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In Eicosanoid Protocols, Elias A. Lianos and a panel of hands-on experts present cutting-edge methods for the study of eicosanoids, including prostaglandins, thromboxanes, and leukotrienes. The readily reproducible methods described hereconcentrate on studying the regulation of expression and function of enzymes, particularly cyclooxygenase (and its two isoforms), phospholipase A2, and lipoxygenases involved in the synthesis of established eicosanoids. Additional chapters are devoted to the characterization and distribution of the thromboxane A2 receptor in tissues and the biological roles of novel eicosanoids. Timely and authoritative, the methods in this book will help their users in exploring the pathobiology of inflammation. Eicosanoid Protocols offers new and established researchers powerful, state-of-the-art tools to probe the regulation and function of eicosanoids.
Polyunsaturated fatty acids are essential for human cell metabolism. As precursors of a very large and extremely versatile family of signaling compounds they play a key role in intracellular communication. Eicosanoids constitute one of the most abundant and prominent subfamilies of these fatty acid derivatives which are formed primarily along oxidative pathways. Prostaglandins, leukotrienes, and related eicosanoids have a modulatory function in mammalian cells and are responsible for tissue responses such as inflammation or wound repair. Increasing activity in eicosanoid research sheds new light on today's most common diseases including atherosclerosis, cancer, Alzheimer's, allergies, and rheumatic diseases. The recent advances already have far-reaching implications in medicine. This detailed account, written by leading experts, covers the ground-breaking developments in recent eicosanoid research. The topics span eicosanoid biogenesis, new aspects of their pathophysiology, for example their influence on the cardiovascular system, as well as the clinical application of synthetic eicosanoids and their antagonists. Researchers and students working in biochemistry or in pharmaceutical, physiological, medicinal and neurochemistry will value this informative introduction to one of the most rapidly developing fields in cell biology.
As a scientist with an interest in proteins you will, at some time in your career, isolate an enzyme that turns out to be yellow—or perhaps you already have. Alternatively, you may identify a polypeptide sequence that is related to known flavin-containing proteins. This may, or may not, be your first encounter with flavoproteins. However, even if you are an old hand in the field, you may not have exploited the full range of experimental approaches applicable to the study of flavoproteins. We hope that Flavoprotein Protocols will encourage you to do so. In this volume we have sought to bring together a range of experimental methods of value to researchers with an interest in flavoproteins, whether or not these researchers have experience in this area. A broad range of techniques, from the everyday to the more specialized, is described by scientists who are experts in their fields and who have ext- sive practical experience with flavoproteins. The wide range of approaches, from wet chemistry to dry computation, has, as a consequence, demanded a range of formats. Where appropriate (particularly for analytical methods) the protocol described is laid out in easy-to-follow steps. In other cases (e. g. , the more advanced spectroscopies and computational methods) it is far more apt to describe the general approach and relevance of the methods. We hope this wide-ranging approach will sow the seeds of many future collaborations - tween laboratories and further our knowledge and understanding of how f- voproteins work.
More than 40 years after the discovery of the nucleosome as the fun- mental unit of chromatin, the multifaceted problem of how variations in ch- matin structure affect the activity of the eukaryotic genome has not been solved. However, during the past few years research on chromatin structure and fu- tion has gained considerable momentum, and impressive progress has been made at the level of concept development as well as filling in crucial detail. The structure of the nucleosome has been visualized at unprecedented reso- tion. Powerful multisubunit enzymes have been identified that alter histone/ DNA interactions in ways that expose regulatory sequences to factors initi- ing and regulating such nuclear processes as transcription. Though the imp- tance of posttranslational modifications of histones, notably their acetylation, has long been known, the finding that a number of bona fide regulators increase transcription by acetylating nucleosomes has lent new support to the old idea that the process of gene regulation is intimately related to the nature of the chromatin environment. A wealth of nonhistone proteins contribute to a continuum of structures with distinct biochemical properties and varying degrees of DNA condensation. Perhaps the most important conclusion from a large number of studies is a fresh appreciation of the dynamic nature of chromatin structure, the built-in flexibility providing the basis for regulation.
The purpose of T Cell Protocols: Development and Activation is to c- lect a series of protocols, particularly those that have been developed within the past few years, to help investigators master new techniques (or improve existing ones) for the study of T-cell Biology. Invariably, in putting together a book like this it is difficult to decide which methods to include and which to leave out. To this end methods were selected from a variety of disciplines, including cellular immunology, b- chemistry, and molecular biology, to try to provide something of interest for everyone who works on T-cell development and activation. I would like to mention that my primary reason for agreeing to put this book together is that, when I was a graduate student, I purchased a copy of Selected Methods in Cellular Immunology by Mishell and Shigii which proved a tremendous help in learning the basics of one-and two- dimensional gel te- niques (and other methods). The cover has long since fallen off, but it still remains one of my most valued reference books for the laboratory. It is my hope that T Cell Protocols: Development and Activation will prove similarly useful to current and future scientists wishing to learn new methods for expl- ing the development and activation of T cells.
As key components of many cell signaling pathways, protein kinases are implicated in a broad variety of diseases, including cancers and neurodegenerative conditions, and offer considerable potential as tractable targets for therapeutic intervention. In Protein Kinase Protocols, a panel of highly skilled laboratory investigators describe both basic and more sophisticated methods for the analysis of kinase-mediated signaling cascades, with emphasis on the identification of proteins according to their interactive relationships and the analysis of their functional properties. Described in step-by-step detail, these readily reproducible techniques offer novices quick access to a complicated field, and provide more experienced investigators many novel time-saving ploys. Emphasis is given to the critical technical steps that are often omitted from methods published in the primary literature. There are also tips on potential pitfalls and copious notes on how to adjust the protocols to work in related systems. Broad in its range of techniques and thoroughly detailed to help ensure experimental success, Protein Kinase Protocols offers both novice and experienced investigators powerful tools for understanding the functional roles of specific protein kinases within signaling cascades and for identification and evaluation of novel therapeutic targets.
In Protein Lipidation Protocols, Michael Gelb brings together a collection of readily reproducible techniques for studying protein lipidation, the covalent attachment of lipids to proteins. These cutting-edge methods-many never published before in a "hands-on" format-deal with glycosyl phosphatidylinositol (GPI)-containing compounds, protein fatty acylation, and protein prenylation. Included are novel techniques for determining the chemical structure of GPI-anchors, for radiolabeling the prenyl groups of protein in eukaryotic cells, a tool for developing inhibitors of the protein farnesyltransferase, and for an exciting lysosomal enzyme that cleaves fatty acyl groups from proteins, the first fatty acylase discovered. Protein Lipidation Protocols offers biochemists, cell and molecular biologists, medicinal chemists, and pharmaceutical researchers state-of-the-art tools for understanding the complex biochemistry of protein lipidation, as well as catalyzing the development of many important new biopharmaceuticals, including anticancer drugs.
Established investigators from around the world describe in step-by-step detail their best techniques for the study of plant hormones and their regulatory activities. These state-of-the-art methods include contemporary approaches to identifying the biosynthetic pathways of plant hormones, monitoring their levels, characterizing the receptors with which they interact, and analyzing the signaling systems by which they exert their effects. Comprehensive and fully detailed for reproducible laboratory success, Plant Hormone Protocols offers plant biologists an indispensable compendium of today's most powerful methods and strategies to studying plant hormones, their regulation, and their activities.
Adhesion molecules are of fundamental importance in the regulation of immunity, inflammation, tissue remodeling, and embryonic development. They comprise different families of homologous proteins, such as selectins, integrins, cadherins, and immunoglobins. In addition, beyond these groups, other str- tures with adhesive properties, such as proteoglycans, occludin, and CD44, have been characterized recently. An understanding of the type and characteristics of adhesive molecules expressed by the different cell types and the possibility of manipulating their activity promises considerable clinical potential. Antibodies, small peptidic and nonpeptidic molecules, have recently been used to inhibit thrombosis by blocking platelet aggregation or inflammation through inhibition of leukocyte infiltration and adhesion. Inhibitors of adhesive molecules are used in expe- mental systems for the study of tumor growth and dissemination. Among major goals in the field are the identification of new members of the known adhesive protein families and of independent new adhesive structures. After structural characterization, even more demanding is the study of the biological activity of the new proteins, and the development of simple, rapid tests for the screening of possible inhibitors. In this regard, the production of such reagents as fragments and antibodies would help define the structure–function relati- ship of individual proteins. Data available in the literature show the complexity of the adhesive process and how different molecular epitopes might contribute to the adhesive properties of a single structure. Finally, a new area of investi- tion is the characterization of the intracellular signaling cascade triggered by the engagement of transmembrane adhesive proteins.