This volume covers methods for the analysis of extracellular vesicles (EV) that can be applied to isolated EVs from a wide variety of sources. This includes the use of electron microscopy, tunable resistance pulse sensing, and nanoparticle tracking analysis. The chapters in this book discuss EV cargoes containing proteins and genomic materials using a number of different approaches, and isolating EVs from platelets and neuronal cells and tissues. 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. Practical and comprehensive, Exosomes and Microvesicles: Methods and Protocols is a valuable resource containing methodologies for anyone interested in researching EVs.

Cell senescence is the process whereby cells permanently lose the possibility to proliferate without undergoing cell death, and occurs in a plethora of distinct model organisms. In Cell Senescence: Methods and Protocols, expert researchers in the field detail the methods that are now commonly used to study cell senescence, in model organisms encompassing bacteria, fungi, worms, flies, zebrafish, and mammalian cells. These techniques cover the study of all the morphological, biochemical and functional manifestations of senescence at the cellular level and include protocols for population analyses and high-throughput approaches in suitable model organisms. 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.

The past decade has witnessed a revolution in the attempts of scientists to under stand the molecular basis of dementia. Although dementia, as defined by global cogni tive decline involving gradual loss of memory, reasoning, judgment, and orientation, presents most commonly in the form of Alzheimer's disease (AD), an assortment of other less common disorders, such as prion and Pick's disease, can also lead to symp toms that are similar to those observed in patients with AD. The primary goal of Molecular Mechanisms of Dementia is to address the various mechanisms and multi faceted approaches currently being employed to more clearly delineate the etiological and pathogenic events responsible for the onset of dementia. Perhaps the greatest boon to obtaining a clearer understanding of the causes of AD has come from genetic and molecular biological studies carried out over the past decade. At the genetic level, it has become increasingly clear that AD is a heteroge neous disorder that can be broadly classified into two categories. "Late onset" (>60 yr) cases, which account for the vast majority of AD, genetically involve "susceptibility" genes representing risk factors for the disease (e. g. , inheritance of the 84 allele of the Apolipoprotein E gene). In many cases, the susceptibility gene can act as a "modifier" that modulates the pathogenic cascade occurring subsequent to a separate etiological event "initiating" or "causing" the disorder.

Grauzone and Completion of Meiosis During Drosophila Oogenesis describes the work behind a major, award winning discovery: the establishment of a new pathway that specifically regulates the female meiosis, a process essential for sexual reproduction. This book chronicles a new gene mapping method and the cloning and documentation of several types of genes that were proven to have significant influence on the cell cycle. It is of interest to anyone doing work with fruit flies, both graduate students and principal investigators.

Oxidation-reduction (i.e. redox) processes at the plasma membrane of any cell have been attracting more and more attention, both in basic and in applied research, since the first workshop dealing with the plasma membrane oxidoreductases was organized in Cordoba, Spain, in 1988. This evolution is evident considering the numerous cell functions performed by plasma membrane redox systems not only in healthy cells but also in cells that escaped from the normal metabolic control (e.g. cancer cells) and cells under attack by pathogens. Plasma membrane redox processes have now been demonstrated to play an essential role in growth control and defense mechanisms of these cells. The great importance of the plasma membrane redox systems originates in the fact that they are located in the membrane which is essentially the site of communication between the living cell and its environment. We may say that the plasma membrane can be considered as the "sensory part" of the cell. No chemical substance can enter the cell interior without interaction with the plasma membrane.

For volume 2 alone:
A gold-standard collection of readily reproducible techniques for the molecular and genetic analysis of germ cells in a variety of different reproductive systems. Volume 2: Molecular Embryo Analysis, Live Imaging, Transgenesis, and Cloning focuses on molecular egg analysis, live egg imaging, nuclear cloning, oocyte cryopreservation, and nuclear transfer. Highlights include the identification and characterization of Oct-4, germline chromatin silencing by RNAi, cDNA subtraction and cloning in the field of trophoblast/placental development, and selective ablation. Each readily reproducible protocol is described in step-by-step detail and contains an introduction outlining the principle behind the technique, lists of equipment and reagents, and tips on troubleshooting and avoiding known pitfalls. Comprehensive and cutting-edge, Germ Cell Protocols offers both novice and established researchers a gold-standard collection of hard-to-find methods of high impact research, diverse procedures that are easy-to-follow, well-illustrated, and allow a cross-species transfer of knowledge from lower vertebrates to higher mammalian systems.
For both volumes:
A gold-standard collection of readily reproducible techniques for the molecular and genetic analysis of germ cells in a variety of different reproductive systems. The methods cover sperm and egg activation, motility, fertilization, nuclear development, nuclear cloning, the molecular characterization of specific events, and the imaging of cell structures. Volume 1: Sperm and Oocyte Analysis focuses on sperm cells, oocyte analysis, oocyte maturation, fertilization, and preparation techniques. Volume 2: MolecularEmbryo Analysis, Live Imaging, Transgenesis, and Cloning contains methods for molecular egg analysis, live egg imaging, nuclear cloning, oocyte preservation, and nuclear transfer.
Comprehensive and cutting-edge, Germ Cell Protocols offers both novice and established researchers a gold-standard collection of hard-to-find methods of high impact research, diverse procedures that are easy-to-follow, well-illustrated, and allow a cross-species transfer of knowledge from lower vertebrates to higher mammalian systems.

Somuchofwhatweknowaboutthepathogenesisofhumandiseasehascomefromthe systematic and careful study of histological material. Indeed, every internal medicine discipline has its landmark papers describing the clinico-pathological correlations. However, increasingly, it is molecular and cellular biology that provides the necessary mechanistic insights. For many years, it was thought that the two skill sets were mutually exclusive, but we hope that this book shows that this is not necessarily so. Implicitinthescienceofhistologyisthepreservationandarchivingoftissue.PartIof the book concentrates on the preparation of tissue, providing an overview of fixation, embedding, and processing (Chapter 1), and in Chapters 2 and 3, the required techniques for the retrieval of RNA from histological sections. Both routine and specialist histological staining techniques are provided in Part II. These include pro- cols for immuno (Chapters 4-7), lectin (Chapter 8), and hybridization (Chapter 9) histochemistry, histologicalstaining (Chapters10and11), aswellasspecificmethods for the in situ identification of hypoxia (Chapter 12) and apoptosis (Chapter 13). Finally, Part III details advances in imaging (Chapters 14-16) and image analysis (Chapter 17). It is hoped that this volume will provide molecular biologists with the basic his- chemical techniques and histologists with the molecular techniques to realise the potential of their resource. We are indebted to the authors for their generosity in sharing these protocol

In the last ten years there has been a considerable increase of interest on the notion of the minimal cell. With this term we usually mean a cell-like structure containing the minimal and sufficient number of components to be defined as alive, or at least capable of displaying some of the fundamental functions of a living cell. In fact, when we look at extant living cells we realize that thousands of molecules are organized spatially and functionally in order to realize what we call cellular life. This fact elicits the question whether such huge complexity is a necessary condition for life, or a simpler molecular system can also be defined as alive. Obviously, the concept of minimal cell encompasses entire families of cells, from totally synthetic cells, to semi-synthetic ones, to primitive cell models, to simple biomimetic cellular systems. Typically, in the experimental approach to the construction of minimal the main ingredient is the compartment, lipid vesicles (liposomes) are used to host simple and complex molecular transformations, from single or multiple enzymic reactions, to polymerase chain reactions, to gene expression. Today this research is seen as part of the broader scenario of synthetic biology but it is rooted in origins of life studies, because the construction of a minimal cell might provide biophysical insights into the origins of primitive cells, and the emergence of life on earth. The volume provides an overview of physical, biochemical and functional studies on minimal cells, with emphasis to experimental approaches. 15 International experts report on their innovative contributions to the construction of minimal cells.

The different aspects of muscle development are considered from cellular, molecular and genetic viewpoints, and the text is supported by black/white and color illustrations. The book will appeal to those studying muscle development and muscle biology in any organism.

The 19 papers include discussions of constructing an integrated genetic and physical map of rice, commonalities and contrasts in the organization of the maize and sorghum nuclear genomes, prospects for comparative genome analyses among mammals, genome analysis in farm animals, sense suppression of p

Updating and building upon previous editions, "Hematopoietic Stem Cell Protocols, Third Edition" provides up-to-date protocols from leading stem cell researchers. This in-depth volume presents a clear view of the landscape of assays available to the stem cell researcher working in the growing hematopoietic stem cell (HSC) field. A robust and active field, it is supported by an abundance of innovative mouse models and molecular tools for analysis of phenotypes and functions in mouse and human cells. Understanding more about hematopoietic stem cell biology is integral if these versatile cells are to be applied effectively to treat and cure a wide range of blood diseases.An introductory chapter puts the major contributions of the book into the proper perspective. Written in the 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 protocols, and notes on troubleshooting and avoiding known pitfalls.

Essential for the laboratory-based researcher, "Hematopoietic Stem Cell Protocols, Third Edition" is a much needed technical resource in the critically important field of hematopoietic stem cell investigation."

Cutting-edge articles review our current understanding of lipid microdomain signaling mechanisms and their physiological and pathological importance. The book describes the role of lipid rafts in learning, memory, and cancer, presents the emerging evidence that lipid rafts play critical roles in signaling pathways and the regulation of synaptic function in the nervous system, and shows how alterations in lipid raft metabolism are implicated in the pathogenesis of neurodegenerative disorders. Techniques are also described for the isolation of lipid rafts, the analysis of the lipid and protein components of lipid rafts, the imaging of lipid rafts in living cells, and the analysis of signal transduction in lipid rafts.

A major mechanism by which cells regulate protein function is to place phosphate groups on serine and threonine residues. Though the steady-state level of protein phosphorylation depends on the relative activities of both kinases and phosphatases, a much greater effort has previously gone into the study of the former that the latter . Today, however, there is an increasing appreciation for the role that protein phosphatases play in the dynamic p- cess of protein phosphorylation . To date, there are four major types of protein serine/threonine phosphatase catalytic subunits, designated protein phosphatase type 1, 2A, 2B, and 2C . Each has been identified by the techniques of protein chemistry and enzymology and can be distinguished from one another by their preference for specific substrates as well as their sensitivity to certain acti- tors and inhibitors . Protein Phosphatase Protocols has been assembled in response to the growing interest these enzymes are receiving . The goal of this compilation is to provide a "how-to" experimental guide to aid newcomers as well as s- soned veterans in their research endeavors, thus further contributing towards our ever increasing knowledge of serine/threonine phosphatases . What you have before you contains contributions by many of the current and emerging leaders in the field . To highlight just a few, these chapters c- tain step-by-step information on how to isolate novel phosphatases and re- latory subunits, assay for activity, and generate immunological reagents for both biochemical and biological characterization of these enzymes .

The human body contains many specialized tissues that are capable of fulfilling an incredible variety of functions necessary for our survival. This volume in the Human Cell Culture Series focuses on mesenchymal tissues and cells. The in vitro study of mesenchymal cells is perhaps the oldest form of human cell culture, beginning with the culturing of fibroblasts. Fibroblasts have long been generically described in the literature, arising from many tissue types upon in vitro cell culture. However, recent studies, many enabled by new molecular biology techniques, have shown considerable diversity in fibroblast type and function, as described within this volume. Mesenchymal tissue types that are described within include bone, cartilage, tendons and ligaments, muscle, adipose tissue, and skin (dermis). The proper function of these tissues is predominantly dependent upon the proper proliferation, differentiation, and function of the mesenchymal cells which make up the tissue. Recent advancements in primary human mesenchymal cell culture have led to remarkable progress in the study of these tissues. Landmark experiments have now demonstrated a stem cell basis for many of these tissues, and, furthermore, significant plasticity and inter-conversion of stem cells between these tissues, resulting in a great deal of contemporary excitement and controversy. Newly-developed mesenchymal cell culture techniques have even lead to novel clinical practices for the treatment of disease.

This book discusses the role of oxidative stress in the reproductive system. The book reviews endogenous sources, methods of determining its levels in body fluid/tissues, the physiological roles of ROS, as well as its negative effects on the human reproductive processes. Also discussed are multiple extrinsic factors that could induce oxidative stress in the reproductive system. This volume covers various clinical pathologies related to the reproductive system that arise from or produce oxidative stress, both in the male and female. The use of antioxidants as a therapeutic measure to keep ROS levels in check are highlighted, describing the outcome of various clinical studies involving antioxidant supplementation in infertile patients. Infertility is a global disease that affects 15-25% of all couples, and oxidative stress arising from a multitude of sources has been implicated as one of the major contributing factors to the decline in human fertility. As such, this book provides an up-to-date review on the significance of ROS in human reproduction.

Membrane proteins play a key role in numerous pathologies such as cancer, cystic fibrosis, epilepsy, hyperinsulinism, and Alzheimer s disease, yet studies on these and other disorders are hampered by a lack of information about the proteins involved. In Heterologous Expression of Membrane Proteins: Methods and Protocols, expert researchers provide an overview of the different heterologous expression systems available to produce these proteins for structural analysis, helping us to understand how these proteins function, and additionally, how their functions can be modified through drug treatment. Chapters examine membrane protein solubilization, purification, and instability in solution, and investigate the strategies that helped to determine the structure of the first heterologously expressed mammalian membrane proteins. Composed in the highly successful Methods in Molecular Biology 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.

Far-reaching and innovative, Heterologous Expression of Membrane Proteins: Methods and Protocols, is an essential guide for novice and experienced researchers alike, providing detailed methods for existing expression systems alongside cutting-edge strategies for future use in the field."

This volume provides a comprehensive and technical presentation of numerous aspects of reproductive cell tissue cryopreservation, and presents readers with current procedures and detailed discussions of novel techniques and the latest innovations. The chapters in this book are divided into five parts and cover subjects such as: immature oocyte cryopreservation, human sperm vitrification and slow-freezing, directional freezing of ovarian tissue, automated vitrification systems, and detailed protocols on popular and commercially available cryopreservation/vitrification systems. 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, Cryopreservation of Mammalian Gametes and Embryos: Methods and Protocols includes chapters written by leading experts in the field and is a valuable resource for anyone interested in the field of cryopreservation.

This volume represents the proceedings of the 9th International Proton Transport Conference, "Mechanisms and Consequences of Proton Transport" held in Leura, Australia, August 19 -21, 200 I. This conference has been held since 1971 every 3 to 4 years with few exceptions in association with the Congress of the International Union of Physiological Sciences. The first meeting was held in Frankfurt, Germany, at the Max-Planck'Institute. Countries hosting the conference have been Germany (twice), Sweden (twice), India, Canada, USA, Great Britain, and now Australia. Over the past 30 years participants at these Proton Transport Conferences have been principal contributors to the major discoveries in the physiology, biochemistry and pharmacology of gastric acid secretion. These include development of the H2-receptor blockers, defining the signaling pathways for the regulation of acid secretion, identifying the gastric proton pump, discovery and development of proton pump inhibitory drugs, and elucidating the physiology and biochemistry of Helicobacter pylori.

Harnessing the sun s energy via photosynthesis is at the core of sustainable production of food, fuel, and materials by plants, algae, and cyanobacteria. Photosynthesis depends on photoprotection against intense sunlight, starting with the safe removal of excess excitation energy from the light-harvesting system, which can be quickly and non-destructively assessed via non-photochemical quenching of chlorophyll fluorescence (NPQ). By placing NPQ into the context of whole-organism function, this book aims to contribute towards identification of plant and algal lines with superior stress resistance and productivity. By addressing agreements and open questions concerning photoprotection s molecular mechanisms, this book contributes towards development of artificial photosynthetic systems. A comprehensive picture from single molecules to organisms in ecosystems, and from leading expert s views to practical information for non-specialists on NPQ measurement and terminology is presented."

At the forefront of life sciences today is the emerging discipline of chembiomolecular science. This new term describes the integration of the frontier fields of chemical biology, chemistry, and pharmacology. Chembiomolecular science aims to elucidate new biological mechanisms as potential drug targets and enhance the creation of new drug therapies. This book comprises the proceedings of the Uehara Memorial Foundation Symposium 2011, which focused on the most recent advances in chembiomolecular science made by leading experts in the field. The book is divided into three main topics. The first is the chemical approach to understanding complex biological systems on a molecular level using chemical compounds as a probe. The second describes the biological approach used to develop new lead drug compounds. The third focuses on the biological system that serves as the potential drug target, the beginning step in the process of developing new drugs. Replete with the latest research, the book will draw the attention of all scientists interested in the synergies between chemistry and biology to elucidate life on a molecular level and to promote drug discovery. Ultimately, the book helps promote the understanding of biological functions at the molecular level and create new pharmaceuticals that can contribute to improving human health.

Death receptors play a central role in directing apoptosis in mammalian cells. This process of active cell death is important for a number of biological processes, e.g. for the regulation of the immune system. Death receptors are cell surface receptors that transmit apoptotic signals initiated by corresponding death ligands. Many complex signaling pathways are activated and apoptosis is the final result of a complex biochemical cascade of events.

Besides their role in the induction of cell death, evidence now exists that death receptors are able to activate several non-apoptotic signaling pathways which, depending on cellular context, may lead to apoptosis resistance, secretion of pro-inflammatory proteins, proliferation and invasive growth of cancer cells.

This book looks at the molecular basis of death receptor signaling and the role of death receptors in cancer development.

Metastatic dissemination of cancer is a main cause of cancer related deaths, therefore biological mechanisms implicated in metastatic process presents an essential object of cancer research. This research requires creation and utilization of adequate laboratory models. The book describes main approaches to model processes of metastatic cancer dissemination and metastases development. The book is structured in according with various metastatic pathways reflecting molecular specificity of metastatic process as well as anatomical specificity of aria of dissemination. Each chapter is introduced by short discussion of clinical aspects of certain metastatic pathway. Especial attention is paid for methods of visualization, quantification and analysis of the modeled metastases. Additional chapter is devoted to methods of mathematic modeling of tumor spread. The data presented in the book may be helpful for cancer researchers and oncologists.

This book highlights recent advances in and diverse techniques for exploring the plasma membrane's structure and function. It starts with two chapters reviewing the history of membrane research and listing recent advances regarding membrane structure, such as the semi-mosaic model for red blood cell membranes and the protein layer-lipid-protein island model for nucleated tissue cell membranes. It subsequently focuses on the localization and interactions of membrane components, dynamic processes of membrane transport and transmembrane signal transduction. Classic and cutting-edge techniques (e.g. high-resolution atomic force microscopy and super-resolution fluorescence microscopy) used in biophysics and chemistry are presented in a very comprehensive manner, making them useful and accessible to both researchers in the field and novices studying cell membranes. This book provides readers a deeper understanding of the plasma membrane's organization at the single molecule level and opens a new way to reveal the relationship between the membrane's structure and functions, making it essential reading for researchers in various fields.

Lipobiology is an interdisciplinary field which incorporates critical aspects of lipid and lipoprotein chemistry into the disciplines of cell biology and physiology. During the last decade, advances in our understanding of the structure and function of lipids, biological membranes and lipid-derived second messengers have underscored the importance of lipids in the regulation of cellular function. This series focuses on salient aspects of the role of lipids in metabolic regulation and cellular activation, with emphasis on emerging concepts and technologies. One goal of this series is to formulate cohesive criteria upon which a foundation for the evaluation of recent work can be based and future directions of research identified.