PAS proteins control numerous physiological and developmental events, and span phylogeny from bacteria to man. Bacterial and plant PAS proteins act as sensors of environmental stimuli, including light, oxygen, and energy status. Not surprising, given these roles, there is intense investigation of the roles of bHLH-PAS proteins in issues of human health including: (1) cancer induction, (2) cancer growth and vascularity, (3) birth defects, including Down syndrome, (4) appetite control and obesity, (5) sleep rhythm disorders, and (6) mental health disorders such as social interactions and learning. PAS proteins encompass many fields of biology, and scientists who work in these fields (circadian rhythms, oxygen regulation, toxin metabolism, bacterial sensors, and development) are an audience, particularly those who actively work on PAS proteins and researchers interested in transcriptional control, signal transduction, and evolution.

LIST OF PARTICIPANTS 11 THERMOPHILIC HYDROLYTIC ENZYMES (PROTEINASES, AMYLASES) 17 Thermal Stability of Homologous Neutral Metalloendopeptidases in Thermo philic and Mesophilic Bacteria: Structural Considerations M. K. PANGBURN, P. L. LEVY, K. A. WALSH and H. NEURATH 19 The Structure and Stability of Thermolysin L. H. WEAVER, W. R. KESTER, K. F. TEN EYCK and B. W. MATTHEWS 31 Studies of the Inhibition of Thermolysin J. FEDER, N. AUFDERHEIDE and B. S. WILDI 41 Effect of EDTA on the Conformational Stability of Thermolysin A. FONTANA, E. BOCCU and F. M. VERONESE 55 Role of a Sulfhydryl Group in the Structure and Function of Alkaline Proteases from a Thermophilic Actinomycete K. MIZUSAWA and F. YOSHIDA 61 Partial Characterization of a Thermophilic Actinomycete Rennin S. LAXER, A. PINSKY and B. BARTOOV 67 Amylase Activity and Stability at High and Low Temperature Depending on Calcium and other Divalent Cations W. HEINEN and A. M. LAUWERS 77 Role of Calcium Ion in the Thermostability of a-Amylase Produced from Bacillus stearothermophilus K. YUTAN I 91 THERMOPHILIC DEHYDROGENASES 105 Thermophilic Glyceraldehyde-3-P Dehydrogenase R. E. AMELUNXEN and R. SINGLETON JR. 107 Glyceraldehyde 3-Phosphate Dehydrogenase from an Extreme Thermophile, Thermus aquaticus 121 J. D. HOCKING and J. I. HARRIS Thermal Properties of Glyceraldehyde 3-Phosphate Dehydrogenase from Escherichia coli A. FONTANA, C. GRANDI, E. BOCCU and F. M. VERONESE 135 Comparative Conformational Properties of Thermophilic and Mesophilic 6-Phosphogluconate Dehydrogenase F. M. VERONESE, C. GRANDI, E. BOCCU and A."

Using genome sequencing, one can predict possible interactions among proteins. There are very few titles that focus on protein-protein interaction predictions in bacteria. The authors will describe these methods and further highlight its use to predict various biological pathways and complexity of the cellular response to various environmental conditions. Topics include analysis of complex genome-scale protein-protein interaction networks, effects of reference genome selection on prediction accuracy, and genome sequence templates to predict protein function.

Physics and the life sciences have established new connections within the past few decades, resulting in biological physics as an established subfield with strong groups working in many physics departments. These interactions between physics and biology form a two-way street with physics providing new tools and concepts for understanding life, while biological systems can yield new insights into the physics of complex systems. To address the challenges of this interdisciplinary area, The Physics of Proteins: An Introduction to Biological Physics and Molecular Biophysics is divided into three interconnected sections. In Parts I and II, early chapters introduce the terminology and describe the main biological systems that physicists will encounter. Similarities between biomolecules, glasses, and solids are stressed with an emphasis on the fundamental concepts of living systems. The central section (Parts III and IV) delves into the dynamics of complex systems. A main theme is the realization that biological systems, in particular proteins, do not exist in unique conformations but can assume a very large number of slightly different structures. This complexity is captured in the concept of a free energy landscape and leads to the conclusion that fluctuations are crucial for the functioning of biological systems. The final chapter of this section challenges the reader to apply these concepts to a problem that appears in the current literature. An extensive series of appendices (Part V) provide descriptions of the key physical tools and analytical methods that have proven powerful in the study of the physics of proteins. The appendices are designed to be consulted throughout the section on protein dynamics without breaking the deductive flow of the logic in the central section of the book.

The standard protocols for the purification of all known cytoskeleton proteins are presented in this manual. Proteins are listed alphabetically and each protocol follows a common format. Thus, the manual provides a quick and easy reference to all relevant procedures for cytoskeleton protein purification.
The isolation procedure for each protein is shown in a clear flowchart, while the source of the protein, equipment and material needed, a list of suppliers, standard references, accession No. of sequences as well as further relevant facts and practical tips are given on a separate page.

In the ten years since the publication of the first edition, great advances in fluorescent labeling, optics, and sample preparation have significantly improved the imaging capability of microscopy, allowing for a continual refinement of our understanding of the cytoskeleton as a dynamic synergy of components. In Cytoskeleton Methods and Protocols, Second Edition, internationally renowned experts present techniques which reflect many of the recent technological advances in experimental tools for cytoskeleton research with emphasis on animal, plant, protist, and fungal model systems. This cutting-edge volume contains methods for live-cell imaging, fluorescence microscopy, electron microscopy, analysis of cell and organelle motility, isolation of cytoskeleton components, and proteomics, amongst other topics. As a volume in the highly successful Methods in Molecular Biology series, chapters incorporate introductions to their respective subjects, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes that provide unpublished technical information on troubleshooting and avoiding known pitfalls.

Up-to-date and comprehensive, Cytoskeleton Methods and Protocols, Second Edition serves as an ideal guide to scientists who wish to continue this fruitful and important biological research."

The book is dedicated to the topical area of biology and medicine and the role of stress proteins HSP70 in the regulation of intracellular protein homeostasis, signaling transduction and cell protection. The book is divided into chapters, which describe the discovery of HSP70 and its molecular structure, the mechanism of the synthesis and function in normal and damaged cells, examine the role of HSP70 in immunity, cancerogenesis, aging, Alzheimer's disease and cardiac surgery. In this book, the author looks at HSP70 as a factor which prevents the transformation of homeostasis mechanisms of intracellular proteins into a link in the pathogenesis of a disease.

The Discovery of Ribonuclease P and Enzymatic Activity of Its RNA Subunit Sydney Brenner and Francis H. C. Crick had a specific project in mind when they offered Sidney Altman a position in their group in 1969 to conduct postdoctoral research at the Medical Research Council Laboratory of Molecular Biology (LMB) in Cambridge, England. At the time, an intense international competition was on- ing in as many as a dozen labs to determine the three-dimensional structure of tRNA. At the LMB, Aaron Klug was attacking the structure by crystallographic analysis with Brian F. C. Clark providing large amounts of purified phenylalanine tRNA. (Eventually, Aaron announced his empirically determined 3-D structure of yeast phenylalanine tRNA, a structure that is generally common to tRNAs, due in part to several conserved, novel three-way nucleotide interactions. ) Concurrently, Michael Levitt, a Ph. D. student of Francis, was visually scrutinizing the cloverleaf secondary structure of the 14 tRNA sequences known at the time. Levitt was searching for nucleotide covariation in different parts of the molecules that were conserved in the 14 sequences known at the time. He identified a possible covariation of an apparent Watson-Crick pairing type between the residues at position 15 from the 5' end of the tRNA and residue 48. This association implied these parts of the tRNA, namely the D loop containing residue 15 and the 5' end of the T stem-adjoining residue 48, folded on one another in a tertiary structure shared by different tRNAs.

Knowledge of cholesterol and its interaction with protein molecules is of fundamental importance in both animal and human biology. This book contains 22 chapters, dealing in depth with structural and functional aspects of the currently known and extremely diverse unrelated families of cholesterol-binding and cholesterol transport proteins. By drawing together this range of topics the Editor has attempted to correlate this broad field of study for the first time. Technical aspects are given considerable emphasis, particularly in relation cholesterol reporter molecules and to the isolation and study of membrane cholesterol- and sphingomyelin-rich "raft" domains. Cell biological, biochemical and clinical topics are included in this book, which serve to emphasize the acknowledged and important benefits to be gained from the study of cholesterol and cholesterol-binding proteins within the biomedical sciences and the involvement of cholesterol in several clinical disorders. It is hoped that by presenting this topic in this integrated manner that an appreciation of the fact that there is much more that needs to be taken into account, studied and understood than the widely discussed "bad and good cholesterol" associated, respectively, with the low- and high-density lipoproteins, LDL and HDL.

Hunger is the world problem Nr 1, overshadowed by an uncontrollable explosion of the human population all over our planet. Lack of food has been one of the most primitive dangers, which animal life had to face at every stage of its evolution. The living body developed in the course of this evolution special emergency reactions against this danger, which is characterised by a lack of food calories, a lack of nitrogen in the form of proteins, a lack of vitamins and oligo elements. Based on an intricate physiological defense pattern man can support complete starvation up to one month, by using up the substance of less important organs in order to maintain the functional matrix of the important organs, mainly the brain and the nervous system. This regulation and the pattern of its mechanisms are of great interest to the physiologist who is aware that they are also responsible for the maintenance of life among millions of human beings who desperately live in a state of permanent hunger. The most serious problem in many developing countries is not the supply of calories (mainly carbohydrate calories) or the supply of vitamins and oligo elements, but the supply of a sufficient amount of protein in order to overcome the protein-calorie malnutrition. This problem must be considered as the most urgent one among all the other problems in the fight against hunger.

A NATO Advanced Study Institute on "Signal Transduction and Protein Phosphorylation" was held to overview recent developments in this area. The participants in the Institute dealt with protein phosphorylation as the most prevalent mode of regulation of cellular processes. First, methods needed to analyze the complex cascade systems involved were reviewed, including protein sequencing, crystallo- graphy, characterization and isolation of membrane proteins, use of monoclonal or polyclonal antibodies and application of fluorescent probes. In great detail the x ray crystallographic structure of glycogen phosphorylase was presented. This enzyme is located at the end of a signal cascade triggered by the hormonal activation of the membrane-bound adenylate cyclase. The interaction of the hormone/receptor with the catalytic subunit of the adenylate cyclase involves GTP-binding proteins. The function of these recently detected intermembrane coupling factors were reviewed, as weIl as the structure and properties of various protein kinases. 2 Major emphasis was placed on Ca + as a second messenger, its metabolism, mechanism of release and uptake from intracellular stores and its role on cell motility and muscle contraction. Two classes of protein phosphatases were discussed. They differ in their subunit structure and substrate specificity and are subject of a highly complex regulatory mechanism as yet not fully under- stood.

A solution to the protein folding problem has eluded researchers for more than 30 years. The stakes are high. Such a solution will make 40,000 more tertiary structures available for immediate study by translating the DNA sequence information in the sequence databases into three-dimensional protein structures. This translation will be indispensable for the analy sis of results from the Human Genome Project, de novo protein design, and many other areas of biotechnological research. Finally, an in-depth study of the rules of protein folding should provide vital clues to the protein fold ing process. The search for these rules is therefore an important objective for theoretical molecular biology. Both experimental and theoretical ap proaches have been used in the search for a solution, with many promising results but no general solution. In recent years, there has been an exponen tial increase in the power of computers. This has triggered an incredible outburst of theoretical approaches to solving the protein folding problem ranging from molecular dynamics-based studies of proteins in solution to the actual prediction of protein structures from first principles. This volume attempts to present a concise overview of these advances. Adrian Roitberg and Ron Elber describe the locally enhanced sam pling/simulated annealing conformational search algorithm (Chapter 1), which is potentially useful for the rapid conformational search of larger molecular systems."

With the invitation to edit this volume, I wanted to take the opportunity to assemble reviews on different aspects of circadian clocks and rhythms. Although most c- tributions in this volume focus on mammalian circadian clocks, the historical int- duction and comparative clocks section illustrate the importance of various other organisms in deciphering the mechanisms and principles of circadian biology. Circadian rhythms have been studied for centuries, but only recently, a mole- lar understanding of this process has emerged. This has taken research on circadian clocks from mystic phenomenology to a mechanistic level; chains of molecular events can describe phenomena with remarkable accuracy. Nevertheless, current models of the functioning of circadian clocks are still rudimentary. This is not due to the faultiness of discovered mechanisms, but due to the lack of undiscovered processes involved in contributing to circadian rhythmicity. We know for example, that the general circadian mechanism is not regulated equally in all tissues of m- mals. Hence, a lot still needs to be discovered to get a full understanding of cir- dian rhythms at the systems level. In this respect, technology has advanced at high speed in the last years and provided us with data illustrating the sheer complexity of regulation of physiological processes in organisms. To handle this information, computer aided integration of the results is of utmost importance in order to d- cover novel concepts that ultimately need to be tested experimentally.

This work is concerned with a group of proteins which were originally consid ered to be an esoteric phenomenon but which have now been shown to play critical roles both in normal and stressed cells as well as being involved in a variety of human diseases. It is the purpose of this work to give a comprehen sive view of these proteins and their various aspects. After an introductory chapter providing an overview of these proteins, the work is divided into four main sections each of which deals with one important aspect of these proteins. Thus, the first section contains a series of chapters which describe individual stress proteins and their roles in particular biological phenomena. Evidently, the induction of these proteins by elevated tempera ture or other stresses is their defining feature and the second section of this book therefore considers the regulation of stress protein gene expression both by stressful stimuli such as elevated temperature or ischaemia and by non stressful stimuli such as cytokines.

The feld of proteomics moves rapidly. New methods, techniques, applications, standards, models and software appear almost on a daily basis. Accompanying this are plenty of texts on the experimental side of the feld and a few appearing on the informatic and data analysis side. This latterly includes one in the Methods in Molecular Biology series tackling the specifc analysis of "Mass spectrometry data in proteomics" in MMB vol. 376. This current collection builds on this, but takes a broader view of proteome data analysis covering data analysis essentials, but also the databases and data models, as well as practical consid- ations for analysing database search results, annotating genomes, and speeding up searches. It also digs deeper into some topics, such as decoy database searching and aspects of signal processing in proteomic mass spectrometry. The aim of the volume is to provide the reader with a mix of reviews and methodology chapters, which build from the essentials of database searching in proteomics, on through specifc data processing challenges to databases, data standards and data models.

The Ottawa '88 meeting of the International Society for Oxygen Transport to Tissue attracted a record number of participants and presentations. We were able to avoid simultaneous sessions and still keep the scientific program to four days by using poster sessions followed by plenary debate on each poster. To paraphrase the British physicist David Bohm, we tried to avoid an ordinary discussion, in which people usually stick to a relatively fixed position and try to convince others to change. This situation does not give rise to anything creative. So, we attempted instead to establish a true dialogue in which a person may prefer and support a certain point of view, but does not hold it nonnegotiab1y. He or she is ready to listen to others with sufficient sympathy, and is also ready to change his or her own view if there is a good reason to do so. Our Society is in its "teen" years, and there are even some arguments about its exact age. Many newer members have raised questions concerning the history of the Society. For this reason, I have asked one of the "founding fathers," D. Bruley, to prepare a brief account of the birth and early history of the Society which appears on the following page.

Trying to address the entire field of presynaptic modulation of neurotransmitter release is a rather daunting undertaking, one that is well beyond the scope of this book. In addition, studies of release modulation, particularly from a biochemical standpoint, have been the subjects of several extensive reviews, meetings, and books (Langer, 1978; Chesselet, 1984; Wessler, 1989; Kalsner and Westfall, 1990), which provide an essential introduction to this subject. What we have focused on, however, are several specific aspects of release modulation that perhaps have not been as extensively discussed. First, we felt that it was important to focus on modulation in the central nervous system; much of the work that has been done in the past has emphasized the peripheral nervous system (e. g. , the autonomic nervous system and the neuromuscular junction), in part because such preparations are more amenable to study. However, it is becoming clear that modulation of release is, if anything, more important in the central nervous system than in the periphery, and that virtually every transmitter system that has been studied shows some type of release modulation. The other way in which we have restricted the scope of this volume has been to try to emphasize studies in which functional (primarily electrophysiological) measures of transmitter release have been used rather than direct biochemical measures of release, and to explore the ways in which release modulation affects the normal physiological function at synapses.

A number of apparently unrelated phenomena in biological systems (e.g., biopolymer aggregation, cell-cell interactions, ion transport across membranes) arise from the special properties of charged surfaces. A sym posium entitled "Electrical Double Layers in Biology," which took place at the Toronto meeting of the Electrochemical Society, 12-17 May 1985, focused on the common features of these phenomena. The papers presented at that symposium are collected here and they illustrate ways in which an under standing of electrical double layers can elucidate a problem in Biology. An example of this approach can be seen from the paper I presented on ion transport and excitation, where the "unusual" ion flows during nerve excitation are actually expected if one includes the effects of electrical double layers at membrane surfaces. Furthermore, the selectivity of the ion channels in these membranes can be better understood on this basis. Other presentations account for such observations as the changes in spacing between muscle proteins during contraction, the interactions of red cells to form rouleaux, the electrical properties of algal cell membranes, electrokinetic potentials during blood flow in arteries, etc. I trust that these papers will indicate the value of electrochemistry in the study of biological systems, an area of research usually called Bioelectrochemistry, and will encourage biologists to use these ideas when approaching related problems."

This volume focuses on the structure, function and regulation of plant signaling G proteins and their function in hormonal pathways, polarity, differentiation, morphogenesis and responses to biotic and abiotic stresses.

Plants are sessile organisms that need to continuously coordinate between external and internal cues. This coordination requires the existence of hubs to allow cross-talk between different signaling pathways. A single family of Rho GTPases, termed either ROPS or RACs, and heterotrimeric G proteins have emerged as the major molecular switches in a multitude of signal transduction pathway in plants.

Recent publications have addressed specific aspects of the endothelins, such as their roles in disease or the importance of endothelin receptors. However, in this book the entire field of endothelins is covered. This includes the pathways of endothelin production and their regulation, the local and systemic actions of endothelins, receptors for the endothelins and the signalling pathways employed, and the involvement of endothelins in a range of diseases. Attention is also paid to the development through chemistry, pharmacology and toxicology of endothelin antagonists and endothelin-converting enzyme inhibitors, with mention of all the important members of these drug classes. This leads to well-rounded discussions of the potential therapeutic benefit of endothelin inhibitors.

A century has already passed since FRIEDRICH MIESCHER, working at Strasbourg and Basel, began his study of protamine, one of the basic nuclear proteins of cells. It was first established by KOSSEL that protamine represents the simplest known protein. In the conviction that research into the nature of protamine would shed light on that of other typical proteins, a group of researchers in Germany followed MIESCHER and laid the foundations of protein chemistry. A general view of prot amines was thus built up by KOSSEL, working at Strasbourg, Berlin, Marburg an der Lahn, and Heidelberg, FELIX at Heidelberg, Munich, and Frankfurt am Main, and WALDSCHMIDT-LEITZ at Prague and Munich. Concepts and techniques established by these studies have been widely utilized for research on other typical proteins. The revolutionary advances in chemical and physical techniques after W orId War II extended the sphere of research to Tokyo in the Far East. Prof. FELIX' visit in 1955 greatly encouraged our research group in Tokyo. His death in August 1960 constituted a sad loss to protein chemistry and stimulated our group to assume responsibility for carrying on the studies. In the following decade we in Tokyo have been able to add a new development to the results on the chemical structure of protamines accumulated by the Eurqpean researchers over a period of about fifty years."

This volume contains the proceedings of the International Conference on Prostaglandins and Lipid Metabolism in Radiation Injury held in Rockville, Maryland, on October 2-3, 1986. Over 200 persons from eight countries attended the program, which consisted of 24 oral presentations and 38 poster presentations. Forty-two of those presentations have been included in this volume. The conference was sponsored by the Armed Forces Radiobiology Research Institute, located in Bethesda, Maryland. The effects of radiation on lipid synthesis and membrane damage are aptly summarized in the first five chapters. These chapters describe the effects of radiation on lipid peroxidation of model membranes, and the role of lipid composition in mammalian cell death and in bacterial radio- and thermosensitivity. In bacteria, lipid peroxidation is not essential for radiation-induced cell death. One of the key points of the conference was the paradoxical nature of the radiobiology of eicosanoids. On the one hand, eicosanoids are mediators of damage; on the other, they are radioprotective agents. It is clear from the literature and from the data presented at the conference that both of these properties may also be observed as a consequence of radiotherapy. Some studies indicate that nonsteroidal anti-inflammatory drugs may minimize or prevent certain radiation induced damage, but other studies show no positive effect."

The initial identification of the Adenomatous polyposis eoli (Ape) gene as the site of mutations in familial adenomatous polyposis (FAP) was described in 1 1992. ,2 A causal relationship between Ape mutations and intestinal tract tumours was confirmed three years later with the establishment ofthe Min mouse model) These mice are heterozygous forApe and develop numerous intestinal tumours that mimic FAP. Subsequently, Ape has emerged as the most commonly mutated gene in colorectal cancerwith reports varying between 50-80% ofsporadic tumours car- rying such mutations. The search for how mutations in Ape initiate and/or support progression oftumours in the intestinal tract has revealed that the Ape protein is a multifunctionalparticipant in a diverse array ofcellular functions. By collecting and assembling the chapters inthisbook, we aimed toprovide an overview of the diverse functions performed by the Ape protein. As summarised in a short final chapter by Trainer,heterozygosityofApe leads to a number ofextracolonic manifestations that further support this emerging picture ofthe Ape protein as an active contributor to many different cellular functions. The first recognised function of Ape was its role in Wnt signalling. o This function is one of the driving forces for how mutations in Ape ensure that cells remain proliferative. Many of the molecular details of this pathway have been discovered and are described in the first chapter by Kennell and Cadigan.

Research in the pharmaceutical industry today is in many respects quite different from what it used to be only fifteen years ago. There have been dramatic changes in approaches for identifying new chemical entities with a desired biological activity. While chemical modification of existing leads was the most important approach in the 1970s and 1980s, high-throughput screening and structure-based design are now major players among a multitude of methods used in drug discov ery. Quite often, companies favor one of these relatively new approaches over the other, e.g., screening over rational design, or vice versa, but we believe that an intelligent and concerted use of several or all methods currently available to drug discovery will be more successful in the medium term. What has changed most significantly in the past few years is the time available for identifying new chemical entities. Because of the high costs of drug discovery projects, pressure for maximum success in the shortest possible time is higher than ever. In addition, the multidisciplinary character of the field is much more pronounced today than it used to be. As a consequence, researchers and project managers in the pharmaceutical industry should have a solid knowledge of the more important methods available to drug discovery, because it is the rapidly and intelligently combined use of these which will determine the success or failure of preclinical projects.