bluuebunny/crossref_metadata_2025_split huggingface dataset:
Use of GFP as a Reporter for the Analysis of Sequence‐Specific Proteases
Authors: Sarath Gautam , Schwartzbach Steven D. 
Published: June 2004

AbstractThis 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

AbstractThis 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

AbstractThis 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.
Digestion of DNA with Restriction Endonucleases
Authors: Bloch Kenneth D. 
Published: June 2004

AbstractRestriction 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.
Characterization of Tachykinin Receptors
Authors: Karpitskiy Vladimir V. 
Published: June 2004

AbstractThe 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

AbstractThis 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

AbstractVoltage‐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

AbstractThis 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.
P1 (Adenosine) Purinoceptor Assays
Authors: Abebe Worku , Mustafa S. Jamal 
Published: June 2004

AbstractP1 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

AbstractNicotinic 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

AbstractCholecystokinin (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

AbstractIn 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

AbstractProstanoids, 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

AbstractBladder 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

AbstractEvaluation 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

AbstractAbnormalities 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.
Overview of Chemical Diversity
Authors: Triggle David J. 
Published: June 2004

AbstractIn 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

AbstractThe 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

AbstractThis 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

AbstractCulture 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

AbstractThis 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

AbstractThis 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

AbstractThis 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

AbstractAstrocytes 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

AbstractEmbryonic 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.