As the amount of information in biology expands dramatically, it becomes increasingly important for textbooks to distill this vast amount of scientific knowledge into concise principles and enduring concepts. Molecular Biology of the Cell, Sixth Edition accomplishes this goal with clear writing and beautiful illustrations. The Sixth Edition has been extensively revised and updated with the latest research in cell biology and it provides an exceptional framework for teaching and learning.

The purpose of Ribozyme Protocols is to provide a helpful compilation of protocols that will be of use- DEGREESnot only to those with some experience of ribozymes- DEGREESbut also to those wishing to use ribozymes for the first time. Although it is usually impossible to cover every aspect of a scientific field, I believe this book approaches that ideal and should help all readers perform meaningful experiments using ribozymes. To design ribozymes, one must consider whether the target site will be accessible; this task can be facilitated by using computer programs that pre dict the folding of the target RNA. Such programs are detailed in Chapters 2 and 3. If the chosen target is an RNA virus that can mutate rapidly, it makes sense to consider those parts of the genome that are least likely to change during viral replication. An example of how this can be done is described in Chapter 4. Although computer analysis may be a useful starting point to select tar get sites, there seems, at the moment, to be no guarantee that any particular chosen site will be efficiently cleaved. Some workers have deliberately bypassed this problem by using libraries of ribozyme sequences and by select ing those that actually hybridize to and/or cleave the target; these methods are described in Chapters 5

The Office of Health and Environmental Research (OHER) has supported and continues to support development of computational approaches in biology and medicine. OHER's Radiological and Chemical Physics Program initiated development of computational approaches to determine the effects produced by radiation of different quality (such as high energy electrons, protons, helium and other heavy ions, etc. ) in a variety of materials of biological interest-such as water, polymers and DNA; these include molecular excitations and sub-excitations and the production of ionization and their spatial and temporal distribution. In the past several years, significant advances have been made in computational methods for this purpose. In particular, codes based on Monte Carlo techniques have .been developed that provide a realistic description of track-structure produced by charged particles. In addition, the codes have become sufficiently sophisticated so that it is now possible to calculate the spatial and temporal distribution of energy deposition patterns in small volumes of subnanometer and nanometer dimensions. These dimensions or resolution levels are relevant for our understanding of mechanisms at the molecular level by which radiations affect biological systems. Since the Monte Carlo track structure codes for use in radiation chemistry and radiation biology are still in the developmental stage, a number of investigators have been exploring different strategies for improving these codes."

The use of molecular biology and biochemistry to study the regulation of gene expression has become a major feature of research in the biological sciences. Many excellent books and reviews exist that examine the experimental methodology employed in specific areas of molecular biology and regulation of gene expression. However, we have noticed a lack of books, especially textbooks, that provide an overview of the rationale and general experimental approaches used to examine chemically or disease-mediated alterations in gene expression in mammalian systems. For example, it has been difficult to find appropriate texts that examine specific experimental goals, such as proving that an increased level of mRNA for a given gene is attributable to an increase in transcription rates. Regulation of Gene Expression: Molecular Mechanisms is intended to serve as either a textbook for graduate students or as a basic reference for laboratory personnel. Indeed, we are using this book to teach a graduate-level class at The Pennsylvania State University. For more details about this class, please visit http: //moltox. cas. psu. edu and select "Courses. " The goal for our work is to provide an overview of the various methods and approaches to characterize possible mechanisms of gene regulation. Further, we have attempted to provide a framework for students to develop an understanding of how to determine the various mechanisms that lead to altered activity of a specific protein within a cell.

I am extremely honored and pleased to have the opportunity to write a few introductory words for this timely volume on Na + /It exchange. This is a field of investigation that I entered into by challenge and necessity, embraced with passion and fmally left in my quest for new discoveries in growth control. Ten years, one third of my scientific life, has been devoted to uncovering the mysteries of intracellular pH (PH;) regulation with respect to growth factor action. I got started on this new topic in 1980, when I heard a rather provocative hypothesis presented by Enrique Rozengurt at an ICN-UCLA Keystone meeting on "Cell Surface and Malignancy." He showed that all mitogens induced amiloride-sensitive Na + entry into resting cells and proposed that, if a compound stimulates Na + influx, it could be a mitogen. In support of his proposal Enrique reported that the amphipathic polypeptide, mellitin, which induced Na+ influx, was indeed mitogenic for 3T3 cells. This was only correlation at this stage. However, I was fascinated by this talk. I immediately approached Enrique to inform him of my skepticism about this beautiful story, and to indicate that I would only be convinced when I succeeded in isolating mutant fibroblasts lacking the amiloride-sensitive Na+ transporter. ''Good luck " was his response.

Immunofluorescence, a suitable laboratory method for the microscopic demonstration of antigens and antibodies in biological materials, useable, for example, to provide evidence for the pathogenesis of disease in histological or cytological preparations and for tumour cell differentiation. For this reason immunofluorescence has a decisive role as the method of choice for the diagnosis of auto-immune diseases. This primer on immunofluorescence techniques, which first appeared in 1979, is a richly illustrated handbook suitable for everyday practical work in the laboratory, useable as both an introduction to the subject as well as an atlas. In hardly any other area of medicine are there so many new findings to report. The second edition of this book is concerned not only with the detection methods which now form an essential and established part of diagnostic techniques, but also with the most recent research results such as the discovery of antibodies against Auerbach's plexus and against podocytes...

Peptide nucleic acids (PNAs) have now existed for slightly more than ten years, with the interest in and applications of this pseudopeptide DNA mimic steadily increasing during the entire period. PNAs have rapidly attracted the attention of scientists from a diversity of fields ranging from (bio)organic and biophysical chemistry to prebiotic evolution, and from molecular biology to genetic diagnostics and drug development. Many of the applications take advantage of the unique properties of PNA-an uncharged pseudopeptide-that distinguish this DNA mimic from more traditional DNA analogs. Rather than trying to create a comprehensive collection of all published methods and protocols involving PNA-many of which have not yet been validated- I have decided to concentrate on select protocols that are either very well established by several groups around the world, such as PCR-clamping and in situ hybridization, or on new methods that may have broader future impact. Basic methods for PNA oligomer synthesis and analyses have also been included. I am very grateful to those friends and colleagues who have enthusiastically contributed their work, discussions, and writing, and thereby made this book possible. Peter E. Nielsen v Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix IINTRODUCTION 1 PNA Technology Peter E. Nielsen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 II CHEMISTRY 2 Solid Phase Synthesis of PNA Oligomers Frederik Beck. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 3 Synthesis of PNA-Peptide Conjugates Satish Kumar Awasthi and Peter E. Nielsen. . . . . . . . . . . . . . . . . . 43 4 Parallel Synthesis of PNA-Peptide Conjugate Libraries Satish Kumar Awasthi and Peter E. Nielsen. . . . . . . . . . . . . . . . . .

Determination of the protein sequence is as important today as it was a half century ago, even though the techniques and purposes have changed over time. Mass spectrometry has continued its recent rapid development to find notable application in the characterization of small amounts of protein, for example, in the field of proteomics. The "traditional" chemical N-terminal sequencing is still of great value in quality assurance of the increasing number of biopharmaceuticals that are to be found in the clinic, checking processing events of recombinant proteins, and so on. It is joined in the armory of me- ods of protein analysis by such techniques as C-terminal sequencing and amino acid analysis. These methods are continually developing. The first edition of Protein Sequencing Protocols was a "snapshot" of methods in use in protein biochemistry laboratories at the time, and this, the second edition, is likewise. Methods have evolved in the intervening period, and the content of this book has similarly changed, the content of some chapters having been superceded and replaced by other approaches. Thus, in this edition, there is inclusion of approaches to validation of methods for quality assurance work, reflecting the current importance of biopharmaceuticals, and also a guide to further analysis of protein sequence information, acknowledging the importance of bioinformatics.

Flavonoids are secondary plant products that have previously been shown to be helpful in determining relationships among plant groups. This work presents comprehensively the occurrence, patterns of variation, and systematic and evolutionary importance of flavonoids in the sunflower family (Asteraceae), the largest family of flowering plants (23,000 species). It gathers together the more than 2500 reports of flavonoids in Asteraceae published between 1950 to the present and interprets these data in context of new taxonomic (especially generic) alignments. The authors discuss flavonoid patterns with reference to modern phylogenetic studies based on morphology and DNA data. This book provides, therefore, the most exhaustive synthesis and evaluation of the systematic and evolutionary import of flavonoids ever accomplished for any large family of angiosperms.

One prerequisite for the evolution of multicellular organisms was the invention of mechanisms by which cells could adhere to one another. At some point in our history, dividing cells no longer went their separate protozoic ways in the primordial oceans, but instead found that by maintaining an association, by sticking together but not fusing, numerous evolutionary advantages became possible. The subsequent development of specialized tissues and organs depended on the elaboration of incredibly sophisticated, regulatable cell-to-cell adhesion mechanisms which are known to operate in biological processes as diverse as the growth of the embryo, the immune response, the establishment of connections between nerve cells, and arteriosclerosis, to name just a few. Although we can only guess at the ancestral mechanisms that fostered the first primitive intercellular unions, some one billion years ago, we now recognize contemporary molecular "themes" with presumably ancient origins that mediate cell-cell interactions.
The chapters in this book serve as useful, thought-provoking, but not exhaustive, commentaries on contemporary topics within the broad field of cell adhesion. If the reader detects a slight tilt toward those adhesion molecules that function in the nervous system, this is merely a reflection of this editor's interests, biases, and of course, limitations.

"Advanced Protocols in Oxidative Stress III" continues the thread of the first two books by covering technology ranging from a portable hand-held detector for remote analysis of antioxidant capacity to sophisticated technology such as shotgun lipidomics, mitochondrial imaging, nano sensors, fluorescent probes, chromatographic fingerprints, computational models and bio statistical applications. Several chapters have shown the effect of pro-oxidation and antioxidants as inflammatory mediators in signaling pathways leading from the initial stimulus to termination through redox cycles. Written for the highly successful "Methods in Molecular Biology" series, 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.

Comprehensive and practical, "Advanced Protocols in Oxidative Stress III" offers to save investigators significant time and effort, allowing them to focus on their own personal topic of interest.

The study of plant development using molecular and genetic techniques is rapidly becoming one of the most active areas of research on flowering plants. The aim of this book is to present a sense of the accomplishments of the past and of the outstanding problems of the future in the study of plant development. An important feature of this book is the integration of results from molecular and genetic studies on various aspects of plant development in a cellular and physiological context. Of particular interest to readers will be the clear treatment of each landmark stage in the life cycle of plants such as seed germination, seedling growth, flowering, gametogenesis, pollen-pistil interactions, embryogenesis, fruit ripening, and seed and bud dormancy. The book also considers the development of individual plant organs such as the shoot, leaf, root and flowers as well as alternate developmental strategies of plants.

In recent years, there have been major advances in the concepts and methodologies used in the study of retinal development at both cellular and molecular levels. These advanced methodologies have allowed and will continue to allow researchers to gain new insights into the molecular mechanisms underlying retinal development. In Retinal Development: Methods and Protocols, expert researchers in the field detail many of the protocols used for a wide range of experiments. These include protocols and techniques for manipulating gene expression in vivo, tracing cell fates with modernized classic blastomere manipulation in Xenopus and with Cre-based technique in mouse and in zebrafish, retinal regeneration and stem cell-based replacement, and ERG (function) recording and non-invasive imaging. 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, Retinal Development: Methods and Protocols provides methodologies crucial to the success of increasingly more complex and often challenging investigations in the fields of retinal development and other biological and biomedical research.

The fungus fusarium is a major plant pathogen that causes disease in nearly every agriculturally important plant. In addition, some strains produce mycotoxins that can cause serious illness in humans and livestock. The enormous economic importance of and health hazards posed by fusarium have fuelled research into its biochemistry, genetics, genomics, proteomics and metabolomics by scientists worldwide. In this book, an international group of researchers critically review the most important research on the genomics and molecular and cellular biology of fusarium.

Describes landmark experiments in cell biology and biochemistry Discusses the "How" and "Why" of historically important experiments Includes primary, original data and graphs Emphasizes biological techniques, which helps understand how many of the experiments performed were possible. Documents, chronologically, how each result fed into the next experiments.

This new edition continues to illustrate the power of biological data in knowledge discovery. It describes biological data types and representations with examples for creating a workflow in bioinformation discovery. The concepts in knowledge discovery from data are illustrated using line diagrams. The principles and concepts in knowledge discovery are used for the development of prediction models for simulations of biological reactions and events. Advanced topics in molecular evolution and cellular & molecular biology are addressed using bioinformation gleaned through discovery. Each chapter contains approximately 10 exercises for practice. This will help students to expand their problem solving skills in Bioinformation Discovery. In this new edition, there are three new chapters covering single nucleotide polymorphism, genes, proteins and disease, and protein functions driven by surface electrostatics.

Solvent Extraction in Biotechnology deals with the reco- very and purification of primary and secondary metabolites by solvent extraction. In the first part the reaction engineering principles: definitions, thermodynamic fundamentals, and system models, the kinetics of mass transfer between two phases without and with chemical reaction as well as extraction equipment, which are important for downstream processing in biotechnology, are considered in detail. The special part of the book describes the recovery of low-molecular metabolites: alcohols, acids and antibiotics with organic solvents, carrier-modifier-solvent systems, supercritical gases as well as with liquid membrane techniques. Several practical examples are given for the recovery of different metabolites as well as for the calculation of the extraction processes necessary for equipment design. Besides solvent extraction, novel separation techniques with liquid membrane, microemulsion and reversed micelles are also presented. This book will introduce the biochemical engineer and process engineer to the recovery of products from complex cultivation broths by modern techniques of solvent extraction and help them with process design.

In recent years, the study of the plant cell cycle has become of major interest, not only to scientists working on cell division sensu strictu, but also to scientists dealing with plant hormones, development and environmental effects on growth. The book The Plant Cell Cycle is a very timely contribution to this exploding field. Outstanding contributors reviewed, not only knowledge on the most important classes of cell cycle regulators, but also summarized the various processes in which cell cycle control plays a pivotal role. The central role of the cell cycle makes this book an absolute must for plant molecular biologists.

Plant Cell and Tissue Culture gives an exhaustive account of plant cell culture and genetic transformation, including detailed chapters on all major field and plantation crops. Part A presents a comprehensive coverage of all necessary laboratory techniques for the initiation, nutrition, maintenance and storage of plant cell and tissue cultures, including discussions on these topics, as well as on morphogenesis and regeneration, meristem and shoot tip culture, plant protoplasts, mutant cell lines, variation in tissue cultures, isogenic lines, fertilization control, cryopreservation, transformation, and the production of secondary metabolites. Part B then proceeds into detail on the specific in vitro culture of specific crops, including cereals, legumes, vegetables, potatoes, other roots and tubers, oilseeds, temperate fruits, tropical fruits, plantation crops, forest trees and ornamentals. Plant Cell and Tissue Culture is, and is likely to remain, the laboratory manual of choice, as well as a source of inspiration and a guide to all workers in the field.

Mass Spectrometry Data Analysis in Proteomics is an in-depth guide to the theory and practice of analyzing raw mass spectrometry (MS) data in proteomics. As MS is a high throughput technique, proteomic researchers must attend carefully to the associated field of data analysis, and this volume outlines available bioinformatics programs, algorithms, and databases available for MS data analysis. General guidelines for data analysis using search engines such as Mascot, Xtandem, and VEMS are provided, with specific attention to identifying poor quality data and optimizing search parameters. Several different types of MS data are discussed, followed by a description of optimal methods for conversion of raw data into peak lists for input to search engines. Choosing the most accurate and complete databases is emphasized, and a report of available sequence databases is included. Methods for assembling expressed sequence tags (ESTs) into assembled nonredundant databases are provided, along with protocols for further processing the sequences into a format suitable for MS data. Mass Spectrometry Data Analysis in Proteomics describes publicly available applications whenever possible.

This volume illustrates how microfluidic approaches can meet the requirement of clinical diagnosis based on molecular or cellular biomarkers. Microchip Diagnostics: Methods and Protocols is divided into four sections describing the business aspects of the microfluidic-based systems for diagnosis, demonstrating how versatile microfluidics can be regarding to protein bioassay integration, presenting microfluidic approaches for nucleic analysis based on mono or diphasic format, and highlighting recent contributions. Written in the highly 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 laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

NMR spectroscopy has found a wide range of applications in life sciences over recent decades. Providing a comprehensive amalgamation of the scattered knowledge of how to apply high-resolution NMR techniques to biomolecular systems, this book will break down the conventional stereotypes in the use of NMR for structural studies. The major focus is on novel approaches in NMR which deal with the functional interface of either protein-protein interactions or protein-lipid interactions. Bridging the gaps between structural and functional studies, the Editors believe a thorough compilation of these studies will open an entirely new dimension of understanding of crucial functional motifs. This in turn will be helpful for future applications into drug design or better understanding of systems. The book will appeal to NMR practitioners in industry and academia who are looking for a comprehensive understanding of the possibilities of applying high-resolution NMR spectroscopic techniques in probing biomolecular interactions.

Inflammation has been described as the basis of many pathologies of human disease. When one considers the updated signs of inflammation, they would be vasodilation, cell migration, and, in the case of chronic inflam- tion, cell proliferation, often with an underlying autoimmune basis. Gen- ally, inflammation may be divided into acute, chronic, and autoimmune, - though the editors believe that most, if not all, chronic states are often the result of an autoimmune response to an endogenous antigen. Thus, a proper understanding of the inflammatory basis may provide clues to new therap- tic targets not only in classical inflammatory diseases, but atherosclerosis, cancer, and ischemic heart disease as well. The lack of advances in classical inflammatory diseases, such as rh- matoid arthritis, may in part arise from a failure to classify the disease into different forms. That different forms exist is exemplified in patients with d- fering responses to existing antiinflammatory drugs, ranging from nonresponders to very positive responders for a particular nonsteroidal an- inflammatory drug (NSAID). Though researchers have progressively unr- eled the mechanisms, the story is far from complete. It should also be noted that the inflammatory response is part of the innate immune response, or to use John Hunter's words in 1795, "inflammation is a salutary response." That may be applied in particular to the defensive response to invading micro- ganisms.

In the post-genomic era, in vitro mutagenesis has emerged as a critically important tool for establishing the functions of components of the proteome. The third edition of In Vitro Mutagenesis Protocols represents a practical toolbox containing protocols vital to advancing our understanding of the connection between nucleotide sequence and sequence function. Fully updated from the previous editions, this volume contains a variety of specialty tools successfully employed to unravel the intricacies of protein-protein interaction, protein structure-function, protein regulation of biological processes, and protein activity, as well as a novel section on mutagenesis methods for unique microbes as a guide to the generalization of mutagenesis strategies for a host of microbial systems. Written in the highly successful Methods in Molecular Biology 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 expert tips on troubleshooting and avoiding known pitfalls.

Authoritative and up-to-date, In Vitro Mutagenesis Protocols, Third Edition offers today's researchers a valuable compendium of reliable and powerful techniques with which to illuminate the proteome and its rich web of biological implications.

Helicobacter pylori is an important human pathogen that infects up to 50% of the human population. As the leading cause of peptic ulcers, gastritis, and gastric cancer worldwide, the organism has been the subject of intensive research to unravel the mysteries of its genetics and cellular biology. In fact, the number of publications in this field has risen dramatically in recent years making it extremely difficult for even the most diligent reader to stay abreast of progress. This book distills the most important cutting-edge findings in the field to produce a timely and comprehensive review. With contributions from leading international helicobacter researchers, topics include: lipopolysaccharides, outer membrane proteins, motility and chemotaxis, type IV secretions systems, metal metabolism, molecular mechanisms of host adaptation, genomotyping, and proteonomics. As a useful introduction to the subject for new researchers and as an invaluable reference for the experienced researcher, this book is essential reading for all researchers working with Helicobacter and related organisms.