bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemical Cleavage of Proteins in Solution
Authors: Crimmins Dan L. , Mische Sheenah M. , Denslow Nancy D.
Published: June 2004

Described in this unit are five basic protocols that are widely used for specific and efficient chemical cleavage of proteins in solution. Cyanogen bromide (CNBr) cleaves at methionine (Met) residues; BNPS‐skatole cleaves at tryptophan (Trp) residues; formic acid cleaves at aspartic acid‐proline (Asp‐Pro) peptide bonds; hydroxylamine cleaves at asparagine‐glycine (Asn‐Gly) peptide bonds, and 2‐nitro‐5‐thiocyanobenzoic acid (NTCB) cleaves at cysteine (Cys) residues. Because the above loci are at relatively low abundance in most proteins, digestion with these agents will yield relatively long peptides.
Amino Acid Analysis
Authors: Crabb John W. , West Karen A. , Dodson W. Scott , Hulmes Jeffrey D.
Published: June 2004

Amino acid analysis (AAA) is one of the best methods to quantify peptides and proteins. Two general approaches to quantitative AAA exist, namely, classical postcolumn derivatization following ion‐exchange chromatography and precolumn derivatization followed by reversed‐phase HPLC (RP‐HPLC). Excellent instrumentation and several specific methodologies are available for both approaches, and both have advantages and disadvantages. This unit focuses on picomole‐level AAA of peptides and proteins using the most popular precolumn‐derivatization method, namely, phenylthiocarbamyl amino acid analysis (PTC‐AAA). It is directed primarily toward those interested in establishing the technology with a modest budget. PTC derivatization and analysis conditions are described, and support and alternate protocols describe additional techniques necessary or useful for most any AAA method‐‐e.g., sample preparation, hydrolysis, instrument calibration, data interpretation, and analysis of difficult or unusual residues such as cysteine, tryptophan, phosphoamino acids, and hydroxyproline.
Phosphoamino Acid Analysis
Authors: Sefton Bartholomew M.
Published: June 2004

It is often valuable to identify the phosphorylated residue in a protein. This unit presents a protocol for partial acid hydrolysis of proteins phosphorylated at serine, threonine, or tyrosine, followed by two‐dimensional thin‐layer electrophoresis of the labeled phosphoamino acid. Phosphothreonine and phosphotyrosine are more stable to hydrolysis in alkali than are RNA and phosphoserine. Therefore, an alternate procedure using mild alkaline hydrolysis of protein samples to enhance the detection of phosphothreonine and phosphotyrosine is also provided.
Detection of Phosphorylation by Immunological Techniques
Authors: Sefton Bartholomew M.
Published: June 2004

Phosphorylation of unlabeled proteins can be detected using immunological or enzymatic techniques. Anti‐phosphotyrosine antibodies are used with immunoblots to detect tyrosine phosphorylation. This unit presents a protocol employing anti‐phosphotyrosine antibodies with detection by either 125I‐labeled protein A or enhanced chemiluminescence (ECL).
Permeabilization Strategies to Study Protein Phosphorylation
Authors: Carter A. Nigel
Published: June 2004

This unit deals with the use of nucleotide triphosphates to label proteins in vitro in permeabilized cells and isolated cellular fractions. Both of these assay formats result in lysates from which the protein of interest may be easily immunoprecipitated; however alternative techniques are described for preparing the final lysate for electrophoretic analysis. A related procedure that does not involve permeabilization is outlined for direct analysis of cytosolic or membrane‐bound kinases. Two different methods for determining the specific radioactivity of 32P‐containing compounds are also included. These experiments generally utilize [γ‐32P]ATP as an exogenously added phosphate donor, although[γ‐32P]GTP can be used in specific cases. The method is very straightforward, although numerous considerations must be made before applying it to each new system.
Overview of Peptide and Protein Analysis by Mass Spectrometry
Authors: Carr Steven A. , Annan Ronald S.
Published: June 2004

One goal of this introductory overview is to try and dispel the still widely held, but now mistaken, belief that mass spectrometry is beyond the technical resources of the typical laboratory or facility engaged in the structural characterization or synthesis of peptides and proteins. Over the last five years, mass spectrometers and associated data systems have become available that can be operated by anyone capable of running a modern amino acid analyzer or Edman sequencer, yet are powerful enough to handle the most demanding analyses that the peptide or protein investigator might require. The remaining goals of this article are to familiarize peptide and protein chemists with the types of mass spectrometers that are appropriate for the majority of their analytical needs, to describe the kinds of experiments that can be performed with these instruments on a routine basis, and to discuss the kinds of information that these experiments provide. The emphasis here is on established tools and techniques that can realistically be used for problem solving. As a result, many useful instruments and techniques employed by the trained mass spectroscopist are not discussed here, because they do not satisfy these criteria or are not yet widely available; however, a few of these are briefly mentioned at the end of the unit. The overview concludes with a tutorial discussion on the meaning and practical importance of a number of fundamental experimental and performance‐related issues that have important implications for interpretation and use of mass spectral data, such as mass accuracy and resolution.
Introducing Samples Directly into Electrospray Ionization Mass Spectrometers Using Microscale Capillary Liquid Chromatography
Authors: Lee Terry D. , Moore Roger E. , Young Mary K.
Published: June 2004

This unit describes the design and operation of a microscale electrospray (ES) interface suitable for the on‐line liquid chromatography (LC) separation and mass spectrometry (MS) analysis of mixtures of peptides and proteins. The interface utilizes an ES needle packed with reversed‐phase support. Such a design has the advantage of minimizing any void volume between the end of the column and point of electrospray ionization, thus maintaining the integrity of the LC separation and maximizing sensitivity. Here, protocols are presented for construction of an integrated LC column ES needle in‐house, packing the ES needle, and mounting and using the microscale ES LC/MS interface assembly. Various options for low‐flow solvent delivery systems are also discussed.
Principles of Macromolecular X‐Ray Crystallography
Authors: Hickman Alison B. , Davies David R.
Published: June 2004

X‐ray crystallography is one of the major tools available for protein structural analysis. This unit provides an introductory review of the principles of X‐ray crystallography that covers how the image is generated and analyzed. The second half of the unit describes strategies for producing crystals of protein, including methods for dealing with proteins that do not easily form crystals.
Probing Protein Structure and Dynamics by Hydrogen Exchange–Mass Spectrometry
Authors: Wang Lintao , Smith David L.
Published: June 2004

Hydrogen exchange rates at peptide backbone amide linkages of proteins are highly sensitive to changes in protein structure and dynamics. Hydrogen exchange‐mass spectrometry (HX‐MS) can be used to measure hydrogen/deuterium exchange in protein fragments and allows the study of large proteins with high sensitivity and at very low concentrations as well as determination of the intermolecular distribution of deuterium. This unit provides a brief discussion of the interplay between protein dynamics and hydrogen exchange, a detailed prescription for designing and performing typical HX‐MS experiments, and common procedures for processing and interpreting HX‐MS data.
Titration Microcalorimetry
Authors: Doyle Michael L.
Published: June 2004

Isothermal titration calorimetry (ITC) is perhaps the most rigorous commercially available method for characterizing protein‐ligand interactions. In this method, interactions are detected by the intrinsic heat (binding enthalpy) change of the reaction. The technique is applicable to native, unmodified proteins in solution. This is important for proteins that lose or change their functional behavior when chemically modified or attached to a surface. ITC is also useful for evaluating qualitative questions such whether a proposed binding interaction occurs at all, or for quantitatively measuring the concentration of functionally active protein. Finally, if executed with proper control experiments, ITC can be a rich source of thermodynamic information about the molecular binding mechanism.
Size‐Exclusion Chromatography with On‐Line Light Scattering
Authors: Arakawa Tsutomu , Wen Jie
Published: June 2004

This unit describes the use of size‐exclusion chromatography with on‐line light scattering, UV absorbance, and refractive index detectors (SEC‐LS/UV/RI) to determine: (a) the molecular weight of simple proteins containing no carbohydrates, (b) the molecular weight of glycoproteins, and (c), most importantly, the molecular weight and stoichiometry of protein‐protein complexes or protein‐carbohydrate complexes. Multiangle light scattering is also discussed.
Serpins (Serine Protease Inhibitors)
Authors: Bauman Susannah J. , Whinna Herbert C. , Church Frank C.
Published: June 2004

Serpins are a class of proteins involved in the regulation of serine and other types of proteases. In humans, the majority of serpins regulate the functions of proteases involved in the body's response to injury. This includes roles in coagulation, fibrinolysis, inflammation, wound healing, and tissue repair. Serpins have been implicated in various animal and human pathologies by the loss of a functional serpin gene through deletion or mutation, which results in a defect in functional protein. Examples of sestorically called antithrombin III) are first described. Then, protocols to determine the second‐order rate constant of AT inhibition of thrombin in the absence and presence of heparin are presented. Also provided is a partial list of other serpins and their purification methods.
Use of GFP as a Reporter for the Analysis of Sequence‐Specific Proteases
Authors: Sarath Gautam , Schwartzbach Steven D.
Published: June 2004

This unit describes a rapid fluorescent assay for sequence‐specific proteases. A recombinant His‐tagged substrate‐GFP fusion protein containing the sequence‐specific protease‐recognition sequence is used as substrate. Batch metal‐chelate chromatography separates uncleaved substrate‐GFP fusion protein from GFP released by proteolysis and proteolytic activity is determined by measuring the fluorescence of GFP remaining in solution.
Overview of Proteome Analysis
Authors: Ali‐Khan Nadeem , Zuo Xun , Speicher David W.
Published: June 2004

This unit reviews the new discipline of proteomics, which includes any large‐scale protein‐based systematic analysis of the proteome or defined sub‐proteome from a cell, tissue, or entire organism. Proteomics originated in the mid‐1990s due to two key enabling advances, availability of complete genome sequences, and mass spectrometry advances that allowed high sensitivity identifications of proteins. Proteome analyses can be broadly categorized into three types of studies: quantitative protein profile comparisons, analysis of protein‐protein interactions, and compositional analysis of simple proteomes or subproteomes such as organelles or large protein complexes. The complexity of different types of proteomes, the merits of targeted versus global proteome studies, and the advantages of alternative separation and analysis technologies are discussed.
Proteomic Analysis Using 2‐D Liquid Separations of Intact Proteins From Whole‐Cell Lysates
Authors: Zhu Kan , Yan Fang , O'Neil Kimberly A. , Hamler Rick , Lubman David M. , Lin Linda , Barder Timothy J.
Published: June 2004

This unit describes procedures for 2‐D liquid separations of proteins from whole‐cell lysates. Protocols for protein isoelectric point (pI) fractionation in the first dimension include the use of liquid isoelectric focusing (IEF) and chromatofocusing. The liquid IEF provides a pI‐based fractionation using a batch‐phase electrophoretic method, while chromatofocusing uses a column‐based chromatographic method to generate the pH gradient. Using either method, a second‐dimension fractionation is provided in the liquid phase using nonporous silica‐based reversed‐phase HPLC (NPS‐RP‐HPLC) to generate a 2‐D liquid map of the protein content of the cell. The eluate of the 2‐D liquid fractionation is directly coupled to a mass spectrometer for on‐line detection of the intact molecular weights of proteins. As a result, a multidimensional map of protein expression is obtained that characterizes cellular proteins by pI, hydrophobicity, and intact molecular weight. Such expression maps are useful for differential proteomic comparison between different cell samples.
Commonly Used Detergents
Authors: Coligan John E.
Published: June 2004

This appendix lists some common detergents along with useful information regarding the type of hydrophilic group (anionic, cationic, amphoteric, or nonionic), the critical micelle concentration (CMC) and the micelle molecular weight.
Protein Precipitation Using Ammonium Sulfate
Authors: Wingfield Paul
Published: June 2004

The basic theory of protein precipitation by addition of ammonium sulfate is presented and the most common applications are listed. Numerous tables are provided for calculating the appropriate amount of ammonium sulfate to add to a particular protein solution.
Agarose Gel Electrophoresis
Authors: Voytas Daniel
Published: June 2004

This appendix presents a protocol for separating and purifying DNA fragments between 0.5 and 25 kb. A describes the use of midigels and minigels.
Digestion of DNA with Restriction Endonucleases
Authors: Bloch Kenneth D.
Published: June 2004

Restriction endonucleases recognize short DNA sequences and cleave double‐stranded DNA at specific sites within or adjacent to the recognition sequences. Restriction endonuclease cleavage of DNA into discrete fragments is one of the most basic procedures in molecular biology. This appendix describes restriction endonucleases and their properties.
Growth and Manipulation of Yeast
Authors: Treco Douglas A. , Reynolds Ann , Lundblad Victoria
Published: June 2004

This unit describes preparation of selected media for growing yeast and also discusses strain storage and revival. Protocols are provided for the assay of β‐galactosidase in liquid culture and for transformation using lithium acetate.
Characterization of Tachykinin Receptors
Authors: Karpitskiy Vladimir V.
Published: June 2004

The mammalian tachykinin peptides are a family of neuropeptides characterized by a common C‐terminal amino acid sequence of the form Phe‐X‐Gly‐Leu‐Met‐NH2, where X is either Phe or Val. Tachykinin peptides have various biological activities, including excitation of both peripheral and central neurons, stimulation of smooth muscle contraction, stimulation of exocrine and endocrine gland secretion, and involvement in immune and inflammatory responses. This unit presents basic methods for the characterization of tachykinin receptors by radioligand binding assay. The protocols detail methods for three major classes of tachykinin receptors and techniques for the generation of high specific activity iodinated radioligands.
Characterization of Benzodiazepine Binding to GABAA Receptors
Authors: Benke Dietmar , Mohler Hanns
Published: June 2004

This unit describes ligand binding assays for the characterization of the benzodiazepine (BZ) site of GABAA receptors in the central nervous system. Compounds, such as diazepam, acting at this site are widely used therapeutically as tranquilizers, hypnotics, anticonvulsants, and muscle relaxants. Originally, this site was termed the BZ receptor. However, since it does not characterize a receptor in its own right, but rather a modulatory site of GABAA receptors, the term BZ site is now frequently used. The basic protocol provides a procedure for analyzing benzodiazepine binding to GABAA receptors from brain. The experimental design of the method is suitable for all commercially available radioligands that interact with the BZ site of central GABAA receptors. By using the appropriate radioligand in combination with particular displacer compounds, individual (or groups of) GABAA receptor subtypes can be identified. Preparation of membrane suspensions for this procedure is described in a support protocol, and five alternate protocols are provided for the identification of diazepam‐sensitive GABAA receptors, the a1‐subtype of diazepam‐sensitive receptors, the a5 subtype of diazepam‐sensitive receptors, both diazepam‐sensitive and diazepam‐insensitive receptors, and diazepam‐insensitive receptors exclusively. Each protocol contains a brief description of the requirements for radioligand binding to these five types of BZ sites.
Characterization of Calcium Channel Binding
Authors: Zheng Wei
Published: June 2004

Voltage‐dependent calcium channels are expressed in a variety of tissues including heart, muscles and brain. Saturation binding of a radioligand to the calcium channel is commonly used to characterize the expression level of the channel protein. Compound competition binding assay is a conventional screening method to determine the affinity of unlabeled compounds for the channel protein. This unit provides detailed experimental methods for two types of radioligand binding assays using [3H]PN200‐100 and [125I](conotoxin MVIIA. Voltage‐dependent calcium channels are expressed in a variety of tissues including heart, muscles and brain
Characterization of Melanocortin Receptors
Authors: Goetz Aaron S. , Ignar Diane M.
Published: June 2004

This unit describes a Scintillation Proximity Assay (SPA) for the measurement of ligand binding to melanocortin receptors (MCRs) using membranes prepared from cell lines stably expressing recombinant MCRs. It provides a facile method for determining the affinity of compounds at MC1R, MC3R, MC4R, or MC5R.
Isolated Cardiac Muscle Assays
Authors: Kenakin Terry
Published: June 2004

Isolated cardiac muscle is one of the major tissues used in pharmacological research. It is compact, easily prepared, and functions as a syncytium yielding rapid responses to a variety of agents. Described in this unit are methods of preparing and handling isolated cardiac muscle, a preparation rich in autonomic receptors.
P1 (Adenosine) Purinoceptor Assays
Authors: Abebe Worku , Mustafa S. Jamal
Published: June 2004

P1 purinoceptors, or adenosine receptors, comprise a group of cell‐surface receptors that mediate the biological effects of adenosine and its analogs. Investigation of the role of purinoceptors may be important in the understanding of certain conditions (e.g., asthma and ischemia) and in the possible development of therapeutic interventions. Four types of P1 purinoceptors—i.e., A1, A2A, A2B, and A3—have been cloned from several species. These receptors are distinguished by pharmacological criteria using selective agonists and antagonists. This unit describes isolated tissue preparations for the pharmacological characterization of purinoceptors based on functional responsiveness. Guinea pig atrium is used as a model to investigate A1 receptors, the porcine coronary artery for A2A receptors, and the guinea pig aorta for A2B receptors. Support protocols describe the procedures used to isolate and prepare the various tissues for response measurements. A summary is provided on how to interpret tissue response to adenosine agonists and antagonists. P1 purinoceptors, or adenosine receptors, comprise a group of cell‐surface receptors that mediate the biological effects of ad
Nicotinic Acetylcholine Receptor Assays
Authors: Canning Brendan J.
Published: June 2004

Nicotinic acetylcholine receptors (nAChRs) in peripheral tissues are localized almost exclusively to autonomic nerves and the motor end plates of striated musculature. Pharmacologic analyses of nicotinic receptor antagonist potencies can be conducted by assessing the ability of these compounds to inhibit responses elicited by preganglionic autonomic nerve stimulation or stimulation of the motor nerves innervating striated muscle in isolated tissue preparations. In addition, in some isolated tissues innervated by autonomic nerves, nicotinic receptor mediated responses can be elicited by exogenously administered agonists, and the effects of antagonists on these responses can be assessed using pharmacologic analyses. This unit describes the guinea pig trachea/esophagus preparation, in which nicotinic receptor pharmacology can be studied at synapses of the parasympathetic and sympathetic nervous system and the striated musculature of the esophagus. In addition, a preparation whereby the nicotinic receptors of the striated musculature of the diaphragm can be studied is described as are techniques for studying exogenous nicotinic agonistmediated effects in two smooth muscle preparations.Nicotinic acetylcholine receptors (nAChRs) in peripheral tissues are localized almost exclusively to autonomic nerves.
Cholecystokinin (CCK) Assays
Authors: Trist David G. , Corsi Mauro
Published: June 2004

Cholecystokinin (CCK) is a peptide that acts as a peripheral hormone and as a central neurotransmitter. To date, two distinct receptors have been identified for CCK using structural and operational criteria; CCK1 and CCK2 (formerly CCKA and CCKB, respectively). In addition, there is the gastrin receptor found in the stomach which has a high structural similarity to the CCK2 receptor, but which displays a different pharmacology. This unit presents two methods for the quantification of selective agonists and antagonists at CCK1 receptors and an assay for CCK2 receptors. In the first tissue, the guinea‐pig ileum longitudinal muscle with myenteric plexus, both CCK1 and CCK2 receptors are present in the same preparation. Each receptor is distinguished in this assay using selective agonists and antagonists against the unwanted receptor subtype. The second preparation, the guinea‐pig gallbladder, is a classical preparation for studying CCK1 receptor‐active compounds in the absence of CCK2 sites
Muscarinic Receptor Assays
Authors: Eglen Richard M. , Choppin Agnes , Watson Nikki , Hegde Sharath S.
Published: June 2004

In the central and peripheral nervous systems, the actions of acetylcholine are mediated by families of ligand gated ion channels and metabotropic receptors. The latter family is composed of muscarinic receptors, since they are selectively activated by muscarine and antagonized by atropine. Five muscarinic receptor subtypes have been identified by molecular biological and cloning studies (M1‐M5), however, currently there are only functional preparations for the M1 through M4 subtypes. This unit describes robust preparations for assessing M1, M2 and M3 receptors in isolated tissues. An assay for the M4 is also described although this has not been evaluated extensively. An endogenous assay has not been identified for the M5 receptor and its physiological role is not well understood. In the central and peripheral nervous systems, the actions of acetylcholine are mediated by families of ligand gated ion chann.
Prostanoid Receptor Assays
Authors: Chen June , Woodward David F. , Coleman Robert A. , Jones Robert L. , Lydford Simon J.
Published: June 2004

Prostanoids, which include the prostaglandins (PGs) and thromboxanes (TXs), interact with a specific family of G‐protein coupled receptors, of which there are known to be five distinct types, DP, EP, FP, IP and TP, each particularly sensitive to one of the five natural prostanoids, PGD2, PGE2, PGF2(, PGI2 and TXA2, respectively. Of these, it is known that the EP receptor comprises four well‐characterized subtypes: EP1, EP2, EP3 and EP4. These receptor subtypes are widely distributed throughout mammals and other species, and show particularly high levels of expression in smooth muscle and blood platelets. Despite the fact that few of these preparations express a single receptor type/subtype in isolation, a range of useful smooth muscle and platelet assays for the various prostanoid receptors are available and are presented in this unit.
Models of Urogenital Dysfunction: Benign Prostatic Hyperplasia (BPH)
Authors: Kenny Barry , Miller Alison , Brunton Neil , Newgreen Donald
Published: June 2004

Bladder outlet obstruction can be associated with benign prostatic hyperplasia (BPH). A reduction in prostatic smooth muscle tone provides a means of improving the obstructive symptoms associated with BPH. This unit presents methods for studying prostatic pressure in both the anesthetized and conscious dog. In both models, the primary determinant is the measurement of experimentally induced changes in intra‐urethral prostatic pressure. The protocols allow measurement of the effects of compounds on agonist‐ or nerve‐stimulated prostatic contraction in vivo. Th conscious dog model has two potential advantages over the anesthetized preparation: it can be used for oral dosing of test compounds and it allows measurement of the effects of test compounds on postural hemodynamics under conditions where reflexes are not compromised by anesthesia. In all the protocols in this unit, measurement of Bladder outlet obstruction can be associated with benign prostatic hyperplasia (BPH)
Intraperitoneal and Subcutaneous Tumor Models for Assessing Anti‐Neoplastic Agents in Rodents
Authors: Hollingshead Melinda
Published: June 2004

Evaluation of compounds for in vivo antineoplastic activity can be achieved in a variety of animal models. Two models commonly employed for these studies are the intraperitoneal challenge survival model and the subcutaneous tumor implant model. The challenge survival model involves intraperitoneal tumor inoculation, test compound treatment and monitoring for survival. This assay can be modulated for stringency by altering the treatment dose, route and schedule. The second model, subcutaneous tumor implants, is more strenuous as the test compound must cross physiologic barriers to reach the target tumor. Additionally, the subcutaneous model is more labor intensive to setup, monitor and evaluate.
Electrocardiographic Toxicity in the Guinea Pig
Authors: Lacroix Pierre
Published: June 2004

Abnormalities of cardiac rhythm are one of the most common clinical problems in cardiology and arise as the result of either disorders of cardiac impulse formation or conduction, or a combination of both. It has been established that some classes of drugs, such as tricyclic antidepressants (e.g., imipramine), cardiac glycosides (e.g., digoxin), and Class I or Class III antiarrhythmic drugs (e.g., quinidine or amiodarone) can produce electrocardiographic toxicity in humans. It is therefore highly advisable to assess the effect of any new compound in this respect, during the early phases of drug development. This unit presents a protocol to detect the electrocardiographic toxicity of compounds in the anesthetized guinea pig.
Nonisotopic Assays for Reporter Gene Activity
Authors: Brasier Allan R. , Fortin John J.
Published: June 2004

This unit describes two nonisotopic systems for reporter gene activity in cells transfected with the firefly luciferase
Overview of Chemical Diversity
Authors: Triggle David J.
Published: June 2004

In the search for new compounds and the identification of compound sources by the pharmaceutical industry, natural products continue to play an extremely important role, principally because they represent a source, real and potential, of remarkable molecular diversity. Although advances in drug discovery have been significantly fueled and paralleled by advances in synthetic organic chemistry, structural biology, and computational chemistry, they continue to be linked to both natural product and biological chemistry. The dramatic progress in synthetic molecular diversity achieved through combinatorial chemistry is heavily influenced by our understanding of biologically driven molecular diversity. Additionally, through advances in genomics, synthetic chemistry is being linked to biochemical systems to create hybrid “natural‐synthetic“ products, “unnatural“ peptides and “self‐evolving“ molecules. This unit discusses specific instances where diversity is generated by exploitation of biological pathways. These strategies are intimately linked to processes of biological screening, and in particular to high‐throughput screening.
Overview on High‐Throughput Screening
Authors: Entzeroth Michael
Published: June 2004

The known effects of drugs from a variety of pharmacological/therapeutic classes on the respiratory system, the life‐threatening consequences of respiratory dysfunction, and world‐wide regulatory safety guidelines all support the need for conducting respiratory evaluations in safety pharmacology. This unit provides an overview of the functional disorders of the respiratory system and presents the techniques and strategies considered to be most appropriate for detecting and characterizing drug‐induced respiratory disorders in safety pharmacology studies.
Use of Electrophysiological Methods in the Study of Recombinant and Native Neuronal Ligand‐Gated Ion Channels
Authors: Thomas Philip , Smart Trevor G.
Published: June 2004

This unit is geared towards investigators wishing to study the effects of externally and internally applied agents on the behavior of ligand‐gated ion channels (LGICs, specifically the GABAA receptor). The reader is taken through a number of electrophysiological techniques applied to whole cell and excised patch recordings of recombinant and native GABAA receptor subtypes used in the generation and analysis of a variety of pharmacological parameters. These data interpretations form the basis for the analysis of potentially novel pharmacological agents active at the GABAA receptor target, but could equally be applied to other LGICs. The analysis is extended to incorporate post‐synaptic inhibitory events in hippocampal neurons. Complementary descriptions of how tissues for such studies are prepared from recombinant and native receptor preparations are included. Attention is given to the physiological phenomena most relevant to everyday scientific literature.
Overview of Cell and Tissue Culture Techniques
Authors: Honegger Paul
Published: June 2004

Culture preparations vary greatly in complexity, ranging from single isolated cells to three‐dimensional histotypic cell structures. Besides cultures obtained directly from animal tissues (primary cultures), permanent cultures of continuously dividing (immortalized) cells have been established. This unit highlights both the advantages and disadvantages of a number in vitro approaches including primary cultures, continuous (permanent or immortal) cell lines, and contrasts culture techniques including suspension, attached (monolayer), and three‐dimensional (aggregate) cultures in addition to explants. The basics of culture maintenance and propagation are covered (i.e., tissue dispersion, cell separation and purification, and passaging), and information is provided on the critical aspects of culturing cells, such as pH of the media, osmolality, humidity, and cell density. Also included is a troubleshooting section on how to cope with problems of contamination by bacteria, mycoplasma, toxic chemicals or foreign cell types.Culture preparations vary greatly in complexity, ranging from single isolated cells to three‐dimensional histotypic cell structure.
Cellular Assays of Chemokine Receptor Activation
Authors: Proudfoot Amanda E.I. , Power Christine A. , Church Dennis J. , Soler Dulce , Mack Matthias
Published: June 2004

This unit describes procedures for measuring the activation of chemokine receptors by their ligands. Chemokines are chemoattractant proteins and two assays are described for analyzing the chemoattractant properties (chemotaxis) of these proteins. Chemokine receptors are coupled to G proteins, and activation of the receptors results in the mobilization of intracellular Ca2+ stores. Two assays for the measurement of this activity are described which employ calcium‐sensitive dyes, one for adherent and one for non‐adherent cells. In addition, an assay for quantitating chemokine receptor downmodulation and subsequent recycling events is described. The assays are applicable to both purified leukocytes and stably expressed recombinant chemokine receptors.This unit describes procedures for measuring the activation of chemokine receptors by their ligands.
Production and Use of HIV‐1 Luciferase Reporter Viruses
Authors: Münk Carsten , Landau Nathaniel R.
Published: June 2004

This unit describes the production and use of HIV‐1 luciferase reporter viruses. These viruses are used to rapidly and accurately quantify HIV‐1 in cell culture. Protocols are also presented for screening and characterization of small molecule inhibitors of HIV‐1 entry into cells.
Receptor Binding Techniques
Authors: Frey Kirk A. , Albin Roger L.
Published: June 2004

This overview first discusses issues relating to the selection of radioligand for receptor binding assays, including the isotopic label and considerations pertaining to the pharmacological and chemical profile of the ligand. This is followed by a section on characterization of ligand‐binding assays, starting with tissue preparation methods, followed by detection of specific binding, determination of incubation and washing conditions and a discussion of saturation and competition assay formats. Quantification of the assay results can be accomplished by autoradiography or film densitometry. Finally, methods and considerations for analysis of the resulting data are presented.
Isolation and Purification of Primary Rodent Astrocytes
Authors: Weinstein David E.
Published: June 2004

Astrocytes are a major cell type in the mammalian central nervous system (CNS). The ability to obtain virtually pure populations of these cells makes it possible to study their function as isolated cells or in mixed populations where they support the growth and survival of surrounding neurons. Unlike other mature CNS cells, mature astrocytes maintain the lifelong ability to reenter the cell cycle. The first isolation procedure described in this unit takes advantage of the proliferative ability of these cells, as does the second, except that no antibody or complement treatment is required. A procedure for detecting glial fibrillary acidic protein (GFAP), which is present in most astrocytes in vivo and virtually all astrocytes in vitro and is a useful marker for assessing the purity of cultures, is also presented.
Differentiation of Embryonic Stem Cells
Authors: Okabe Shigeo
Published: June 2004

Embryonic stem (ES) cells are pluripotent cells whose developmental state is equivalent to cells of the inner cell mass in the blastocyst‐stage embryo. This unit presents a method of producing differentiated ES cells in which the cells are first aggregated on a less adhesive surface to form embryoid bodies (EBs). EBs are allowed to attach to a permissive substrate and then differentiate into neural cells in a serum‐free medium. The resulting cell population can be maintained for several passages in serum‐free medium in the presence of basic fibroblast growth factor (bFGF) or allowed to differentiate into postmitotic neurons by plating onto a glial monolayer followed by culture with serum. Also included in the unit are procedures for preparing feeder cells from embryonic fibroblasts and for preparing glial monolayer cultures, both for use in the main ES production protocol.
Immortalizing Central Nervous System Cells
Authors: Whittemore Scott R.
Published: June 2004

This unit presents methods for isolating clonal, neural‐derived cell lines. One approach for isolating such neural cell lines involves a replication‐deficient retrovirus encoding a specific oncogene and a selectable marker which are used to infect dissociated CNS cells dissected at a developmental stage at which the cell population of interest has not undergone its terminal mitotic division. Also presented is a method for cloning by limiting dilution, which may be necessary to obtain a pure population of cells. Following growth under appropriate selection conditions, clones are isolated and tested for their ability to differentiate with the desired phenotypic properties. A method is also provided for coating tissue culture dishes, which is necessary for successful culture of CNS neurons.
Isolation and Purification of Primary Schwann Cells
Authors: Weinstein David E. , Wu Rina
Published: June 2004

Schwann cells are the glial cells of the peripheral nervous system (PNS) which both myelinate and provide trophic support for their associated axons. Their functions are critical for proper development, homeostasis, and regeneration of the PNS. Schwann cells can be isolated and expanded in culture. Such a culture can be used as a source of myelinating glia in culture or as a source of Schwann cells for various cell and molecular biological experiments. The proliferative ability of these cells is exploited in the isolation procedures presented here.
Interaction Trap/Two‐Hybrid System to Identify Interacting Proteins
Authors: Golemis Erica A. , Serebriiskii Ilya , Gyuris Jeno , Brent Roger
Published: June 2004

The parameters and experimental conditions for this important system are constantly undergoing improvement. This newest version includes expanded tables describing interaction trap components and additional libraries compatible with the interaction trap system. It also features a new protocol on performing a hunt by interaction mating. Some of the commercial vendors selling yeast two‐hybrid reagents recommend using interaction mating to perform a hunt, so this procedure should be of great interest.
Selection of Transfected Mammalian Cells
Authors: Mortensen Richard , Chestnut Jonathan D. , Hoeffler James P. , Kingston Robert E.
Published: June 2004

Analysis of gene function frequently requires the formation of mammalian cell lines that contain the studied gene in a stably integrated form. Approximately one in 104 cells in a transfection will stably integrate DNA (the efficiency can vary depending on the cell type). Therefore, a dominant, selectable marker is used to permit isolation of stable transfectants. In the first part of this unit, the procedure for determining selection conditions and the resulting stable transfection is presented and the most commonly used selectable markers are discussed. The second protocol includes conditions for thirteen markers commonly used for selection of mammalian cells. A third protocols describes selection of transfected cells from the total population soon after transfection with plasmids that express both the gene of interest and a selection tag. Optimization of transfection conditions can be facilitated by a simple staining assay detailed in a support protocol.
Production of a Heterozygous Mutant Cell Line by Homologous Recombination (Single Knockout)
Authors: Mortensen Richard
Published: June 2004

Gene targeting by homologous recombination allows the introduction of specific mutations into any cloned gene. This unit provides a protocol in which the gene of interest is inactivated by interrupting its coding sequence with a positive selectable marker. A negative selectable marker is included in the construct outside the region of target gene homology in order to enrich for clones in which the target gene has undergone homologous recombination. The altered target gene is then expressed in embryonic stem cells. A support protocol describes a method for transient expression of Cre recombinase to remove sequences between lox sites, which can also be used as a selection method.
Differential Display of mRNA by PCR
Authors: Livesey F.J. , Hunt C.P.
Published: June 2004

This unit outlines the polymerase chain reaction (PCR)‐based technique of mRNA differential display, which identifies genes that are differentially expressed between cells or tissues. The approach described here is a modification of the original method, referred to as single base‐anchored differential display. The basic protocol describes the actual differential display PCR reaction along with details of the identification, reamplification, and cloning of candidate differentially expressed genes. The support protocol provides instructions on removing contaminating genomic DNA from the RNA samples and reverse transcribing the purified RNA to produce the cDNA used in the subsequent PCR reactions.
Production of Antipeptide Antisera
Authors: Coligan John E. , Tam James P. , Shao Jun
Published: June 2004

This unit describes methods used for the chemical coupling of synthetic peptides to carrier proteins, required for the preparation of peptide immunogens. The carrier protein described here is keyhole limpet hemocyanin (KLH) because it is the one most commonly used. However, other proteins may be used in place of KLH, including bovine serum albumin (BSA) and ovalbumin. Coupling may be accomplished as described with MBS, which requires a Cys residue in the peptide, or with glutaraldehyde, EDCI, or BDB. Also included are an assay for detecting free sulfhydryl (SH) groups, a means of calculating coupling efficiency, an immunization schedule, an indirect ELISA, and a method for preparing a peptide affinity column. The final methodology described is the multiple antigen (MAP) system.
Using Phage Display in Neurobiology
Authors: Bradbury Andrew , Sblaterro Daniele , Marzari Roberto , Rem Louise , Hoogenboom Hennie
Published: June 2004

In this unit some of the basic protocols involved in the manipulation of phage display libraries are described, including the rescue and amplification of such libraries, selection and screening from them and testing of derived clones.
Preparation and Maintenance of Organotypic Cultures for Multi‐Electrode Array Recordings
Authors: Karpiak Veronica C. , Plenz Dietmar
Published: June 2004

Preparation and Maintenance of Organotypic Cultures for Multi‐Electrode Array Recordings (Veronica C. Karpiak and Dietmar Plenz, National Institutes of Mental Health, NIH, Bethesda, Maryland). Recording from neuronal cultures with multi‐electrode arrays (MEAs) provides a powerful tool for studying neuronal activity with many neurons simultaneously in vitro. This unit describes the detailed steps necessary for growing organotypic cultures on MEAs and the typical neuronal activity that is obtained with this methodology.
Imaging Nervous System Activity with Voltage‐Sensitive Dyes
Authors: Zecevic Dejan , Djurisic Maja , Cohen Lawrence B. , Antic Srdjan , Wachowiak Matt , Falk Chun X. , Zochowski Michal R.
Published: June 2004

Optical recording with a voltage‐sensitive dye is advantageous where membrane potential must be recorded in many sites at once. This unit describes methods for making voltage‐sensitive dye measurements on different preparations to study (1) how a neuron integrates its synaptic input into its action potential output by measuring membrane potential everywhere synaptic input occurs and where spikes are initiated; (2) how a nervous system generates a behavior in Aplysia abdominal ganglion; and (3) responses to sensory stimuli and generation of motor output in the vertebrate brain by simultaneous measurement of population signals from many areas. The approach is three‐pronged: (1) find the dye with the largest signal‐to‐noise ratio; (2) reduce extraneous sources of noise; and (3) maximize the number of photons measured to reduce the relative shot noise. A discussion of optical recording methods including the choice of dyes, light sources, optics, cameras, and minimizing noise is also provided.
Measurement of NO and NO Synthase
Authors: Weissman Ben Avi , Gross Steven S.
Published: June 2004

Nitric oxide (NO) is a key biosignaling molecule produced in both peripheral tissues and the central nervous system by a family of enzymes known as nitric oxide synthases (NOSs). NOSs convert L‐arginine to stoichiometric quantities of NO and L‐citrulline using molecular oxygen and NADPH as cofactors. Techniques for measurement of NO and NOS activity are essential to demonstrate the role of NO and NO‐derived species in biological systems. This unit describes two methods for detection of NO: a direct method employing chemiluminescent detection and one based on quantification of the stable oxidation products with detection using the Greiss reagent. Additionally, NOS activity can be quantified by measuring the conversion of radiolabeled L‐arginine to radiolabeled L‐citrulline.
Measurement of Oxygen Radicals and Lipid Peroxidation in Neural Tissues
Authors: Hall Edward D. , Andrus Paula K.
Published: June 2004

An important role for oxygen radical‐mediated neuronal damage has been implicated in a number of acute and chronic neurodegenerative disorders. Particular interest has centered upon oxygen radical‐induced, iron‐catalyzed lipid peroxidation (LP) as the principal mechanism of the neuronal injury associated with oxygen radicals. Thus, there has been a growing interest in methods for monitoring increased oxygen radical levels as an index of oxidative stress as well as markers of LP‐associated oxidative injury in a number of in vitro and in vivo model systems. This unit provides a detailed description of the salicylate trapping method for the measurement of the most highly reactive oxygen radical, the hydroxyl radical, as well as several direct or indirect methods for assessment of cellular LP in either cell cultures or in in vivo models.
Animal Tests of Anxiety
Authors: File Sandra E. , Lippa Arnold S. , Beer Bernard , Lippa Morgen T.
Published: June 2004

Animal tests of anxiety are used to screen novel compounds for anxiolytic or anxiogenic activity, to investigate the neurobiology of anxiety, and to assess the impact of other occurrences such as exposure to predator odors or early rearing experiences. This unit presents protocols for the most commonly used animal tests of anxiety. The Geller‐Seifter conflict test, the social interaction test, light/dark exploration, the elevated plus‐maze, defensive burying, and the thirsty rat conflict. The protocols are described in terms of drug screening tests, but can be modified easily for other purposes.
Assessment of Spatial Memory Using the T Maze
Authors: Wenk Gary L.
Published: June 2004

This unit describes the use of the T maze to assess spatial memory, and takes into account the alternating behavior of rats in searching for food. The task is based on the premise that animals have evolved an optimal strategy to explore their environment and obtain food with a minimum amount of effort. The T maze has been most extensively used to investigate specific aspects of spatial working memory, which is operationally defined as information that is only useful to a rat during the current experience with the task. A modification of the maze allows for the assessment of reference memory, defined as information that is useful across all exposures to the task (i.e., on any day of testing). Finally, in the absence of food‐deprivation, a simple T maze can be used as described to assess spontaneous alternation.
Macronutrient Selection in Experimental Animals
Authors: Kanarek Robin B.
Published: June 2004

To address the question of how animals choose diets from an array of nutritionally different foods, researchers have designed experiments in which animals are provided with separate sources of the three macronutrients, protein, fat and carbohydrate. These experiments are useful for investigating the neuroanatomical and neurochemical mechanisms involved in food choice, as well as for assessing the effects of changes in physiological status (e.g., pregnancy; lactation and increased energy expenditure) or disease states (e.g., diabetes; and obesity) on nutrient intakes.