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The process whereby a single cell, the fertilized egg, develops into an adult has fascinated for centuries. Great progress in understanding that process, h- ever, has been made in the last two decades, when the techniques of molecular biology have become available to developmental biologists. By applying these techniques, the exact nature of many of the interactions responsible for forming the body pattern are now being revealed in detail. Such studies are a large, and it seems ever-expanding, part of most life-science groups. It is at newcomers to this field that this book is primarily aimed. A number of different plants and animals serve as common model org- isms for developmental studies. In Molecular Methods in Developmental Bi- ogy: Xenopus and Zebrafish, a range of the molecular methods applicable to two of these organisms are described, these are the South African clawed frog, Xenopus laevis, and the zebrafish, Brachydanio rerio. The embryos of both of these species develop rapidly and externally, making them particularly suited to investigations of early vertebrate development. However, both Xenopus and zebrafish have their own advantages and disadvantages. Xenopus have large, robust embryos that can be manipulated surgically with ease, but their pseudotetraploidy and long generation time make them unsuitable candidates for genetics. This disadvantage may soon be overcome by using the diploid Xenopus tropicalis, and early experiments are already underway. The transp- ent embryos of zebrafish render them well-suited for in situ hybridization and immunohistochemistry, and good for observing mutations in genetic screens.
Scientific Frontiers in Developmental Toxicology and Risk Assessment reviews advances made during the last 10-15 years in fields such as developmental biology, molecular biology, and genetics. It describes a novel approach for how these advances might be used in combination with existing methodologies to further the understanding of mechanisms of developmental toxicity, to improve the assessment of chemicals for their ability to cause developmental toxicity, and to improve risk assessment for developmental defects. For example, based on the recent advances, even the smallest, simplest laboratory animals such as the fruit fly, roundworm, and zebrafish might be able to serve as developmental toxicological models for human biological systems. Use of such organisms might allow for rapid and inexpensive testing of large numbers of chemicals for their potential to cause developmental toxicity; presently, there are little or no developmental toxicity data available for the majority of natural and manufactured chemicals in use. This new approach to developmental toxicology and risk assessment will require simultaneous research on several fronts by experts from multiple scientific disciplines, including developmental toxicologists, developmental biologists, geneticists, epidemiologists, and biostatisticians.
Basic Science Methods for Clinical Researchers addresses the specific challenges faced by clinicians without a conventional science background. The aim of the book is to introduce the reader to core experimental methods commonly used to answer questions in basic science research and to outline their relative strengths and limitations in generating conclusive data. This book will be a vital companion for clinicians undertaking laboratory-based science. It will support clinicians in the pursuit of their academic interests and in making an original contribution to their chosen field. In doing so, it will facilitate the development of tomorrow's clinician scientists and future leaders in discovery science. - Serves as a helpful guide for clinical researchers who lack a conventional science background - Organized around research themes pertaining to key biological molecules, from genes, to proteins, cells, and model organisms - Features protocols, techniques for troubleshooting common problems, and an explanation of the advantages and limitations of a technique in generating conclusive data - Appendices provide resources for practical research methodology, including legal frameworks for using stem cells and animals in the laboratory, ethical considerations, and good laboratory practice (GLP)
Developmental biology is a newly-developing subject that is attracting much interest because of exciting findings being made using a combination of classical embryo manipulation with more modern techniques. This book brings together for the first time, and in comprehensive, easy-to-follow protocols, practical instructions for all of the main techniques, from traditional embryology to cellular and molecular methods. It includes complete reprints of all the stage tables in common use for the main laboratory species. It will become an essential addition to laboratory benches of those using any technique as applied to embryos, cells, or tissues.
This volume details a wide range of methods, ranging from beginner through advanced, used to further study zebrafish and fish germline. Chapter guide readers through cultivating and manipulating germ cells, imaging of germline processes and the molecular analysis of their, protein, and RNA. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, application details for both the expert and non-expert reader, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Germline Development in the Zebrafish: Methods and Protocols aims to ensure successful results in the further study of this vital field.
The purpose of Calpain Methods and Protocols is quite straightf- ward: it is to present the actual experimental methods used in many different laboratories for the study of calpain. It will provide the vital experimental detail, and the discussion of possible pitfalls, for which the standard journals no longer provide space. This will make it as easy as possible for investi- tors interested in calpain to adopt established methods without repeating old mistakes, and to adapt and apply these methods in novel approaches to the many outstanding calpain questions. These questions range from purely biochemical problems of protein structure and enzyme regulation at the molecular level, through large areas of cell biology, to applied and clinical aspects of calpain function in human d- ease. Within this panoply of topics, a wide range of investigators will find many fascinating and as yet unanswered questions about calpain. Calpain Methods and Protocols will provide instant access to many essential te- niques, while saving them the time and effort involved in developing a new method. In addition to questions relating to the normal physiological roles of the calpains, there is considerable evidence that inappropriate calpain activity may have pathological effects in many tissues, for example, following ischemia. This provides a major stimulus for the development of specific calpain inhi- tors for therapeutic purposes, and for the development of methods to evaluate such inhibitors.
In Protein Structure, Stability, and Folding, Kenneth P. Murphy and a panel of internationally recognized investigators describe some of the newest experimental and theoretical methods for investigating these critical events and processes. Among the techniques discussed are the many methods for calculating many of protein stability and dynamics from knowledge of the structure, and for performing molecular dynamics simulations of protein unfolding. New experimental approaches presented include the use of co-solvents, novel applications of hydrogen exchange techniques, temperature-jump methods for looking at folding events, and new strategies for mutagenesis experiments. Unique in its powerful combination of theory and practice, Protein Structure, Stability, and Folding offers protein and biophysical chemists the means to gain a more comprehensive understanding of some of this complex area by detailing many of the major techniques in use today.
Dr. Tom Moss assembles the new standard collection of cutting-edge techniques to identify key protein-DNA interactions and define their components, their manner of interaction, and their manner of function, both in the cell and in the test tube. The techniques span a wide range, from factor identification to atomic detail, and include multiple DNA footprinting analyses, including in vivo strategies, gel shift (EMSA) optimization, SELEX, surface plasmon resonance, site-specific DNA-protein crosslinking, and UV laser crosslinking. Comprehensive and broad ranging, DNA-Protein Interactions: Principles and Protocols, 2nd Edition, offers a stellar array of over 100 up-to-date and readily reproducible techniques that biochemists and molecular, cellular, and developmental biologists can use successfully today to understand DNA-protein interactions.
The past decade has seen an extraordinary growth in research interest in neurotrophic factors, and the study of the neurotrophin family has led this activity. Nevertheless, this area of research has often struggled as a result of techniques that were either inadequate or just emerging from other research fields and disciplines. Neurotrophin Protocols has brought together many leaders in the neurotrophin field who detail their special expertise in a wide variety of techniques. Though most procedures are valid across many diff- ent fields of research, some of those described here have been developed to address particular issues within the neurotrophic factor field. The protocols cover a broad range of biochemical, histological, and biological techniques that are often required by the modern laboratory. However, all have been written with sufficient detail to allow any laboratory to achieve proficiency without need of reference to other texts. Neurotrophin Protocols is divided into four sections dealing with p- tein, RNA, recombinant, and in vivo techniques. Protein techniques have in general been less successfully employed than those dealing with RNA or DNA. However, procedures that achieve localization and quantification of the neurotrophins are now being used more extensively. Their inclusion here should assist further studies at the protein level. Transgenic cell lines and animals are commonplace in the scientific research literature, but their inc- sion in several chapters in this book provide some novel uses that are not readily available elsewhere.
Research in the matrix metalloproteinase field began with the demonstration by Gross and Lapière, in 1962, that resorbing tadpole tail expressed an enzyme that could degrade collagen gels. These humble beginnings have led us to the elucidation of around twenty distinct vertebrate MMPs, along with a variety of homologs from such diverse organisms as sea urchin, plants, nematode worm, and bacteria. This, coupled with four known specific inhibitors of MMPs, the TIMPs, gives a complex picture. Part I of Matrix Metalloproteinase Protocols provides the reader with a selective overview of the MMP arena, and a chance to come to grips with where the field has been, where it is, and where it is going. I hope that this complements all of the methodology that comes later. Part II presents the reader with a diverse set of methods for the expression and purification of MMPs and TIMPs, bringing together the long and often hard-earned experience of a number of researchers. Part III allows the reader to detect MMPs and TIMPs at both the protein and mRNA level, whereas Part IV gives the ability to assay MMP and TIMP activities in a wide variety of circumstances.