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.

The past decade has seen an extraordinary growth in research interest in neurotrophic factors, and the study of the neurotrophin family has led this activity. Nevertheless, this area of research has often struggled as a result of techniques that were either inadequate or just emerging from other research fields and disciplines. Neurotrophin Protocols has brought together many leaders in the neurotrophin field who detail their special expertise in a wide variety of techniques. Though most procedures are valid across many diff- ent fields of research, some of those described here have been developed to address particular issues within the neurotrophic factor field. The protocols cover a broad range of biochemical, histological, and biological techniques that are often required by the modern laboratory. However, all have been written with sufficient detail to allow any laboratory to achieve proficiency without need of reference to other texts. Neurotrophin Protocols is divided into four sections dealing with p- tein, RNA, recombinant, and in vivo techniques. Protein techniques have in general been less successfully employed than those dealing with RNA or DNA. However, procedures that achieve localization and quantification of the neurotrophins are now being used more extensively. Their inclusion here should assist further studies at the protein level. Transgenic cell lines and animals are commonplace in the scientific research literature, but their inc- sion in several chapters in this book provide some novel uses that are not readily available elsewhere.

The chemokines family of small proteins are involved in numerous b- logical processes ranging from hematopoiesis, angiogenesis, and basal l- kocyte trafficking to the extravasation and tissue infiltration of leukocytes in response to inflammatory agents, tissue damage, and bacterial or viral infection. Chemokines exert their effects through a family of seven G-protein coupled transmembrane receptors. Worldwide interest in the chemokine field surged dramatically early in 1996, with the finding that certain chemokine receptors were the elusive coreceptors, required along with CD4, for HIV infection. Today, though over 40 human chemokines have been described, the n- ber of chemokine receptors lags behind—only 17 human chemokine receptors have been identified so far. What has emerged over the years is that most chemokine receptors bind several distinct ligands, and indeed the majority of chemokines are able to bind to multiple chemokine receptors, explaining to some extent the apparent disparity in the numbers of chemokines and rec- tors. Yet in spite of the apparent redundancy in chemokine/chemokine rec- tor interactions, it is clear that in vivo, spatial, temporal, and indeed cell- and tissue-specific expression of both chemokines and their receptors are imp- tant factors in determining the precise nature of cellular infiltrates in phy- ological and pathological processes.

Mycotoxins produced by molds are common contaminants of many important crops, including wheat, corn, rice, and peanuts. Some mycotoxins are found in fruits and vegetables. These contaminants have a broad range of toxic effects, including carcinogenicity, neurotoxicity, and reproductive and developmental toxicity. The occurrence of mycotoxins in foods is an unavoidable worldwide problem. About 80 countries have imposed regulatory limits to minimize human and animal exposure to mycotoxins. Regulatory limits, including international standards, have tremendous economic impact and must be developed using science-based risk assessments. The purpose of Mycotoxin Protocols is to provide the scientific and technological basis for analytical methods for use in obtaining the exposure data needed for risk assessments. Mycotoxin Protocols is divided into four sections, which are interc- nected. The first section: Chapters 1–5 describe the general techniques for mycotoxin analysis with emphasis on the importance of method validation based on statistical parameters; sampling procedures for collecting a sample as representative as possible of a bulk lot; the isolation of mycotoxins for use as analytical standards or for toxicological studies; the evaluation of purity and preparation of standards; and the detection and identification of impu- ties in isolated mycotoxins. Sections 2–4: Chapters 6–19 describe the most current chromatographic and immunochemical methods for studies on the major mycotoxins.

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.

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).

As the major task of sequencing the human genome is near completion and full complement of human genes are catalogued, attention will be focused on the ultimate goal: to understand the normal biological functions of these genes, and how alterations lead to disease states. In this task there is a severe limitation in working with human material, but the mouse has been adopted as the favored animal model because of the available genetic resources and the highly conserved gene conservation linkage organization. In just of ten years since the first gene-targeting experiments were p- formed in embryonic stem (ES) cells and mutations transmitted through the mouse germline, more than a thousand mouse strains have been created. These achievements have been made possible by pioneering work that showed that ES cells derived from preimplantation mouse embryos could be cultured for prolonged periods without differentiation in culture, and that homologous rec- bination between targeting constructs and endogenous DNA occurred at a f- quency sufficient for recombinants to be isolated. In the next few years the mouse genome will be systematically altered, and the techniques for achi- ing manipulations are constantly being streamlined and improved.