Amino acid analysis is a technique that has become commonplace in biotechnology, biomedical, and food analysis laboratories. This book describes a variety of amino acid analysis techniques and how each technique can be used to answer specific biological questions. The first two chapters in Amino Acid Analysis Protocols introduce the concepts, basic theory, and practice of amino acid analysis. The following chapters give detailed instructions on various methods and their applications. As highlighted, there are many different approaches to amino acid ana- sis, but in all cases the results depend heavily on the quality of the sample. Therefore a new way to desalt samples prior to hydrolysis is covered as an introductory chapter (Chapter 3), and most authors have devoted a section to sample preparation, especially to the collection and storage of bodily fluids. Some of the amino acid analysis methods described in this book are based on HPLC separation and analysis after precolumn derivatization. The precolumn derivatization techniques described use (a) 6-aminoquinolyl-N-hydro- succinimidyl carbamate (AQC) (Chapters 4 and 8); (b) 1-fluoro-2- dinitrophenyl-5-L-alanine amide (Marfey's reagent), which allows sepa- tion and analysis of enantiomeric amino acids (Chapter 5); (c) O-phthalal- hyde (OPA) (Chapters 6 and 10); (d) butylisothiocyanate (BITC) and benzylisothiocyante (BZITC) (Chapter 11); (e) phenylisothiocyanate (PITC) (Chapters 12 and 13); (f) ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-s- fonate (SBD-F) (Chapter 17); and (g) 9-fluorenylmethyl-chloroformate (FMOC-Cl) (Chapter 10).

The current book describes the chemical and physical behaviour of polymers and biopolymers that form highly associating structures in equilibrium solution. It summons the established results known of polymer complexes in solution, taking into account also the recent developments in biotechnology concerning this topic, in technological applications of polymer-protein interactions, in fluorescence and scattering techniques for the study of intra- and interpolymer association and in the study of ionomers in solution. The book covers the whole range from synthesis and fundamental aspects to applications and technology of associated polymers.

Viruses and RNAi share an intricate relationship at many levels. RNAi is an important antiviral defense mechanism in plants and invertebrates, microRNAs of viral or cellular origin affect many aspects of virus biology, and replication of many, if not all, mammalian viruses can be suppressed by RNAi." Antiviral RNAi: Concepts, Methods, and Applications "provides a collection of protocols for the analysis of viral small RNAs and natural antiviral RNAi responses as well as for the development and optimization of RNAi-based antiviral drugs. As RNAi is a central regulatory mechanism in the cell, the methods in this volume can also be applied out of the context of a virus infection. Divided into five convenient parts, this detailed volume reviews important basic concepts in the field of antiviral RNAi, provides experimental and bio-informatic tools for the analysis of small silencing RNAs, covers methods to biochemically dissect RNAi-based antiviral defense and viral counter-defense mechanisms, describes methods for the design, expression, and delivery of therapeutic antiviral siRNAs, and finally presents genome-wide RNAi approaches for the identification of factors involved in virus replication. Written in the highly successful "Methods in Molecular Biology " series format, chapters contain 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 accessible, "Antiviral RNAi: Concepts, Methods, and Applications" serves as an ideal guide for both novice and experienced researchers alike striving to dissect the role of RNAi in the viral life cycle or to further boost the development of novel therapeutics and experimental tools based on RNAi technology.

Many compounds of biological and pharmacological interest are as- metric and show optical activity. Approximately 40% of the drugs in use are known to be chiral and only about 25% are administered as pure enantiomers. It is well established that the pharmacological activity is mostly restricted to one of the enantiomers (eutomer). In several cases, unwanted side effects or even toxic effects may occur with the inactive enantiomer (distomer). Even if the side effects are not that drastic, the inactive enantiomer has to be meta- lized, which represents an unnecessary burden for the organism. The admin- tration of pure, pharmacologically active enantiomers is therefore of great importance. The ideal way to get to pure enantiomers would be by enantioselective synthesis. However, this approach is usually expensive and not often practicable. Usually, the racemates are obtained in a synthesis, and the separation of the enantiomers on a preparative scale is necessary. On the other hand, there is also a great demand for methods of enantiomer separation on an analytical scale for controlling synthesis, checking for racemization p- cesses, controlling enantiomeric purity, and for pharmacokinetic studies. C- ventional methods for enantiomer separation on a preparative scale are fractionated crystallization, the formation of diastereomeric pairs followed by repeated recrystallization, and enzymatic procedures. In recent years, ch- matographic methods such as gas chromatography and, especially, liquid ch- matography have attracted increasing interest for chiral separation, both on analytical and preparative scales.

The previous edition of Transmembrane Signaling Protocols was published in 1998. Since then the human genome has been completely sequenced and new methods have been developed for the use of microarrays and proteomics to analyze global changes in gene expression and protein profiles. These advances have increased our ability to understand transmembrane signaling processes in much greater detail. They have also simultaneously enhanced our ability to determine the role of a large number of newly identified molecules in signaling events. In addition, novel video microscopy methods have been developed to image transmembrane signaling events in live cells in real time. In view of these major advances, it is time to update the previous edition. Because of the success of that volume, we have chosen to keep the essential character of the book intact. Introductory chapters from experts have been included to provide overall perspective and an overview of recent advances in signal transduction pathways. The individual chapters now include comp- hensive detailed methods, studies in genetically tractable systems, fluorescence microscopy in live single cells, ex vivo analysis of primary cells from tra- genic mice, as well as genomic and proteomic approaches to the analysis of transmembrane signaling events. We would like to express our deep gratitude to the coauthors of this publi- tion. We hope that Transmembrane Signaling Protocols, Second Edition will serve as a valuable resource for future progress in the study of signal transd- tion pathways.

This volume of the Methods in Molecular Biology series is entirely devoted to the study of steroid receptor biology. Steroid hormone receptors represent a powerful system for the study of both the most fundamental molecular mec- nisms of gene regulation and control and the gross physiological responses of organisms to steroid hormones. Research in this field has brought forth advances in the treatment of cancer, endocrine disorders, and reproductive biology, and allowed elucidation of the fundamental biological mechanisms of gene expr- sion. In Steroid Receptor Methods: Protocols and Assays, the reader will find a collection of methods and protocols submitted by many fine steroid receptor researchers from throughout the world. These authors have been instructed to create a highly informative cross-section of the latest research techniques ava- able. The resulting work is timely, useful, and approachable for both the ex- rienced researcher and the novice to the field. Because the steroid receptor family is represented by a wonderfully diverse, yet strongly interrelated set of steroid receptor proteins, Steroid Receptor Methods contains protocols for the prod- tion and purification of a variety of receptor forms, including the progesterone, glucocorticoid, and androgen receptors. These procedures provide the raw ma- rial needed to conduct sophisticated biochemical analysis of receptor properties. Other techniques presented allow the reader to perform biochemical experiments on DNA binding characteristics, hormone binding assays, and protocols using combinatorial chemistry for drug discovery.

In the last few years, significant breakthroughs in transcription research expanded our appreciation for the complexity of molecular controls on gene expression in mammalian cells. In Transcription Factors: Methods and Protocols, experts in the field describe state-of-the-art approaches that investigators can use to probe critical mechanisms underlying transcription factor nuclear-cytoplasmic trafficking as well as to assess the functional impact of post-translational modifications on transcription factor function. The chapters are written by prominent scientists, many of whom developed these methods, and highlight protocols that focus on specific transcription factor family members with particular relevance to human disease. Composed in the highly successful Methods in Molecular Biology(TM) series format, each chapter contains a brief introduction, step-by-step methods, a list of necessary materials, and a Notes section which shares tips on troubleshooting and avoiding known pitfalls.

Comprehensive and current, Transcription Factors: Methods and Protocols compiles the latest techniques for elucidating controls on transcription factor intracellular localization and activity, and consequently is unlike any other methods-based text on transcriptional regulation today.

In this volume, international experts discuss the following topics: molecular principles of the genesis of prostate cancer and the involvement of oncogenes and tumour suppressor genes; changes of cell-cell contacts; defects in androgen receptors and their effect on treatment with antiandrogens; drug resistance mechanisms and new therapeutic principles; and molecular diagnosis of prostate cancer.

This volume compiles a broad range of step-by-step protocols, complementary to the ones published in the first edition of this book, to study various aspects of mitochondrial structure and function in different model organisms, both in vitro and in vivo. 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, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Mitochondria: Practical Protocols, Second Edition aims to be useful for beginners as well as for experienced researchers in the field.

This volume presents detailed laboratory protocols for in vitro synthesis of mRNA with favorable properties, its introduction into cells by a variety of techniques, and the measurement of physiological and clinical consequences such as protein replacement and cancer immunotherapy. Synthetic techniques are described for structural features in mRNA that provide investigational tools such as fluorescence emission, click chemistry, photo-chemical crosslinking, and that produce mRNA with increased stability in the cell, increased translational efficiency, and reduced activation of the innate immune response. Protocols are described for clinical applications such as large-scale transfection of dendritic cells, production of GMP-grade mRNA, redirecting T cell specificity, and use of molecular adjuvants for RNA vaccines. 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 laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Synthetic mRNA: Production, Introduction into Cells, and Physiological Consequences is a valuable and cutting-edge resource for both laboratory investigators and clinicians interested in this powerful and rapidly evolving technology.

This volume is made up of papers presented at the Second International Altschul Symposium: Biology and Pathology of Astrocyte-Neuron Interactions. The symposium was held in Saskatoon, Canada at the University of Saskatchewn in May, 1992 in memory of Rudolf Altschul, a graduate of the University of Prague and a pioneer in the fields of the biology of the vascular and nervous systems. Dr. Altschul was Professor and Head of the Department of Anatomy at the University of Saskatchewan from 1955 to 1963. The Altschul Symposia were made possible by an endowment left by Anni Altschul and by other contributions. The symposia are held biennially. One of the greatest challenges for present day scientists is to uncover the mechanisms of brain function. Although cellular anatomy of the nervous system has already been well outlined and indeed was delineated by the beginning of the century, experimental analysis of the function of the brain is relatively recent. The framework of the brain is made up of stellate cells, the astrocytes, which are interconnected by means of their processes, thus presenting a meshwork through which the neurons send their axons, accompanied by oligodendrocytes. Microglia are distributed throughout the brain.

Recently there have been many advances in the understanding of the genetic basis of development and regular breakthroughs are being made in the field of tumour cell targeting. Both these areas of research are coming together in terms of their perception of programmed cell death. A broad introduction of the biological significance of cell death is followed by a major review of the significance of physiological cell death in tumours and the factors that influence it. The volume includes a consideration of the role and functions of lymphokines and their bearing on tumour cell targeting and cell killing. The interleukins, interferons and tumour necrotic factor (TNF) are presented in terms of their functional significance rather than in a sequential or systematic manner. This volume concentrates on the bases of biological or programmed cell death. Aspects of necrosis are, however, of necessity covered in comparative and technical terms. The central section on cell death in tumours is followed by a resume of the techniques employed in demonstrating cell death and this section closes with an appendix giving practical details of selected methods. It is hoped that the book may help potential research workers focus not only on the underlying molecular biology of programmed cell death but also provide impetus for the development of appropriate therapeutic regimes in tumour research.

This book introduces the reader to modern computational and statistical tools for translational epigenomics research. Over the last decade, epigenomics has emerged as a key area of molecular biology, epidemiology and genome medicine. Epigenomics not only offers us a deeper understanding of fundamental cellular biology, but also provides us with the basis for an improved understanding and management of complex diseases. From novel biomarkers for risk prediction, early detection, diagnosis and prognosis of common diseases, to novel therapeutic strategies, epigenomics is set to play a key role in the personalized medicine of the future. In this book we introduce the reader to some of the most important computational and statistical methods for analyzing epigenomic data, with a special focus on DNA methylation. Topics include normalization, correction for cellular heterogeneity, batch effects, clustering, supervised analysis and integrative methods for systems epigenomics. This book will be of interest to students and researchers in bioinformatics, biostatistics, biologists and clinicians alike. Dr. Andrew E. Teschendorff is Head of the Computational Systems Genomics Lab at the CAS-MPG Partner Institute for Computational Biology, Shanghai, China, as well as an Honorary Research Fellow at the UCL Cancer Institute, University College London, UK.

In our first protocols book, Free Radical and Antioxidant Protocols (1), r- erence to in vivo, ex vivo, or in situ techniques were few compared to classical biochemical assays and only 6 of the 40 chapters were concerned with these applications. In our second book, Oxidative Stress Biomarkers and Antioxidant Protocols (2), which is being published concurrently with this third volume, Oxidants and Antioxidants: Ultrastructure and Molecular Biology Protocols, the number of such chapters has increased. The literature dealing with histoche- cal/cytochemical and immunohistochemical techniques and staining to identify cellular/subcellular sites of oxidative stress has expanded rapidly, as has the molecular biology methodology used to analyze free radical and antioxidant (AOX) reactions, as well as the monitoring of living tissue. A two-way search was performed for each technique listed in Table 1, coupled with "oxidative stress" using the PUBMED search engine from the National Library of Medicine at NIH. Most of the techniques involved in m- suring oxidative stress employ molecular biology or ultrastructural approaches. Of these techniques, histology, polymerase chain reaction, and Western blotting are the most widely used. Several forms of therapy are now available for patients with increased oxidative stress. In addition to standard antioxidant therapy supplementation in vivo and in vitro, photodynamic therapy (PDT) employs excitation of a photon-emitting compound delivered systemically for free radical-mediated necrosis of affected tissues, and stem cells are also being used to induce signaling events or replace antioxidant enzymes.

General compendium of HDAC inhibitors with deep emphasis on toxicity issues of synthetic HDAC inhibitors Various groups of natural HDAC inhibitors, their representatives and premier sources Cyclic tetrapeptides of natural origin and their importance as cancer chemotherapeutic agents Hydroxamates and depsipeptides from natural sources and their promising role in cancer therapy Natural Flavonoids, their HDAC inhibitory tendency and marvellous anticancer activity Non-flavonoid natural HDAC inhibitors and their pleasing cytotoxic effects towards cancer models Combined therapy involving natural flavonoids with other anticancer molecules for synergistic and additive benefits against cancer models Non-flavonoid HDAC inhibitors and conventional drugs in collaborative mode against aggressive malignancies Nanotechnology based delivery of natural HDAC inhibitors for greater therapeutic efficacy over traditional combinatorial therapy

"Methods of Clinical Epidemiology" serves as a text on methods useful to clinical researchers. It provides a clear introduction to the common research methodology specific to clinical research for both students and researchers. This book sets out to fill the gap left by texts that concentrate on public health epidemiology and focuses on what is not covered well in such texts. The four sections cover methods that have not previously been brought together in one text and serves as a second level textbook of clinical epidemiology methodology. This book will be of use to postgraduate students in clinical epidemiology as well as clinical researchers at the start of their careers.

The complement system, first described more than a century ago, was for many years the ugly duckling of the immunology world, but no more. Complement in recent years has blossomed into a fascinating and fast moving field of immediate relevance to clinical scientists in fields as diverse as transplantation biology, virology, and inflammation. Despite its emergence from the shadows, complement retains an unwarranted reputation for being "difficult." This impression derives in large part from the superficially complicated nomenclature, a relic of the long and tortuous process of unraveling the system, of naming components in order of discovery rather than in a syst- atic manner. Once the barrier of nomenclature has been surmounted, then the true simplicity of the system becomes apparent. Complement comprises an activation system and a cytolytic system. The former has diverged to focus on complement to distinct targets-bacteria, - mune complexes, and others-so that texts now describe three activation pa- ways, closely related to one another, but each with some unique features. The cytolytic pathway is the same regardless of the activation process and kills cells by creating pores in the membrane. Complement plays an important role in killing bacteria and is essential for the proper handling of immune complexes. Problems occur when complement is activated in an inappropriate manner-the potent inflammation-inducing products of the cascade then cause unwanted tissue damage and destruction.

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 agric- tural 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 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. Ag- bacterium-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 devel- ments indicating that Agrobacterium can also be used to deliver DNA to non-plant species including bacteria, fungi, and even mammalian cells.

Signaling networks are composed of numerous signaling pathways and each has its own intricate component parts. Signaling outputs are dynamic, extraordinarily complex and yet highly specific. In, Computational Modeling of Signaling Networks: Methods and Protocols, expert researchers in the field provide key techniques to study signaling networks. Focusing on Systems of ODEs, parameterization of signaling models, signaling pathways, mass-action kinetics and ODEs, and how to use modeling to plan experiments. 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, Computational Modeling of Signaling Networks: Methods and Protocols aids scientists in continuing study of how signaling networks behave in space and time to generate specific biological responses and how those responses impact biology and medicine.

This volume includes comprehensive descriptions of miRNA biogenesis and their role in the development and progression of various human diseases. The first few chapters of MicroRNA Profiling: Methods and Protocols discuss the effects of over-expressing and repressing of a target miRNA and their effects on cell viability and proliferation. The next few chapters explore the protocols for total RNA isolation from cells and cell-derived product including formalin fixed paraffin embedded tissue and plant tissue. The last few chapters discuss isolation and characterization of exosomes from medium conditioned by cell lines, serum, and plasma specimens. This book also includes discussions of several software tools, such as miRandola, PicTar, DIANA, and miRWalk. 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. Comprehensive and cutting-edge, MicroRNA Profiling: Methods and Protocols is a valuable resource for anyone interested in the field of Micro RNAs.

Eight years have elapsed since the first International Meeting on Plant Mitochondria was held in Marseilles. Since this date numerous important developments have occurred within the field and hence a further conference on this fundamental area of research was considered well overdue. This volume summarises the lecture and poster sessions of the second International Meeting on Plant Mitochondria held in Aberystwyth, July 20- 24th, 1986. The meeting was held not only to bring together plant scientists interested in the bioenergetics of plant mitochondria but also those who are interested in the regulatory role of mitochondria in plant growth and respiration. A further important aspect of this conference was to introduce plant physiologists and biochemists to the plant molecular biologists in an attempt to not only discuss problems of mutual interest but to also learn much more about the real questions which the biochemists and physiologists wish to answer. Hopefully the volume reflects much of the current excitement and advances being made in the field. Although many of the participants of the first meeting were present the expertise of Walter Bonner, Jack Hanson and Gaston Ducet, to name but a few, was sorely missed. The conference consisted of forty-five minute review lectures followed by thirty minute research lectures, the summaries of which are found in the longer articles. The meeting was divided into four seSSions, namely, organisation of the electron transport chain; mitochondrial interactions; mitochondrial biogenesis and plant growth and development.

The first protocols book, Free Radical and Antioxidant Protocols (1) was published in late 1998. Sections were divided into three parts, covering selected biochemical techniques for measuring oxidative stress, antioxidant (AOX) activity, and combined applications. In choosing the 40 methods to be included in that book, I realized there were considerably more of equal value than that which we could have presented in a single volume. To produce a comprehensive resource, this book and a third are being compiled to expand coverage of the field. A summary of papers (2) published on this important subject emphasizes the continuing rapid growth in oxidative stress investigations relating to our understanding of biochemical reactions, their relevance to pathophysiological mechanisms, how disease may arise, and how therapeutic intervention may be achieved(3). Although there is some overlap between the categories, the ana- sis shown below illustrates where current studies are concentrated and are almost evenly distributed between free radicals and AOX. Over the last 4 yr, there has been a 55% increase in the number of papers published in the area.

This book provides a versatile and lucid treatment of classic as well as modern probability theory, while integrating them with core topics in statistical theory and also some key tools in machine learning. It is written in an extremely accessible style, with elaborate motivating discussions and numerous worked out examples and exercises. The book has 20 chapters on a wide range of topics, 423 worked out examples, and 808 exercises. It is unique in its unification of probability and statistics, its coverage and its superb exercise sets, detailed bibliography, and in its substantive treatment of many topics of current importance. This book can be used as a text for a year long graduate course in statistics, computer science, or mathematics, for self-study, and as an invaluable research reference on probabiliity and its applications. Particularly worth mentioning are the treatments of distribution theory, asymptotics, simulation and Markov Chain Monte Carlo, Markov chains and martingales, Gaussian processes, VC theory, probability metrics, large deviations, bootstrap, the EM algorithm, confidence intervals, maximum likelihood and Bayes estimates, exponential families, kernels, and Hilbert spaces, and a self contained complete review of univariate probability.

The purpose of these volumes is to provide a reference work for the methods of purifying many of the receptors we know about. This be comes increasingly important as full-length receptors are overexpressed in bacteria or in insect cell systems. A major problem for abundantly expressed proteins will be their purification. In addition to purification protocols, many other details can be found concerning an individual receptor that may not be available in standard texts or monographs. No book of this type is available as a compendium of purification procedures. Receptor Purification provides protocols for the purification of a wide variety of receptors. These include receptors that bind: neurotransmit ters, polypeptide hormones, steroid hormones, and ligands for related members of the steroid supergene family and others, including receptors involved in bacterial motion. The text of this information is substantial, so as to require its publication in two volumes. Consequently, a division was made by grouping receptors by the nature of their ligands. Thus, in Volume One there are contributions on serotonin receptors, adrenergic receptors, the purification of GTP-binding proteins, opioid receptors, neurotensin receptor, luteinizing hormone receptor, human chorionic gonadotropin receptor, follicle stimulating hormone receptor, thyro tropin receptor, prolactin receptor, epidermal growth factor receptor, platelet derived growth factor receptor, colony stimulating factor recep tor, insulin-like growth factor receptors, insulin receptor, fibronectin receptor, interferon receptor, and the cholecystokinin receptor."

This volume aims to describe a complementary range of molecular, cell biological, and in vivo protocols used to investigate the structure-function of nuclear receptors, together with experimental approaches that may lead to new drugs to selectively target nuclear receptor-associated diseases. The Nuclear Receptor Superfamily, Second Edition will benefit those starting out in the nuclear receptor research field as well as to more established researchers who wish to apply different methods to a particular receptor or research problem. 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. Authoritative and cutting-edge, The Nuclear Receptor Superfamily, Second Edition aims to ensure successful results in the further study of this vital field.