This volume provides readers with a wide collection of the latest and readily reproducible technical protocols available in the field of non-viral gene delivery vectors. The chapters in this book are organized into three major parts: Part I is a section on conventional bolus gene delivery vectors that introduces typical transfection approaches relying on the addition of transfectants to the cell culture medium where the cells are grown in; Part II covers stimuli-responsive bolus transfectants and topics on gene delivery complexes made of smart polymers or stimuli-responsive polymers that change according to the environment they are in and delivered by dripping into cells; Part III discusses examples of substrate-mediated gene delivery-also termed reverse transfection-and the immobilization of a gene delivery vector onto a surface as opposed to more typical bolus delivery from the medium. 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. Cutting-edge and practical, Non-Viral Gene Delivery Vectors: Methods and Protocols is written for experimentalists, and is an essential part of many laboratory bookshelves. This book will help novice and professionals alike succeed in their research in this field.

Principles of Neurobiology, Second Edition presents the major concepts of neuroscience with an emphasis on how we know what we know. The text is organized around a series of key experiments to illustrate how scientific progress is made and helps upper-level undergraduate and graduate students discover the relevant primary literature. Written by a single author in a clear and consistent writing style, each topic builds in complexity from electrophysiology to molecular genetics to systems level in a highly integrative approach. Students can fully engage with the content via thematically linked chapters and will be able to read the book in its entirety in a semester-long course. Principles of Neurobiology is accompanied by a rich package of online student and instructor resources including animations, figures in PowerPoint, and a Question Bank for adopting instructors.

New and exciting biological functions are still being discovered for vitamin A derivatives, including the vast number of physiological activities of retinoids. In Retinoids: Methods and Protocols, expert researchers in the field present the most recent technical tools with diverse techniques for both in vitro and in vivo studies. Combining biochemical, biophysical, and cell biological techniques, the book addresses topics such as the detection and quantitation of retinoids using HPLC, mass spectrometry, and fluorescence, fluorescence anisotropy of retinol binding protein, cell culture models for studying retinoid transport and the role of retinol in embryonic stem cell culture, as well as many other detailed procedures. Written in the highly successful Methods in Molecular Biology(TM) series format, chapters include introductions to their respective subjects, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and notes highlighting tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Retinoids: Methods and Protocols seeks to aid beginning and experienced researchers from widely varied fields in the search to uncover even more vital aspects of vitamin A's impact on the human body.

This second edition provides updated and new chapters on selected genetic, molecular, biochemical, and cell biological techniques. Chapter's guide readers through methods and principles on primordial germ cells and germline stem cells, however many of these principles can be applied to different types of adult stem cells. 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, Germline Stem Cells: Second Edition aims to present the new findings and techniques that have developed since the first edition.

Alzheimer's disease is the most common cause of senile dementia. Since the discovery in 1984 of the amyloid ?-peptide (A?) as the core protein of the senile plaques present in the brains of Alzheimer's disease sufferers, an immense amount of research has gone into mapping out the molecular basis of this debilitating disease. The aim of Alzheimer's Disease: Methods and Protocols is to bring together the main biochemical, cell biological, and molecular biological techniques and approaches that are being used to investigate the molecular basis of Alzheimer's disease. This volume begins with chapters of an introductory/ review nature. Chapter 1 provides a historical introduction to Alzheimer's d- ease with particular emphasis on the central role played by A? and its re- tion to tau. Chapter 2 examines the genetics underlying this neurodegenerative disease, covering the amyloid precursor protein, apolipoprotein E, and the presenilins. Chapter 3 presents an overview of currently available therapeutic agents and prospects for drugs of the future.

The last few years have seen an explosion of new information and resources in the areas of plant molecular genetics and genomics. As a result of developments such as high throughput sequencing, we now have huge amounts of information available on plant genes. But how does this help people charged with the task of improving crop species to create products with altered functions or improved characteristics? This volume considers ways in which the new information, resources and technology can be exploited by the plant breeder. Examples in current use will be quoted wherever possible.

In 1868, Ernst Neumann recognized that blood cells re quire continuous replenishment during postnata1life. Before him, the assumption was that cells of the blood, like nerves once formed in the embryo, remain in the body throughout life. Neumann also recognized that this process occurred within the bone marrow, because this tissue provided a fa vorable environment for proliferation and differentiation of blood cell precursors. Vera Danchakoff, the Russian embryologist working in the US, in 1916 made an analogy to the soil and the seed. Bone marrow forms the soil, providing a favorable environment for the growth of seed, the hemopoietic stem cell, and other progenitor cells. Imagine in the remote past a heap of similar tree seeds. These seeds develop in our moderate climate into a tall and many branched tree. Suppose the wind bears a part of the seeds away and brings them to a land possessing different environmental conditions, we will say the arc tic lands. There the seeds may develop but they may pro duce trees no higher than our moss."

The incentive for putting together Volume 4 of this series was to review the wealth of new information that has become available in prokaryotic organisms in protein export and membrane biogenesis. Just in the last several years, protein translocation has now been efficiently reconstituted using defined components and the mechanism by which proteins are moved across membrane bilayers is now being examined at a higher resolution. In addition, because of a new technical breakthrough using osmolytes, it is now possible to reconstitute a number of channel proteins, ATPase, receptors, and transporters. In many cases, it is possible to successfully predict the membrane topology of these types of proteins using both "hydrophobicity analysis" and the "positive inside" rule.
In this volume, two chapters focus on protein translocation across membranes ("Biochemical Analyses of Components Comprising the Protein Translocation Machinery of E. Coli; Protein Translocation Genetics"), while several others on how proteins assemble into the ineer membrane of E. Coli ("Membrane Protein Assembly; Membrane Insertion of Small Proteins: Evolutionary and Functional Aspects; Pigment-Protein Complex Assembly in Rhodobacter sphaeroides and Rhodobacter Capsulatus"). Other sections review recent progress on transporters ("Identification and Reconstitution of Anion Exchange Mechanisms in Bacteria; Helic Packing in the C-Terminal Half of Lactose Permease") and signal transduction ("Mechanism of Transmembrane Signaling in Osmoregulation") as well as the assembly of prints into the outer membrane ("Export and Assembly of Outer Membrane Proteins in E. coli"). Although the emphasis of the book is on proteins, the role of phospholipids in controlling various cell surface processes is reviewed ("Role of Phospholipids in coli Cell Function"). I should point out the reason for the rapid progress in bacteria research is because of the possibility to apply biochemistry and genetics in this organism.

A diverse team of researchers, technologists, and engineers describe, in simple and practical language, the major current and evolving technologies for improving the biocatalytic capabilities of mammalian, microbial, and plant cells. The authors present state-of-the-art techniques, proven methods, and strategies for industrial screening, cultivation, and scale-up of these cells, and describe their biotech and industrial uses. Special emphasis is given to the solving critical issues encountered during the discovery of new drugs, process development, and the manufacture of new and existing compounds. Other topics include recombinant protein expression, bioinformatics, high throughput screening, analytical tools in biotechnology, DNA shuffling, and genomics discovery.

Interest in retinoic acid, the main biologically active derivative of vi- min A or retinol, increased dramatically between 1989 and 1993, following the cloning of nuclear receptors or RARs reported in 1987 (Fig. 1). Important discoveries since then have shown how RARs work as all-trans retinoic ac- dependent heterodimers with related nuclear receptors for 9-cis retinoic acid called RXRs. This has stimulated the development of synthetic analogs s- cific for each type of receptor, and opens the way to develop new methods for regulating pharmacologically the activity ofretinoic acid-dependent pathways of gene activation. The potential for the development of new drugs by the pharmaceutical industry is now a maj or factor driving forward our understa- ing of vitamin A-regulated pathways in animal development and homeostasis. However, apart from the real potential ofretinoid analogs as novel pharma- logical agents, there remains the considerable intellectual challenge of und- standing the way in which vitamin A and its derivatives function in cell development and differentiation. Retinoid Protocols is an attempt to bring together various methodologies that will be vital for rising to this challenge in the future. Retinoid molecular biology has few methods of its own, but is reliant on standard molecular biology methods applied to this particular research area.

In this contribution, several specialists describe the early-21st century knowledge on the molecular networks that regulate cell cycle progression, with an emphasis on the G1 phase of the cell cycle. The first part of Regulation of G1 Phase Progression is concerned with the individual molecules that form the network, including cyclins, cyclin-dependent kinases, inhibitors of these kinases and retinoblastoma and p53. The second section describes the signaling cascades by which external factors influence the cell cycle network, including mitogens, the extracellular matrix, nutrients and oxygen radicals. The last section describes the effects of specific external conditions on cell cycle progression and are presented such as serum starvation and subsequent re-addition and stress conditions (heat, osmolarity). The final two chapters describe the relation between cell cycle progression with cell differentiation and with apoptosis.

Cell Surface Receptors: A Short Course on Theory and Methods, Second Edition is a primer for the study of cell surface receptors. The simplified discussion of methods and their underlying principles removes the usual intimidation caused by the specialized vocabulary or sophisticated mathematics that characterize many of the primary papers in this field. In this way, the basic concepts become emphasized. This volume is a starting point: a textbook as well as a manual to which the investigator can return for a refresher course, when needed.

All three peroxisome proliferator-activated receptor (PPAR) subtypes share a high degree of structural homology while exhibiting differences in function, tissue distribution, and ligand specificity. In Peroxisome Proliferator-Activated Receptors: Discovery and Recent Advances, the authors trace the history of PPAR discovery and detail the receptor structure and its posttranslational modifications. Furthermore, endogenous ligands as well as various classes of exogenous ligands, subtype-selective, dual and pan agonists as well as antagonists, are discussed. In addition, the tissue distribution and versatile functions of PPAR subtypes in major organs are described. As PPARs play critical roles as regulators of numerous physiological as well as pathophysiological pathways, Peroxisome Proliferator-Activated Receptors: Discovery and Recent Advances aims to help researchers to develop safer and more effective PPAR modulators as therapeutic agents to treat a myriad of diseases and conditions.

The three-dimensional organization of the DNA inside the eukaryotic cell nucleus has emerged a critical regulator of genome integrity and function. Increasing evidence indicates that nuclear pore complexes (NPCs), the large protein channels that connect the nucleus to the cytoplasm, play a critical role in the establishment and maintenance of chromatin organization and in the regulation of gene activity. These findings, which oppose the traditional view of NPCs as channels with only one: the facilitation of nucleocytoplasmic molecule exchange, have completely transformed our understanding of these structures. This book describes our current knowledge of the role of NPCs in genome organization and gene expression regulation. It starts by providing an overview of the different compartments and structures of the nucleus and how they contribute to organizing the genome, then moves to examine the direct roles of NPCs and their components in gene expression regulation in different organisms, and ends by describing the function of nuclear pores in the infection and genome integration of HIV, in DNA repair and telomere maintenance, and in the regulation of chromosome segregation and mitosis. This book provides an intellectual backdrop for anyone interested in understanding how the gatekeepers of the nucleus contribute to safeguarding the integrity and function of the eukaryotic genome.

Since the pioneering discoveries of Hodgkin, Huxley, and Katz, it has been clear that specific ion conductance pathways underlie electrical act- ity. Over the ensuing 50 years, there has been ever increasing, and occasi- ally explosive, changes in the scope of efforts to understand ion channel behavior. The introduction of patch clamp technology by Erwin Neher and Bert Sakmann about 20 years ago led to the realization of the great variety of novel ion channel species, and the subsequent revolution in cl- ing has revealed an even greater diversity of the underlying molecular entities. Today, advances in the study of ion channel structure and function c- tinue at a high pace, from angstrom resolution imaging of crystallized ch- nels to their genetic manipulations in animals. In this regard, the field is a balanced one that inquires not only what ion channel entities are there, or how they operate, but also where are these molecular electronic switches? However, this balance is not particularly well presented to the general sci- tific audience or to specialists in the field. There are plenty of wonderful and useful books and monographs, as well as conferences and meetings on v- tually every aspect of ion channel structure and function. However, we are unaware that the channel localization theme has been considered in a u- fied forum.

Leading experts critically summarize the state of knowledge concerning the molecular, anatomical, physiological, and behavioral aspects of NPY and its congeners. Each article provides a comprehensive and in-depth survey, an overview of the role of NPY in the discipline covered, a discussion of the likely future direction that the field will take, and an up-to-date bibliography.
Chapters include a treatment of the evolution of the PP family of genes, the structure of the NPY gene, and the distribution of NPY on the cardiovascular system, actions of NPY on the electrophysiological properties of nerve cells, and the effects of NPY on feeding and behavior. The chapters are written in an accessible style and serve both as an introduction to the field and as an extensive and detailed treatment of the current state of knowledge.

The Fifth Ir Gene Workshop was held at the Chase-Park Plaza Hotel, St. Louis, MO, August 28-31, 1982; 240 scientists participated in the Workshop. The man uscripts compiled in this book describe the state of the art concerning Ir genes. Although the notion of Ir Genes: Past, Present, and Future has not been ad dressed specifically by each author, the reader is certain to get this flavor from the contributions. In this Preface, we have tried to summarize some of the salient ob servations and discussions from the Workshop. The mUltiple genes of the I region have been defined traditionally by serolog ical analysis of intra-H-2 recombinant mice and the pattern of immune responses to certain antigens developed by these recombinant mice. The application of sev eral new techniques, such as gene cloning and DNA sequencing, production of T and B cell hybridomas, and development of cloned T cell lines has changed this tradition and introduced a new phase into the analysis of the I region, Ia antigens, and Ir genes."

Cancerremainstobeoneofthemostdevastatingdiseasesworldwidesincelong ago. Thepoorprognosisofcancerislargelyduetometastasis. Metastasisisoften depictedasamultistageprocessinwhichmalignantcellsspreadfromtheprimary locustodistantorgansviacirculation. Whereasgeneticalterationsweresuggested tobeessentialfortransformationofprimarytumorcellsintometastaticphenotype, epigeneticeventsareequallyimportant,whichmaybetriggeredbymetastaticf- torswhereverintheprimarytumorlocus,bloodcirculationandthesecondaryloci. Signaltransductionsinitiatedbythemetastaticfactorsareresponsibleformediating themolecularandcellularprocessesleadingtometastasis. Blockadeoftherelevant molecularpathwaysisoneofthemosteffectivestrategiesforpreventionoftumor metastasis. Clinicaltrialsareunderwaywithpromisingoutcome. Inthisbook, wetakecomprehensive review inregard withthisexciting eld ofcancerresearch. Chapter1takesabriefoverviewofrecentlyidenti edsignal mechanismsforeachstepoftumormetastasisincludingtheinitiationstage,intra- sation,anti-anoikisinbloodcirculation,homing,extravasationand nalsurvivalin themetastaticsite. Chapter2makesacompletedreviewforthemolecularandcel- lareventsinvolvedininitiationofmetastasis. Especially,thesignalingmechanisms formediatingtumorprogressioninducedbysomeimportantmetastaticfactorsare described. InChapters3and4,thecentralrolesofMAPKanditsdownstreameff- torsMAPKAPKplayineachstepoftumormetastasisarewelldelineated. Chapter5 furtherdescribesdetailedlyabouthowGrb2andotheradaptorproteins,upstreamof MAPK cascade, contribute tometastasis. InChapter 6, therole ofreactive o- genspecies(ROS)intumorprogressionarehighlighted. Moreover,thepotential contribution of ROS to cross talk between major signaling cascades that lead to sustainedMAPKactivationareproposedinChapter7. Chapter8takesaninsight intothesignalingmechanismsfordynamictraf ckingandturnoveroffocalad- sionproteinsinregulationoftractionandretractionforces,whichareneededfor celllocomotionandinvasion. Chapter9describestheinvolvementofNotchsign- ingpathwaywhichisnotonlyessentialforembryonicdevelopmentbutalsoplays importantroleintumorprogression. Chapter10reviewedtherecentlyidenti ed cancer- and metastasis-initiating cells involved in tumor progression. Especially, signal pathways that are frequently deregulated in cancer stem/progenitor cells v vi Preface duringcancerprogressionarehighlighted. Chapter11describestheroleoflipid rafts, a special component within membrane lipid domain, in signal transd- tion triggered by growth factor receptors leading to tumor metastasis. Finally, Chapters12,Chapters13,andChapters14presentthesignalingpathwaysresp- sibleformetastaticprogressionofspeci ctumorsincludingovariancancer,uveal melanomaandhepatoma,respectively. WethankallthecontributorsofeveryChapterinthebookincludingJia-RuWu, Chi-TanHu,LaureVoisin,StephanieDuhamel,SylvainMeloche,AlexeyShiryaev, MarijkeVanGhelue,UgoMoens,AlessioGiubellino,PraveenR. Arany,Moulay A. Alaoui-Jamali, Krikor Bijian, Panagiota Toliopoulos, Pingyu Zhang, Patrick A. Zweidler-McKay,MurielleMimeault,SurinderK. Batra,SamirKumarPatra, LydiaW. T. Cheung,CarmanK. M. Ip,AliceS. T. Wong,CecileLaurent,Jerome Couturier,XavierSastre-Garau,LaurenceDesjardins,EmmanuelBarillot,Sophie Piperno-Neumann,SimonSauleandRajagopalN. Aravalli. Wehopethisbookmightstimulatemorecancerbiologiststoemphasizethis eld whichbene tsdevisingmoreeffectivemoleculartargetingstrategiesforprevention ofcancermetastasis. Hualien,Taiwan Wen-ShengWu Chi-TanHu Contents 1 Overview of Signal Transduction in Tumor Metastasis...1 Wen-ShengWuandJia-RuWu 2 Microenvironment Triggers EMT, Migration and Invasion of Primary Tumor via Multiple Signal Pathways ...9 Wen-ShengWuandChi-TanHu 3 The ERK1/2 MAP Kinase Signaling Pathway in Tumor Progression and Metastasis ...25 LaureVoisin,StephanieDuhamel,andSylvainMeloche 4 Mitogen-Activated Protein Kinase-Activated Protein Kinases and Metastasis...41 AlexeyShiryaev,MarijkeVanGhelue,andUgoMoens 5 Grb2 and Other Adaptor Proteins in Tumor Metastasis ...77 AlessioGiubellinoandPraveenR. Arany 6 The Role of ROS Signaling in Tumor Progression...103 Wen-ShengWuandJia-RuWu 7 Signal Cross Talks for Sustained MAPK Activation and Cell Migration Mediated by Reactive Oxygen Species: The Involvement in Tumor Progression...

This volume introduces different concepts and methods of detecting RNA in biological material in a variety of model systems. The chapters in this book discuss methods that will answer numerous biological questions that arise in the study of RNAs. Some of the topics covered in this book are single mRNA molecule detection in embryos and neurons; detection of mRNA and associated molecules by ISH-IEM on frozen sections; optimizing molecular beacons for intracellular analysis of RNA; imaging translation dynamics of single mRNA molecules in live cells; preparation of high-throughput sequencing libraries; and capturing RNA binding proteins in embryos and in cell-culture. 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. Cutting-edge and comprehensive, RNA Detection: Methods and Protocols is a valuable resource for novel and experiences scientist in the expanding field of RNAs.

Living beings require constant information processing for survival. In cells, information is being processed and propagated at various levels, from the gene regulatory network to chemical pathways, to the interaction with the environment. How this is achieved and how information is coded is still poorly understood. For example, what a cell interprets as information in the temporal level of an mRNA and what is interpreted as noise remains an open question. Recently, information theoretical methods and other tools, developed in the context of engineering and natural sciences, have been applied to study diverse biological processes. This book covers the latest findings on how information is processed in various biological processes, ranging from information processing and propagation in gene regulatory networks to information processing in natural language. An overview is presented of the state-of-the-art in information processing in biological systems and the opinion of current leaders in this research field on future research directions.

This fully updated edition explores methods involving sphingosine-1-phosphate (S1P), a bioactive lysophospholipid which has become the focus of much research interest as it has widespread developmental and physio-pathological actions, controlling events within the nervous, reproductive, gastrointestinal, vascular, respiratory, and immune systems, in addition to having a prominent role in cancer, early mammalian embryogenesis, and stem cells. Here, worldwide experts in the S1P field describe in-depth techniques in an array of cell types and with various physiological applications, showcasing the important effects of S1P in development and in physiopathology. As a volume in the Methods in Molecular Biology series, chapters contain introductions to their respective topics, lists of the relevant materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips for troubleshooting and avoiding known pitfalls. Authoritative and timely, Sphingosine-1-Phosphate: Methods and Protocols, Second Edition is another key resource for scientists working in this fast-moving and dynamic field.

The past decade has witnessed an explosion of our knowledge on the structure, coding capacity and evolution of the genomes of the two DNA-containing cell organelles in plants: chloroplasts (plastids) and mitochondria. Comparative genomics analyses have provided new insights into the origin of organelles by endosymbioses and uncovered an enormous evolutionary dynamics of organellar genomes. In addition, they have greatly helped to clarify phylogenetic relationships, especially in algae and early land plants with limited morphological and anatomical diversity. This book, written by leading experts, summarizes our current knowledge about plastid and mitochondrial genomes in all major groups of algae and land plants. It also includes chapters on endosymbioses, plastid and mitochondrial mutants, gene expression profiling and methods for organelle transformation. The book is designed for students and researchers in plant molecular biology, taxonomy, biotechnology and evolutionary biology.

Animal cell technology is a growing discipline of cell biology which aims not only to understand structures, functions and behaviors of differentiated animal cells but also to uncover their abilities for industrial and medical purposes. The goal of animal cell technology includes clonal expansion of differentiated cells with useful abilities, optimization of their culture conditions on the industrial scale, modulation of their ability in order efficiently to produce medically and pharmaceutically important proteins, and application of animal cells to gene therapy and formation of artificial organs. This Volume gives the readers a complete review of the present state of the art in Japan, a country where this field is well advanced, as well as in Asia, Europe and the United States. The Proceedings will be useful for cell biologists, biochemists, molecular biologists, biochemical engineers and those in other disciplines related to animal cell culture, working in academic environments as well as in the biotechnology and pharmaceutical industries.

This detailed volume provides a comprehensive resource covering the existing and state-of-the-art tools in the field of profiling chromatin accessibility and its dynamics. Beginning with a section on bulk-cell methods for profiling chromatin accessibility and nucleosome positioning that rely on enzymatic cleavage of accessible DNA and produce information about relative accessibility, the book continues with methods that use single-molecule and enzymatic approaches to solving the problem of mapping absolute occupancy/accessibility, emerging tools for mapping DNA accessibility and nucleosome positioning in single cells, imaging-based methods for visualizing accessible chromatin in its nuclear context, as well as computational methods for the processing and analysis of chromatin accessibility datasets. 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 and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and up-to-date, Chromatin Accessibility: Methods and Protocols serves as an extensive and useful reference for researchers studying different facets of chromatin accessibility in a wide variety of biological contexts. Chapter 6 is available open access under a Creative Commons Attribution 4.0 International License via link.springer.com.

This book presents the latest findings in mechanosensitive ion channel research compiled by a number of internationally recognized experts in the field of mechanosensitivity, specializing in mechanosensitive or mechanically gated ion channels and the underlying mechanosensitive signaling cascades. The book provides a comprehensive description of mechanotransduction, which is defined as the biochemical response of cells to mechanical stimulation, and provides the reader with the latest vision of signaling cascades. Descriptions of the most widely used experimental models, the methods of choice and references to experimental protocols, the latest experimental findings and current vision, and potential problems in data interpretation, will be of considerable use to all those interested in this field of research.