Cystic Fibrosis Methods and Protocols

ISBN-13: 9781617372445 / Angielski / Miękka / 2011 / 615 str.

William R. Skach

Opis

Since the cloning of the cystic fibrosis transmembrane conductance re- lator (CFTR) nearly a decade ago, cystic fibrosis (CF) research has witnessed a dramatic expansion into new scientific areas. Basic researchers, clinicians, and patients increasingly rely on fundamental techniques of genetics, molecular biology, electrophysiology, biochemistry, cell biology, microbiology, and immunology to understand the molecular basis of this complex disease. Research into the pathophysiology of CF has established numerous paradigms of ion channel dysfunction that extend from inflammation and infection in the airways of patients to basic mechanisms of protein processing and regulation in intracellular components. With these rapid advances has come an increasing need for research scientists to understand and utilize a growing array of basic laboratory tools. This volume of Methods in Molecular Medicine, Cystic Fibrosis Methods and Protocols satisfies that need by providing detailed protocols for the laboratory techniques used throughout CF research. From electrophysiology and cell biology, to animal models and gene therapy, the comprehensive set of methods covered here provide step-by-step instructions needed for investigators to incorporate new approaches into their research programs. Contributions have been chosen to reflect the rich diversity of techniques and to provide a cohesive framework for understanding challenges that are currently at the forefront of CF research. It is hoped that this volume will serve as a valuable reference that will not only foster interdisciplinary investigations into current problems encountered in CF, but also facilitate the translation of new scientific discoveries into clinical solutions.

Recenzje

"The coverage of methodologies is extremely broad and will be useful for those wanting to find out more about some of the less routine techniques used in the analysis of the CFTR protein/chloride channel. The style of protocol-based chapters enables in manuscripts due to considerations of space or potential repetition of previously published techniques. As such many of the chapters are valuable." -Molecular Biotechnology

"Undoubtedly this book will be regarded as essential reading for scientists in the field. It provides details of research methodology, which are rarely published in peer reviewed journals. It will also enable researchers to identify subtle differences in techniques in their own laboratories from those described in these chapters. As such, it will be a very important book for all those who conduct laboratory-based research into cystic fibrosis." -Journal of Paediatrics and Child Health

Spis treści

Part I. Genetics of Cystic Fibrosis CFTR Mutation Detection by Multiplex Heteroduplex (mHET) Analysis on MDE Gel Julian Zielenski, Isabel Aznarez, Tuncer Onay, John Tzounzouris, Danuta Markiewicz, and Lap-Chee Tsui cDNA Microarrays for Pharmacogenomic Analysis of Cystic Fibrosis, Meera Srivastava Ofer Eidelman, and Harvey B. Pollard Natural Animal Models of Human Genetic Diseases Jeffrey J. Wine, Michael Dean, and Damjan Glavac Part II. CFTR Structure and Function: Structure, Gating, and Regulation Electrophysiological Approach to Studying CFTR Horst Fischer Quantitative Analysis of ATP-Dependent Gating of CFTR Allan Powe, Zhen Zhou, Tzyh-Chang Hwang, and Georg Nagel CFTR Regulation by Phosphorylation Tang Zhu, Deborah A. R. Hinkson, David Dahan, Alexandra Evagelidis, and John W. Hanrahan Transepithelial Measurements of Bicarbonate Secretion in Calu-3 Cells Robert J. Bridges Transepithelial Impedance Analysis of Chloride Secretion Ashvani K. Singh, Sangeeta Singh, Daniel C. Devor, Raymond A. Frizzell, Willy van Driessche, and Robert J. Bridges Studies of the Molecular Basis for Cystic Fibrosis Using Purified Reconstituted CFTR Protein Ilana Kogan, Mohabir Ramjeesingh, Canhui Li, and Christine E. Bear Probing CFTR Channel Structure and Function Using the Substituted-Cysteine-Accessibility Method Myles H. Akabas Methods for the Study of Intermolecular and Intramolecular Interactions Regulating CFTR Function Anjaparavanda P. Naren Fluorescent Indicator Methods to Assay Functional CFTR Expression in Cells Alan S. Verkman and Sujatha Jayaraman Expression, Folding, and Degradation Immunolocalization of CFTR in Intact Tissue and Cultured Cells Christopher R. Marino Analysis of CFTR Trafficking and Polarization Using Green Fluorescent Protein and Confocal Microscopy Bryan D. Moyer and Bruce A. Stanton CFTR Folding and Maturation in Cells Mohamed Benharouga, Manu Sharma, and Gergely L. Lukacs Isolation of CFTR: Chaperone Complexes by Co-Immunoprecipitation Geoffrey C. Meacham and Douglas M. Cyr CFTR Expression and ER-Associated Degradation in Yeast Yimao Zhang, Susan Michaelis, and Jeffrey L. Brodsky Manipulating the Folding Pathway of -F508 CFTR Using Chemical Chaperones Marybeth Howard and William J. Welch CFTR Degradation and Aggregation Michael J. Corboy, Philip J. Thomas, and W. Christian Wigley In Vitro Reconstitution of CFTR Biogenesis and Degradation Jon Oberdorf and William R. Skach In Vitro CFTR Folding Assays Rhesa D. Stidham, W. Christian Wigley, and Philip J. Thomas Analysis of CFTR Endocytosis by Cell Surface Biotinylation Kelly Weixel and Neil A. Bradbury Regulatory Complexes CFTR Regulation of ENaC Scott H. Donaldson, Elaine G. Poligone, and M. Jackson Stutts Yeast Two-Hybrid Identification and Analysis of Protein Interactions with CFTR Viswanathan Raghuram, Kenneth R. Hallows, and J. Kevin Foskett Biochemical Assays for Studying Indirect Interactions Between CFTR and the Cytoskeleton Peter J. Mohler, Patricia L. Kultgen, M. Jackson Stutts, and Sharon L. Milgram CFTR-Associated ATP Transport and Release Marie E. Egan Part III. Pathophysiology of Cystic Fibrosis Inflammatory Mediators in CF Patients, Jay B. Hilliard, Michael W. Konstan, and Pamela B. Davis Bacterial Colonization and Infection in the CF Lung Scott D. Sagel, Elaine B. Dowell, and Frank J. Accurso Antimicrobial Peptides and Proteins in the CF Airway, Alexander M. Cole and Tomas Ganz Bacterial-Epithelial Interactions Ruth Bryan and Alice Prince

Blurb

Since the cloning of the cystic fibrosis transmembrane conductance regulator (CFTR) nearly a decade ago, cystic fibrosis researchers, clinicians, and patients have come to rely increasingly on a diverse array of fundamental techniques to understand the molecular basis of this complex disease. Cystic Fibrosis Methods and Protocols consolidates a broad range of detailed and readily reproducible in vitro, cellular, and whole animal laboratory protocols into an indispensable resource. From electrophysiology and cell biology, to animal models and gene therapy, this comprehensive set of methods provides the step-by-step instructions needed for investigators to incorporate new approaches into their research programs. Specific protocols describe new techniques for diagnosis, in vitro methods for the expression and functional analysis of CFTR, novel biochemical and cellular systems to determine how mutations subvert CFTR function, and in vivo protocols to examine how CFTR dysfunction produces multisystem pathology in human and animal models.
Comprehensive, multidisciplinary, and highly practical, Cystic Fibrosis Methods and Protocols makes accessible to today's cystic fibrosis investigator the powerful new scientific techniques required to investigate the basic science of the disease and to translate this into effective clinical solutions.