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 past few years, the scientific community has witnessed significant progress in the study of ion channels. Technological advancement in biophysics, molecular biology, and immunology has been greatly ac celerated, making it possible to conduct experiments which were deemed very difficult if not impossible in the past. For example, patch-clamp techniques can now be used to measure ionic currents generated by almost every type of cell, thereby allowing us to analyze whole-cell and single channel events. It is now possible to incorporate purified ion channel components into lipid bilayers to reconstitute an "excitable membrane." Gene cloning and monoclonal antibody techniques provide us with new approaches to the study of the molecular structure of ion channels. A variety of chemicals have now been found to interact with ion channels. One of the classical examples is represented by tetrodotoxin, a puffer fish poison, which was shown in the early 1960s to block the voltage-activated sodium channel in a highly specific and potent manner.

Membrane fusion is an important molecular event which plays a pivotal role in many dynamic cellular processes, such as exocytosis, endocytosis, membrane biogenesis, fertilization, etc. While many reports on the physico-chem1cal process involved in membrane fusion have appeared in the literature and much information has accumulated, there has been no comprehensiv meeting of workers in the field. A recent symposium which took place at the Center for Tomorrow, State University of New York at Buffalo, New York, April 27-29, 1987, was the first meeting of its kind to specifically address the molecular mechanisms of membrane fusion. The Symposium consisted of oral, workshop and poster presentation sessions. The papers presented in the oral and workshop sessions are collected here and arranged approximately in the order presented at the Symposium. These papers are the most up-to-date and representative work at the for front of each aspect of membrane fusion. Although the readers may find some differences in interpretations regarding the molecular mechanisms of membrane fusion, there is an over all concensus that increased hydrophobicity and dehydration of the membrane surface are essential physico-chemical factors for membrane fusion to occur. We trust that these papers will contribute to your further understanding of the mechanisms of membrane fusion."

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.

Mitosis: Methods and Protocols provides state-of-the-art overviews on the most important approaches currently used in mitosis research spanning from the analysis of single molecules in isolation to their utilization within the complex environment of the cell. The volume is divided into four parts, each focused on methods pertaining to distinct aspects of mitosis research. Part I presents approaches for visualizing and analyzing the dynamic behaviors of the spindle apparatus, the microtubule based machine that drives chromosome segregation. Part II focuses more generally on methods for studying and manipulating the microtubule cytoskeleton in cells and complex cell free extracts. Part III provides state of the art biophysical and high resolution microscopy approaches for assessing complex interactions between microtubules and microtubule-associated proteins in isolation as well as microtubule structure in cells. Part IV provides methods for studying the effects of cell shape on cell division and methods for quantifying aneuploidy (aberrant chromosome number) which frequently results from mitotic defects and has been linked to human maladies ranging from birth defects to cancer. 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 key tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Mitosis: Methods and Protocols seeks to provide diverse methods and new techniques to address new or old questions related to the mechanisms of mitosis.

A general review of lipid bilayer structure and dynamics is given, including such current topics as the hydration of lipid bilayers, the superstructural behaviour of bilayers at different states of hydration and external conditions, the role and behaviour of lipid bilayers on fusion and rupture and the interaction of lipid bilayers with small organic molecules and additives and of protein lipid bilayer interactions. In addition, recent research on lipid interaction with proteins and other molecules in monolayers is reviewed, and the use of highly aligned samples under biologically relevant conditions and the benefits derived from such preparations are addressed. Finally, the latest approach in simulation of impurities within a lipid bilayer is introduced. This book will be a comprehensive review of the current state of biologically relevant model membrane systems which will become an indispensible reference for the "working biophysicist."

This book provides an update of recent advances in the basic and clinical applications of cell-based therapies for myocardial repair and regeneration in ischemic heart disease (IHD) and heart failure (HF). The first sections of the book illuminate basic aspects of stem cells such as definitions, isolation criteria and characterization of embryonic and adult stem cells, as well as pluripotent stem cells and tissue specific progenitor and stem cells. In the following section, the text examines the role of critical regulators of stem cell differentiation in myocardial regeneration, that include circadian rhythyms, microRNAs, epigenetics, microvescicles, and exosomes. The text concludes with a review of the use of stem/progenitor cells in cardiac regeneration and discuss current controversies, unresolved issues, challenges, and future directions. Stem Cells and Cardiac Regeneration is addressed to a wide audience consisting of cardiologists, clinicians, and research scientists, who will learn new concepts in cardiovascular regeneration and repair with special focus on molecular mechanisms and therapeutic potential for cardiovascular regeneration.

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.

Soil salinity is a key abiotic-stress and poses serious threats to crop yields and quality of produce. Owing to the underlying complexity, conventional breeding programs have met with limited success. Even genetic engineering approaches, via transferring/overexpressing a single 'direct action gene' per event did not yield optimal results. Nevertheless, the biotechnological advents in last decade coupled with the availability of genomic sequences of major crops and model plants have opened new vistas for understanding salinity-responses and improving salinity tolerance in important glycophytic crops. Our goal is to summarize these findings for those who wish to understand and target the molecular mechanisms for producing salt-tolerant and high-yielding crops. Through this 2-volume book series, we critically assess the potential venues for imparting salt stress tolerance to major crops in the post-genomic era. Accordingly, perspectives on improving crop salinity tolerance by targeting the sensory, ion-transport and signaling mechanisms were presented in Volume 1. Volume 2 now focuses on the potency of post-genomic era tools that include RNAi, genomic intervention, genome editing and systems biology approaches for producing salt tolerant crops.

This new volume reviews current progress on different approaches of in vivo reprogramming technology. Leaders in the field discuss how in vivo cell lineage reprogramming can be used for tissue repair and regeneration in different organs, including brain, spinal cord, pancreas, liver and heart. Recent studies on in vivo cell reprogramming towards pluripotency are reviewed; examples are given to show its potential in regenerative medicine. In each chapter, the regenerative potential of different in vivo reprogramming approaches is discussed in detail. More specifically, how different tissue failures or damages can be treated with this technology is explained. Examples from various animal models are given and the regenerative potential of in vivo reprogramming is compared to that of cell transplantation studies. The last chapter discusses current challenges of these preclinical studies and gives suggestions in order to improve the current strategies. Future directions are indicated for the transition of in vivo reprogramming technology to clinical settings. This is among the first books in the literature which specifically focuses on the in vivo reprogramming technology in regenerative medicine and these chapters collectively cover one of the most important and exciting topics of regenerative medicine.

Defining and understanding cellular and molecular mechanisms that are relevant to women's health has become a critical area of scientific pursuit. Until recently, very little effort has been place on defining or understanding critical differences between women and men that may be critical to the overall health of the woman. In 1990, the National Institutes of Health recognized this gap in knowledge resulting in the creation of the Office of Research on Women's Health. One of the purposes of this office was to advance the understanding of health issues from the women's perspective from both a basic and clinical scientific perspective. From a scientific evolution of understanding, the existence of this office is new and thus there has not been enough time for new information to integrate itself in our current scientific thought process. This book will seek to capture and disseminate our current understanding of scientific advancements relevant to women's health and provide the information to a broad audience. The purpose of this work is to discuss recent advancements in basic science across three areas of concern for women's health. In addition, the book will provide "translational" chapters that attempt to place the basic science work in context within our current understanding of the human. Although it is well acknowledge that gender differences exist across organ function which translates into differences in whole body function, until recently little effort has been made to define basic mechanisms within various tissues within the woman. This work will focus on recent scientific findings that are relevant to women's health and to provide novel and relevant information to interested scientists and clinicians.

This book serves as an introduction to targeted genome editing, beginning with the background of this rapidly developing field and methods for generation of engineered nucleases. Applications of genome editing tools are then described in detail, in iPS cells and diverse organisms such as mice, rats, marine invertebrates, fish, frogs, and plants. Tools that are mentioned include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas9, all of which have received much attention in recent years as breakthrough technologies. Genome editing with engineered nucleases allows us to precisely change the target genome of living cells and is a powerful way to control functional genes. It is feasible in almost all organisms ranging from bacteria to plants and animals, as well as in cultured cells such as ES and iPS cells. Various genome modifications have proven successful, including gene knockout and knock-in experiments with targeting vectors and chromosomal editing. Genome editing technologies hold great promise for the future, for example in biomedical research, clinical medicine, and generation of crops and livestock with desirable traits. A wide range of readers will find this book interesting, and with its focus on applications in a variety of organisms and cells, the book will be valuable for life scientists in all fields.

Cytokines are pleiotropic regulatory proteins involved in essentially all biological processes and associated with a wide variety of diseases, including inflammatory disorders as well as many types of cancer and leukemia. Knowledge about the quantitative and qualitative nature of cytokine production is critical in the understanding of normal and pathological processes. The cytokine detection in biological and clinical samples faces many challenges including their low abundance, the need to distinguish between active and latent cytokine forms, and the need to measure multiple cytokines in a single assay. This volume will provide a comprehensive collection of classic and cutting-edge methodologies that are currently used to analyze and quantify cytokines and their biological activities in complex biological and clinical samples. The chapters are divided into four main categories. The first group focuses on the immunodetection of released cytokines in tissue culture supernatants, plasma, serum, and whole blood samples by immunoassays.These immunoassays measure the total concentrations of released cytokines regardless of their biological activity, and include ELISA, flow cytometry, ELISPOT, and the antibody-based proximity ligation. . The second group will focus on the analysis of biologically active cytokines by bioassays using neutralizing antibodies, chemotaxis assay, cytokine-induced cell degranulation assay, cell proliferation and differentiation, cytokine-induced cytokine production, and the radioreceptor cytokine assay. The third group focuses on the analysis of intracellular cytokines by flow cytometry, western blotting and fluorescence and confocal microscopy. In addition, this category includes protocols for quantitative analysis of cytokine gene expression by real time RT-PCR and analysis of the cytokine promoter occupancy by chromatin immunoprecipitation. The fourth group focuses on the recently developed multiplex arrays that can measure multiple cytokines in the same sample at the same time.This group includes quantification of multiple cytokines using cytometric bead arrays, ELISPOT assays, proteomics cytokine evaluation, multiplexed proximity ligation assays for high-throughput cytokine analysis, and finally, cytokine gene expression analysis by gene arrays. The protocols will be written by experienced basic and clinical researchers with hands-on knowledge of the described protocols. By covering a broad variety of methods used in cytokine detection and analysis, this book will be of interest not only to biochemists, molecular biologists and immunologists but also to physician-scientists working in the field of cytokine research.

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.

The ovary is a suitable organ for studying the processes of cell death. Cell death was first described in the rabbit ovary (Graaffian follicles), the phenomenon being called 'chromatolysis'. To date, it is recognized that various forms of cell death (programmed cell death, apoptosis and autophagy) are essential components of ovarian development and function. Programmed cell death is responsable for the ovarian endowment of primordial follicles around birth; in the prepuberal and adult period, apoptosis is a basic mechanism by which oocytes are eliminated by cancer therapies and environmental toxicants; in the ovarian cycle, follicular atresia and luteal regression involve follicular cell apoptosis. Finally, abnormalities in cell death processes may lead to ovarian disease such as cancer and chemoresistance. In this book, after an introductory description of various forms of cell death and of the ovary development and function in mammals, the processes of cell death in ovarian somatic cells and oocytes are described at cytological, physiological and molecular levels and analyzed in the embryonic, prepuberal and adult ovary. A complex array of molecular pathways triggered by extrinsic and intrinsic signals able tor induce or suppress cell death in the same cell, according to cell type and ovary developmental stage, emerges. Physiological interactions with the axis hypothalamus-hypophysis as well as ovarian internal functional signal are also critically reviewed to explain the abortive development of follicles before the beginning of the ovarian cycle. The book conveys information useful to the updating of biologists and physicians who are interested to the ovary biology and functions. Hopefully it should provide also clues for stimulating novel experiments in the study of cell death in the mammalian ovary still at an early stage.

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.

Modern cell biology has brought improvements in therapy for advanced malignant diseases through immunomodulation, hematopoietic stem cell transplantation, and other advanced techniques. Collected here are selected papers from the Fifth International Symposium of Keio University for Life Sciences and Medicine on Cell Therapy.
All chapters include innovative basic research for clinical application: immunotherapy, cancer vaccination, molecular biology of hematopoietic stem cells, stem cell processing, and gene therapy.
The book is divided into three parts: Immunotherapy for malignant diseases; Hematopoietic stem cell biology and clinical application; and International collaboration in hematopoietic stem cell transplantation. Included in the third part is information on bone marrow registries from around the world.
The book thus presents up-to-date information on biological and clinical aspects of treatment, with insight into the future of cell therapy.

Computers have revolutionized the analysis of sequencing data. It is unlikely that any sequencing projects have been performed in the last few years without the aid of computers. Recently their role has taken a further major step forward. Computers have become smaller and more powerful and the software has become simpler to use as it has grown in sophistication. This book reflects that change since the majority of packages described here are designed to be used on desktop computers. Computer software is now available that can run gels, collect data, and assess its accuracy. It can assemble, align, or compare multiple fragments, perform restriction analyses, identify coding regions and specific motifs, and even design the primers needed to extend the sequencing. Much of this soft ware may now be used on relatively inexpensive computers. It is now possible to progress from isolate d DNA to database submission without writing a single base down. To reflect this progression, the chapters in our Sequence Data Analysis Guidebook are arranged, not by software package, but by fimction. The early chapters deal with examining the data produced by modem automated sequenc ers, assessing its quality, and removing extraneous data. The following chap ters describe the process of aligning multiple sequences in order to assemble overlapping fragments into sequence contigs to compare similar sequences from different sources. Subsequent chapters describe procedures for compar ing the newly derived sequence to the massive amounts of information in the sequence databases."

During the last decade, an increased interest in somatic stem cells has led to a flurry of research on one of the most accessible tissues of the body: skin. Much effort has focused on such topics as understanding the heterogeneity of stem cell pools within the epidermis and dermis, and their comparative utility in regenerative medicine applications. In Skin Stem Cells: Methods and Protocols, expert researchers in the field detail many of the methods which are now commonly used to study skin stem cells. These include methods and techniques for the isolation, maintenance and characterization of stem cell populations from skin. 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, Skin Stem Cells: Methods and Protocols seeks to aid scientists in the further understanding of these diverse cell types and the translation of their biological potential to the in vivo setting.

This volume is a collection of chapters contributed by leading experts in the emerging field of analytical morphology. Its contents cover a wide range of techniques for morphologic research and diagnosis and it is intended for anyone who wants to keep abreast of the rapid development in this field. Analytical morphology is a contemporary science dealing with the analy- sis of shape, size and color arrangement of cell and tissue components by means of a variety of analytical maneuvers. It differs from conventional mor- phology and histopathology in that it employs methods beyond routine hema- toxylin and eosin or histochemical staining. To a great extent, the advance- ment of analytical morphology is based on new advances in other disciplines, such as immunology, molecular biology, laser technology, microwave tech- nology and computer science. Using these new methods, particular cellular components that would otherwise remain invisible, such as peptides, pro- teins, or nucleotide sequences, are highlighted by visible markers through chemical, physical, immunological or molecular biological reactions. These methods include immunocytochemistry, in situ hybridization, in situ poly- merase chain reaction, antigen retrieval, image analysis, and the like. Analytical morphology is the foundation of contemporary medicine. It provides concrete and visible evidence for many conceptual deductions of other life science disciplines.