Over the past 10 years great progress has been made in the development of efficient techniques for both gene isolation and mapping. The identifica- tion and isolation of transcribed sequences from large chromosomal regions are central to the human genome mapping project. Techniques for isolating novel cDNAs have applications both in the overall construction and integra- tion of long-range physical and transcription maps and in the identification of disease genes. A number of different techniques for the isolation of cDNAs from mam- malian genomes have been developed, including screening "zoo" blots, the use of large genomic clones (YACs or cosmids) for hybridization against cDNA libraries, and CpG island mapping. More recently two highly efficient tech- niques have been introduced: exon trapping, based on the presence of exon splice sites, and direct selection, based on the enrichment of selected cDNAs using immobilized YACs or cosmids. Leading researchers in the field have contributed chapters detailing the practical procedures for these and other widely used methods. The most rapid progress presently being made in the field of gene isolation concerns the partial sequencing of cDNA clones from one or both ends to produce expressed sequence tags (ESTs). Indeed, by Octo- ber 1995, the EST division of Genbank (dbEST) contained a total of approxi- mately 270,000 human EST sequences accounting for almost half the number of sequence entries in Genbank.

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

This book features a special subsection of Nanomedicine, an application of nanotech nology to achieve breakthroughs in healthcare. It exploits the improved and often novel physical, chemical and biological properties of materials only existent at the nanometer scale. As a consequence of small scale, nanosystems in most cases are efficiently uptaken by cells and appear to act at the intracellular level. Nanotechnology has the potential to improve diagnosis, treatment and follow-up of diseases, and includes targeted drug delivery and regenerative medicine; it creates new tools and methods that impact sig nificantly upon existing conservative practices. This volume is a collection of authoritative reviews. In the introductory section we define the field (intracellular delivery). Then, the fundamental routes of nanode livery devices, cellular uptake, types of delivery devices, particularly in terms of localized cellular delivery, both for small drug molecules, macromolecular drugs and genes; at the academic and applied levels, are covered. The following section is dedicated to enhancing delivery via special targeting motifs followed by the introduction of different types of intracellular nanodelivery devices (e.g. a brief description of their chemistry) and ways of producing these different devices. Finally, we put special emphasis on particular disease states and on other biomedical applications, whilst diagnostic and sensing issues are also included. Intracellular delivery / therapy is a highly topical which will stir great inter est. Intracellular delivery enables much more efficient drug delivery since the impact (on different organelles and sites) is intracellular as the drug is not supplied externally within the blood stream. There is great potential for targeted delivery with improved localized delivery and efficacy.

In aerobic tissues such as heart, brain, kidney, liver and brown fat, mitochon- dria account for more than 20% of cell protein and play an essential role in res- piration, ATP formation, ketogenesis, gluconeogenesis, amino acid metabolism, ureagenesis, thermogenesis and a variety of other metabolic activities. The mecha- nisms by which these activities are integrated and regulated within the overall context of cellular physiology is of much current research interest. In order to bring together scientists examining the various diverse aspects of this overall pro- blem, an International Conference on INTEGRATION OF MITOCHONDRIAL FUNC- TION was held June 4-7, 1987 at the Hanes Art Center on the campus of the Uni- versity of North Carolina at Chapel Hill. The chapters of this volume derive from presentations made at this conference. The focus of INTEGRATION OF MITOCHONDRIAL FUNCTION is on signifi- cant new experimental and theoretical advances concerning integration of mito- chondrial function at the organelle, cell, tissue and whole organism levels of organization.

An understanding of the mechanisms by which plants perceive environmental cues, both physical and chemical, and transduce the signals that influence specific expression of genes, is an area of intensive scientific research. With the completion of the genome sequence of Arabidopsis it is understood now that a larger number of genes encode for proteins involved in signalling cascades and transcription factors. In this volume, different chapters deal with plant receptors, second messengers like calcium ions, phosphoinositides, salicylic acid and nitrous oxide, calcium binding proteins and kinases. In addition to dealing with the response of plants to light, hormones, pathogens, heat, etc. on cellular activity, work currently going on in apoptosis, cell division, and plastid gene expression is also covered in this book.

The goal of Biological Aging: Methods and Protocols is to present some of the most promising and important tools that are currently used in biological aging research. These tools include established protocols such as aging cell culture as well as many more contemporary approaches such as nuclear transfer, microarray and proteomics technologies and the use of ribozymes in aging research. Collectively, these powerful tools combined with the many other techniques that are presented are rapidly advancing the exciting and expanding field of biological aging.

Lysosomes are membrane-surrounded organelles which are present in all animal cells. The importance of this organelle is underlined by an increasing number of human diseases, which are associated with an impaired function of the lysosomal compartment.

This book summarizes the current state-of-the art knowledge about this unique organelle. It addresses the biogenesis of this compartment, the transport of lysosomal proteins, the role of the lysosomal membrane in lysosomal stability and transport, the function of lysosomal proteases and hydrolases, lysosomal storage disorders, and new concepts on how to treat these diseases. In addition to these classical topics, new insights into lysosomal functions are covered by chapters dealing with specialized lysosomes involved in bone resorption and plasma membrane repair, the lysosomal transciptome, and proteome and the emerging role of lysosomes in special forms of autophagy.

This book will provide readers with a comprehensive overview into how this fascinating organelle works and how research in the field is developing.

The first libraries of complementary DNA (cDNA) clones were con structed in the mid-to-late 1970s using RNA-dependent DNA polymerase (reverse transcriptase) to convert poly A* mRNA into double-stranded cDNA suitable for insertion into prokaryotic vectors. Since then cDNA technology has become a fundamental tool for the molecular biologist and at the same time some very significant advances have occurred in the methods for con structing and screening cDNA libraries. It is not the aim of cDNA Library Protocols to give a comprehensive review of all cDNA library-based methodologies; instead we present a series of up-to-date protocols that together should give a good grounding of proce dures associated with the construction and use of cDNA libraries. In deciding what to include, we endeavored to combine up-to-date versions of some of the most widely used protocols with some very usefiil newer techniques. cDNA Library Protocols should therefore be especially useful to the investigator who is new to the use of cDNA libraries, but should also be of value to the more experienced worker. Chapters 1-5 concentrate on cDNA library construction and manipula tion, Chapters 6 and 7 describe means of cloning difficult-to-obtain ends of cDNAs, Chapters 8-18 give various approaches to the screening of cDNA libraries, and the remaining chapters present methods of analysis of cDNA clones including details of how to analyze cDNA sequence data and how to make use of the wealth of cDNA data emerging from the human genome project."

Systematic investigations of the structure, mechanics, and dynamics of biological surfaces help us understand more about biological processes taking place at cell and bacteria surfaces. Presented here is a study of the role membrane-bound saccharides play in the modulation of interactions between cells/bacteria and their environments. In this thesis, membrane structures were probed perpendicular and parallel to the surface, and sophisticated planar models of biomembranes composed of glycolipids of various structural complexities were designed. Furthermore, specular and off-specular X-ray and neutron scattering experiments were carried out. This research has led to the development of several new methods for extracting information on the structure and mechanics of saccharide-rendered biomembranes from the measured scattering signals. In fact, more is now known about the influence of the saccharide structure. These results demonstrate that the study of planar model systems with X-ray and neutron scattering techniques can provide comprehensive insight into the structure and mechanics of complex biological surfaces.

Sodium channels confer excitability on neurons in nociceptive pathways and exhibit neuronal tissue specific and injury regulated expression. This volume provides recent insights into the control of expression, functioning and membrane trafficking of nervous system sodium channels and reviews why sodium channel sub-types are potentially important drug targets in the treatment of pain. The roles of sodium channels in dental and visceral pain are also addressed. The emerging role of sodium channel Nav1.3 in neuropathic states is another important theme.

Authors from the pharmaceutical industry discuss pharmacological approaches to the drug targeting of sodium channels, and in particular Nav1.8, exclusively expressed in nociceptive neurons. The final chapter highlights the functional diversity of sodium channels in part provided by post-transcriptional processing and the insights into sodium channel function that are being provided by tissue specific and inducible gene knock-out technology.

In the past few years there has been the increased recognition that the effects of oxidative stress are not limited to the damage of cellular constituents. There is now evidence that reactive oxygen species (ROS) can alter cell function by acting upon the intermediates, or second messengers, in signal transductions. Such effects on signaling mechanisms probably account for the role of oxidative stress in inflammation, aging, and cancer. This volume brings together internationally recognized researchers in both the major areas covered by the book, oxidative stress and signal transduction. The work is organized in three sections. The first deals with the immediate cellular responses to oxidative stress and the production of second messengers. The second details the connection between second messengers and the gene. The third part looks more closely at the level of the gene.

MicroRNA research and development is the billion-dollar baby and most lucrative option for drug discovery in gene therapy industries worldwide. Personalized microRNA treatments are in many cases the only remedy for viral diseases that have no cure in conventional drugs and offer to bring us closer than ever to "personalized medicine." They also counteract cancer and other infectious and neuro-diseases. Early diagnosis, prognosis, staging, and sub-classification of various cancers can easily be facilitated by microRNA-based biomarkers. MicroRNA surveys recent advances in RNA and RNA-protein components that highlight RNA delivery, its stability, and applications of RNA-based drugs for the modulation of gene/protein expression and gene editing. The book not only focuses on the modern medicines of microRNA-based early diagnostic and therapy development, but also works as a hidden treasure for drug discovery of multiple rare diseases worldwide. It offers indispensable learning materials for academic researchers, graduate, and medical students, and offers a powerful practical guide for RNA-Pharma and gene therapy industries.

This detailed volume encompasses new technological developments that specifically address questions related to adenosine 3',5'-monophosphate (cAMP) compartmentalization, that probe relevant protein-protein interactions, that increase the spatial and temporal resolution of cAMP signal detection, and that can facilitate integration of the mounting complexity of the information that is becoming available on this signaling system. cAMP, the prototypical intracellular second messenger, regulates a large variety of cellular functions and biological processes, including gene transcription, cell metabolism, proliferation, development, as well as more specialized functions depending on the cell type, so the realization of its extremely complex spatial organization and local regulation is providing novel mechanistic insight into cell physiology and is producing a novel framework for the identification of disease mechanisms. Written in the highly successful Methods in Molecular Biology series format, chapters include introduction 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. Practical and authoritative, cAMP Signaling: Methods and Protocols serves as a vital resource for researchers working in this expanding, complex field.

This book is a broadly historical account of a remarkable and very exciting scientific story-the search for the number of human chromosomes. It covers the processes and people, culminating in the realization that discovering the number of human chromosomes brought as much benefit as unraveling the genetic code itself. With the exception of red blood cells, which have no nucleus and therefore no DNA, and sex cells, humans have 46 chromosomes in every single cell. Not only do chromosomes carry all of the genes that code our inheritance, they also carry them in a specific order. It is essential that the number and structure of chromosomes remains intact, in order to pass on the correct amount of DNA to succeeding generations and for the cells to survive. Knowing the number of human chromosomes has provided a vital diagnostic tool in the prenatal diagnosis of genetic disorders, and the search for this number and developing an understanding of what it means are the focus of this book.

Of all scientific instruments, probably none has had more applications in the life sciences than the light microscope. In Light Microscopy: Methods and Protocols, expert researchers explore the basics and the latest advances in microscope instrumentation, sample preparation, and imaging techniques, all of which have been producing fundamental insights into the functions of cells and tissues. Chapters cover a variety of bright field and fluorescence microscopy-based approaches that are central to the study of a range of biological questions, providing information on how to prepare cells and tissues for microscopic investigations, covering detailed staining procedures, and exploring methods to analyze images and interpret the results accurately. 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, Light Microscopy: Methods and Protocols is an essential handbook for all researchers who are exploring the intriguing microscopic world of the cell.

Products from Cells - Cells as Products This book ist he "lasting" product, a resource ofup to date information in the scientific literature fort he field ofanimal cell tec hnology, as it was presented during a pleasant and s timulating mee ting that was held in Lugano Switzerland in April 1999. "Products" appeartwice int he title oft he conference. This clearly indicates the fact that the focus oft he papers presented during this meeting was really the application ofn ew technologies (novel reactors or novel vectors, for example for the preparation and/ort he more efficient generation ofproducts ) that could be used, mainly, int he medical field. Classical approaches forthe use ofa nimal cells, for example forthe p r oduction of virus vaccines for human and animal health, still remain an important technology and still have, surprisingly, quite significant potential for further development and improvement. How ever, it appears that major technological advances an d major growth from an economical point ofview are occurring in other areas. Most importantly, protein production on the basis of recombinant DNA molecules transferred into a nimal cells, appears to be an ever increasing field of interest and innovation, even though the first production scheme with this technology was approved more than 15 years ago.

Epithelia are one of the commonest tissue types in the animal kingdom. Chapters from leading scientists in the major international research laboratories use examples from different systems to illustrate the form and function of epithelia. An important theme is the way in which epithelial cells differentiate to specialized tissue - reversal of this process occurs when cells become tumorigenic.

The plant' is often the most neglected part of plant-based medicine. Throughout time, humans have searched, collected, and effectively used plants for healing. Currently, the medicinal plant-based business is flourishing at a dramatic pace and at the expense of an already declining population of plant species, many of which are on the verge of extinction. In spite of this history and popularity, the mystery of what transforms a plant into a medicinal plant persists, and there are chronic problems with ensuring the safety and efficacy of medicinal plant products. Therefore, there is a real need for a full characterization of medicinal plant species and for the development and application of novel technologies for the production of plant-based medicines. This book highlights some of the recent advances and new approaches to the development of technologies for plant-based medicines and is intended to stimulate new discussions among researchers, regulatory authorities, and pharmaceutical organizations, leading to significant advancements in the field.

Despite many technological challenges faced by the xenotransplantation field, many major advances have been made in the last two decades. The field seeks to overcome the limitations and difficulties in organ procurement, which also apply to human cells and tissues, and facilitate the development of new therapies based on cell and engineered-tissue. Xenogeneic cells are simpler than solid organs and seem to pose less hurdles to attain long-term graft survival. In, Xenotransplantation: Methods and Protocols expert researchers study characterizations of xenogeneic interactions at the cellular and molecular levels and describe the use of relevant small-animal and pig-to-primate models. Related ethical and legal considerations are also covered. 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, Xenotransplantation: Methods and Protocols aids scientists in continuing to study xenotransplantation and its multiple aspects.

In situ hybridization has developed as a means of localizing specific DNA and RNA sequences within tissues. The great strength of this approach is the ability to relate the distribution of specific nucleic acids with cell structures and the protein products of the target gene by means of immunohistochemistry. Complementary DNA, RNA or oligonucleotide probes, suitably labelled, are hybridized to specific DNA or RNA targets within tissues. The spatial information thus obtained has contributed greatly to our understanding of such diverse areas of research as gene mapping, viral infection, cytogenetics, protein synthesis, prenatal diagnosis and tissue grafting. This book is not intended as another recipe book, although it does describe theoretical and practical aspects of the technology. Rather, the authors critically describe the contribution made by in situ hybridization to specific areas of medical research.

The current explosive progress in molecular biological research can be definitively traced to the development of molecular cloning technology. The ability to insert specific gene sequences into cloning vectors and their subse quent expansion is the cornerstone of modem molecular biology. A direct practical outcome of molecular cloning technology is its application to ex press specific recombinant genes. Currently, recombinant gene products are used in a wide spectrum of applications, including gene therapy, production of bioactive pharmaceuticals, synthesis of novel biopolymers, in agriculture and animal husbandry, and so on. A fundamental requirement for successful recombinant gene expression is the design of the cloning vector and the choice of the host organism for expression. Recombinant Gene Expression Protocols grows out of the need for a laboratory manual that provides the reader the background and rationale, as well as the practical protocols for the preparation of "expression constructs" and their introduction into appropriate host cells and/or organisms. The chap ters in this book are grouped by their expression hosts, including E. coli, yeast, mammalian cells, nonmammalian eukaryotes such as plants, Xenopus, and insects, as well as in transgenic organisms. In-depth information is presented on the important characteristics of expression cloning vectors and the various methods for efficiently introducing expression constructs into target cells and/ or organisms. Throughout Recombinant Gene Expression Protocols, the authors have consistently striven for a balanced presentation of both background informa tion and actual laboratory details.

''Interesting with many useful ideas and references. It covers a broad range and it is a good introduction to this field.'' ---Analyst

Since the initial establishment of Robert Koch's postulates in the nineteenth century, microbial protein toxins have been recognized as a major factor of bacterial and fungal virulence. An increasing number of proteins produced and secreted by various bacteria, yeasts and plants are extremely toxic and most of them developed remarkably "intelligent" strategies to enter, to penetrate and to finally kill a eukaryotic target cell by modifying or blocking essential cellular components.

This book describes the strategies employed by protein toxins to render their pro- and eukaryotic producers a selective growth advantage over competitors. In providing an up-to-date overview on the mode of protein toxin actions, it accommodates biomedically and biologically relevant toxin model systems. As a result, it significantly broadens our perspective on biochemical architecture and molecular ploy behind the lethal principles of pro- and eukaryotic toxins.

Research into and interest in the role of stromal cells in immunology has exploded over the past 15 years. The conventional view that placed non-hematopoietic stromal cells as passive, structural, and supportive entities has now been replaced with an appreciation that these cells have active, dynamic roles during immune responses, and thus impact on the pathophysiology of multiple immune-mediated diseases. This book serves to provide solid grounding in the fundamentals of stromal immunology, focusing on the biological aspects of their function in addition to highlighting key areas for the development of the field in the future. The book is also a unique source of information on emerging concepts that place stromal cells from outside lymphoid organs as major contributors to the biology of diverse conditions, such as rheumatoid arthritis, chronic parasitic infection, inflammatory bowel disease, and cancer.