bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of P1 (Adenosine) Purinoceptors
Authors: Jarvis Michael F. 
Published: June 2004

AbstractThe purine nucleoside adenosine (ADO) is an important modulator of cellular function in mammalian tissues. ADO modulates neuronal excitability through an interaction with different cell surface receptor subtypes that are heterogeneously distributed in the mammalian central nervous system, as well as in many peripheral tissues. Four different ADO receptor subtypes have been cloned and characterized. Described in this unit are three radioligand binding assays for pharmacological characterization of the high‐affinity ADO receptor subtypes A1, A2A, and A3 receptors. Pharmacological characterization of the low‐affinity A2B receptor using radioligand binding has been limited by the paucity of potent and selective ligands for this subtype. Since receptor localization is an important criterion for differentiation of receptor subtypes, a support protocol that describes the methodology for the localization of ADO receptors in rat brain tissue using autoradiography is also included.
Characterization of Angiotensin II Receptors
Authors: Smith Ronald D. , McCall Dale E. , Timmermans Pieter B.M.W.M. 
Published: June 2004

AbstractAngiotensin II (Ang II) is the primary mediator of the renin angiotensin system and acts primarily as a circulating hormone, but may also act as a paracrine or autocrine factor. Ang II binds specifically to G protein‐coupled receptors and triggers a number of characteristic cellular responses, including vascular smooth muscle contraction and adrenal cellular aldosterone secretion. There are multiple receptor subtypes for Ang II; however, the distinction between binding sites versus receptors with their associated functional manifestations has been controversial. There are two principal human receptor subtypes, designated AT1 and AT2, that are distinguished by the selective affinity of the nonpeptide antagonists losartan and PD123177. In rodents, additional isoforms of the Ang II receptor have been cloned (e.g., AT1A and AT1B), although their functional distinctions are undetermined. The focus of this unit is the identification of Ang II binding sites and receptor subtypes. Radioligand competition binding assays are provided for determining the affinity of Ang II for receptors on adrenal cortical microsomes, whole adrenal membranes, or CHO cells transfected with the AT1A receptor.
Characterization of Cholecystokinin (CCK) Receptors
Authors: Miller Laurence J. 
Published: June 2004

AbstractCholecystokinin (CCK) and gastrin are structurally related peptide hormones synthesized and secreted by endocrine cells in the gastric antrum, the proximal small intestine, and the central and enteric nervous systems. A broad spectrum of cells express receptors and represent physiologic targets for these peptides. The major physiologic targets for gastrin in the oxyntic gastric mucosa, including the parietal cells and select enterochromaffin cells, while CCK has a broader range of targets, with most involved in nutrient assimilation and appetite control, and the regulation of anxiety. This unit describes binding assays for the determination of receptor number, ligand affinity, and inhibition constants for both CCKA and CCKB receptors, in addition to procedures detailing the preparation of tissues from various sources.
Characterization of Corticotropin‐Releasing Factor (CRF) Receptors
Authors: Grigoriadis Dimitri E. , Lorang Marge T. , Duggan Nicola , De Souza Errol B. 
Published: June 2004

AbstractCorticotropin‐releasing factor (CRF) and its receptors play a major role in regulating an organism's response to physical, emotional and environmental stress. While the primary role of CRF is the regulation of adrenocorticotropin hormone (ACTH) secretion from the pituitary and modulation of the hypothalamic‐pituitary adrenal axis, CRF is also widely distributed in the central nervous system where it produces a broad spectrum of autonomic, electrophysiological and behavioral effects consistent with a neurotransmitter or neuromodulator role in the brain. Methods are provided for characterizing the receptor proteins through which CRF exerts its function. A well‐characterized radioligand receptor binding assay is provided that yields quantitative information about the affinity and density of receptors in a variety of tissues, while another procedure utilizes similar kinetic theories but, in contrast to the homogenized or whole cell suspension approach, makes use of slide‐mounted tissue sections.
Characterization of Wild‐Type Excitatory Amino Acid Ion Channel Receptors
Authors: Ferkany John W. 
Published: June 2004

AbstractThe ubiquitous distribution of EAA receptors results from the fact that EAA‐mediated neurotransmission is the most prevalent type of fast, chemically‐mediated communication in the brain and spinal cord. Indeed, EAA‐mediated neurotransmission is thought to participate in a host of physiological and pathological events including learning and memory, epilepsy, neurodegenerative conditions such as Alzhiemer's, Parkinson's and Huntington's Diseases, and the neurodegeneration associated with stroke and head trauma and neuropathic pain. Described in this unit are receptor binding assays for studying ion‐channel forming excitatory amino acid (EAA) receptors in the mammalian central nervous system. One of the three major types of ionotropic (channel‐forming) EAA receptors are the AMPA receptors, and the other two types are classified on the basis of the original agonists identified for these sites: kainic acid (kainate; KA) and N‐methyl‐D‐aspartic acid (NMDA). Thus, the three recognized ionotropic receptors are the AMPA, KA and NMDA recognition sites. Like GABA receptors, the AMPA and NMDA receptors have multiple regulatory sites.
Characterization of the Picrotoxin Site of GABAA Receptors
Authors: Mehta Ashok K. , Ticku Maharaj K. 
Published: June 2004

AbstractThis protocol describes an in vitro assay for characterization of the picrotoxin site of GABAA receptors in rat brain membranes using various radioligands. Methods and representative data for Scatchard analysis (Kd, Bmax determination), association kinetics, dissociation kinetics and competition assays (IC50, Ki determination) are included in the protocol.
[3H](+)MK801 Radioligand Binding Assay at the N‐Methyl‐D‐Aspartate Receptor
Authors: Reynolds Ian J. 
Published: June 2004

AbstractThe N‐methyl‐D‐aspartate subtype of glutamate receptor is unusual in that it requires two endogenous agonists for activation. Thus, in addition to glutamate, the amino acid glycine (or possibly D‐serine) is an essential co‐agonist. This unit presents a radioligand binding protocol that detects ligand activity at the NMDA receptor‐associated glycine site. This is a convenient approach that exploits the ability of NMDA receptor modulators to alter the kinetics of ligands that bind to the channel‐blocking site of the NMDA receptor. This protocol takes advantage of one of the most potent and specific ligands that bind to this receptor, in this case [3H](+)MK801. Importantly, this assay can detect and differentiate agonists and antagonists that bind to the glycine site. A protocol for the measurement of glycine site activity with [3H](+)MK801 binding is provided, along with support protocols that provide information to aid in the design of assays of agonists and antagonists of the glycine site, and data analysis.
Overview of Receptor Allosterism
Authors: Christopoulos Arthur 
Published: June 2004

AbstractStudies of the behavior of many ligand‐gated ion channels have long provided evidence that more than one molecule of ligand was able to bind to each receptor complex, a phenomenon termed cooperativity. The conclusion that some receptors possessed more than one binding site for ligands thus invoked another phenomenon that was originally described in the field of enzymology, that is, the concept of allosteric binding sites. The biological activity of enzymes can be modified, either in a positive or negative fashion, by the binding of ligands to sites that are topographically distinct from the substrate‐binding site. These accessory binding sites are termed allosteric sites, in contrast to the substrate‐binding (active) site (the isosteric site). Thus, allosteric interactions arise because the binding of a ligand to the allosteric site induces a conformational change in the protein that modulates the binding of the substrate to the isosteric site, and vice versa. The biological activity of the enzyme arises from the subsequent (modified) properties of the substrate‐binding site, and not through a direct effect of the allosteric modulator itself. This overview unit discusses definitions of various terms used in the field of receptor allosterism, and explains in clear terms how allosteric transitions and interactions can affect receptor activation. In addition, the functions of allosteric modulators are discussed along with explanations of how these modulators can be attractive tools for drug development.
Characterization of 5‐HT1A,B and 5‐HT2A,C Serotonin Receptor Binding
Authors: Clarke William P. , Berg Kelly A. , Gould Georgianna , Frazer Alan 
Published: June 2004

AbstractThis unit describes assays for measuring the binding of radioligands to two major types of receptors for 5‐hydroxytryptamine (5‐HT or serotonin), 5‐HT1 and 5‐HT2 receptors, in homogenates of brain tissue or cloned into cells in culture. The specific receptor subtypes covered are 5‐HT1A, 5‐HT1B, 5‐HT2A, and 5‐HT2C. In addition, methodology for using quantitative autoradiography to measure radioligand binding to serotonin receptors in brain slices is described. Protocols are provided for characterization of both saturation and competition binding assays, and instructions for data analysis of these assays is also described. In addition, methodology is provided for the quantification (image analysis) of radioligand binding in brain tissue sections to determine receptor density, preparation of rat brain sections for quantitative autoradiography, and thionin staining of thaw‐mounted tissue sections to define certain brain regions.
Characterization of Chemokine Receptors
Authors: Scheer Alexander , Proudfoot Amanda E.I. 
Published: June 2004

AbstractThis unit describes the procedures for measuring binding of a radiolabeled chemokine to chemokine receptors in cells or cell membranes. The whole‐cell binding assay can be used for both purified leukocytes as well as transfected cell lines expressing chemokine receptors. Two basic protocols are described. The first is applicable to all cells expressing chemokine receptors, both primary cultures as well as recombinantly transfected cell lines expressing a single chemokine receptor. The second is used principally for transfected cell lines and measures chemokine binding to cell membranes using scintillation proximity assay (SPA) methodology, but does not require washing steps and thus is applicable to automated high‐throughput screening strategies. An alternative procedure is also described which also uses SPA methodology to measure GTP‐gamma‐S binding to chemokine receptors in cell membranes. The two basic protocols can be used to determine the binding of compounds to chemokine receptors, while the alternate protocol determines the effects of compounds on the chemokine‐stimulated activation of the receptor.
Overview of Signal Transduction
Authors: Esbenshade Timothy A. , van Biesen Tim 
Published: June 2004

AbstractSignal transduction involves translating extracellular stimuli into appropriate intracellular events that mediate the intended physiological response. The aim of this overview is to present some common features of cellular signaling pathways and to discuss how they interact to mediate responses to environmental stimuli. In particular, it focuses on several common aspects of signal transduction, including the regulation of signaling pathways by protein functional domain interactions as well as the intracellular proteins that mediate signal transduction. Among the proteins covered are those associated with G protein receptor signaling (G proteins, phospholipases, nucleotide cyclases, and phosphatases); protein kinases (tyrosine kinases, serine/threonine kinases) and their respective phosphatases; and, very briefly, several common ion channels (calcium and potassium).
Adenylate and Guanylate Cyclase Assays
Authors: Kelly John J. , Stevens Troy , Thompson W. Joseph 
Published: June 2004

AbstractThis unit presents two basic protocols to determine adenylate cyclase and guanylate cyclase activity in tissue and cell homogenates, permeabilized cells, or subcellular fractions. Two parts are presented for each method. First, the enzyme reaction that causes the formation of the labeled cyclic nucleotide is performed. Second, the separation of cyclic nucleotide products from unreacted nucleotide triphosphates and metabolites is performed using Dowex 50 resin and aluminum oxide chromatographies. In the case of guanylate cyclase, alternative separation protocols are also provided. Additionally, support protocols are provided that describe both the preparation of columns used in the assays and the preparation of tissue or cells to be assayed.
Overview of Phosphoinositide Hydrolysis
Authors: Kendall David 
Published: June 2004

AbstractPhosphoinositide hydrolysis is a ubiquitous, multifunctional, intracellular signaling mechanism induced (via receptor activation) by a wide variety of signaling molecules such as hormones and neurotransmitters. Phosphoinositide hydrolysis generates intracellular second messengers that regulate cell function. This overview describes the major molecular players involved in phosphoinositide signaling along with experimental strategies for measuring phosphoinositide hydrolysis, including inositol radiolabeling methodologies, membrane phospholipase C assays, cytidine prelabeling, and mass measurements of phosphoinositide cycle products.
Nitric Oxide Synthase Assays
Authors: Zhang Jie 
Published: June 2004

AbstractNitric oxide (NO) is a widespread messenger molecule that regulates cardiovascular tone, immune responses, and neuronal function. This unit provides a simple protocol for measuring NO production based on the stoichiometric formation of the byproduct L‐citrulline by nitric oxide synthase (NOS) when NO is produced. Support protocols describe the preparation and purification of NOS from tissues or cultured cells, and for the purification of the radiolabeled L‐arginine substrate by thin‐layer chromatography (TLC).
Analysis of Early Gene Responses
Authors: Duman Ronald S. 
Published: June 2004

AbstractEarly response genes, also referred to as immediate early genes, are rapidly induced in response to a variety of extracellular stimuli, and have been used as markers of cellular activation. Expression of immediate early genes, such as c‐fos, can be used to determine the influence of specific drugs on receptor regulation of intracellular signaling pathways. This unit describes two techniques for the analysis of mRNA from immediate early genes. Northern blot analysis provides a method for size‐fractionation of native mRNA by gel electrophoresis and visualization of a specific mRNA in a pool of total RNA isolated from a tissue of interest. Thus, this analysis provides important information on the size and abundance of the mRNA of interest and on the specificity of the hybridization probe. However, this approach does not provide information on the cellular localization of the mRNA. Such data may be obtained by in situ hybridization of mRNA in tissue sections. In addition to the northern and in situ hybridization techniques, this unit provides two support protocols for radiolabeling either RNA or DNA probes along with information on the selection of an appropriate probe and hybridization conditions.
Assay of Receptor‐Stimulated Phosphoinositide Turnover
Authors: Kendall David A. , Alexander Stephen P.H. 
Published: June 2004

AbstractThe stimulation of phosphoinositide turnover is one of the key means by which receptors evoke responses in target cells and tissues. This is true for both G protein‐coupled receptors and receptors that couple via tyrosine kinase activity. The protocols in this unit allow for pharmacological analysis of receptors coupled to phosphoinositide turnover. In general, the [3H]myo‐inositol prelabeling methodology (described for both tissue slices and cultured cells) is the more widely applicable, since it requires fewer experimental steps and typically gives rise to a better signal‐to‐noise ratio. Individual inositol phosphates can also be determined as described by chromatographic separation on ion‐exchange columns. In some circumstances (for example, when rapid responses to receptor stimulation are to be investigated or when the absolute levels of the active inositol phosphate are to be examined), it is preferable to use the mass assay described here for inositol (1,4,5)‐trisphosphate from either tissue slices and cultured cells. This unit also provides support protocols for the preparation of [3H]myo‐inositol, chromatography columns, tissue slices, and the IP3‐binding protein.
HIV Protease Assays
Authors: Furfine Eric S. 
Published: June 2004

AbstractThe fluorescence‐based assay described in this unit is used not only to screen large numbers of compounds in a 96‐well format for the ability to inhibit human immunodeficiency virus‐1 (HIV) protease, but also to determine accurately the affinity of inhibitors for the enzyme. A method for performing an active site titration, using tight‐binding inhibitors, and determining the Km of a substrate is also provided. Because numerous potent inhibitors of HIV protease have been identified, information is provided on the analysis required to deal with these potent compounds and with fluorometric enzyme assay data in general.
Topoisomerase Assays
Authors: Nitiss John L. 
Published: June 2004

AbstractTopoisomerases are nuclear enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of topoisomerases: the type I enzymes, which make single‐stranded cuts in DNA, and the type II enzymes, which cut and pass double‐stranded DNA. In this unit, an in vitro assay for topoisomerase I activity based on relaxation of supercoiled DNA by that enzyme is described. This is followed by an assay for topoisomerase II based on the decatenation of double‐stranded DNA. In conjunction with these assays, the preparation of mammalian cell extracts for assaying topoisomerase activity is described. Procedures are also detailed for the assay of topoisomerase covalent complexes in vivo and for measuring DNA cleavage caused by topoisomerase I and topoisomerase II in vitro. A related electrophoretic method for mapping topoisomerase II cleavage sites is finally included.
Protein Farnesyltransferase Assays
Authors: Zhang Fang L. , Bishop W. Robert 
Published: June 2004

AbstractThe enzyme protein farnesyltransferase (FPT) transfers a farnesyl group from the prenyl donor farnesyl diphosphate (FPP) to a cysteine residue on substrate proteins which contain a C‐terminal CaaX motif, where C is cysteine, a is an aliphatic amino acid, and X is methionine, serine, or glutamine. Known substrates for FPT include nuclear lamin B and the small GTP‐binding proteins H‐, K‐, and N‐Ras. Short peptides encompassing the CaaX motif of these proteins are also farnesylated by FPT. In this unit, two methods for assaying FPT activity and testing inhibitors are described. Both are based on measuring the transfer of [3H]farnesyl from FPP to a protein or peptide substrate. The first method, the TCA assay, uses native protein substrates of FPT (a support protocol details procedures for expressing and purifying histidine‐tagged H‐Ras in bacteria), and the prenylated product is collected by trichloroacetic acid (TCA) precipitation in the presence of carrier protein. A support protocol is also provided for preparing bovine brain membrane extract for use as the carrier. The second method employs scintillation proximity assay (SPA) technology in which a biotinylated peptide is used as a substrate, and streptavidin SPA beads are used to capture the farnesylated peptide. These procedures can be easily modified to measure prenylation of other protein substrates by FPT and related enzymes.
Protein Tyrosine Kinase Activity Assays
Authors: Hawes Brian E. , van Biesen Tim 
Published: June 2004

AbstractProtein tyrosine kinases (PTKs) are ubiquitous enzymes that are integrally involved in the regulation of transformation mechanisms, normal and pathological growth, cell cycle regulation, immune responses, and a variety of intracellular signaling mechanisms. This rapidly growing family of enzymes is generally divided into two groups: receptor PTKs (with more than twelve distinct families) and nonreceptor PTKs (with more than nine distinct families). PTKs mediate the enzymatic transfer of the gamma phosphate of ATP to the phenolic groups on tyrosine residues to generate phosphate monoesters. In this unit, several assays are provided to measure the ability of PTKs to transphosphorylate protein and peptide substrates, and to autophosphorylate. Phosphorylation of exogenous substrates or autophosphorylation is detected using a 32P‐ or 33P‐phosphorylated protein. Alternatively, antibodies recognizing phosphorylated tyrosine residues can be used to quantify PTK activity. In some cases, antibodies are available for context‐specific phosphotyrosine residues, thereby enabling the detection of PTK‐specific substrate phosphorylation.
Monoamine Oxidase Assays
Authors: Tipton Keith F. , Davey Gavin , Motherway Martha 
Published: June 2004

AbstractMonoamine oxidase catalyzes the oxidative deamination of primary aliphatic and aromatic amines and some secondary and tertiary amines, including the hormone and neurotransmitter amines epinephrine, dopamine, norepinephrine and serotonin. The two isoenzymes, MAO‐A and MAO‐B, differ according to substrate and inhibitor specificities. For example, selective inhibitors of MAO‐A have been shown to be effective antidepressants whereas some MAO‐B inhibitors have been reported to be beneficial in the treatment of Parkinson's and perhaps Alzheimer's diseases. Thus, it is important to have accurate procedures for determining the activities of each MAO isoenzyme in tissues that may contain both. In this unit, several MAO assay procedures are described, along with support protocols for equilibrating ion‐exchange resin, preparing aldehyde dehydrogenase, preparing dose‐response curves for determining the relative proportions of isoenzyme activities, determining concentrations of each isoenzyme, assessing new compounds as putative MAO inhibitors, and determining protein concentration of membrane‐bound proteins.