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Leading scientists offer an unprecedented suite of master protocols for analyzing neuropeptide structure and function using optimized classic methods and the latest state-of-the-art techniques. This comprehensive collection includes valuable techniques for extracting and purifying neuropeptides from biological sources, for their sequence determination by Edman degradation and PCR, and for the solid-phase synthesis and purification of peptides and peptide analogs. Also covered are physical techniques for the characterization of synthetic or natural peptides, methods for immunocytochemical sample preparation and visualization at both light and electron levels, and techniques for neuropeptide-receptor interaction. Chapters on molecular modeling, radioisotopic labeling, production of antisera, the measurement of efflux from brain to blood, and the assay and inhibition of neuropeptidases complete this indispensable collection of readily reproducible protocols.
The observation that neuropeptide Y (NPY) is the most abundant peptide present in the mammalian nervous system and the finding that it elicits the most powerful orexigenic signal have led to active investigations of the properties of the NPY family of hormones, including peptide YY (PYY) and pancreatic polypeptide (PP). Nearly two decades of research have led to the identification of several NPY receptor subtypes and the development of useful receptor selective ligands. Moreover, these investigations have imp- cated NPY in the pathophysiology of a number of diseases, including feeding disorders, seizures, memory loss, anxiety, depression, and heart failure. Vigorous efforts are therefore continuing, not only to understand the bioche- cal aspects of NPY actions, but also toward developing NPY-based treatments for a variety of disorders. To facilitate these efforts, it was decided to produce the first handbook on NPY research techniques as part of the Methods in Molecular Biology Series. In compiling Neuropeptide Y Protocols, I have gathered contributions on techniques considered critical for the advancement of the NPY field from experts in various disciplines. Each chapter starts with a brief introduction, with Materials and Methods sections following. The latter sections are presented in an easy to follow step-by-step format. The last section of the chapter, Notes, highlights pitfalls and the maneuvers employed to overcome them. This information, not usually disseminated in standard research pub- cations, may prove extremely useful for investigators employing these te- niques in NYP research.
Since the early days, the field of neuropeptide biology has dramatically widened, and today the ultimate frontiers in neuropeptide research lie in the development of pharmacologically active compounds that are capable of crossing the blood-brain barrier to exert their biological role(s) in vivo and in the construction of genetic vectors to be employed in gene therapy. Neuropeptides: Methods and Protocols presents a readily reproducible collection of established and emerging techniques for neuropeptide research as contributed by expert researchers in the field. The detailed methods presented cover subjects such as immunocytochemical localization, biochemical characterization, functional analysis, development and production of genetic probes, and the design of neuropeptide derivatives for cellular neurobiology as well as the potential therapeutic applications. Written in the highly successful Methods in Molecular BiologyTM series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Authoritative and easy to follow, Neuropeptides: Methods and Protocols is intended for a large audience of scientists, including histologists, biochemists, cellular and molecular biologists, and electrophysiologists that are currently active in the field or are willing to enter this exciting and still expanding area of neurobiology.
After 20 years of intensive effort, novel neuropeptides continue to be discovered, and the field of neuropeptide research is still expanding. As new analytical techniques become available, their applicability to the study of neu ropeptides brings fresh insights into the properties and functions of these ubiq uitous chemical messengers. Presented in this single volume, Neuropeptide Protocols, are 33 chapters covering these new techniques, together with more established methods. Each contributor is actively engaged in neuropeptide research and so brings to his or her description an awareness of the practical problems inher ent in the method, and provides sound advice on how to overcome them. The format conforms to the style of previous books in the Methods in Molecular Biology series. Each chapter provides an instruction to the technique, and item ized list of equipment and reagents, and a step-by-step set of instructions to enable practitioners to reproduce the method. The Notes section gives insights into pitfalls or critical stages, tips to overcome these obstacles, and sugges tions for extensions or modifications of the basic protocol. Neuropeptide Protocols is intended as a benchtop manual providing the entire gamut of techniques that form the essential tool kit of the practicing neuropeptide researcher. It will be useful for those new to the field, as well as for established workers who wish to try a new technique for the first time.
The observation that neuropeptide Y (NPY) is the most abundant peptide present in the mammalian nervous system and the finding that it elicits the most powerful orexigenic signal have led to active investigations of the properties of the NPY family of hormones, including peptide YY (PYY) and pancreatic polypeptide (PP). Nearly two decades of research have led to the identification of several NPY receptor subtypes and the development of useful receptor selective ligands. Moreover, these investigations have imp- cated NPY in the pathophysiology of a number of diseases, including feeding disorders, seizures, memory loss, anxiety, depression, and heart failure. Vigorous efforts are therefore continuing, not only to understand the bioche- cal aspects of NPY actions, but also toward developing NPY-based treatments for a variety of disorders. To facilitate these efforts, it was decided to produce the first handbook on NPY research techniques as part of the Methods in Molecular Biology Series. In compiling Neuropeptide Y Protocols, I have gathered contributions on techniques considered critical for the advancement of the NPY field from experts in various disciplines. Each chapter starts with a brief introduction, with Materials and Methods sections following. The latter sections are presented in an easy to follow step-by-step format. The last section of the chapter, Notes, highlights pitfalls and the maneuvers employed to overcome them. This information, not usually disseminated in standard research pub- cations, may prove extremely useful for investigators employing these te- niques in NYP research.
The volumes in this series include contemporary techniques significant to a particular branch of neuroscience. They are an invaluable aid to the student as well as the experienced researcher not only in developing protocols in neuroscience but in disciplines where research is becoming closely related to neuroscience. Each volume of Methods in Neurosciences contains an index, and each chapter includes references. Dr. Conn became Editor-in-Chief of the series beginning with Volume 15, so each subsequent volume could be guest-edited by an expert in that specific field. This further strengthens the depth of coverage in Methods in Neurosciences for students and researchers alike. - Comprehensive protocols included for: Enzymes involved in the activation of bioactive peptidases and proteins - Prohormone/neuropeptide processing pathways - Enzymes involved in peptide metabolism - Posttranslational processing enzymes
Despite being known and studied for years, peptides have never before attracted enough attention to necessitate the invention of the term "peptidomics" in order to specify the study of the complement of peptides from a cell, organelle, tissue or organism. In Peptidomics: Methods and Protocols, expert researchers present a comprehensive range of analytical techniques for the analysis of the peptide contents of complex biological samples with an emphasis often on higher throughput techniques, suitable for the analysis of large numbers of peptides typically present in the peptidomes. Encompassing a number of species ranging from bacteria to man, the methods presented intensively cover topics such as organism handling, tissue and organ dissection, cellular and subcellular fractionation, peptide extraction, fractionation and purification, structural characterization, molecular cloning, and sequence analysis. Written in the highly successful Methods in Molecular BiologyTM series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls. Comprehensive and cutting-edge, Peptidomics: Methods and Protocols brings this ten year old field fully up-to-date in order to inspire novices and experts alike with the easy-to-follow practical advice needed to set up and carry out analysis of the peptide contents of complex biological samples.
Neuropeptides exhibit multiple functions as hormones and regulators. As such, many techniques are required to understand the contribution and functions of neuropeptides in health and disease processes. Research in the neuropeptide field is aimed toward a better understanding of the physiology of the human body and toward the development of better diagnostics and therapeutics. This book will familiarize the audience with forefront techniques in the field.
Methods in Neurosciences, Volume 6: Neuropeptide Technology: Synthesis, Assay, Purification, and Processing describes procedures and tools of assay useful for the identification, purification, and quantification of neuropeptides and their receptors. This volume is divided into four sections— chemical synthesis and biosynthesis; measurement of neuropeptides; purification and characterization; and neuropeptide degrading and processing enzymes. In these sections, this book specifically discusses the synthesis of peptide substrates for protein kinase C; synthesis of glycosyl neuropeptides; and ultrastructural localization of peptides. The measurement of neurokinin B by radioimmunoassay; purification and characterization of neuroendocrine peptides from rat brain; and preparation of glia maturation factor ß are also elaborated. This text likewise covers the assays for arginine/lysine carboxypeptidases and enzymes that metabolize atrial natriuretic peptide. This publication is beneficial to neuroscientists and students researching on the synthesis, assay, purification, and processing of neuropeptides.