Methods in Molecular Medicine – serie

Leading researchers and clinicians describe their state-of-the-art findings and hypotheses arising from a variety of different approaches to this group of diseases. Their approaches include clinical presentations, epidemiology, transgenic methods, and diagnostic tests via transmission electron microscopy and immunoblotting. The diseases treated range from human to animal spongiform encephalopathies and include Creutzfeldt-Jakob disease, mad cow disease, and scrapie.

An outstanding collection of state-of-the-art experimental methods for herpes virology. These procedures range from the more biological in vivo maneuvers to the purely molecular, and are described in detail by selected experts to ensure reproducibility. Most of the protocols rely on the herpes simplex virus as prototype, so that it then becomes relatively easy to extrapolate and make the necessary modifications required for application to other herpesviruses, especially members of the alpha group, such as PRV and EHV.

An unprecedented collection of core PCR techniques useful for the study and diagnosis of human diseases. Cutting-edge and essential for today's diagnostic laboratories, these techniques heavily utilize nonisotopic, solution phase, and in situ amplification methods. A significant number of chapters describe applications exploiting the eggwhite sensitivity of PCR in the detection of rare or single cells, as in identifying fetal cells circulating in maternal blood, preimplantation embryo diagnosis, or finding circulating cancer cells. The book demonstrates over and over again the power of PCR - its high sensitivity, specificity, and ability to rapidly discriminate sequence variations.

A much-needed collection of cutting-edge methods for the detection and discovery of new cancer markers, particularly molecular tumor markers. Each method is fully described by a hands-on expert who regularly uses it in the laboratory and has perfected it to a high degree of ready reproducibility. Special emphasis is given to statistical considerations in the analysis of tumor markers, recent discoveries in the field of serum and tissue marker proteins, cytogenetic markers, molecular markers, and markers for the early detection of cancer. The well-honed procedures detailed here will enable cancer researchers and clinical oncologists alike to realize the promise of these powerful new diagnostic strategies for the detection, therapy, and successful prevention of cancer.

Helicobacter pylori Protocols offers an outstanding collection of state-of-the-art protocols for the identification and molecular manipulation of H. pylori. The authoritative contributors supply detailed and readily reproducible protocols for the culturing of H. pylori, for the isolation and restriction endonuclease digestion of H. pylori chromosomal DNA, and for the transformation and insertional mutagenesis of H. pylori. They also provide molecular epidemiological techniques, including ribotyping, PCR-RFLP, and RAPD-PCR. These procedures have been developed by leading practitioners to solve the difficult technical problems created by the application of the powerful bacterial genetic and molecular cloning techniques to H. pylori.

Lectins have in the past been regarded by many scientists as curious proteins of uncertain structure and specificity that bind to carbohydrates of dubious significance themselves. All this is rapidly changing. The functional importance of glycosylation in cell-cell and cell-pathogen interactions, as well as intracellular events, has been recognized by the explosion of the science of glycobiology. This has been paralleled by the realization that lectins, once they have been well characterized, can be extremely useful tools for exam- ing structural changes in glycosylation and their functional consequences for human pathophysiology. Different lectins vary considerably in their degree of specificity. Some, such as wheatgerm agglutinin, have fairly broad specificity (for glucosamine or sialic acid), whereas others, such as Maackia amurensis, are specific not only for a single carbohydrate, but also for its linkage (2-3 linked sialic acid). Lectins with relatively broad specificity may be very useful as an adjunct to isolation or quantification of soluble glycoproteins, whereas lectins of known, and precise, specificity will be more useful for characterization of carbo- drate structure.We have included an appendix in Lectin Methods and Pro- cols that provides the known specificities of all lectins cited in the text.

A collection of cutting-edge techniques for detecting most of the major viruses that afflict mankind, including influenza, hepatitis, herpes, polio, mumps, HIV, and many more. The techniques are well-tested, easily reproducible, and readily employ all the new technologies - PCR, RIA, ELISA, and latex-agglutination - that have revolutionized the field. These methods not only make it possible to do the necessary analysis in hours instead of days, but can also be automated in a laboratory havng only low levels of biological containment. Frequently, the protocols for viruses causing human diseases can be adapted to similar viruses of veterinary importance. Through its state-of-the-art methods a physician can, for the first time, determine early in a viral infection which antiviral drug should be used and minimize the period of treatment to avoid unnecessary side effects.

Laboratory studies in hemostasis have traditionally focused on abn- malities of platelet function or the quantitative and qualitative disorders that affect the proteins involved in blood coagulation. However, over the last 10 years there has been an explosion in our understanding of the molecular bases that underlie many of the inherited and acquired disorders of hemostasis. Many of these disorders are now routinely diagnosed and assessed by methods that involve genotypic analysis. Indeed in the late 1990s the distinction between molecular methods for research and for routine diagnosis is becoming incre- ingly blurred. The techniques and approaches that are used in hemostasis are manifold and published in isolation in a variety of publications. The aim, therefore, of this volume Hemostasis and Thrombosis Protocols is to pull together, into a single volume, the variety of techniques that are frequently used in the field of hemostasis. We have targeted this volume at laboratories who wish to move into the field of molecular hemostasis or who may already have some expe- ence in this area but wish to develop new areas of research and diagnosis. The chapters are wide-ranging and hopefully provide a broad overview of the d- fering applications in which these standard techniques can be used. Though the articles may appear relatively specific, the techniques contained within them are applicable to the study of many different disorders and we hope that they provide a series of ideas and concepts well-suited to problem solving.

Expert laboratory scientists describe in detail their methodologies for constructing ribozymes designed to elucidate the role of specific genes as key routes to the development of novel therapies for a wide variety of diseases. The authors review the many sites targeted with ribozymes in various diseases and provide specific accounts of the practical techniques required for the proper use of ribozymes in these systems. Their cutting-edge protocols demonstrate how to achieve ribozyme expression in distinct cellular systems, the preparation and use of high-efficiency ribozyme DNA or RNA delivery, and the studies required to prove the efficacy of ribozyme-mediated inhibition. Therapeutic Applications of Ribozymes contains all the technical support needed to make ribozyme technology a widely used and significant methodology.

A collection of cutting-edge techniques for detecting most of the major viruses that afflict mankind, including influenza, hepatitis, herpes, polio, mumps, HIV, and many more. The techniques are well-tested, easily reproducible, and readily employ all the new technologies - PCR, RIA, ELISA, and latex-agglutination - that have revolutionized the field. These methods not only make it possible to do the necessary analysis in hours instead of days, but can also be automated in a laboratory havng only low levels of biological containment. Frequently, the protocols for viruses causing human diseases can be adapted to similar viruses of veterinary importance. Through its state-of-the-art methods a physician can, for the first time, determine early in a viral infection which antiviral drug should be used and minimize the period of treatment to avoid unnecessary side effects.

In Molecular Bacteriology: Protocols and Clinical Applications, Woodford and Johnson bring together in a single volume a state-of-the-art collection of detailed molecular methods for the diagnosis and clinical investigation of bacterial infections. These wide ranging techniques - each presented by recognized experts who have fine-tuned their methods for optimum performance - will permit readers to establish them successfully in their own laboratories. An accompanying series of minireviews highlights and explains the clinical and epidemiological relevance of the techniques and shows how they may be applied to major pathogen species and various types of antibiotic resistance. Molecular Bacteriology: Protocols and Clinical Applications enlarges our current understanding of the role that molecular methodology can play in modern medical bacteriology, fully explaining both the practical and theoretical aspects of the cutting-edge methods that are increasingly finding diagnostic use in laboratories throughout the world.An authoritative laboratory resource for everyone working with infectious diseases today, this book's insightful reviews and detailed protocols will soon win many devotees.

An unprecedented collection of core PCR techniques useful for the study and diagnosis of human diseases. Cutting-edge and essential for today's diagnostic laboratories, these techniques heavily utilize nonisotopic, solution phase, and in situ amplification methods. A significant number of chapters describe applications exploiting the eggwhite sensitivity of PCR in the detection of rare or single cells, as in identifying fetal cells circulating in maternal blood, preimplantation embryo diagnosis, or finding circulating cancer cells. The book demonstrates over and over again the power of PCR - its high sensitivity, specificity, and ability to rapidly discriminate sequence variations.

A state-of-the-art collection of detailed synthetic procedures that lead to a variety of scaffolds, turn mimetics, peptide-bound replacements, and enzyme inhibitors. Topics range from unusual syntheses of amino acids to the use of a variety of linear and heterocyclic scaffolds in place of the peptide backbone. Important chemical procedures and methods include the transient protection of charged peptides as neutral prodrugs for improved blood-brain penetration and the replacement of peptide bonds with heterocyclic rings, olefins and fluoroolefins, and ketomethylenes. Synthetic protocols towards the transition-state mimics and reactive "warheads," applicable in enzyme inhibitors, are also disclosed.

G. E. Francis and Cristina Delgado have assembled a comprehensive collection of leading-edge protocols for drug targeting studies. In addition to the detailed step-by-step instruction, the protocols review the principal current targeting strategies, discuss how to build the constructs, examine theoretical considerations relevant to solid tumors, and describe experimental and clinical applications of targeted drug delivery systems. Also present are new chemical and biological approaches to the preparation of targeted biopharmaceuticals, including ribonuclease antibody chimeras, bispecific antibodies, enzyme antibody conjugates, and folate-bearing conjugates. Applications include the targeting of myocardial infarctions using liposomal systems, the use of monoclonal antibodies to target malaria and HIV-infected cells, the targeting of fusion proteins to leukemias and lymphomas, and the use of antioxidant enzyme antibody conjugates to target the pulmonary endothelium.Comprehensive and innovative, Drug Targeting: Strategies, Principles, and Applications documents in practical detail the major new chemical and biological targeting methods now being used to produce today's emerging rich array of novel diagnostic agents and promising biopharmaceuticals.

In recent years, molecular techniques have enhanced our ability to detect sexually transmitted infections and to conduct research to further our und- standing of sexually transmitted diseases. Molecular methods to quantitate pathogen load have also been shown to be useful for the management of HIV and other viral STDs. Existing laboratory manuals for the clinical microbi- ogy laboratory often do not include molecular methods for STDs. Sexually Transmitted Diseases: Methods and Protocols is intended to fill the need for a dedicated manual that covers all the fundamental aspects of molecular pro- cols for laboratory diagnosis, as well as research methodology for STDs, - cluding HIV. There are more than types of molecular techniques described in this book covering nine major sexually transmitted pathogens. Although mole- lar methods for the detection of such pathogens as Trichomonas vaginalis are available in the published literature, they have not been included since they are not yet widely used for laboratory diagnosis or research. Sexually Transmitted Diseases: Methods and Protocols is one of a series of books treating Methods Molecular Medicine, published by Humana Press.

In Hematologic Malignancies: Methods and Techniques, a panel of acknowledged experts review many of the key molecular methods used for the diagnosis and subsequent management of hematologic malignancies. These clinically relevant techniques range from routine test procedures to highly sophisticated methods currently offered only by specialized reference laboratories, and fall into five major groups: cytogenetics, polymerase chain reaction, flow cytometry, cytochemistry and immunochemistry, and apoptosis and cytokine receptors. Serving both clinical and experimental needs, Hematologic Malignancies: Methods and Techniques provides an array of powerful tools that will guide clinicians - especially hematologists, oncologists, and pathologists - to better diagnose and manage their patients with hematologic malignancies, and enable researchers to assess the anticancer effect of agents that impact cancer cells at the molecular level.

Experienced scientists describe - in an easily-followed format - their state-of-the-art techniques for evaluating antiviral compounds. The assays described include systems for investigating drugs used against herpesviruses, hepatitis viruses, human immunodeficiency viruses, human papillomaviruses, and influenza viruses. These well-tested methods range from cellular assays to some of the most up-to-date molecular approaches for identifying compounds that are active against viral enzymes and the development of viral resistance against drugs currently in use. Timely and comprehensive, Antiviral Methods and Protocols offers today's researchers in academia, clinical departments, and the pharmaceutical industry the powerful, reproducible, and novel methods needed to evaluate compounds effective against both acute and chronic infections.

State-of-the-art review articles by leading experts summarize how to develop and employ the highly promising new DNA vaccines, what clinical results can be expected from their use, and what is known about how they work. Key topics range from vaccine design and construction to preparation and delivery methods, including the use of classical adjuvants, "genetic adjuvants," and the immunostimulatory properties of DNA and selected oligonucleotide sequences. Several contributors provide strategic ideas on antigen engineering and describe the novel applications of DNA vaccine methodology that have recently emerged. Cutting-edge and comprehensive, DNA Vaccines: Methods and Protocols provides a snapshot of the methods and thinking from which the vaccines of tomorrow will evolve.

Yvonne and Christopher Barnett and a team of recognized international experts detail key biochemical, analytical, and molecular techniques for the investigation of aging at the cellular, tissue, organ, and whole system levels. These cutting-edge methods address a wide range of research needs, from uncovering the factors associated with cell senescence and death, to exploring alterations in the body's ability both to metabolize xenobiotics and to defend itself against biomolecular damage. State-of-the-art protocols also measure the morphological, functional, and molecular changes that accumulate within mitochrondria over time, and permit the genetic and functional characteristics of the immune system to be determined. Wide ranging and highly practical, Aging Methods and Protocols provides today's molecular gerontologists, pharmacologists, and clinical investigators with a gold-standard collection of readily reproducible techniques for identifying those key cellular and molecular processes that might one day make it possible to regulate the aging process.

Over one hundred well-respected scientists comprehensively review every aspect of the biology underlying ovarian cancer and present a remarkably detailed collection of easy-to-follow methods for illuminating basic mechanisms underlying all forms of cancer. Here today's investigators will find the broadest imaginable array of molecular biology techniques specially designed to determine not only tumor genetics, expression, and protein function, but also to elucidate the genetic mechanisms by which gene and immunotherapies may be perfected. Versatile and comprehensive, Ovarian Cancer Methods and Protocols constitutes a veritable treasury of well-proven expert methods that will help basic scientists and clinical researchers alike master the powerful technologies required for effective cancer research today.

Leading clinical and laboratory scientists describe cutting-edge methods for examining the mechanisms of cellular resistance to anticancer cytotoxics in human tumors. The protocols contain detailed instructions and extensive troubleshooting tips that allow researchers effectively to study a wide variety of drug resistance mechanisms, including aspects of drug-induced cell death, drug uptake/efflux, drug metabolism, and DNA repair. Each method is designed to help identify the correlation between molecular and biochemical data and the clinical responses of the patient. Cytotoxic Drug Resistance Mechanisms illuminates all the clinically relevant mechanisms used as markers of the biological response to anticancer biotherapeutics today.

Leading experts comprehensively review all aspects of the delivery of therapeutic molecules to cells using electrical impulses - i.e., the extraordinarily promising new areas of electrogenetherapy, electrochemotherapy, and transdermal drug delivery. Their survey ranges from outlines of the basic physical principles that govern cell permeabilization by pulsed electric fields, to descriptions of the current state-of-the-art in instrumentation and electrodes, to summaries of preclinical and clinical trial results. The authors focus on drug, gene, and transdermal delivery techniques, providing detailed examples of drug delivery using electric fields from a variety of pulse generators and electrodes. Comprehensive and authoritative, Electrochemotherapy, Electrogenetherapy, and Transdermal Drug Delivery: Electrically Mediated Delivery of Molecules to Cells provides entre to an immensely practical set of in vivo electroporation techniques, including electrogenetherapy, electrochemotherapy, and transdermal drug delivery - techniques holding great promise for all those working toward better therapies for cancer, metabolic diseases, and vaccination.

An outstanding collection of detailed, state-of-the-art techniques for studying many aspects of nervous system cell biology. The methods span a multidisciplinary range of cellular and molecular approaches to both normal and injured nervous system function, and particularly to neurodegenerative processes. Included are basic RT-PCR techniques, cell culture systems, second-messenger signaling methods, and patch-clamp techniques. These powerful techniques will illuminate nervous system function, injury, degeneration, and the repair/regenerative process.

Leading researchers describe in step-by-step detail their best state-of-the art molecular techniques for elucidating the biochemical processes associated with human cancer. Among the techniques presented for identifying chromosomal rearrangements are fluorescent in situ hybridization (FISH), spectral karyotyping (SKY), comparative genomic hybridization (CGH), microsatellite analysis, PCR and RT-PCR, and DNA fingerprinting. Additional methods include SSCP, DGGE, the nonisotopic RNase cleavage assay, and the protein truncation assay and DNA sequencing for identifying mutations.

K. Fan Chung and Ian Adcock bring together the first collection of laboratory methods for studying the molecular and cellular mechanisms of asthma. Leading experts describe highly effective methods for obtaining cells from asthmatic airways, analyzing gene and protein expression in these clinical samples, and using molecular and cellular tools for studying cytokine expression and release in this disease. There are also cutting-edge techniques for studying asthma-related genes and genetic polymorphisms, and for understanding the effects of asthma treatment. User-friendly and state-of-the-art, Asthma: Mechanisms and Protocols provides all asthma researchers - whether novice or experienced, whether in experimental or clinical research - with a first-class collection of readily reproducible pharmacological, cellular, molecular, biochemical, and genetic methods for elucidating not only the mechanisms of the disease itself, but also of the drugs for asthma's treatment.

The term "electrophoresis" was first used by Michaelis in 1909, to - scribe the migration of colloids in an electric field. The first practical elect- phoresis method was described by Tiselius in 1937. He used a U-tube filled with buffer layered on top of sample; migration could be monitored using Schlieren optics. In zone electrophoresis, the U-tube was replaced by paper, a support material employed simply to prevent or minimize diffusion of ions, so that ions applied in a narrow strip to the paper will separate and remain as relatively discrete zones. Paper was superceded by a variety of other media, - cluding cellulose acetate, hydrolyzed starch (starch gel), agarose, and polyacry- mide. The latter, in addition to being a support medium, has size-sieving properties. From the basic zone electrophoresis, other means of separation have been dev- oped. These include, isoelectric focusing, isotachophoresis, density gradient el- trophoresis, and various forms of immunoelectrophoresis. In some ways Capillary Electrophoresis (CE) has gone full circle back to the original method of Tiselius.In its simplest form, separations occur in a buffer solution within a glass (fused silica) tube and detection occurs as sample moves past an optical window. CE has rapidly developed into a technique that rivals HPLC in its versatility. All the classical electrophoretic separations-zone, IEF, and isotachophoresis-have their counterparts in CE. Excitingly so, and - thoritatively treated in Clinical Applications of Capillary Electrophoresis.

Professor Michael N. Marsh, a recognized world authority on this condition, together with a team of other expert laboratory/clinical investigators from around the world, present a collection of the state-of-the-art techniques for studying the biology and immunopathology of celiac disease. Both the novice and experienced researcher will find in this collection detailed step-by-step methods for cloning lymphocytes, creating gene/peptide libraries, and performing genotyping, linkage, and positional cloning. Also included are techniques for determining the peptide structure of HLA-bound material (tandem mass spectroscopy), computerized morphometry, in situ hybridization, organ culture, and monoclonal AB assays. Comprehensive and cutting-edge, Celiac Disease: Methods and Protocols provides all experimental and clinical investigators with the essential core of readily reproducible methods necessary for successful work on celiac disease today.

Anthony Killeen has assembled a collection of readily reproducible molecular pathology techniques that are either frequently performed or recognized for their significant diagnostic utility. Each method is described in step-by-step detail by a leading molecular pathologist or laboratory scientist who has developed it or used it extensively. These clinical laboratory techniques can be used for the diagnosis or monitoring of cancer, hematological malignancies, infectious diseases, and selected genetic disorders. Comprehensive and path-breaking, Molecular Pathology Protocols will enable clinical laboratories to introduce new molecular pathology tests and lay the groundwork for a much-needed standardization in this rapidly developing field.

In Melanoma Techniques and Protocols: Molecular Diagnosis, Treatment, and Monitoring, a broadly diverse group of researchers and clinicians offer not only reviews of the most important recent advances in melanoma oncology, tumor immunology, and pathology, but also state-of-the-art molecular techniques for probing its basic biology. These readily reproducible methods can be used to develop new diagnostic approaches, therapeutics, and status monitoring of patients suffering both early- and late-stage melanoma. With emphasis on the experimental details critical to experimental success, each method is described in step-by-step fahion by hands-on masters, often those who have developed hem in their own laboratories. Comprehensive and highly instructive, Melanoma Techniques and Protocols: Molecular Diagnosis, Treatment, and Monitoring offers both clinicians and basic scientists concerned with malignant melanoma both new clinical perspectives and the wide ranging experimental protocols essential for achieving more rapid breakthroughs in this near-epidemic disease.