benefit of combination thera... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4682899 Sun, 29 Aug 2010 23:00:00 +0100 4682899 http://www.medworm.com/index.php?rid=4682898&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-803-4_6 Rivaroxaban is a small-molecule, direct factor Xa inhibitor that is under investigation for the prevention and treatment of venous and arterial thrombosis. To date, oral anticoagulants have been limited largely to vitamin K antagonists. Despite their remarkable benefits, vitamin K antagonists are limited by their narrow therapeutic window, the existence of multiple food and drug interactions, and the need for frequent monitoring and dose-adjustment. Rivaroxaban represents a potentially attractive alternative to warfarin, as it could enable simplified once-daily dosing, requires no therapeutic monitoring, and has a lower potential for drug interactions. At present, the safety and efficacy of rivaroxaban for the prophylaxis and treatment of venous thromboembolism has been evaluated in phase-... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4682898 Sun, 29 Aug 2010 23:00:00 +0100 4682898 http://www.medworm.com/index.php?rid=4682897&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-803-4_8 Novel adenosine diphosphate (ADP) P2Y12 antagonists such as prasugrel, ticagrelor, cangrelor, and elinogrel are in various phases of clinical development. These ADP P2Y12 antagonists have advantages over clopidogrel ranging from faster onset to greater and less variable inhibition of platelet function. Novel ADP P2Y12 antagonists are under investigation to determine whether their use can result in improved antiplatelet activity, faster onset of action, and/or greater antithrombotic effects than clopidogrel without an unacceptable increase in hemorrhagic or other side effects. Prasugrel (CS-747; LY-640315), a novel third-generation oral thienopyridine, is a specific, irreversible antagonist of the platelet ADP P2Y12 receptor. Pre-clinical and early phase clinical studies have shown prasugre... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4682897 Sun, 29 Aug 2010 23:00:00 +0100 4682897 http://www.medworm.com/index.php?rid=4682896&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60761-803-4_9 To date, there have been few systematic studies of the antiplatelet and/or anticoagulant effects of natural products. According to the Natural Medicines Comprehensive Database, approximately 180 dietary supplements have the potential to interact with warfarin, and more than 120 may interact with aspirin, clopidogrel, and dipyridamole. These include anise and dong quai (anticoagulant effects); omega 3-fatty acids in fish oil, ajoene in garlic, ginger, ginko, and vitamin E (antiplatelet properties); fucus (heparin-like activity); danshen (antithrombin III-like activity and anticoagulant bioavailability); and St. John’s Wort and American Ginseng (interference with drug metabolism). Other supplements, such as high doses of vitamin E (vitamin K antagonist activity), alfalfa (high-vitamin ... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=4682896 Sun, 29 Aug 2010 23:00:00 +0100 4682896 http://www.medworm.com/index.php?rid=2819092&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_9 The necessity of genotyping high number of variations in extended sample sets has become apparent in the era of large genomic studies of common complex disorders, in cancer and in pharmacogenomics. The single nucleotide polymorphisms’ (SNPs) apparent advantages over other genetic markers such as high frequency, relative stability, and statistically random distribution across the genome have made them a method of choice for most of these genome-wide oriented applications. The requirement for simultaneous genotyping of high number of SNPs, keeping at the same time reasonable price and reliable accuracy, triggered the rise of the genotyping throughput, and led to the development of the array-based technologies. The present chapter briefly reviews the methodological and historical aspect... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=2819092 Fri, 31 Jul 2009 23:00:00 +0100 2819092 http://www.medworm.com/index.php?rid=2819091&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_8 We describe here in detail a protocol for adenoviral gene transfer into isolated mouse islets of the pancreas. Effective gene transfer into pancreatic islets using recombinant adenoviruses can be achieved with a multiplicity of infection (MOI) of 10. However, if the islets are not dispersed, adenoviral gene transfer is limited only to the cells on the periphery of the islets, which represent the glucagon-producing alpha cells in rodents. Dispersion of pancreatic islets with EGTA increases the efficiency of gene transfer into the cells within the core of the islets, which consist of insulin-producing beta cells. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=2819091 Fri, 31 Jul 2009 23:00:00 +0100 2819091 http://www.medworm.com/index.php?rid=2819090&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_7 In situ hybridization represents a powerful technique to localize DNA or RNA of interest at the chromosomal or cellular level. In endocrine tissues composed of diverse and varied cell types, in situ hybridization has allowed the identification of specific cells responsible for the expression of genes controlling the function of the tissue. Our laboratory has routinely used this approach to understand the cellular expression of genes associated with the growth of the ovarian follicle, rupture of the follicle, and transformation of the ruptured follicle into the corpus luteum. The current study outlines the procedural details of in situ detection of mRNA in tissues and illustrates the utility of this approach in identifying the ovarian cells expressing the matrix metalloproteinases and their... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=2819090 Fri, 31 Jul 2009 23:00:00 +0100 2819090 http://www.medworm.com/index.php?rid=2819089&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_6 Microarray technology has been used widely in gynecology. Numerous studies have used this method to address biological questions related to human endometrium. The cyclic changes of endometrium confer special characteristics that should be considered before genomic analysis. The present study reviews these considerations and the principles of transcriptomic analysis through an example of a comparison of three different phases of the menstrual cycle. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=2819089 Fri, 31 Jul 2009 23:00:00 +0100 2819089 http://www.medworm.com/index.php?rid=2819088&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_5 DNA microarray is an important discovery technology that allows the analysis of the expression of thousands of genes at a time. Data from DNA microarrays elucidate fundamental biological processes through discovery of differential expression of genes not previously known or predicted to be involved in a particular process. In the ovary and other hormone-responsive tissues, the technology can be used to examine the effects of gene mutations, pharmaceutical agents, disease, hormones, developmental changes, or changes in gene expression related to aging. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=2819088 Fri, 31 Jul 2009 23:00:00 +0100 2819088 http://www.medworm.com/index.php?rid=2819087&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_4 Lipid second messengers play important roles in many cell signaling cascades. Lipid signaling molecules allow for high specificity, rapid transduction, and rapid reversibility of localized stimulation events. Fluorescent sensors capable of detecting individual signaling lipids enable their production and degradation to be followed, revealing the nature and dynamics of signaling pathways. The following sections outline a method for using lipid sensors to monitor the production of signaling lipids on the plasma membrane of C2C12 myotubes in response to insulin signaling. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=2819087 Fri, 31 Jul 2009 23:00:00 +0100 2819087 http://www.medworm.com/index.php?rid=2819086&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_3 Glucocorticoids regulate a broad spectrum of physiologic functions essential for life and play an important role in the maintenance of basal and stress-related homeostasis. At the cellular level, the actions of glucocorticoids are mediated by the human glucocorticoid receptor α (hGRα), a ligand-dependent transcription factor ubiquitously expressed in almost all tissues and cells. The molecular mechanisms of hGRα action involve (a) binding to glucocorticoids, (b) cytoplasmic to nuclear translocation, (c) binding/association to DNA/chromatin, and (d) transcriptional activation or repression by interacting with cofactors and other transcription factors. Mutations or polymorphisms in the hGR gene may impair these molecular mechanisms of hGRα action, thereby altering tis... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=2819086 Fri, 31 Jul 2009 23:00:00 +0100 2819086 http://www.medworm.com/index.php?rid=2819085&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_2 The ability to precisely measure circulating levels of hormones is a foundation of modern endocrinology. For assisted reproductive technologies such as in vitro fertilization (IVF), accurate determination of circulating levels of estradiol are crucial for patient management, retrieval of fertilizable oocytes, and successful pregnancy outcome. For many years, circulating levels of estradiol were determined by radioimmunoassay. More recently, nonradioactive techniques such as ELISAs or chemiluminescent approaches have replaced traditional radioimmunoassays. In the current chapter, we outline the procedures for analysis of circulating levels of estradiol by both radioimmunoassay and chemiluminescent techniques. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=2819085 Fri, 31 Jul 2009 23:00:00 +0100 2819085 http://www.medworm.com/index.php?rid=2819084&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_25 In recent years, genome-wide association approaches have proven a powerful and successful strategy to identify genetic contributors to complex traits, including a number of endocrine disorders. Their success has meant that genome wide association studies (GWAS) are fast becoming the default study design for discovery of new genetic variants that influence a clinical trait or phenotype. This chapter focuses on a number of key elements that require consideration for the successful conduct of a GWAS. Although many of the considerations are common to any genetic study, the greater cost, extreme multiple testing, and greater openness to data sharing require specific awareness and planning by investigators. In the section on designing a GWAS, we reflect on ethical considerations, study design, s... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=2819084 Fri, 31 Jul 2009 23:00:00 +0100 2819084 http://www.medworm.com/index.php?rid=2819083&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_24 Oxidative stress (OS) is an imbalance between the amount of reactive oxygen species (ROS) produced and the ability of the antioxidants to scavenge these. OS has been established as a major etiological cause of male infertility. High levels of ROS are harmful and cause damage to sperm nuclear DNA. Evaluation of OS-related damage to spermatozoa is therefore highly relevant in assisted reproductive techniques (ART) such as intracytoplasmic sperm injection (ICSI). ICSI is an effective therapy for severe male factor infertility that bypasses the majority of reproductive tract deficiencies. Despite the controversial findings in the existing literature, there is now enough evidence to show that sperm DNA damage is detrimental to reproductive outcomes. In addition to impairment of fertility, such ... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=2819083 Fri, 31 Jul 2009 23:00:00 +0100 2819083 http://www.medworm.com/index.php?rid=2819082&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_23 In recent years, evidence has emerged supporting the hypothesis that cancer is a stem cell disease. The cancer stem cell field was led by the discovery of leukemia stem cells (Tan, B.T., Park, C.Y., Ailles, L.E., and Weissman, I.L. (2006) The cancer stem cell hypothesis: a work in progress. Laboratory Investigation. 86, 1203–1207), and within the past few years cancer stem cells have been isolated from a number of solid tumor including those of breast and brain cancer among others (Al-Hajj M., Wicha M.S., Benito-Hernandez A., Morrison, S.J., and Clarke, M.F. (2003) Prospective identification of tumorigenic breast cancer cells. Proc. Natl. Acad. Sci. USA 100, 3983–3988; Singh, S.K., Clarke, I.D., Terasaki, M., Bonn, V.E., Hawkins, C., Squire, J., and Dirks, P.B. (2003) Id... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=2819082 Fri, 31 Jul 2009 23:00:00 +0100 2819082 http://www.medworm.com/index.php?rid=2819081&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_22 Introduction of foreign DNA into the mouse germ line is considered a major technical advancement in the fields of developmental biology and genetics. This technology now referred to as transgenic mouse technology has revolutionized virtually all fields of biology and provided new genetic approaches to model many human diseases in a whole animal context. Several hundreds of transgenic lines with expression of foreign genes specifically targeted to desired organelles/cells/tissues have been characterized. Further, the ability to spatio-temporally inactivate or activate gene expression in vivo using the “Cre-lox” technology has recently emerged as a powerful approach to understand various developmental processes including those relevant to molecular endocrinology. In this chapter,... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=2819081 Fri, 31 Jul 2009 23:00:00 +0100 2819081 http://www.medworm.com/index.php?rid=2819080&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_21 ES cells are a potential source for insulin producing cells (IPCs). However, two major handicaps are establishing reliable differentiation protocols and the lack of imaging techniques that allow monitoring of these cells post-transplantation. Here, we describe a new approach for monitoring the in vitro differentiation and real-time, non-invasive imaging of ES cell-derived IPCs in vivo. ES cells were molecularly engineered so that the rat insulin promoter (RIP) driven luciferase (Luc) expression was specifically turned on and up regulated following their differentiation into IPCs. The rationale underlying this approach is that the transcriptional activation of RIP leads to Luc expression in IPCs providing an extremely sensitive reporter for monitoring the earliest differentiation events in ... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=2819080 Fri, 31 Jul 2009 23:00:00 +0100 2819080 http://www.medworm.com/index.php?rid=2819079&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_20 During the past decade the remarkable progress in molecular genetics and the possibility to engineer cells to express genes reporting on the activity of specific promoters has produced major changes in biological research. The description and validation of reporter mice for non-invasive assessment of biological and biochemical processes in living subjects and the results obtained with the models reporting on the activity of estrogen and peroxisome proliferator receptors clearly showed that such technologies have the potential to enhance our understanding of disease and drug activity. Although reporter-gene technology is in its infancy, reporter animals already represent a valuable tool for biomedical investigation. The present chapter aims at critically illustrating the methodology to be a... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=2819079 Fri, 31 Jul 2009 23:00:00 +0100 2819079 http://www.medworm.com/index.php?rid=2819078&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_1 Characterization of the binding of a hormone to its cognate receptor is a cornerstone of many studies in molecular and cellular endocrinology since this event represents the beginning of a specific cellular response, generally from a highly regulated extracellular messenger. The premise of hormone−receptor interaction follows from the law of mass action describing a reversible second-order reaction, hormone plus receptor, to give a non-covalently associated hormone-receptor complex. From this basic principle, a host of useful experimental parameters are available to the interested investigator. This chapter is focused on development of the experimental and mathematical underpinning of hormone−receptor interaction, with emphasis on a gonadotropin, chorionic gonadotropin (or lute... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=2819078 Fri, 31 Jul 2009 23:00:00 +0100 2819078 http://www.medworm.com/index.php?rid=2819077&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_19 Endometriosis, a common gynecological disorder responsible for infertility and pelvic pain, is defined as the presence of endometrial glands and stroma within extra-uterine sites. Gene expression studies performed on endometriotic tissue homogenates have yielded results reflecting mRNA abundance in a mixture of cell types (including epithelial cells, stromal cells, fibrotic tissue, and muscle tissue). Therefore, a method for quantifying gene expression separately in individual cell populations is essential for identifying genetic markers. Laser capture microdissection is a technique for obtaining pure populations of cells from heterogeneous tissues. This chapter provides methods to obtain high-quality RNA suitable for a variety of different down stream applications from frozen endometrial ... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=2819077 Fri, 31 Jul 2009 23:00:00 +0100 2819077 http://www.medworm.com/index.php?rid=2819076&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_18 Many organisms, including humans, have many more proteins than are actually coded for by their genes. This discrepancy is partially explained by the existence of alternative transcripts produced by the same gene. Multiple isoforms of the same gene sometimes perform completely different functions, and as such, knowing the sequence of one of the transcripts is not enough. Rapid Amplification of cDNA Ends (RACE) provides an inexpensive and powerful tool to quickly identify alternative transcripts of a gene when the partial or complete sequence of only one transcript is known. In the following sections, we outline details for rapid amplification of 5’ and 3’ cDNA ends using the “New Race” technique. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=2819076 Fri, 31 Jul 2009 23:00:00 +0100 2819076 http://www.medworm.com/index.php?rid=2819075&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_17 Small ubiquitin-related modifier (SUMO) is an ubiquitin-like protein that is covalently attached to a variety of target proteins. Unlike ubiquitination, sumoylation does not target proteins for proteolytic breakdown, but is instead involved in regulating multiple protein functional properties including protein−protein interactions and subcellular targeting, to name a few. Protein sumoylation has been particularly well characterized as a regulator of many nuclear processes as well as nuclear structure, making the characterization of this modification vital for understanding nuclear structure and function. Consequently, there has been intense interest in identifying new proteins that are targets of this modification and determining what role it plays in regulating their functions. This... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=2819075 Fri, 31 Jul 2009 23:00:00 +0100 2819075 http://www.medworm.com/index.php?rid=2819074&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_16 Bioluminescent Resonance Energy Transfer is a naturally occurring phenomenon that can be exploited to explore protein−protein interactions in real-time in intact cells and cellular extracts. It detects energy transferred between a bioluminescent donor enzyme (Renilla luciferase) fusion protein and a fluorescent (GFP2, a mutant of Green Fluorescent Protein) acceptor fusion protein. Optimal detection of BRET2 energy transfer relies on the distance and orientation generated by the fusion proteins. This chapter describes in detail the BRET2 assay as it is used to examine the physical interaction between the nuclear receptor ERα and the transcriptional coactivator SRC-1. Description of methods include selection of donor and acceptor combinations, fusion construct generation and vali... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=2819074 Fri, 31 Jul 2009 23:00:00 +0100 2819074 http://www.medworm.com/index.php?rid=2819073&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_15 Chromatin immunoprecipitation (ChIP) is a well-established procedure for protein−DNA interaction research. ChIP-chip, combining chromatin immunoprecipitation (ChIP) and microarray technology (Chip), enables scientists to survey genome-wide DNA binding sites for a given protein. The ChIP-chip technique has been used to identify transcription factor binding sites, explore epigenomic information and investigate factors in DNA replicate/repairs. Here we describe a protocol for ChIP-chip to study Pituitary Tumor Transforming Gene (PTTG1) in mammalian cells. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=2819073 Fri, 31 Jul 2009 23:00:00 +0100 2819073 http://www.medworm.com/index.php?rid=2819072&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_14 In order to fully understand the functions of a DNA-binding protein it is necessary to identify all of its binding sites in chromosomes and assess the role of each site in the overall biological function of the factor. An approach ChIP-on-Chip which combines the chromatin immunoprecipitation technique with chromosomal DNA microarray analysis, has proven to be a powerful means for the chromosome-wide identification of protein binding sites. This approach can also be used to characterize chromosome-wide variations in patterns of post-translational protein modifications, for example histone modifications. This chapter presents methodologies for the ChIP-on-Chip analysis, using as an example the identification of chromosome-wide binding sites for the TATA-binding protein in mitotic cells. (Sou... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=2819072 Fri, 31 Jul 2009 23:00:00 +0100 2819072 http://www.medworm.com/index.php?rid=2819071&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_13 Regulating gene expression is a complex process requiring the interaction of multiple transcription factors with their cognate recognition sequences. While these DNA-bound transcription factors are the primary drivers of gene expression, the capacity of a transcription factor to alter gene expression is tempered by its association with a host of coregulatory proteins that are recruited to the DNA-bound transcription factor. We have developed a novel approach to isolate large complexes of proteins associated with the DNA-bound estrogen receptor α (ERα) using an agarose-based electrophoretic mobility shift assay (EMSA). This method should be readily adapted to a variety of cultured cell lines, DNA sequences, and transcription factors and has the potential to provide valuable info... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=2819071 Fri, 31 Jul 2009 23:00:00 +0100 2819071 http://www.medworm.com/index.php?rid=2819070&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_12 As a transcriptional regulator, the androgen receptor (AR) regulates the expression of many genes that are essential for male sexual differentiation, including the development of both normal prostate and prostate cancer. The AR acts by binding to regulatory DNA sequences found on the promoters of regulated genes. The study of AR activity on such responsive promoters is greatly facilitated by the use of the reporter gene assay, which provides a quantitative and reproducible method for studying the activity of such promoters. Among the several reporter genes that can be used, the genes encoding luciferase (Luc) and chloramphenicol acetyltransferase (CAT) have been used most widely and successfully by researchers interested in AR-regulated promoters. Such studies have led to the identificatio... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=2819070 Fri, 31 Jul 2009 23:00:00 +0100 2819070 http://www.medworm.com/index.php?rid=2819069&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_11 Use of reporter genes provides a convenient way to study the activity and regulation of promoters and examine the rate and control of gene transcription. Many reporter genes and transfection methods can be efficiently used for this purpose. To investigate gene regulation and signaling pathway interactions during ovarian follicle development, we have examined promoter activities of several key follicle-regulating genes in the mouse ovary. In this chapter, we describe use of luciferase and β-galactosidase genes as reporters and a cationic liposome mediated cell transfection method for studying regulation of activin subunit- and estrogen receptor α (ERα)-promoter activities. We have demonstrated that estrogen suppresses activin subunit gene promoter activity while activin inc... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=2819069 Fri, 31 Jul 2009 23:00:00 +0100 2819069 http://www.medworm.com/index.php?rid=2819068&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-60327-378-7_10 The methylated DNA immunoprecipitation microarray (MeDIP-chip) is a genome-wide, high-resolution approach to detect DNA methylation in whole genome or CpG (cytosine base followed by a guanine base) islands. The method utilizes anti-methylcytosine antibody to immunoprecipitate DNA that contains highly methylated CpG sites. Enriched methylated DNA can be interrogated using DNA microarrays or by massive parallel sequencing techniques. This combined approach allows researchers to rapidly identify methylated regions in a genome-wide manner, and compare DNA methylation patterns between two samples with diversely different DNA methylation status. MeDIP-chip has been applied successfully for analyses of methylated DNA in the different targets including animal and plant tissues (1, 2). Here we pres... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=2819068 Fri, 31 Jul 2009 23:00:00 +0100 2819068 http://www.medworm.com/index.php?rid=1902758&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_9 The mammalian inner ear has very limited ability to regenerate lost sensory hair cells. This deficiency becomes apparent when hair cell loss leads to hearing loss as a result of either ototoxic insult or the aging process. Coincidently, with this inability to regenerate lost hair cells, the adult cochlea does not appear to harbor cells with a proliferative capacity that could serve as progenitor cells for lost cells. In contrast, adult mammalian vestibular sensory epithelia display a limited ability for hair cell regeneration, and sphere-forming cells with stem cell features can be isolated from the adult murine vestibular system. The neonatal inner ear, however, does harbor sphere-forming stem cells residing in cochlear and vestibular tissues. Here, we provide protocols to isolate sphere-... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1902758 Fri, 24 Oct 2008 15:23:10 +0100 1902758 http://www.medworm.com/index.php?rid=1902757&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_8 The mammalian inner ear forms from a thickened patch of head ectoderm called the otic placode. The placodal ectoderm invaginates to form a cup whose edges cinch together to establish a fluid-filled sac called the otic vesicle or otocyst. The progenitor cells lining the otocyst lumen will give rise to sensory and non-sensory cells of the inner ear. These formative stages of inner ear development are initiated during the first week of postimplantation embryonic development in the mouse. The inaccessibility of the inner ear in utero has hampered efforts to gain insight into the molecular mechanisms regulating essential developmental processes. An experimental embryological method to misexpress genes in the developing mammalian inner ear is presented. Expression plasmid encoding a gene of inte... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1902757 Fri, 24 Oct 2008 15:23:10 +0100 1902757 http://www.medworm.com/index.php?rid=1902756&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_7 Helios® Gene Gun–mediated transfection is a biolistic method for mechanical delivery of exogenous DNA into cells in vitro or in vivo. The technique is based on bombardment of a targeted cellular surface by micron- or submicron-sized DNA-coated gold particles that are accelerated by a pressure pulse of compressed helium gas. The main advantage of Helios® Gene Gun–mediated transfections is that it functions well on various cell types, including terminally differentiated cells that are difficult to transfect, such as neurons or inner ear sensory hair cells, and cells in internal cellular layers, such as neurons in organotypic brain slices. The successful delivery of mRNA, siRNA, or DNA of practically any size can be achieved using biolistic transfection. This chapter provi... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1902756 Fri, 24 Oct 2008 15:23:09 +0100 1902756 http://www.medworm.com/index.php?rid=1902755&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_6 Gene manipulation, specifically in the hair cells of the inner ear during development and adulthood in mice, is crucial for understanding the physiology of hearing and the pathology of deafness in humans. Recent advances have demonstrated that gene expression can be manipulated in developing mouse hair cells in a spatially and temporally controlled manner. The Cre–loxP system has been widely used for such purposes. Many laboratories, including ours, have developed and characterized transgenic mouse lines that express or induce Cre activity specifically in inner ear hair cells. These Cre lines have been used with high efficiency to inactivate several genes such as Rb in hair cells. Here we discuss the use of these Cre lines in inner ear research with emphasis on practical issues for r... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1902755 Fri, 24 Oct 2008 15:23:09 +0100 1902755 http://www.medworm.com/index.php?rid=1902754&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_5 Greatly impacting the field of neural development are new technologies for generating fate maps in mice and thus for illuminating relationships between embryonic and adult brain structures. Until now, efforts in mammalian models such as the mouse have presented challenges because their in utero development limits the access needed for traditional methods involving tracer injection or cell transplantation. But access is no longer an obstacle. It is now possible to deliver cell lineage tracers via noninvasive genetic, rather than physical, means. The hinge-pin of these new “genetic fate mapping” technologies is a class of molecule called a site-specific recombinase. The most commonly used being Cre and Flp. Through the capacity to produce precise DNA excisions, Cre or Flp can act... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1902754 Fri, 24 Oct 2008 15:23:04 +0100 1902754 http://www.medworm.com/index.php?rid=1902753&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_4 To understand the mechanisms of development of the inner ear, it is important to know the lineal relationships among the different cell types and the migrational boundaries of individual clones within the inner ear. This chapter details the basic methods for performing lineage analysis of the inner ear using replication-defective retroviral vectors in chicken embryos. Protocols are provided for generating avian retroviruses pseudotyped with vesicular stomatitis virus (VSV) envelopes to improve infectivity in early embryos. Moreover, we include the pioneering methods of the Cepko laboratory, whereby a library of DNA tags was developed to allow clonal relationships to be confirmed by PCR amplification and sequencing of the tag in dispersed clonal progeny. By varying the site and time of vira... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1902753 Fri, 24 Oct 2008 15:23:04 +0100 1902753 http://www.medworm.com/index.php?rid=1902752&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_3 In situ hybridization is well suited to obtaining specific topological information on gene transcripts and thereby to relating such observations to a particular function. In spite of the technical and practical difficulties, the application of molecular biological techniques such as in situ hybridization to the cochlea can provide important insights. However, the rarity of gene products (mRNA and proteins) in the cochlea and its fragile structure require the refinement and adaptation of in situ hybridization methods. The present chapter provides a detailed in situ hybridization protocol adapted to frozen tissue sections collected from adult and neonatal stages of the vertebrate cochlea. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1902752 Fri, 24 Oct 2008 15:23:03 +0100 1902752 http://www.medworm.com/index.php?rid=1902751&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_24 The high sensitivity of mammalian hearing is achieved by amplification of the motion of the cochlear partition. The origin of this cochlear amplification is the elongation and contraction of outer hair cells (OHCs) in response to acoustical stimulation. This motility is made possible by a membrane protein embedded in the lateral membrane of OHCs. The gene of this protein has been identified and termed prestin. We, herein, present a method for observation by atomic force microscopy (AFM) of prestin expressed in the Chinese hamster ovary (CHO) cell plasma membrane. To obtain a stable sample for AFM imaging in liquid, we used as an example in the protocol provide here, CHO cells transfected with prestin or FLAG-tagged prestin, and untransfected CHO cells (1). The cells attached to a substrate... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1902751 Fri, 24 Oct 2008 15:23:03 +0100 1902751 http://www.medworm.com/index.php?rid=1902750&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_23 Regulation of important cellular functions via signaling pathways is a fundamental property of the cell. Intracellular Ca2+ is probably a best known second messenger in cell biology. In mechanosensory cells of the inner ear, the hair cells, intracellular Ca2+ participates in a variety of functions including mechano-electrical transduction, synaptic transmission, and efferent regulation of the outer hair cells, one of two types of hair cells in the mammalian cochlea. The outer hair cells are responsible for the amplification of sound-induced vibrations within the cochlea, which determines the sensitivity of mammalian hearing. Besides Ca2+, another intracellular ion, Cl- may have very specific function in the same outer hair cells. Intracellular Cl- is required for the motor function of pres... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1902750 Fri, 24 Oct 2008 15:23:02 +0100 1902750 http://www.medworm.com/index.php?rid=1902749&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_22 As more and more proteins specific to hair cells are discovered, it becomes imperative to understand their structure and how that contributes to their function. The fluorescence microscopic methods described here can be employed to provide information on protein-protein interactions, whether homomeric or heteromeric, and on protein conformation. Here, we describe two fluorescence microscopic methodologies applied to the outer hair cell-specific membrane protein prestin: the intensity and fluorescence lifetime (FLIM) variants of FRET (Fluorescence Resonance Energy Transfer), used in the study of protein-protein interactions, and the Scanning Cysteine Accessibility Method (SCAM), used for the determination of protein conformation. The methods are readily adaptable to other proteins. (Source:... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1902749 Fri, 24 Oct 2008 15:23:02 +0100 1902749 http://www.medworm.com/index.php?rid=1902748&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_21 Modern biologists have at their disposal a large array of techniques used to assess the existence and relative or absolute quantity of any molecule of interest in a sample. However, implementing most of these procedures can be a daunting task for the first time, even in a lab with experienced researchers. Just choosing a protocol to follow can take weeks while all of the nuances are examined and it is determined whether a protocol will (a) give the desired results, (b) result in interpretable and unbiased data, and (c) be amenable to the sample of interest. We detail here a robust procedure for labeling proteins in a complex lysate for the ultimate differential quantification of protein abundance following experimental manipulations. Following a successful outcome of the labeling procedure... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1902748 Fri, 24 Oct 2008 15:23:01 +0100 1902748 http://www.medworm.com/index.php?rid=1902747&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_20 Surface plasmon resonance is an optical technique utilized for detecting molecular interactions. Binding of a mobile molecule (analyte) to a molecule immobilized on a thin metal film (ligand) changes the refractive index of the film. The angle of extinction of light, reflected after polarized light impinges upon the film, is altered, monitored as a change in detector position for the dip in reflected intensity (the surface plasmon resonance phenomenon). Because the method strictly detects mass, there is no need to label the interacting components, thus eliminating possible changes of their molecular properties. We have utilized surface plasmon resonance to study the interaction of proteins of hair cells. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1902747 Fri, 24 Oct 2008 15:23:00 +0100 1902747 http://www.medworm.com/index.php?rid=1902746&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_2 Using gene arrays, it is currently possible to simultaneously measure mRNA levels of many genes in any tissue of interest. Undoubtedly, comprehensive measurements of gene expression as part of carefully designed experiments will continue to further our understanding of audition and have the potential to open up new avenues of research. This chapter describes a reliable protocol to prepare high-quality biotin-labeled RNA target, specifically for oligonucleotide array experiments. The procedure includes isolation of high-quality total RNA, synthesis of double-stranded cDNA engineered for in vitro transcription with T7 RNA polymerase, subsequent in vitro transcription in the presence of biotin-labeled ribonucleotides, and fractionation of the RNA to ∼ 500 bp fragments, suitable for... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1902746 Fri, 24 Oct 2008 15:22:57 +0100 1902746 http://www.medworm.com/index.php?rid=1902745&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_19 Expression of almost every gene is regulated at the transcription level. Therefore, transcriptional factor Transcription factors, consequently, have marked effects on the fate of a cell by establishing the gene expression patterns that determine biological processes. In the auditory and vestibular systems, transcription factors have been found to be responsible for development, cell growth, and apoptosis. It is vital to identify the transcription factor target genes and the mechanisms by which transcription factors control and guide gene expression and regulation pathways. Compared with earlier methods devised to study transcription factor–DNA interactions, the advantage of the chromatin immunoprecipitation (ChIP) assay is that the interaction of a transcription factor with its targe... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1902745 Fri, 24 Oct 2008 15:22:56 +0100 1902745 http://www.medworm.com/index.php?rid=1902744&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_18 Genomics has provided us with vast amounts of data and thus, the challenge to identify and characterize gene products. Proteomics analysis, using methods such as yeast two-hybrid screenings, isoelectric focusing, and mass spectroscopy, generate potentially useful information. To determine functional relationships between and among proteins, however, the initial data for putative protein interactions must first be validated. One technique, which is considered the gold standard, is coimmunoprecipitation. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1902744 Fri, 24 Oct 2008 15:22:56 +0100 1902744 http://www.medworm.com/index.php?rid=1902743&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_17 Structure-function studies of membrane proteins present a unique challenge to researchers due to the numerous technical difficulties associated with their expression, purification and structural characterization. In the absence of structural information, rational identification of putative functionally important residues/regions is difficult. Phylogenetic relationships could provide valuable information about the functional significance of a particular residue or region of a membrane protein. Evolutionary Trace (ET) analysis is a method developed to utilize this phylogenetic information to predict functional sites in proteins. In this method, residues are ranked according to conservation or divergence through evolution, based on the hypothesis that mutations at key positions should coincid... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1902743 Fri, 24 Oct 2008 15:22:55 +0100 1902743 http://www.medworm.com/index.php?rid=1902742&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_16 Functional proteomics comprises a wide range of technologies for the identification of novel protein-protein interactions and biological markers. Studies of protein-protein interactions have gained from the development of techniques and technologies such as immunoprecipitation, preparative two-dimensional (2-D) gel electrophoresis for peptide mass fingerprinting (PMF), using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). These applications enabled the discovery of putative protein partners without a priori knowledge of which one(s) might be relevant. Here, we report the methods by which membrane proteins are isolated from cochlear tissues and prepared for identification by mass spectro... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1902742 Fri, 24 Oct 2008 15:22:55 +0100 1902742 http://www.medworm.com/index.php?rid=1902741&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_15 We describe a protocol to screen for protein-protein interactions using the Gal-4 based yeast two-hybrid system. In this protocol, we describe serial transformation of bait into an already constructed cDNA library in yeast AH109 cells. We find this method to gives the most number of true interactions. Where a pre-made library in yeast cells is not available, the method outlined can be quickly adapted. AH109 cells can be first transformed with bait containing a vector followed by selection of yeast containing the bait. A second transformation of yeast cells is then accomplished with the cDNA library. The method is quick and can lead to the discovery of significant interactions. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1902741 Fri, 24 Oct 2008 15:22:54 +0100 1902741 http://www.medworm.com/index.php?rid=1902740&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_14 We describe here the “twist-off” method of bundle isolation, where dissected inner-ear organs are embedded in agarose, then subjected to a mechanical disruption that shears off bundles and leaves them in agarose blocks. With care in the dissection and in clean-up of the isolated bundles, contamination from cell bodies can be kept to a minimum. Isolated bundles can be analyzed by a variety of techniques, including immunocytochemistry, SDS-PAGE, immunoblotting, and mass spectrometry. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1902740 Fri, 24 Oct 2008 15:22:54 +0100 1902740 http://www.medworm.com/index.php?rid=1902739&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_13 The identification of the majority of the known causative genes involved in nonsyndromic sensorineural hearing loss (NSHL) started with linkage analysis as part of a positional cloning procedure. The human and mouse genome projects in combination with technical developments on genotyping, transcriptomics, proteomics, and the creation of animal models have greatly enhanced the speed of disease gene identification. In the present chapter, we first discuss the possibilities for exclusion of known NSHL loci and genes. Subsequently, methods are described to determine the genomic regions that contain the genetic defects. These include linkage analysis with genotyping and statistical evaluation and the determination of copy number variations. In the case of a large genomic region, candidate genes... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1902739 Fri, 24 Oct 2008 15:22:53 +0100 1902739 http://www.medworm.com/index.php?rid=1902738&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_12 Most chronic bacterial infections are associated with biofilm formation wherein the bacteria attach to mucosal surfaces, wound tissue, or medical device surfaces in the human body via the formation of an extracellular matrix. Biofilms assume complex three-dimensional structures dependent on the species, the strain, and the prevailing environmental conditions and are composed of both the bacteria and the extracellular slime-like matrices, which surround the bacteria. Bacteria deep in the biofilm live under anaerobic conditions and must use alternatives to O2 as a terminal electron acceptor. Thus, the metabolic rates of these deep bacteria are greatly reduced, which renders them extremely resistant to antibiotic treatment, and for reasons not clearly understood, it is often very difficult to... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1902738 Fri, 24 Oct 2008 15:22:52 +0100 1902738 http://www.medworm.com/index.php?rid=1902737&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_11 Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis are the three leading bacteria species associated with otitis media. Defining the molecular epidemiology of bacteria known to cause otitis media is of great importance, in both clinical and research settings. PFGE and MLST provide data for the characterization of isolates’ genetic relatedness, yet they differ in the types of studies for which they are most useful. Consequently, knowledge of both techniques is important for laboratories intending to study the molecular epidemiology of otitis media–associated bacterial pathogens. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1902737 Fri, 24 Oct 2008 15:22:50 +0100 1902737 http://www.medworm.com/index.php?rid=1902736&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_10 Recent advances in molecular biology have led to a better understanding of the etiology of vestibular schwannomas. The underlying purpose of vestibular schwannoma research is the development of new treatment options; however, such options have not yet been established. A fundamental understanding of the underlying molecular events leading to tumor formation began when mutations in the neurofibromatosis type 2 (NF2) tumor suppressor gene were identified in vestibular schwannomas. The clinical characteristics of vestibular schwannomas and neurofibromatosis type 2 (NF2) syndromes have both been related to alterations in the NF2 gene. Genetic screening for NF2 is now available. When utilized with clinical screening, such as magnetic resonance imaging (MRI), conventional audiometry, and auditor... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1902736 Fri, 24 Oct 2008 15:22:49 +0100 1902736 http://www.medworm.com/index.php?rid=1902735&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-523-7_1 The amphibian Xenopus offers a unique model system for uncovering the genetic basis of auditory and vestibular function in an organism that is well-suited for experimental manipulation during animal development. However, many procedures for analyzing gene expression in the peripheral auditory and vestibular systems mandate the ability to isolate intact RNA from inner ear tissue. Methods presented here facilitate preparation of high quality inner ear RNA from larval and post-metamorphic Xenopus specimens that can be used for a variety of purposes. We demonstrate that RNA isolated with these protocols is suitable for microarray analysis of inner ear organs, and for cloning of large transcripts, such as those for ion channels. Genetic sequences cloned with these procedures can be used for tra... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1902735 Fri, 24 Oct 2008 15:22:48 +0100 1902735 http://www.medworm.com/index.php?rid=2504171&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-448-3_9 Transdifferentiation is defined as an irreversible switch in postnatal life of one differentiated cell to another. Transdifferentiation from different cellular origins into pancreatic-like β-cells is of clinical significance since this approach may offer a potential cure for diabetes. In order to achieve this goal, the liver is considered as a suitable candidate due to its close developmental relationship to the pancreas, its large size and a well-documented regenerative capacity that could provide enough original tissues to initiate the transdifferentiation procedure. In this chapter, we describe a protocol to overexpress Pdx1, a master regulator essential for pancreas development in the cultured human liver cell line, HepG2. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=2504171 Thu, 31 Jul 2008 23:00:00 +0100 2504171 http://www.medworm.com/index.php?rid=1548055&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_9 Role of specific reactive lipids as well as amino acids in control of insulin signalling in adipose tissue is well recognized. Since it is practically impossible to measure the levels of all metabolites in the biological sample simultaneously with a single analytical platform, we utilize multiple platforms to study the lipids and metabolites of relevance to adipose tissue metabolism and insulin signalling. Two screening platforms cover a broad range of lipid molecular species (UPLC/MS based lipidomics platform) as well as organic acids and sterols (GCxGC-TOF platform). A targeted platform for amino acids (UPLC) is also applied. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1548055 Sun, 01 Jun 2008 04:00:00 +0100 1548055 http://www.medworm.com/index.php?rid=1548054&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_8 Brown and white adipose tissues in mammals have a number of similar properties, such as lipid storage and adipokine production, but also distinctive properties. The energy-storing white adipose tissue has few mitochondria and low oxidative capacity. The heat-producing brown adipose tissue has a high density of mitochondria and high oxidative capacity. Mitochondrial function can be investigated in cells and organelles isolated from both brown and white adipose tissues. This chapter describes methods for successful isolation of suitable preparations of adipose tissues and their subsequent use. Questions concerning thermogenic capacity of the tissues, their potential influence on whole body metabolism, and specific properties of the mitochondria and their mode of function may be addressed usi... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1548054 Sun, 01 Jun 2008 04:00:00 +0100 1548054 http://www.medworm.com/index.php?rid=1548053&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_7 Regulation of blood flow in tissues such as skeletal muscle, liver, and adipose tissue is needed to meet the changing local metabolic and physiological demands under varying conditions. In healthy individuals, adipose tissue blood flow (ATBF) is remarkably responsive to meal ingestion, but changes in ATBF in response to other physiological stimuli, such as stress and physical exercise, have also been noted. The ATBF response to nutrient intake may be of particular importance in the regulation of metabolism by facilitating transport of nutrients as well as signaling between adipose tissue and other metabolically active tissues. A reduction in both fasting and postprandial ATBF has been observed in obesity; this impairment is associated with insulin resistance. A better understanding of the ... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1548053 Sun, 01 Jun 2008 04:00:00 +0100 1548053 http://www.medworm.com/index.php?rid=1548052&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_6 Brown and white adipose tissue have recently gained prominence as key players in obesity and related health problems, such as type-2 diabetes and cardiovascular disease. Brown adipose tissue-dependent nonshivering thermogenesis significantly affects the body's energy balance. Originally considered as a passive store of lipids, white adipose tissue has recently been found to secrete a number of hormones and cytokines and to be thus involved in the control of body metabolism and energy balance at multiple sites. These findings have renewed the interest in adipose organ biology, including its innervation by the autonomic nervous system and sensory nerves. Here, we describe our protocols for detecting different types of adipose tissue nerves by light microscopy using peroxidase immunostaining ... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1548052 Sun, 01 Jun 2008 04:00:00 +0100 1548052 http://www.medworm.com/index.php?rid=1548051&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_5 Angiogenesis is required for the growth and expansion of both healthy and pathological tissues. The plasticity of the adipose tissue is reflected by its remarkable ability to expand or to reduce in size throughout the adult lifespan. We, and others, have recently shown that expansion of fat mass is dependent on angiogenesis, and suppression of angiogenesis might provide a novel therapeutic approach for prevention and treatment of obesity. Here, we outline two technical procedures for assessment of angiogenesis in adipose tissues. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1548051 Sun, 01 Jun 2008 04:00:00 +0100 1548051 http://www.medworm.com/index.php?rid=1548050&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_4 Adipose tissue plays a critical role in energy homeostasis, not only in storing triglycerides, but also in responding to nutrient, neural, and hormonal signals, and secreting adipokines that control feeding, thermogenesis, immunity, and neuroendocrine function. It is conceivable that adipose tissue-specific gene expression would influence the aforementioned functions. A feasible approach to prepare adipose tissue-specific transgenic mouse models is necessary for such studies. Here, we report the preparation of adipose tissue-specific cholesteryl ester transfer protein transgenic mice. The general principle might apply to the establishment of other adipose tissue-specific transgenic mice models. (Source: Springer protocols feed by Molecular Medicine) Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1548050 Sun, 01 Jun 2008 04:00:00 +0100 1548050 http://www.medworm.com/index.php?rid=1548049&cid=s_37127_67_f&fid=37127&url=http%3A%2F%2Fwww.springerprotocols.com%2FAbstract%2Fdoi%2F10.1007%2F978-1-59745-245-8_3 In vivo adipose tissue quantification is an important tool to characterize phenotypes of obesity, especially in the human. The amount and distribution of adipose tissue is associated with many of the adverse consequences of obesity. Recent studies suggest that adipose tissue is not a single homogeneous compartment. Regional adipose tissue depots vary in biological functions and individual adipose tissue compartments have stronger associations with metabolic conditions than does total adipose tissue mass. Currently there is intense and increasing interest in regional adipose tissue compartments. Computed tomography and magnetic resonance imaging often are used to quantify adipose tissue volumes or cross-sectional adipose tissue areas. Other modalities, including dual-energy absorpti-ometry ... Springer protocols feed by Molecular Medicine news http://www.medworm.com/rss/comments.php?id=1548049 Sun, 01 Jun 2008 04:00:00 +0100 1548049