Antigen processing and presentation, as a field, explores a broad range of protein interactions and functions, both intracellular (in the cytoplasm and in the endoplasmic reticulum) and at the cell surface (between T cells and MHC molecules). To investigate such a diverse array, it is necessary that biochemical, cell biology, and immunological techniques all be employed. The purpose of Antigen Processing and Presentation Protocols is therefore to detail the most up-to-date techniques being used in this burgeoning field. Such techniques include those used to question how MHC-binding peptides are generated, to test how peptides are delivered to MHC molecules, to analyze MHC peptide-binding patterns, and to assay the T-cell response to MHC/peptide. Antigen Processing and Presentation Protocols should aid both those new and those experienced in this area of research in extending the questions that can be asked and answered by the application of these current methods. For editorial assistance, I would like to thank Angela Beninga and Rachael Turnquist.

With the advent of high-throughput technologies following completion of the human genome project and similar projects in model organisms, the number of genes of interest has expanded and the traditional methods for gene function analysis cannot achieve the throughput necessary for large-scale exploration. Gene Function Analysis brings together a number of techniques that have developed recently for looking at gene function, including computational, biochemical and biological methods and protocols.

Since the first international meeting on Vitamin B6 involvement in catalysis took place in 1962, there have been periodic meetings every three or four years. In 1990, scientists studying another cofactor, PQQ, which had already attracted the scientific community's interest for its possible involvement in amino acid decarboxylation and reactions involving amino groups, joined forces with those investigating pyridoxal phosphate-dependent enzymes. Since then, the international PQQ/quinoproteins meetings have been held jointly. In the years following the original meeting 37 years ago in Rome, Italy, the scientific gatherings have taken place in Moscow, Russia (1966); Nagoya, Japan (1967); Leningrad (St. Petersburg), Russia (1974); Toronto, Canada (1979); Athens, Greece (1983); Turku, Finland (1987); Osaka, Japan (1990); and Capri, Italy (1996). For the first time in the history of these symposia, the international meeting was held in the United States, from October 31 through November 5, 1999, in Santa Fe, New Mexico. The scientific program focus shifted significantly beyond the original emphasis on catalysis to aspects such as cellular and genetic regulation of events involving proteins that require pyridoxal phosphate or quinoproteins. The growing awareness of the involvement of these proteins in biotechnology processes and fundamental physiological events, as well as their implication in diseases, was also represented, with emphasis on the molecular basis of these events. The meeting was symposium S278, sponsored by the International Union of Biochemistry and Molecular Biology (IUBMB).

Putrescine and spermidine are ubiquitous in living organisms. Spermine, third of the three most commonly occurring natural polyamines, is probably present in all eukaryotes but is rare (or nonexistent) in prokaryotes. Polyamine residues are constituents of many compounds found in plants and insects. Putrescine, spermidine, or spermine-containing alkaloids are found in many plants, nonproteinaceous spider and wasp toxins contain polyamine residues, and glutathionyl-spermidine conjugates have been found in some pathogenic microorganisms. In most cells polyamines are the products of a highly regulated bios- thetic pathway. It is not clear whether the elaborate regulation of polyamine synthesis is a consequence of their essential role(s) in cellular differentiation and development, or part of a defense mechanism to prevent overaccumulation of compounds that are toxic in excess. In addition to their biosynthetic capa- bility, many cells also possess transport systems for polyamines that respond to intracellular polyamine levels, and other stimuli, and are regulated by mecha- nisms that are at present incompletely defined. Two routes of polyamine catabolism have been identified in mammalian cells, a biodegradative route and a recycling pathway. The relative impor- tance of these pathways and their overall regulation is only partially resolved. What is clear is the widespread occurrence of a variety of polyamine-oxid- ing enzymes in animals, plants, bacteria, and fungi. Polyamine catabolism, by whichever route, results in the formation of aminoaldehydes as intermediates.

Cardiovascular Proteomics: Methods and Protocols presents cutting-edge protocols and strategies for proteomic evaluation of cardiovascular disease written by pioneering researchers in the field. Topics explored in this comprehensive volume include obtaining specific heart proteins, cutting-edge techniques for identifying risk biomarkers of atherome plaque rupture, analyzing the secretome of explanted endarterectomies cultured in vitro, and phage display techniques for deciphering the molecular diversity of blood vessels. Detailed protocols for the isolation of short- and long-term culture of the adult mouse cardiac myocytes are also included.

Numerous advanced proteomic techniques are addressed, including protein separation by two-dimensional electrophoresis and two-dimensional difference gel electrophoresis, liquid chromatography coupled to mass spectrometry, and the SELDI-TOF approach to searching for biomarkers of stroke in human serum or of hypertension in the serum of animal models. Proteomic strategies and protocols for studying the proteome of endothelial, arterial smooth muscle cells, foam cells, and circulating blood monocytes constitute a major element of this text. Cutting-edge techniques for the analysis of subproteomes from isolation to final characterization and the characterization of posttranslational modifications are also addressed. This useful snapshot of current proteomic techniques is a single source for protocols for the identification and validation of novel biomarkers and targets in cardiovascular diseases.

Years ago when we were asked to write a book on the present-day knowledge of the molecular biology of poliovirus, we did not expect that such an apparently simple task could involve so much time and effort. Our writing was hampered by the fact that both of us are full time "workers," so that this monograph is mainly a spare time expedience. The main attention of this book focuses on a detailed review of the molecular biology of poliovirus and especially on the advances of the last decade; medical and environmental aspects are only briefly mentioned. Observations from older studies are considered in view of more recent information. Some of the older ob servations provided fundamental insights and paved the way for present day research; too often such data has been neglected or independently rediscovered. Today, poliovirus research has again attracted considerable interest. High points gained within the last few years were the elucidation of the complete nucleotide sequences of the RNAs of the three poliovinls serotypes and the corresponding vaccine strains, the demonstration of genome evolution during transmission of poliovirus in an epidemic, further characterization of the antigenic sites on the virus particle and of the antigenic drift, characterization of alternate conforma tional states of the virion capsid, the development of monoclonal antibodies against some of the virus proteins, observations on the role of the plasma membrane, cytoskeleton, and cytoplasmic membranes as mediators in the virus induced redirection of the synthetic machinery of the host cell, and characteriza"

In this second edition of a widely used classic laboratory manual, leading experts utilize the tremendous progress and technological advances that have occurred to create a completely new collection of not only the major basic techniques, but also advanced protocols for yeast research and for using yeast as a host to study genes from other organisms. The authors provide detailed methods for the isolation of subcellular components-including organelles and macromolecules, for the basic cellular and molecular analysis specific for yeast cells, and for the creation of conditional mutant phenotypes that lend themselves to powerful genome manipulation. Additional protocols offer advanced approaches to study genetic interactions, DNA and chromatin metabolism, gene expression, as well as the foreign genes and gene products in yeast cells.

Though great advances in public health are witnessed world over in recent years, infectious diseases, besides insect vector-borne infectious diseases remain a leading cause of morbidity and mortality. Control of the epidemics caused by the non-vector borne diseases such as tuberculosis, avian influenza (H5N1) and cryptococcus gattii, have left a very little hope in the past. The advancement of research in science and technology has paved way for the development of new tools and methodologies to fight against these diseases. In particular, intelligent technology and machine-learning based methodologies have rendered useful in developing more accurate predictive tools for the early diagnosis of these diseases. In all these endeavors the main focus is the understanding that the process of transmission of an infectious disease is nonlinear (not necessarily linear) and dynamical in character. This concept compels the appropriate quantification of the vital parameters that govern these dynamics. This book is ideal for a general science and engineering audience requiring an in-depth exposure to current issues, ideas, methods, and models. The topics discussed serve as a useful reference to clinical experts, health scientists, public health administrators, medical practioners, and senior undergraduate and graduate students in applied mathematics, biology, bioinformatics, and epidemiology, medicine and health sciences.

Agrobacterium tumefaciens is a soil bacterium that for more than a century has been known as a pathogen causing the plant crown gall disease. Unlike many other pathogens, Agrobacterium has the ability to deliver DNA to plant cells and permanently alter the plant genome. The discovery of this unique feature 30 years ago has provided plant scientists with a powerful tool to genetically transform plants for both basic research purposes and for agricultural development. Compared to physical transformation methods such as particle bomba- ment or electroporation, Agrobacterium-mediated DNA delivery has a number of advantages. One of the features is its propensity to generate a single or a low copy number of integrated transgenes with defined ends. Integration of a single transgene copy into the plant genome is less likely to trigger "gene silencing" often associated with multiple gene insertions. When the first edition of Agrobacterium Protocols was published in 1995, only a handful of plants could be routinely transformed using Agrobacterium. Agrobacterium-mediated transformation is now commonly used to introduce DNA into many plant species, including monocotyledon crop species that were previously considered non-hosts for Agrobacterium. Most remarkable are recent developments indicating that Agrobacterium can also be used to deliver DNA to non-plant species including bacteria, fungi, and even mammalian cells.

This second edition expands upon and updates the vital research covered in its predecessor, by presenting state-of-the-art multidisciplinary and systems-oriented approaches to complex diseases arising from and driven by the acute inflammatory response. The chapters in this volume provide an introduction to different types of computational modeling, and how these methods can be applied to specific inflammatory diseases, with a focus on providing readers a roadmap for integrating advanced mathematical and computational techniques with traditional experimental methods. In this second edition, we cover both well-established and emerging modeling methods, especially state-of-the-art machine learning approaches and the integration of data-driven and mechanistic modeling. This volume introduces the concept of Model-based Precision Medicine as an alternative approach to the current view of Precision Medicine, based on leveraging mechanistic computational modeling to decrease cost while increasing the information value of the data being obtained. By presenting the role of computational modeling as an integrated component of the research process, Complex Systems and Computational Biology Approaches to Acute Inflammation: A Framework for Model-based Precision Medicine offers a window into the recent past, the present, and the future of computationally-augmented biomedical research.

Over the past decade, there has been an explosive development of research of intrinsically disordered proteins (IDPs), which are also known as unfolded proteins. Structural biologists now recognize that the functional diversity provided by disordered regions complements the functional repertoire of ordered protein regions. In Intrinsically Disordered Protein Analysis :Methods and Experimental Tools, expert researchers explore the high abundance of IDPs in various organisms, their unique structural features, numerous functions, and crucial associations with different diseases. Volume 1 includes sections on assessing IDPs in the living cell,NMR based techniques, vibrational spectroscopy, and other spectroscopic techniques. Written in the highly successful Methods in Molecular Biology (TM) series format, the chapters include the kind of detailed description and implementation advice that is crucial for getting optimal results in the laboratory. Thorough and intuitive, Intrinsically Disordered Protein Analysis: Methods and Experimental Tools helps scientists further their investigations of these fascinating and dynamic molecules.

The main application of genomic markers is the mapping, diagnosis, understanding, manipulation and eventual molecular cloning of loci of medical, biologic or economic interest. These achievements are likely to have a practical impact not only in basic science, e.g. in the elucidation of the basic processes involved in a particular pathway or in retracing the history of plant speciation, but also in breeding practice. In agriculture this translates into the exploitation of this knowledge for further genetic improvement since traits of agronomic interest are often polygenic. The elucidation of the genetic basis of polygenic inheritance and the subsequent efficient utilization of this knowledge in breeding schemes require the availability of an adequate network of genetic markers spanning the entire genome. The purpose of the present manual is to provide new investigators in this area with an introduction and a basic practical state-of-the-art description of how to proceed.

Biosensors for Single-Cell Analysis explores a wide range of biosensor technologies and their applications in single-cell characterization and analysis. Sections cover key biophysical and chemical single-cell properties that consider proteomic, metabolic, electrical, mechanical and optical properties. Each chapter features key definitions and case studies, providing detailed guidance for researchers who want to replicate covered solutions in their work. Tutorial sections, evaluations of the current state-of-the-field and future developments are also included. Microfluidic approaches to characterization, such as microfluidic impedance flow cytometry and microfluidic flow cytometry are considered alongside more conventional approaches, such as mass spectroscopy, fluorescent and mass flow cytometry. Additionally, key types of biosensors are covered, including atomic force microscopy, micropipette aspiration, optical tweezers, microfluidic hydrodynamic stretchers, microfluidic constriction channel and microfluidic optical stretchers.

The double-stranded (ds)RNA viruses represent a diverse group of viruses that vary widely in host range (humans, animals, plants, fungi, and bacteria), genome segment number (one to twelve), and virion organization (T-number, capsid layers, or turrets). Members of this fascinating group include the rotaviruses, renowned globally as the commonest cause of gastroenteritis in young children, and bluetongue virus, an economically important pathogen of cattle and sheep. In recent years, remarkable progress has been made in determining, at atomic and subnanometeric levels, the structures of a number of key viral proteins and of the virion capsids of several dsRNA viruses, highlighting the significant parallels in the structure and replicative processes of many of these viruses. By providing unique insights into fundamental aspects of structure-function relationships in virus particles, virus particle assembly, virus-cell interactions, and viral pathogenesis, approaches for the development of

The innate immune response is a crucial component of early resistance to infection, and it is now revealing increasing levels of complexity. The ability to modify the genome in vivo, has facilitated understanding of complex interactions between leucocytes and other components of the immune system, and phenotype-driven strategies using chemical mutagenesis have placed another powerful weapon in the armamentarium. In, Leucocytes: Methods and Protocols provides detailed protocols and practical advice on a variety of modern approaches to the study of leucocytes and their products. Written in the highly 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 laboratory protocols, and key tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Leucocytes: Methods and Protocols seeks to aid scientists in further study into the immune system and leucocytes.

During the past decade as the data on gene sequences and expression patterns rapidly accumulated, cell-free protein synthesis technology has also experienced a revolution, becoming a powerful tool for the preparation of proteins for their functional and structural analysis. In Cell-Free Protein Production: Methods and Protocols, experts in the field contribute detailed techniques, the uses of which expand deep into the studies of biochemistry, molecular biology, and biotechnology. Beginning briefly with basic methods and historical aspects, the book continues with thorough coverage of protein preparation methods, the preparation of proteins that are generally difficult to prepare in their functional forms, applications of the cell-free technologies to protein engineering, as well as some methods that are expected to constitute a part of future technologies. Written in the highly successful Methods in Molecular Biology series format, the 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.

Authoritative and cutting-edge, Cell-Free Protein Production: Methods and Protocols aims to help researchers continue the growth of the vital exploration of cell-free sciences and technologies in order to better understand the dynamic lives of cells.

advanced metastatic disease of solid tumors, dictates that each tumor mass, indeed each individual metastasis, will have a unique antigen and cytokine environment and hence unique response to immune modu lation. A differential response to immunotherapy is thus inevitable. 4. Many of the human trials described are not randomized and report survival or response against historical controls. Most tumors described are immunogenic human tumors: renal cell cancer and melanoma are most common. In order to avoid the well-described inter-patient vari ation and rare incidence of spontaneous response among patient samples as well as selection bias and changes in practice over time, randomized trials are required. 5. Immunological treatment is unlike conventional chemotherapy in its endpoint. Most chemotherapeutic regimes require a complete response or a good partial response for cure or good palliation. There are now many cases where immunotherapy has provided long-term palliation without massive tumor reduction. Immunity may be stimulated to a degree which holds tumorigenicity in check and most importantly, pro vides good palliation for the patient in a manner that differs essentially from chemotherapy."

The aim of Plant Proteomics: Methods and Protocols is to present up-- date methods and protocols used by recognized scientists in the world of plant proteomics. If this world was a very small one twenty-five years ago when the first papers were published, it has since experienced exponential growth, and in most countries around the world there are laboratories working on plant proteomics. Two-dimensional gel electrophoresis is still the basic method used, but it has been improved greatly with IPG in the first dimension (Chapter 13) and with new detection methods with fluorochromes (Chapters 14 and 15). Signi- cant progress has been achieved in protein extraction, which is particularly difficult with plant tissues containing phenols, proteases, and other secondary metabolites that interfere with proteins. Standard procedures have been op- mized (Chapters 1 and 2) for peculiar tissues (Chapters 3, 4, and 5) and cellular compartments (Chapters 6 to 10). These methods rely on improvements made in the solubilization of proteins from membranes (Chapters 11 and 12). Mass spectrometry was a revolution that permitted the high throughput identifi- tion of proteins separated by 2D gels (Chapters 19 and 20) but also from blue native 1D gels (Chapters 27 and 28) despite the fact that Edman sequencing can still be useful (Chapter 18). Associated with other techniques such as 2DLC or LC of intact proteins, mass spectrometry also permits the identification of polypeptides from complexes (Chapters 21 and 22).

Mathematical Approaches to Molecular Structural Biology offers a comprehensive overview of the mathematical foundations behind the study of biomolecular structure. Initial chapters provide an introduction to the mathematics associated with the study of molecular structure, such as vector spaces and matrices, linear systems, matrix decomposition, vector calculus, probability and statistics. The book then moves on to more advanced areas of molecular structural biology based on the mathematical concepts discussed in earlier chapters. Here, key methods such as X-ray crystallography and cryo-electron microscopy are explored, in addition to biomolecular structure dynamics within the context of mathematics and physics. This book equips readers with an understanding of the fundamental principles behind structural biology, providing researchers with a strong groundwork for further investigation in both this and related fields.

Contents. List of Contributors. Preface. T.J. Mitchell and T.J. Williams: The role of eotaxin and related CC-chemokines in asthma and allergy. Roger J. Davis: Signal transduction by the JNK group of MAP kinases. Marie Chabot-Fletcher: TNF and IL-1 signaling to NF-kB. Anthony M. Manning: Small molecule regulators of AP-1 and NF-kB. Robert T. Abraham: Mammalian target of rapamycin: Immunosuppressive drugs offer new insights into cell growth regulation. Catherine A. Burton, John Boylan, Candy Robinson, Janet Kerr and Pamela Benfield: Constitutive expression of a tumor suppressor leads to tumor regression in a xenograft model. Steven D. Shapiro: Macrophage metalloproteinases in destructive inflammatory diseases. Nancy H. Ruddle: Lymphotoxin in inflammation and lymphoid organ development: Variations on a theme. Steven L. Kunkel, Sem H. Phan, Nicholas W. Lukacs, Cory Hogaboam and Stephen W. Chensue: Chemokine/cytokine biology during the evolution of fibrotic disease. Long Gu, Susan C. Tseng and Barrett J. Rollins: The role of MCP-1 in disease. Lisa A. Beck, Cristiana Stellato, Syed Shahabuddin, Renate Nickel and Robert P. Schleimer: The role of chemokines in allergic diseases of the airways. James Winkler and Ken Tramposch (coordicators): Ninth International Conference of the Inflammation Research Association, November 1-5, 1998: Summaries of workshops and poster discussions. William Williams and Elizabeth Arner (chair persons): Targets in rheumatoid and osteoarthritis. Lawrence Wennogle and Nancy Cusack (chair persons): Signal transduction and regulation of gene expression. James Burke and Floyd Chilton (chair persons): Mediators in inflammation and their enzymes. Denis Schrier and Fandrew Issekutz (chair persons): Cell adhesion molecules and leukocyte trafficking. Robert M. Strieter and David Underwood (chair persons): Pulmonary inflammation, fibrosis, and disease. W. Hunter and E. Turley (chair persons): Angiogenesis, wound repair and skin inflammation. Index.

In Molecular Embryology: Methods and Protocols, Second Edition, expert investigators provide a comprehensive guide to the cutting-edge methods used across the dramatically growing field of vertebrate molecular embryology. Time-tested techniques take advantage of the most commonly used vertebrate experimental models: murine embryos for their genetics, chick embryos for in vivo manipulation, zebrafish for mutagenesis, amphibian embryos, and nonvertebrate chordates. The second edition collects classic protocols which have become standard techniques in the laboratory and presents them in a complementary fashion with novel and emerging approaches, allowing researcher to become more familiar with commonly studied embryos used in biomedical research. Insightful to the experienced professional, Molecular Embryology: Methods and Protocols, Second Edition, presents cutting-edge findings of perhaps the greatest period in growth and productivity in the field of developmental biology.

Epigenetics fine-tunes the life processes dictated by DNA sequences, but also kick-starts pathophysiological processes including diabetes, AIDS and cancer. This volume tracks the latest research on epigenetics, including work on new-generation therapeutics.

When setting out to decide on the content of DNA Repair Protocols: Prokaryotic Systems, I was conscious of the need to portray the vast array of pathways and enzymatic activities that are part of the discipline of DNA repair. In addition to the classical DNA repair activities, I wanted to convey the significant interest that has been generated in recent years in the use of the proteins and repair systems as research tools, much like the use of restriction enzymes over the last few decades. Therefore, in addition to chapters deta- ing protocols for investigating specific repair activities, I have included s- eral chapters in this book on the applied use of DNA repair proteins and systems. The many years of research on bacterial DNA repair systems have allowed us to really understand the majority of DNA repair pathways in bac- rial cells. Building on this knowledge, research has lead to major advances in understanding mammalian DNA repair and uncovered its links to human d- ease, such as DNA mismatch repair and colon cancer, nucleotide excision repair and xeroderma pigmentosum, DNA helicase function in Bloom's s- drome, and so on. Such have been the advances that Science magazine iden- fied the collective DNA repair systems as its "Molecule of the Year" in 1994.

This volume provides an exhaustive review of the most current knowledge of the composition, architecture and dynamics of the nuclear envelope. In contrast to other comprehensive works on cellular membranes in general, this book is the first to specifically address the nuclear envelope. Experts in the field relate the different domains of the nuclear envelope, their biogenesis, their composition, their mode of targeting to chromosomes and how they dissociate from chromosomes upon cell division. A wealth of information from several experimental systems is provided, such as dividing somatic cells, embryos and nuclear assembly assays in cell-free extracts, in a wide range of vertebrate and invertebrate organisms ranging from humans to yeast. The book also contains up-to-date reviews on the relationship between mutations in proteins of the nuclear envelope and Emery-Dreifuss muscular dystrophy. The breadth of topics covered in what may seem to be a field as narrow as the nuclear envelope makes this volume a valuable reference.

Geneticists and molecular biologists have been interested in quantifying genes and their products for many years and for various reasons (Bishop, 1974). Early molecular methods were based on molecular hybridization, and were devised shortly after Marmur and Doty (1961) first showed that denaturation of the double helix could be reversed - that the process of molecular reassociation was exquisitely sequence dependent. Gillespie and Spiegelman (1965) developed a way of using the method to titrate the number of copies of a probe within a target sequence in which the target sequence was fixed to a membrane support prior to hybridization with the probe - typically a RNA. Thus, this was a precursor to many of the methods still in use, and indeed under development, today. Early examples of the application of these methods included the measurement of the copy numbers in gene families such as the ribosomal genes and the immunoglo bulin family. Amplification of genes in tumors and in response to drug treatment was discovered by this method. In the same period, methods were invented for estimating gene num bers based on the kinetics of the reassociation process - the so-called Cot analysis. This method, which exploits the dependence of the rate of reassociation on the concentration of the two strands, revealed the presence of repeated sequences in the DNA of higher eukaryotes (Britten and Kohne, 1968). An adaptation to RNA, Rot analysis (Melli and Bishop, 1969), was used to measure the abundance of RNAs in a mixed population."