SV40 Protocols


Book Description

Simian virus 40 gained notoriety in the 1960s because it was found to be a contaminant of polio and adenovirus vaccines that had been administered to millions of healthy individuals worldwide. The public health implications of this revelation provided the initial impetus for an in-depth study of SV40 biology. Later work showed that SV40 DNA sequences as well as infectious virus are in fact found in human tumors and may have contributed to oncog- esis. It also turned out that SV40 uses mostly cellular machinery to carry out many steps in viral infection, which makes it a powerful probe for examining many fundamental questions in eukaryotic molecular biology. SV40 Pro- cols consolidates a number of well-tested step-by-step techniques in one v- ume; experts with hands-on experience in particular methods give detailed accounts of their optimized experimental protocols, so that the beginner, as well as more experienced researchers, may readily overcome problems of ambiguity often present in the literature. As with other DNA tumor viruses, the response of cultured cells to SV40 infection depends upon the species being infected. Monkey cells s- port virus production, which leads to their death, whereas rodent cells p- duce only the early proteins and acquire a transformed phenotype. Thus, SV40 Protocols is organized in two sections. The first relates to assays of the lytic cycle of the virus, and the second deals with transformation.




Immunotoxin Methods and Protocols


Book Description

Immunotoxins represent a new class of human therapeutics that have widespread applications and a potential that has not yet been fully recognized since they were first conceived of by Paul Ehrlich in 1906. The majority of advances in the development and implementation of immunotoxins has occurred over the last 20 years. The reasons for this use of immunotoxins in basic science and clinical research are the powerful concurrent advances in genetic engineering and receptor physiology. Recombinant technology has allowed investigators to produce sufficient quantities of a homogeneous c- pound that allows clinical trials to be performed. The identification of specific receptors on malignant cell types has enabled scientists to generate immunotoxins that have had positive results in clinical trials. As more cellular targets are identified in coming years, additional trials will be conducted in different disease states affecting still larger patient populations. Modulation of the immune system to decrease the humoral response to immunotoxins may improve their overall efficacy. As increasingly more effective compounds are generated, it will be necessary to decrease the local and systemic toxicity - sociated with these agents, and methods for doing so are presently being - veloped. The work presented in Immunotoxin Methods and Protocols focuses on three specific areas of immunotoxin investigation that are being conducted by experts throughout the world. The first section describes the construction and development of a variety of immunotoxins.




Nuclease Methods and Protocols


Book Description

Nucleases, enzymes that restructure or degrade nucleic acid polymers, are vital to the control of every area of metabolism. They range from “housekeeping” enzymes with broad substrate ranges to extremely specific tools (1). Many types of nucleases are used in lab protocols, and their commercial and clinical uses are expanding. The purpose of Nuclease Methods and Protocols is to introduce the reader to some we- characterized protein nucleases, and the methods used to determine their activity, structure, interaction with other molecules, and physiological role. Each chapter begins with a mini-review on a specific nuclease or a nuclease-related theme. Although many chapters cover several topics, they were arbitrarily divided into five parts: Part I, “Characterizing Nuclease Activity,” includes protocols and assays to determine general (processive, distributive) or specific mechanisms. Methods to assay nuclease products, identify cloned nucleases, and determine their physiological role are also included here. Part II, “Inhibitors and Activators of Nucleases,” summarizes assays for measuring the effects of other proteins and small molecules. Many of these inhibitors have clinical relevance. Part III, “Relating Nuclease Structure and Function,” provides an overview of methods to determine or model the 3-D structure of nucleases and their complexes with substrates and inhibitors. A 3-D structure can greatly aid the rational design of nucleases and inhibitors for specific purposes. Part IV, “Nucleases in the Clinic,” summarizes assays and protocols suitable for use with t- sues and for nuclease based therapeutics.




Immunization Safety Review


Book Description

The Immunization Safety Review Committee was established by the Institute of Medicine (IOM) to evaluate the evidence on possible causal associations between immunizations and certain adverse outcomes, and to then present conclusions and recommendations. The committee's mandate also includes assessing the broader societal significance of these immunization safety issues. While all the committee members share the view that immunization is generally beneficial, none of them has a vested interest in the specific immunization safety issues that come before the group. The committee reviews three immunization safety review topics each year, addressing each one at a time. In this fifth report in a series, the committee examines the hypothesis that exposure to polio vaccine contaminated with simian virus 40 (SV40), a virus that causes inapparent infection in some monkeys, can cause certain types of cancer.




Connexin Methods and Protocols


Book Description

Direct cell–cell communication is a common property of multicellular organisms that is achieved through membrane channels which are organized in gap junctions. The protein subunits of these intercellular channels, the connexins, form a multigene family that has been investigated in great detail in recent years. It has now become clear that, in different tissues, connexins speak several languages that control specific cellular functions. This progress has been made possible by the availability of new molecular tools and the improvement of basic techniques for the study of membrane channels, as well as by the use of genetic approaches to study protein function in vivo. More important, connexins have gained visibility because mutations in some connexin genes have been found to be linked to human genetic disorders. Connexin Methods and Protocols presents in detail a collection of te- niques currently used to study the cellular and molecular biology of connexins and their physiological properties. The field of gap junctions and connexin research has always been characterized by a multidisciplinary approach c- bining morphology, biochemistry, biophysics, and cellular and molecular biology. This book provides a series of cutting-edge protocols and includes a large spectrum of practical methods that are available to investigate the fu- tion of connexin channels. Connexin Methods and Protocols is divided into three main parts.




Cytoskeleton Methods and Protocols


Book Description

Over the past two decades experimental studies have solidified the int- pretation of the cytoskeleton as a highly dynamic network of microtubules, actin microfilaments, intermediate filaments, and myosin filaments. Rather than a network of disparate fibers, these polymers are often interconnected and display synergy, which is the combined action of two or more cytoskeletal polymers to achieve a specific cellular structure or function. Cross-commu- cation among cytoskeletal polymers is thought to be achieved through cytoskeletal polymer accessory proteins and molecular motors that bind two or more cytoskeletal polymers. Development of the modern concept of the cytoskeleton is a direct o- growth of advances in experimental tools and reagents that are available to cell and molecular biologists. Technological advances and refinements in cell imaging have made it possible to selectively image a single cytoskeletal po- mer and monitor its dynamics through the use of fluorescence probes in vitro and in vivo. Two decades ago, cytoskeletal research was limited to a few perturbation reagents that included colchicine and cytochalasin. Today, the perturbation arsenal has expanded to a highly selective group of reagents that includes Taxol, nocodazole, benomyl, latrunculin, jasplakinolide, and such endogenous proteins as gelsolin. These reagents enable the investigator to selectively perturb or destroy a cytoskeletal polymer while leaving other cytoskeletal polymers intact. Site-specific monoclonal antibodies that target a specific cytoskeletal polymer have proven to be highly selective affinity tools for cytoskeletal research.




DNA Repair Protocols


Book Description

The first edition of this book, published in 1999 and called DNA Repair Protocols: Eukaryotic Systems, brought together laboratory-based methods for studying DNA damage and repair in diverse eukaryotes: namely, two kinds of yeast, a nematode, a fruit fly, a toad, three different plants, and human and murine cells. This second edition of DNA Repair Protocols covers mammalian cells only and hence its new subtitle, Mammalian Systems. There are two reasons for this fresh emphasis, both of them pragmatic: to cater to the interests of what is now a largely mammalocentric DNA repair field, and to expedite editing and prod- tion of this volume. Although DNA Repair Protocols: Mammalian Systems is a smaller book than its predecessor, it actually contains a greater variety of methods. Fourteen of the book’s thirty-two chapters are entirely new and areas of redundancy present in the first edition have been eliminated here (for example, now just two chapters describe assays for nucleotide excision repair [NER], rather than seven). All eighteen returning chapters have been revised, many of them ext- sively. In order to maintain a coherent arrangement of topics, the four-part p- titioning seen in the first edition was dispensed with and chapters concerned with ionizing radiation damage and DNA strand breakage and repair were re- cated to near the front of the book. Finally, an abstract now heads each chapter.




Proteoglycan Protocols


Book Description

Proteoglycans are some of the most elaborate macromolecules of mammalian and lower organisms. The covalent attachment of at least five types of glycosami- glycan side chains to more than forty individual protein cores makes these molecules quite complex and endows them with a multitude of biological functions. Proteoglycan Protocols offers a comprehensive and up-to-date collection of prepa- tive and analytical methods for the in-depth analysis of proteoglycans. Featuring st- by-step detailed protocols, this book will enable both novice and experienced researchers to isolate intact proteoglycans from tissues and cultured cells, to establish the composition of their carbohydrate moieties, to generate strategies for prokaryotic and eukaryotic expression, to utilize methods for the suppression of specific proteoglycan gene expression and for the detection of mutant cells and degradation products, and to study specific interactions between proteoglycans and extracellular matrix proteins as well as growth factors and their receptors. The readers will find concise, yet comprehensive techniques carefully drafted by leading experts in the field. Each chapter commences with a general Introduction, followed by a detailed Materials section, and an easy-to-follow Methods section. An asset of each chapter is the extensive notation that includes troubleshooting tips and practical considerations that are often lacking in formal methodology papers. The reader will find this section most valuable because it is clearly provided by experienced scientists who have first-hand knowledge of the techniques they outline. In addition, most of the chapters are well illustrated with examples of typical data generated with each method.




Genomics Protocols


Book Description

We must unashamedly admit that a large part of the motivation for editing Genomics Protocols was selfish. The possibility of assembling in a single volume a unique and comprehensive collection of complete protocols, relevant to our work and the work of our colleagues, was too good an opportunity to miss. We are pleased to report, however, that the outcome is something of use not only to those who are experienced practitioners in the genomics field, but is also valuable to the larger community of researchers who have recognized the potential of genomics research and may themselves be beginning to explore the technologies involved. Some of the techniques described in Genomics Protocols are clearly not restricted to the genomics field; indeed, a prerequisite for many procedures in this discipline is that they require an extremely high throughput, beyond the scope of the average investigator. However, what we have endeavored here to achieve is both to compile a collection of procedures concerned with geno- scale investigations and to incorporate the key components of “bottom-up” and “top-down” approaches to gene finding. The technologies described extend from those traditionally recognized as coming under the genomics umbrella, touch on proteomics (the study of the expressed protein complement of the genome), through to early therapeutic approaches utilizing the potential of genome programs via gene therapy (Chapters 27–30).




Neuropeptide Y Protocols


Book Description

The observation that neuropeptide Y (NPY) is the most abundant peptide present in the mammalian nervous system and the finding that it elicits the most powerful orexigenic signal have led to active investigations of the properties of the NPY family of hormones, including peptide YY (PYY) and pancreatic polypeptide (PP). Nearly two decades of research have led to the identification of several NPY receptor subtypes and the development of useful receptor selective ligands. Moreover, these investigations have imp- cated NPY in the pathophysiology of a number of diseases, including feeding disorders, seizures, memory loss, anxiety, depression, and heart failure. Vigorous efforts are therefore continuing, not only to understand the bioche- cal aspects of NPY actions, but also toward developing NPY-based treatments for a variety of disorders. To facilitate these efforts, it was decided to produce the first handbook on NPY research techniques as part of the Methods in Molecular Biology Series. In compiling Neuropeptide Y Protocols, I have gathered contributions on techniques considered critical for the advancement of the NPY field from experts in various disciplines. Each chapter starts with a brief introduction, with Materials and Methods sections following. The latter sections are presented in an easy to follow step-by-step format. The last section of the chapter, Notes, highlights pitfalls and the maneuvers employed to overcome them. This information, not usually disseminated in standard research pub- cations, may prove extremely useful for investigators employing these te- niques in NYP research.