Bidirectional traffic of macromolecules across the nuclear envelope is an active and essential transport process in all eukaryotic cells. Work on various model systems has led to a tremendous increase in our understanding of nuclear transport in recent years.This volume summarizes our current knowledge of protein and RNA transport into and out of the nucleus. It contains nine up-to-date reviews which cover various aspects of nucleocytoplasmic transport, including the structure and function of the nuclear pore complex, the role of soluble transport factors in protein and RNA transport, and the regulation of protein transport through the nuclear pore.

RAN: AN ATYPICAL GTPASE Mark G. Rush and Peter D'Eustachio New York University School o/Medicine. Department,o/Biochemistry New York NY 10016 ABSTRACT GTPases, proteins that bind and hydrolyze GTP (guanosine triphos- phate) are critical regulators of many metabolic pathways. Although these proteins are enzymes that catalyze the hydrolysis of GTP to GDP + Pi, their primary function is not the hydrolysis of GTP per se, but rather the coupling of this hydrolysis to metabolic regulation. Such coupling is gen- erally achieved through the interaction of the GTP-bound form of the GTPase with proteins known as *effectors. Effectors are often enzymes whose activities are modulated by the GTPase. However, effectors can also be structural proteins involved in assembling intracellular macromo- lecular complexes, such as actin filaments and microtubules, as well as proteins involved in the intracellular transport of proteins and RNAs. In- deed, the subject of this anthology, the small GTPase Ran, may exert most or all of its regulatory functions by interacting with non-enzyme effectors. This property of Ran distinguishes it from other well studied GTPases, and has resulted in the elucidation of novel mechanisms of Ran action that are quite distinct from previously established paradigms of GTPase function. 1. INTRODUCTION The Ras-related nuclear protein Ran is a highly conserved (80% identity among yeasts and humans) member of the Ras superfamily of small GTP binding and hydrolyzing proteins.

One ofthe major drivers in biological research is the establishment ofstructures and functions of the 50,000 or so proteins in our bodies. Each has a characteristic- dimensional structure, highly "ordered" yet "disordered"! This structure is essential for a protein's function and, significantly, it must be sustained in the competitive and complex environment of the living cell. It is now being recognised that when a cell loses control, proteins can se- assemble into more complex supermolecular structures such as the amyloid fibres and plaques associated with the pathogenesis of prion (CJD) or age-related (Alzheimer's) diseases. This is a pointer to the wider significance of the self-assembling properties of polypeptides. It has been long known that, in silk, polypeptides are assembled into- sheet structures which impart on the material its highly exploitable properties of flexibility combined with high tensile strength. But only now emerging is the recognition that peptides can Self-assemble into a wide variety of non-protein-like structures, including fibrils, fibres, tubules, sheets and monolayers. These are exciting observations and, more so, the potential for materials and medical exploitations is so wide ranging that over 80 scientists from Europe, USA, Japan and Israel. met 1-6 July 1999 in Crete, to discuss the wide-ranging implications of these novel developments. There was a spirit of excitement about the workshop indicative of an important new endeavor. The emerging perception is that of a new class of materials set to become commercially viable early in the 21st century.

This volume presents the response of the eukaryotic translational apparatus to cellular stress and apoptosis, including kinases activated through both the ERK and stress-activated pathways. It further explores two agents that inhibit protein synthesis, calcium and the immunosuppressant rapamycin. Six chapters written by leading experts in the field provide both new data and comprehensive literature reviews. Both the regulation of initiation and elongation are discussed, and the mechanisms of apoptosis are related to changes in the protein synthesis machinery.

Proteoglycans are some of the most elaborate macromolecules of mammalian and lower organisms. The covalent attachment of at least five types of glycosami- glycan side chains to more than forty individual protein cores makes these molecules quite complex and endows them with a multitude of biological functions. Proteoglycan Protocols offers a comprehensive and up-to-date collection of prepa- tive and analytical methods for the in-depth analysis of proteoglycans. Featuring st- by-step detailed protocols, this book will enable both novice and experienced researchers to isolate intact proteoglycans from tissues and cultured cells, to establish the composition of their carbohydrate moieties, to generate strategies for prokaryotic and eukaryotic expression, to utilize methods for the suppression of specific proteoglycan gene expression and for the detection of mutant cells and degradation products, and to study specific interactions between proteoglycans and extracellular matrix proteins as well as growth factors and their receptors. The readers will find concise, yet comprehensive techniques carefully drafted by leading experts in the field. Each chapter commences with a general Introduction, followed by a detailed Materials section, and an easy-to-follow Methods section. An asset of each chapter is the extensive notation that includes troubleshooting tips and practical considerations that are often lacking in formal methodology papers. The reader will find this section most valuable because it is clearly provided by experienced scientists who have first-hand knowledge of the techniques they outline. In addition, most of the chapters are well illustrated with examples of typical data generated with each method.

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.

Direct cell-cell communication is a common property of multicellular organisms that is achieved through membrane channels which are organized in gap junctions. The protein subunits of these intercellular channels, the connexins, form a multigene family that has been investigated in great detail in recent years. It has now become clear that, in different tissues, connexins speak several languages that control specific cellular functions. This progress has been made possible by the availability of new molecular tools and the improvement of basic techniques for the study of membrane channels, as well as by the use of genetic approaches to study protein function in vivo. More important, connexins have gained visibility because mutations in some connexin genes have been found to be linked to human genetic disorders. Connexin Methods and Protocols presents in detail a collection of te- niques currently used to study the cellular and molecular biology of connexins and their physiological properties. The field of gap junctions and connexin research has always been characterized by a multidisciplinary approach c- bining morphology, biochemistry, biophysics, and cellular and molecular biology. This book provides a series of cutting-edge protocols and includes a large spectrum of practical methods that are available to investigate the fu- tion of connexin channels. Connexin Methods and Protocols is divided into three main parts.

Recent advances in large-scale DNA sequencing technology have made it possible to sequence the entire genome of an organism. Attention is now turning to the analysis of the product of the genome, the proteome, which is the set of proteins being expressed by a cell. Two-dimensional gel electrophoresis can be used to create cellular protein maps which give a quantitative and qualitative picture of the proteome. Mass spectrometry is the method of choice for the rapid large-scale idenfification of these proteomes and their modifications. An understanding of these methods is critical for scientists in the "Post-Genome" era.

Recent advances in large scale DNA sequencing technology have made it possible to sequence the entire genome of an organism. Attention is now turning to the analysis of the product of the genome, the proteome, which is the set of proteins being expressed by a cell. Mass spectrometry is the method of choice for the rapid large-scale identification of these proteomes and their modifications. This is the first book to extensively cover the applications of mass spectrometry to proteome research.

Actin is an extremely abundant protein that comprises a dynamic polymeric network present in all eukaryotic cells, known as the actin cytoskeleton. The structure and function of the actin cytoskeleton, which is modulated by a plethora of actin-binding proteins, performs a diverse range of cellular roles. Well-documented functions for actin include: providing the molecular tracks for cytoplasmic streaming and organelle movements; formation of tethers that guide the cell plate to the division site during cytokinesis; creation of honeycomb-like arrays that enmesh and immobilize plastids in unique subcellular patterns; supporting the vesicle traffic and cytoplasmic organization essential for the directional secretory mechanism that underpins tip growth of certain cells; and coordinating the elaborate cytoplasmic responses to extra- and intracellular signals. The previous two decades have witnessed an immense accumulation of data relating to the cellular, biochemical, and molecular aspects of all these fundamental cellular processes. This prompted the editors to put together a diverse collection of topics, contributed by established international experts, related to the plant actin cytoskeleton. Because the actin cytoskeleton impinges on a multitude of processes critical for plant growth and development, as well as for responses to the environment, the book will be invaluable to any researcher, from the advanced undergraduate to the senior investigator, who is interested in these areas of plant cell biology.

The Fifth Chinese Peptide Symposium, hosted by Lanzhou University, was held at Lanzhou, China July 14-17, 1998, with 156 participants, including 30 scientists from abroad, representing nine countries. The four-day conference was both intense and spiritually rewarding. Our goal for CPS-98 was to provide a forum for the exchange of knowledge, cooperation and friendship between the international and Chinese scientific communities, and we believe this goal was met. The symposium consisted of 8 sessions with 42 oral and 90 poster presentations, including synthetic methods, molecular diversity and peptide libraries, structure and conformation of peptides and proteins, bioactive peptides, peptide immunology, De Novo design and synthesis of proteins and peptides, ligand-receptor interactions, the chemistry-biology-interface and challenging problems in peptides. The enthusiastic cooperation and excellent contributions were gratifying and the active response of the invited speakers contributed to the success of the symposium. The presentations were of excellent caliber and represented the most current and significant aspects of peptide science. Dr. Kit Lam of the University of Arizona and Dr. Yun-Hua Ye of Peking University were the recipients of "The Cathay Award" sponsored by the H. H. Liu Education Foundation, offered for their seminal contribution in peptide science and the Chinese Peptide Symposium. Four outstanding young scientists were selected by the organizing committee to receive awards sponsored by Haikou Nanhai Pharmaceutical Industry Co. Ltd. (Zhong He Group).

Wheat is the world's most important agricultural commodity. In Europe, where wheat is the main staple, bread wheat (Triticum aestivum) covers the majority of land on which wheat is cropped. Wheat breeders and technologists have contributed greatly to the continued success of bread wheat and its products. The bread-making quality' of a wheat variety can be described in relation to the processing its kernels must undergo to make a good bread. Bread wheat kernels must be suitable for proper milling into a flour that can produce a dough capable of becoming fine bread. The type of bread varies depending on local bread-making practices. Part I of this book contains a study of the anatomy and chemical composition of wheat kernels, and of the fundamental difference between soft' and hard' kernelled varieties. It relates these characteristics to the processes of milling, dough-making and manufacturing of bread, and to biscuit and pasta making. The genetic basis for these characteristics is illustrated, and assay methods for characterizing wheat varieties - ranging from Saunders' chewing test to the most recent developments in glutenin and gliadin research - are evaluated. Part II briefly describes - country by country - how bread-making quality has been integrated into wheat-breeding programmes throughout Europe, and how breeders have attempted to resolve the conflict between yield and quality. It describes how quality wheats travelled' around the world - from their endogenic source in Eastern Europe to North America, and back again to Europe. This explains how specific genetic material can appear in the pedigrees of varieties grown in a wide range of agro-ecological zones. In addition to givingan interesting historical survey, the book points the way forward for breeders' efforts in the future. Bread-Making Quality updates and interprets knowledge in a way that makes it particularly accessible for food technologists, breeders, students, and teachers.

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).

Although the term redox covers an important number of chemical reactions, biochemists are more familiar with reactions involving the reactions mediated by electron transfer chains associated with respiration, the thiol-disulfide exchanges and the reactions occurring in the presence of free radicals. More recently, the importance of these reactions in the living world and in medicine has been recognized by biochemists, biologists, physiologists, physicians, etc. The importance of the subject in both fundamental and is reflected by the abundance of interesting reviews applied science concerning the subject (Cadenas, 1989, Del Maestro, 1991) and books (Dreosti, 1991; Rice-Evans and Burdon, 1994; Armstrong, 1994) The aim of this chapter is to describe basic reactions known with references to reviews covering special subjects related to redox reactions. Transformation of energy in living organisms is mediated by complex biological systems such as electron transfer chains where the succession of redox reactions provides energy to the organisms. Molecular oxygen or dioxygen is an essential molecule and is the terminal acceptor of electrons during respiration in eukaryotes. In these organisms, the electron transfer chain is located in the mitochondrial membranes and produces adenosine triphosphate (ATP). In anaerobes, the electron acceptor is C0 , S, sulphate or nitrate ions 2 instead of 02.

A team of expert investigators and clinical researchers comprehensively review complement's basic biology, its role in disease, methods to measure its activity, and strategies for its inhibition in patients. Each chapter focuses on a specific area of basic and applied complement biology, spelling out the activation pathways and complement receptors. Informative animal models are discussed in detail, including the relative values of each model and the important interspecies differences that can distort the interpretation of preclinical studies. The emphasis throughout is on the pros and cons of the therapeutic use of recombinant complement inhibitors in specific diseases. Cutting-edge and innovative, Therapeutic Interventions in the Complement System highlights for today's researcher and biotechnologist effective strategies of drug discovery and development that are producing valuable new complement inhibitors for the treatment of a wide variety of clinically important diseases.

Considerable effort and time is allocated to introducing cell culture and fermentation technology to undergraduate students in academia, generally through a range of courses in industrial biotechnology and related disciplines. Similarly, a large number of textbooks are available to describe the appli- tions of these technologies in industry. However, there has been a general lack of appreciation of the significant developments in downstream processing and isolation technology, the need for which is largely driven by the stringent re- latory requirements for purity and quality of injectable biopharmaceuticals. This is particularly reflected by the general absence of coverage of this s- ject in many biotechnology and related courses in educational institutions. For a considerable while I have felt that there is increasing need for an introductory text to various aspects of downstream processing, particularly with respect to the needs of the biopharmaceutical and biotechnology ind- try. Although there are numerous texts that cover various aspects of protein purification techniques in isolation, there is a need for a work that covers the broad range of isolation technology in an industrial setting. It is anticipated that Downstream Processing of Proteins: Methods and Protocols will play a small part in filling this gap and thus prove a useful contribution to the field. It is also designed to encourage educational strategists to broaden the coverage of these topics in industrial biotechnology courses by including accounts of this important and rapidly developing element of the industrial process.

This book is based on an advanced course of lectures on ribosome structure and protein biosynthesis that I offer at the Moscow State University. These lectures have been part of a general course on molecular biology for almost three decades, and they have undergone considerable evolution as knowledge has been pro gressing in this field. The progress continues, and readers should be prepared that some facts, statements, and ideas included in the book may be incomplete or out of-date. In any case, this is primarily a textbook, but not a comprehensive review. It provides a background of knowledge and current ideas in the field and gives ex amples of observations and their interpretations. I understand that some interpre tations and generalizations may be tentative or disputable, but I hope that this will stimulate thinking and discussing better than if I left white spots. The book has a prototype: it is my monograph "Ribosome Structure and Pro tein Biosynthesis" published by the Benjamin/Cummings Publishing Company, Menlo Park, California, in 1986. Here I have basically kept the former order of pre sentation ofthe topics and the subdivision into chapters. The contents ofthe chap ters, however, have been significantly revised and supplemented. The newly writ ten chapters on translational control in prokaryotes (Chapter 16) and eukaryotes (Chapter 17) are added."

Philip H. Howe and a group of well-versed experimentalists present the first major volume a collection of indispensable classic and cutting-edge TGFss assays. Described in great detail to ensure robust and successful results, these readily reproducible techniques range from the growth inhibition assay for TGFss to methods for monitoring its interactions with the mediating proteins. Extensive notes discuss potential pitfalls and provide tips on how to avoid failures, and throughout, emphasis is given to detailing those technical steps critical for experimental success that are often omitted in the primary literature.
Concise and highly practical, Transforming Growth Factor-Beta Protocols provides today's molecular and cell biologists-both expert and novice-with time-tested methods for the identification and analysis of the signal transduction pathways by which TGFss induces and modulates physiological behavior."

A wealth of information has accumulated over the last few years on the human genome. The new insights have completely changed the focus of protein analysis. It is no longer time-consuming analysis of unknown products, but rather selective identifications of individual forms, modifications and processings, and overall analysis of global protein outputs from cells and tissues in health and disease. This book gears to the rising need of sensitive, accurate, and fast separation and identification techniques in proteomics. It discusses current methodologies of modern protein analysis, from isolation and sample preparation, over analysis and identification, to final characterization. Several evaluations concentrate on the now productive approaches of two-dimensional gel electrophoresis and mass spectrometry, but alternative methods and further perspectives are also outlined. The book includes an overlook over current databases to connect protein analysis data with all available information, ...

The mucins (mucus glycoproteins) have long been a complex corner of glycoprotein biology. While dramatic advances in the separation, structural an- ysis, biosynthesis, and degradation have marked the progress in general glycop- tein understanding, the mucins have lagged behind. The reasons for this lack of progress have always been clear and are only now being resolved. The mucins are very large molecules; they are difficult to separate from other molecules present in mucosal secretions or membranes; they are often degraded owing to natural protective functions or to isolation methodology and their peptide and oligos- charide structures are varied and complex. Understanding these molecules has demanded progress in several major areas. Isolation techniques that protect the intact mucins and allow dissociation from other adsorbed but discrete molecules needed to be developed and accepted by all researchers in the field. Improved methods for the study of very large molecules with regard to their aggregation and polymerization were also needed. Structural analysis of the peptide domains and the multitude of oligosaccharide chains was required for smaller sample sizes, for multiple samples, and in shorter time. In view of these problems it is perhaps not surprising that the mucins have remained a dilemma, of obvious biological importance and interest, but very difficult to analyze.

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.

This book collates and reviews recent advances in the microbial metabolism of amino acids, emphasizing diversity - in terms of the range of organisms under investigation and their natural ecology - and the unique features of amino acid metabolism in bacteria, yeasts, fungi, protozoa and nematodes. As well as studying the individual amino acids, including arginine, sulfur amino acids, branched-chain amino acids and aromatic amino acids, a number of themes are explored throughout the work. These include: - Comparative issues between the metabolism of microbes and those of higher organisms, including plants and mammals - Potential for drug targets in pathways of both biosynthesis and degradation of amino acids - Relationship between amino acids or associated enzymes and virulence in parasitic pathogens - Practical implications for food microbiology and pathogen characterization - Future priorities relating to fundamental biochemistry of microrganisms, food quality and safety, human and animal health, plant pathology, drug design and ecology As the volume of research into the metabolism of amino acids grows, this comprehensive study of the subject is a vital tool for researchers in the fields of biological, medical and veterinary sciences, including microbiology, biochemistry, genetics and pathology. This book is also essential for corporate organizations with active research and development programmes, such as those in the pharmaceutical industry.

This volume contains the proceedings of the Ninth Meeting of the "International Study Group for Tryptophan Research" (lSTRY), held at the University of Hamburg, Germany, from October 10 to 14, 1998. At this meeting the recent developments in the field of tryptophan research were presented by leading researchers from all over the world in 81 oral and 48 poster contri butions. Research on tryptophan and its derivatives provides an inexhaustible subject. At the conference we tried to compose a multifacetted picture of the recent investiga tions through contributions from the major disciplines involved. Thus, we tried to strike a balance between basic research topics and clinical, nutritional or industrial applica tions. We offered workshops on tryptophan (in sleep and mood), melatonjn, IDO-acti vation and the eosinophilia-myalgia syndrome (EMS) as a platform for intensive discussion for the participants. In these proceedings many contributions are multidisciplinary and have practical or theoretical implications for different research fields. Hence, we have organized this volume in nine main chapters according to basic disciplines and subjects. We are aware that this classification is artificial, but we hope that it is the best compromise for contributors and readers."

The effort to sequence the human genome is now moving toward a c- clusion. As all of the protein coding sequences are described, an increasing emphasis will be placed on understanding gene function and regulation. One important aspect of this analysis is the study of how transcription factors re- late transcriptional initiation by RNA polymerase II, which is responsible for transcribing nuclear genes encoding messenger RNAs. The initiation of Class II transcription is dependent upon transcription factors binding to DNA e- ments that include the core or basal promoter elements, proximal promoter elements, and distal enhancer elements. General initiation factors are involved in positioning RNA polymerase II on the core promoter, but the complex - teraction of these proteins and transcriptional activators binding to DNA e- ments outside the core promoter regulate the rate of transcriptional initiation. This initiation process appears to be a crucial step in the modulation of mRNA levels in response to developmental and environmental signals. Transcription Factor Protocols provides step-by-step procedures for key techniques that have been developed to study DNA sequences and the protein factors that regulate the transcription of protein encoding genes. This volume is aimed at providing researchers in the field with the well-detailed protocols that have been the hallmark of previous volumes of the Methods in Molecular (TM) Biology series.

With the end of the Human Genome Project in sight, the next important step is to determine the function of genes. Proteome Research is an important approach to this study and is the first book to comprehensively cover the application of two-dimensional electrophoresis, the central methodology in proteome research. The state-of-the-art is described in detail and the available detection methods are extensively covered. Sufficient detail is given to allow readers to apply these technologies to their own particular requirements.