This text concerns the computer-based design and modelling, computational approaches and instrumental methods for elucidating molecular mechanisms of protein folding. Ligand-acceptor interactions are included in volumes 202 and 203 as are genetic and chemical methods for the production of functional molecules including antibodies and antigens, enzymes, receptors, nucleic acids and polysaccharides and drugs.

This book brings together the major techniques used in the isolation or enrichment of individual populations of organelles and other subcellular structures from plants with the goal that, by being able to isolate subcellular structures, the research and understanding of various facets of compartmentalized function in plant cells can be advanced. Written for the highly successful Methods in Molecular Biology series, expert contributors provide chapters that contain introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Isolation of Plant Organelles and Structures: Methods and Protocols will greatly aid those who regularly isolate subcellular components as well as those whose research has lead them to focus on a subcellular compartment or a particular process for the first time, thus producing the need to be able to isolate it or enrich it for study.

In a rapidly evolving and extremely important area of medical science, it is often difficult for the student, teacher, and researcher to keep abreast of all the important advances. The purpose of Molecular Biology ofDiabetes, Parts I and II is to bring to these individuals the latest knowledge of diabetes-related research in a comprehensive, yet concise manner. To this end, we have assembled chapters, written by most of the world's experts in the field, that we believe compre hensively survey and synthesize a coherent understanding of the subject. Studies of the etiology of type I and type II diabetes are extremely exciting and essential, since we hope to one day prevent the disease using gene therapy. These aspects are covered in Molecular Biology of Diabetes: I. Autoimmunity and Genetics; Insulin Synthesis and Secretion. In type II diabetes, an abnormality in pancreatic secretion exists concomitantly with peripheral insulin resistance. This abnor mality of insulin secretion is believed to be related to a defect(s) in glucose sensing. Uncoupling of glucose sensing from insulin secre tion may be the crucial step in the pathogenesis of noninsulin-depen dent diabetes. In this volume, we have invited authors to describe their studies on all known factors affecting -cell function, including autoimmunity and genetics of diabetes, as well as molecular mecha nisms of insulin synthesis and secretion. In the last few years, the most rapidly advancing area of research in diabetes has been, in fact, related to insulin action."

Epithelia are one of the commonest tissue types in the animal kingdom. Chapters from leading scientists in the major international research laboratories use examples from different systems to illustrate the form and function of epithelia. An important theme is the way in which epithelial cells differentiate to specialized tissue - reversal of this process occurs when cells become tumorigenic.

The work introduces the fundamentals concerning the measure of discrete information, the modeling of discrete sources without and with a memory, as well as of channels and coding. The understanding of the theoretical matter is supported by many examples. One particular emphasis is put on the explanation of Genomic Coding. Many examples throughout the book are chosen from this particular area and several parts of the book are devoted to this exciting implication of coding.

As the major task of sequencing the human genome is near completion and full complement of human genes are catalogued, attention will be focused on the ultimate goal: to understand the normal biological functions of these genes, and how alterations lead to disease states. In this task there is a severe limitation in working with human material, but the mouse has been adopted as the favored animal model because of the available genetic resources and the highly conserved gene conservation linkage organization. In just of ten years since the first gene-targeting experiments were p- formed in embryonic stem (ES) cells and mutations transmitted through the mouse germline, more than a thousand mouse strains have been created. These achievements have been made possible by pioneering work that showed that ES cells derived from preimplantation mouse embryos could be cultured for prolonged periods without differentiation in culture, and that homologous rec- bination between targeting constructs and endogenous DNA occurred at a f- quency sufficient for recombinants to be isolated. In the next few years the mouse genome will be systematically altered, and the techniques for achi- ing manipulations are constantly being streamlined and improved.

In 1970, Manfred Eigen initiated the study of the origin of self-reproducing systems of macromolecules and their evolution. Large-scale nucleotide sequencing (with computer methods) was introduced from 1977. The authors of this book, the first edition of which appeared (in Russian) in 1985, have been engaged in the research of the evolution of molecular genetic regulatory systems ever since those pioneering years. The book considers many fundamental problems of molecular biology, evolution, molecular genetic organization, the structure and function of macromolecules, always with the underlying motive of developing a unified theory. It describes many original, theoretical results as well as computational methods.

Recombinant viruses provide an efficient mechanism for the transfer and expression of DNA in eukaryotic cells. First, the transfer of DNA by viral infection-utilizing specific cell surface receptors and cellular intern- ization mechanisms-occurs much more readily than DNA transfer via uptake induced by such physical methods as calcium phosphate coprecipitation or electroporation. Second, the novel strategies employed by the virus to express its own genes can then be "hijacked" in the recombinant virus to express the researcher's gene of interest The purpose of Practical Molecular Virology isthus to compile a coll- tion of readily repeatable gene transfer and expression methods from wo- ers expert in the use of a variety of recombinant viral vectors . These include those designed for the production of recombinant antigens, such as pol- virus and yeast Ty-VLPs; those giving very high levels of recombinant protein expression, for example, baculovirus, vaccinia virus, and SV40; and finally viral vectors used for efficient, stable gene transfer to eu otic cells, such as retroviruses and herpesviruses . The first chapter describes the viral life cycle for each virus, and explains how this can be adapted to allow construction of recombinant vectors. Subsequent chapters deal with methods for producing and char- terizing recombinant viruses . I make no apology for the hyperproliferation of chapters dealing with recombinant retroviral methods and applications, since I believe this is clearly proportional to the recent expansion of interest in these techniques.

Recent work has revealed that stabilizing G-quadruplexes in telomeric DNA inhibits telomerase activity, providing impetus for the development of G-quartet-interacting drugs, while G-quartet-containing oligonucleotides have been recognized as a potent class of aptamers effective against STAT3 and other transcription factors implicated in oncogenesis, proving these guanine-quartets to be a vital and rich area for future study. In "G-Quadruplex DNA: Methods and Protocols", experts in the field present a collection of detailed techniques for studying G-quartet formation, dynamics, and molecular recognition. Written in the highly successful Methods in Molecular Biology (TM) series format, chapters include brief 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. Authoritative and cutting-edge, "G-Quadruplex DNA: Methods and Protocols "promises to be a useful resource for those familiar with G-quartets as well as an easy entry point for those researchers from diverse fields who are just developing an interest in the exciting implications of G-quadruplex DNA.

Corynebacteria are a diverse group Gram-positive bacteria found in a range of different ecological niches such as soil, vegetables, sewage, skin, and cheese smear. Some, such as Corynebacterium diphtheriae, are important pathogens while others, such as Corynebacterium glutamicum, are of immense industrial importance. In fact, C. glutamicum is one of the biotechnologically most important bacterial species in use today with an annual production of more than two million tons of amino acids, mainly L-glutamate and L-lysine. Due to its industrial importance, C. glutamicum has been studied extensively over the years, and the publication of the C. glutamicum genome sequence in 2003 provided renewed impetus to these studies. To date, the complete genome sequences of four different species have been published, and sequencing of at least two more species is ongoing. These genomic data have enabled a dramatic improvement in our understanding of the corynebacterial genome architecture, metabolic p

PCR Cloning Protocols, Second Edition, updates and expands Bruce White's best-selling PCR Cloning Protocols (1997) with the newest procedures for DNA cloning and mutagenesis. Here the researcher will find readily reproducible methods for all the major aspects of PCR use, including PCR optimization, computer programs for PCR primer design and analysis, and novel variations for cloning genes of special characteristics or origin, with emphasis on long distance PCR and GC-rich template amplification. Also included are both conventional and novel enzyme-free and restriction site-free procedures to clone PCR products into a range of vectors, as well as state-of-the-art protocols to facilitate DNA mutagenesis and recombination, and to clone the challenging uncharacterized DNA flanking a known DNA fragment.

The current explosive progress in molecular biological research can be definitively traced to the development of molecular cloning technology. The ability to insert specific gene sequences into cloning vectors and their subse quent expansion is the cornerstone of modem molecular biology. A direct practical outcome of molecular cloning technology is its application to ex press specific recombinant genes. Currently, recombinant gene products are used in a wide spectrum of applications, including gene therapy, production of bioactive pharmaceuticals, synthesis of novel biopolymers, in agriculture and animal husbandry, and so on. A fundamental requirement for successful recombinant gene expression is the design of the cloning vector and the choice of the host organism for expression. Recombinant Gene Expression Protocols grows out of the need for a laboratory manual that provides the reader the background and rationale, as well as the practical protocols for the preparation of "expression constructs" and their introduction into appropriate host cells and/or organisms. The chap ters in this book are grouped by their expression hosts, including E. coli, yeast, mammalian cells, nonmammalian eukaryotes such as plants, Xenopus, and insects, as well as in transgenic organisms. In-depth information is presented on the important characteristics of expression cloning vectors and the various methods for efficiently introducing expression constructs into target cells and/ or organisms. Throughout Recombinant Gene Expression Protocols, the authors have consistently striven for a balanced presentation of both background informa tion and actual laboratory details.

The purpose of Applications Therapeutic of Ribozymes is to provide an overview of the utility of ribozymes to selectively inhibit the expression of RNA. The ribozyme applications appearing in this book should benefit not only those experienced in ribozymes, but also those applying this ribozyme technology for the first time. It is hoped that a better understanding of the therapeutic application ofribozymes will have a significant impact on human disease in the near future. The field ofribozyme biochemistry has come a long way since the initial observation that RNA is capable of catalysis, initially in cis during processing of larger molecules and ultimately the use of ribozymes in trans to achieve cleavage of target sequence. The fundamental observation that hammerhead (and subsequently hairpin) ribozymes could be designed to cleave a target messenger RNA, and the clarification of sequence restrictions in both the ribozyme and the target RNA, have paved the ways for the use of ribozymes as a tool to manipulate gene expression at the molecular level. This had pre- ously been a domain of antisense oligonucleotides and triplex DNA. Sub- quent studies have explored the myriad biological systems in which ribozyme-mediated inhibition of genes involved in various cellular processes may be used to uncouple important signaling pathways or to reverse the p- notypic expression of a pathologic process.

Molecular Toxicology Protocols, Second Edition aims to bring together a series of articles describing validated methods to elucidate specific molecular aspects of toxicology, the emphasis being on the application of molecular methods to genetic toxicology. The volume is divided into ten parts, roughly corresponding to the spectrum of biomarkers intermediate between exposure and disease outcomes as proposed in molecular epidemiology models. Subjects of these new chapters range from preparation of fluid specimens for analysis of cellular inflammatory responses to genotoxic insults to sensitive methods for proteomic analysis and aberrant DNA methylation patterns. Written in the successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, Molecular Toxicology Protocols, Second Edition addresses not only the needs of molecular biologists and toxicologists, but also those of individuals interested in applying molecular methods to clinical applications, such as geneticists, pathologists, biochemists, and epidemiologists.

Since its invention and subsequent development nearly 20 years ago, po- merase chain reaction (PCR) has been extensively utilized to identify numerous gene probes in vitro and in vivo. However, attempts to generate complete and full-length complementary cDNA libraries were, for the most part, fruitless and remained elusive until the last decade, when simple and rapid methods were developed. With current decoding and potential application of human genome information to genechips, there are urgent needs for identification of functional significance of these decoded gene sequences. Inherent in bringing these app- cations to fruition is the need to generate a complete and full-length cDNA library for potential functional assays of specific gene sequences. Generation of cDNA Libraries: Methods and Protocols serves as a laboratory manual on the evolution of generation of cDNA libraries, covering both ba- ground information and step-by-step practical laboratory recipes for which p- tocols, reagents, operational tips, instrumentation, and other requirements are detailed. The first chapter of the book is an overview of the basics of generating cDNA libraries, which include the following: (a) the definition of a cDNA library, (b) different kinds of cDNA libraries, (c) differences between methods for cDNA library generation using conventional approaches and novel stra- gies, including reverse generation of RNA repertoires from cDNA libraries, and (d) the quality of cDNA libraries.

With the increasing complexity of software systems and their widespread growth into many aspects of our lives, the need to search for new models, paradigms, and ultimately, technologies, to manage this problem is evident. The way nature solves various problems through processes evolving during billions of years was always an inspiration to many computational paradigms; on the other hand, the complexity of the problems posed by the investigation of biological systems challenged the research of new tractable models. Molecular Computational Models: Unconventional Approaches is looking into new computational paradigms from both a theoretical perspective which offers a solid foundation of the models developed, as well as from a modeling angle, in order to reveal their effectiveness in modeling and simulating, especially biological systems. Tools and programming concepts and implementation issues are also discussed in the context of some experiments and comparative studies.

This volume reviews the new potential treatments and research in the area of Alzheimer's disease. Special attention is given to international developments in all fields relevant to new drug development. Topics discussed include: progress in the international harmonization of drug development guidelines for dementia drugs; bioethics and law; development of rating instruments; behavioural treatments; and the activities of the Reagan Foundation. The text integrates basic and clinical research findings, and provides evaluation of new approaches to therapy by world leaders in the field. The potential benefit for Alzheimer patients and families resulting from these research programmes, from molecular biology to clinical pharmacology, is reviewed and evaluated.

Koval provides an interdisciplinary forum for the diverse studies involved in the stress biology of eukaryotic cells. Readers gain access to the most recent information available for eukaryotic systems ranging from plants to humans. For the student, this format introduces a source of potentially unifying concepts and hypotheses. Scientists will find a unique opportunity to conveniently examine the similarities among inducible responses initiated by a variety of agents.

This volume contains selected papers presented at the Sendai International Sympo sium on Molecular and Cellular Mechanisms of Cardiovascular Regulation held from May 10-12, 1995, to honor the contributions ofProfessorNorio Taira, Chairman of the Department of Pharmacology (1972-1995), Tohoku University School of Medicine, Sendai, Japan. The Department of Pharmacology at Sendai has a long tradition of significant contribution to the development of drug therapy for cardiovascular diseases. The late Professor Koroku Hashimoto, the predecessor of Professor Norio Taira, first suggested the mode of action of calcium antagonists and their potential usefulness in therapy of ischemic heart disease and hypertension at an early stage of their development. The need for greater understanding of the pathophysiology of cardiovascular dis eases is more critical now than ever before because modern advances in basic and clinical sciences have prolonged the average life expectancy. Using a wide range of molecular and electrophysiological techniques, major advances are occurring frequently in the field of cardiovascular physiology and pharmacology. Such multifaceted approaches are preferred because human cardiovascular diseases are complex, requiring multiple interventions and an in-depth understanding of molecular mechanisms underlying the disease. The first section of this book focuses on molecular mechanisms of ion channel regulation. Eight of ten chapters in this section are devoted to the recent advances in molecular characterization and regulation of various types of potassium channels in cardiac, vascular, and neuronal tissues. A discussion of the structure and function of sodium and calcium channels is also included.

Since the beginning of agricultural production, there has been a continuous effort to grow more and better quality food to feed ever increasing popula tions. Both improved cultural practices and improved crop plants have allowed us to divert more human resources to non-agricultural activities while still increasing agricultural production. Malthusian population predictions continue to alarm agricultural researchers, especially plant breeders, to seek new technologies that will continue to allow us to produce more and better food by fewer people on less land. Both improvement of existing cultivars and development of new high-yielding cultivars are common goals for breeders of all crops. In vitro haploid production is among the new technologies that show great promise toward the goal of increasing crop yields by making similar germplasm available for many crops that was used to implement one of the greatest plant breeding success stories of this century, i. e., the development of hybrid maize by crosses of inbred lines. One of the main applications of anther culture has been to produce diploid homozygous pure lines in a single generation, thus saving many generations of backcrossing to reach homozygosity by traditional means or in crops where self-pollination is not possible. Because doubled haploids are equivalent to inbred lines, their value has been appreciated by plant breeders for decades. The search for natural haploids and methods to induce them has been ongoing since the beginning of the 20th century."

The receptor-associated JAK protein kinases and their substrates, the STAT transcriptional activators, transmit signals following cytokine and growth factor binding to receptors expressed on the cell surface, to result in specific transcriptional and cellular responses. Over the last two decades, the field has progressed from identification of the individual components through to an understanding of the activation and deactivation mechanisms, and the complex structural detail of the proteins involved. We now know that these pathways are important in many biological processes, including growth and development, hematopoiesis, and the innate and adaptive immune response. JAK-STAT Signalling: Methods and Protocols provides detailed methodologies for examining many aspects of the pathway. Divided into four sections, topics include JAK and STAT specific approaches, the negative regulators of the pathway (SOCS proteins), and the production and crystallization of JAK and STAT proteins, among others. Written in the successful Methods in Molecular Biology (TM) series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls. Authoritative and easily accessible, JAK-STAT Signalling: Methods and Protocols will be of use not only to those working in the area but also to new investigators who are led to delve into the complexities of JAK and STAT responses.

Since the beginning of agricultural production, there has been a continuous effort to grow more and better quality food to feed ever increasing popula tions. Both improved cultural practices and improved crop plants have alIowed us to divert more human resources to non-agricultural activities while still increasing agricultural production. Malthusian population predictions continue to alarm agricultural researchers, especially plant breeders, to seek new technologies that will continue to allow us to produce more and better food by fewer people on less land. Both improvement of existing cultivars and development of new high-yielding cultivars are common goals for breeders of alI crops. In vitro haploid production is among the new technologies that show great promise toward the goal of increasing crop yields by making similar germplasm available for many crops that was used to implement one of the greatest plant breeding success stories of this century, i. e., the development of hybrid maize by crosses of inbred lines. One of the main applications of anther culture has been to produce diploid homozygous pure lines in a single generation, thus saving many generations of backcrossing to reach homozygosity by traditional means or in crops where self-pollination is not possible. Because doubled haploids are equivalent to inbred lines, their value has been appreciated by plant breeders for decades. The search for natural haploids and methods to induce them has been ongoing since the beginning of the 20th century."

David Kuter and a host of leading international researchers summarize in one volume all the knowledge of thrombopoietins (TPO) available today. The distinguished experts review the history of the search to discover TPO, describe the molecular and biological characteristics of this new molecule, and present the results of the preclinical animal experiments that will guide clinical use of this new hormone. Along the way they provide the most recent and comprehensive guide to the biology of megakaryocytes and platelets.

Target Discovery and Validation: Reviews and Protocols, Volumes 1 and 2 review the most progressive and current methods for drug target discovery and validation. These volumes explore how recent improvement in understanding the molecular mechanisms of human pathology is impacting drug target discovery in the laboratory and in real therapeutics, specifically for cancers and autoimmune disorders.
Volume 1 focuses on novel and innovative techniques, and presents the most up-to-date protocols available for maximizing the likelihood of achieving target-selective inhibition in vivo while minimizing side effects. The profound impact of genomics, proteomics and bioinformatics on target discovery is explored, and specific attention is given to the role of transgenic and knockout animals in functional genomics and target validation. Cancer researchers will find tremendous value in the molecular classification of breast cancers and the review of protocols for tumor antigens and cancer vaccines. The methods and protocols collected here, all reviewed by leading scientists and clinicians, present the practical details necessary for translating the enormous discovery potential of the genome into real therapeutic products.
Volume 2 collects all the practical details required for efficient translation of discovered targets into real pharmaceutical drugs. Specific targets in cancers and autoimmunity are described and the potential of using siRNA's, antisense oligonucleotides and RNA aptimers in patients is reviewed. This volume explores the tremendous impact of the application of genotyping and gene expression profiling on the future of healthcare, and presents cutting-edge protocols to aid inbringing agents against specific targets closer to application in the clinic.
Collectively, these volumes provide a thorough review of the most cutting-edge methods available for each step in drug target identification, validation, and clinical application. For researchers, an understanding of available methods aids in the creation of innovative experiments in the laboratory, and the successful translation of target discovery to real therapeutics.

This detailed book arrives as there is an increasing need for multiplex biomarker readouts for improved clinical management and to support the development of new drugs by pharmaceutical companies, due to continuous technical developments and new insights into the high complexity of many diseases. Chapters explore the basic technology platforms being applied in the fields of genomics, proteomics, transcriptomics, metabolomics, and imaging, which are currently the methods of choice in multiplex biomarker research. The book also describes the chief medical areas in which the greatest progress has been made and highlight areas where further resources are required. Written for the highly successful Methods in Molecular Biology series, methodology chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Multiplex Biomarker Techniques: Methods and Applications serves as an ideal resource for a wide variety of researchers interested in these vital multiplex techniques.