bluuebunny/crossref_metadata_2025_split huggingface dataset:
Measuring Protein Interactions by Optical Biosensors
Authors: Schuck Peter , Boyd Lisa F. , Andersen Peter S. 
Published: June 2004

AbstractThis unit gives an introduction to the basic techniques of optical biosensing for measuring equilibrium and kinetics of reversible protein interactions. Emphasis is given to the description of simple and robust approaches that will provide reliable results. How to avoid the most commonly encountered problems and artifacts is also discussed.
Assaying Proteases in Cellular Environments
Authors: Wang Kevin K.W. 
Published: June 2004

AbstractTo better study protease activation, regulation, and inhibition physiologically or pharmacologically, it is often advantageous to use cell‐based systems. The introduction of the cellular environment poses additional challenges for the assay of the protease of interest. In this unit, various methods for monitoring protease activity in cell‐based systems are discussedctivity is discussed. The scope of the unit is restricted to mammalian cell system. It is also stressed that specific considerations or requirements for a particular protease or peptidase are often needed in order to optimize the assay.
Monitoring Metalloproteinase Activity Using Synthetic Fluorogenic Substrates
Authors: Troeberg Linda , Nagase Hideaki 
Published: June 2004

AbstractFluorogenic synthetic substrates are commonly used to monitor the activity of peptidases in vitro. This unit presents a representative protocol that employs (7‐methoxycoumarin‐4‐yl)acetyl‐Pro‐Leu‐Gly‐Leu‐(3‐[2,4‐dinitrophenyl]‐L‐2,3‐diaminopropionyl)‐Ala‐Arg‐NH2 (Mca‐Pro‐Leu‐Gly~Leu‐Dpa‐Ala‐Arg‐NH2) as a substrate to assay matrix metallopeptidases (MMPs). This substrate was first described for the assay of MMP‐1, ‐2 and ‐3 and it is now widely used as a general MMP substrate. Protocols are given for both stopped‐time assays (suitable for assaying MMP activity in a large number of samples) and continuous assays (commonly used when establishing an assay protocol or investigating kinetic aspects of enzyme behavior). Other fluorogenic peptides and protein substrates, together with non‐fluorogenic alternatives, are also discussed.
Receptors as Drug Targets
Authors: Williams Michael 
Published: June 2004

AbstractThis overview introduces the basic terminology and theory of receptor‐ligand interactions. Receptors are envisaged as cell surface recognition sites for endogenous hormones, neurotransmitters, and neuromodulators. Receptor selectivity, binding saturability and reversibility, and functionality are discussed in this unit, with special emphasis placed on receptors being targets for drug development. The term Receptor is now used in a broad sense to describe any recognition site for a compound, the latter being defined as an exogenous chemical or biological substance that acts through a binding site or receptor in tissues. Thus, enzymes, uptake sites, ligand‐binding proteins, voltage‐gated ion channels, and intracellular targets are also envisaged as receptors. The use of recombinant receptor systems, receptor mutations and chimeras and allosteric ligands are also described in the setting of drug discovery and development.
Characterization of Opioid Receptors
Authors: DeHaven Robert N. , DeHaven‐Hudkins Diane L. 
Published: June 2004

AbstractThis unit presents two convenient radioligand binding assay methods for opioid receptors. These are applicable to all three of the opioid receptors that have been cloned to date (the mu, kappa and delta receptors), and can be used with numerous commercially available radiolabeled ligands. Moreover, they can serve as reasonable starting points in the development of new assays. A method for determining the binding of radioligand to cloned opioid receptors expressed on the surface of cultured cells is detailed along with a similar method to study radioligand binding to receptors from tissue homogenates. Also included is a procedure for titrating inhibitors of opioid receptor‐ligand binding, along with guidelines for analysis of the resulting data.
Characterization of Potassium Channel Binding
Authors: Scott Victoria E.S. 
Published: June 2004

AbstractThis unit provides an introduction to the binding of specific ligands to K+channels that are expressed in a number of different preparations. The assays described can be used for screening a large number of compounds for interaction at specific binding sites and provides, in selected cases, details of expected results for unlabeled ligands that are known to bind to certain K+channels.
Characterization of Histaminergic Receptors
Authors: Hancock Arthur A. , Esbenshade Timothy A. 
Published: June 2004

AbstractHistamine mediates vascular smooth muscle relaxation and bronchial smooth muscle contraction, but also acts as a neurotransmitter, mediating gastric acid release and modulating the release other neurotransmitters in addition to histamine. In this unit, radioligand binding protocols are described for each of the known subtypes of histamine receptors, namely, H1, H2, and H3. These receptors demonstrate low amino acid sequence homology at the protein level and have divergent pharmacological characteristics such that the conditions described for assaying each receptor are dissimilar.
Quantification of Allosteric Interactions at G Protein Coupled Receptors Using Radioligand Binding Assays
Authors: Christopoulos Arthur 
Published: June 2004

AbstractAllosteric interactions are non‐competitive in nature and involve the simultaneous binding of two ligands to the same receptor. These interactions are also commonly referred to as being cooperative. The effect of an allosteric modulator is to cause a conformational change in the receptor protein that yields a decrease or increase in the equilibrium binding affinity of a classical (orthosteric) agent, such as an agonist or competitive antagonist, for its binding site on the receptor, and vice versa. Because of the complex nature of allosteric phenomena, their detection and quantification relies on the use of both equilibrium and non‐equilibrium binding assays, otherwise the phenomenon may be misinterpreted. This unit outlines the most common experimental approaches to measuring allosterism at G protein‐coupled receptors, and also discusses the analysis of experimental data derived from such assays.
Cyclooxygenase Assays
Authors: Gierse James K. , Koboldt Carol M. 
Published: June 2004

AbstractCyclooxygenase (COX, also known as prostaglandin H2 synthase, PGH2) is one of the most significant enzymes in pharmacology since COX inhibition is the mechanism of action of most nonsteroidal anti‐inflammatory drugs (NSAIDs). There are two forms: COX‐1 is a constitutive form of the enzyme that has been linked to the production of physiologically important prostaglandins that may play a role in homeostasis (gastric, renal, etc.). COX‐2 is inducible by cytokines and growth factors, and induction of COX‐2 is linked to inflammatory cell types and tissues, so COX‐2 is believed to be the target for the anti‐inflammatory actions of NSAIDs. COX‐2 also exists in a constitutive form in kidney and brain. Thus, there is an ongoing effort to identify compounds that will inhibit COX‐2 in preference to COX‐1 since such an agent may be a safer, and perhaps more efficacious, NSAID. Three assays are provided in this unit for measuring COX activity: one is to measure the final product of the reaction, PGE2, by ELISA; another is to measure directly the oxygen uptake that occurs during the first step of the reaction using an oxygen sensor; and the third is to measure the second reaction (peroxidase function) spectrophotometrically.
Characterization of Matrix Metalloproteinase Inhibitors: Enzymatic Assays
Authors: Marcotte Patrick A. , Davidsen Steven K. 
Published: June 2004

AbstractThe matrix metalloproteinases (MMPs) are a family of tightly regulated proteases that are involved in the catabolic aspect of remodeling and maintenance of normal tissue, and more than 20 human MMPs have been identified thus far. The MMPs collectively degrade a broad range of protein components of the extracellular matrix. While some substrate overlap exists, individual MMPs have been shown to process certain substrates more efficiently than others. These differences raise the critical issue of whether broad‐spectrum inhibitors, active against all MMPs, or selective inhibitors, targeted to a subset of enzymes, represent the optimal therapeutic strategy for a given disease. This suggests the need to assess the inhibition potency of test compounds across a range of MMP family members. Described in this unit is a method for the in vitro characterization of MMP inhibitors. The is used to determine the potency of test compounds as inhibitors of 8 representative MMPs through the measurement of their inhibition of cleavage of a fluorogenic substrate. Since this substrate is efficiently hydrolyzed by all MMPs in the screening assays presented here, the method is convenient for assessing the selectivity of inhibitors against multiple enzymes. A describes the activation of MMP zymogens.
Cytochrome P450 Assays
Authors: Delaporte Enock , Rodrigues A. David 
Published: June 2004

AbstractCytochrome P450s (CYPs) play a major role in drug detoxification, and inhibition of CYP‐mediated metabolism may lead to accumulation of toxic drug levels in the plasma. To prevent adverse drug‐drug interactions, new drug candidates are routinely tested for their ability to inhibit these enzymes. This unit describes a variety of protocols for evaluating new chemical entities as inhibitors of CYP activity. An example protocol illustrates a high‐throughput screening format using a fluorogenic probe, and a method for evaluating a test compound as a time‐dependent inhibitor of CYP is also described.
Histamine Receptor Assays
Authors: Shankley Nigel P. , Morton Magda F. , Watt Gillian F. 
Published: June 2004

AbstractThis unit describes three standard in vitro bioassays for studying histamine H1, H2 and H3 receptors in isolated intact tissues removed from the guinea pig. Both the H1 and H3 receptor assays are based on preparations of the ileum, whereas the spontaneously beating right atrium assay is used for the H2‐receptor.This unit describes three standard in vitro bioassays for studying histamine H1, H2 and H3 receptors in isolated intact tissue.
Models of Cardiovascular Disease: Measurement of Arrhythmogenic/Antiarrhythmic Activity in the Guinea Pig and Antihypertensive Activity in the Conscious Rat
Authors: Lacroix Pierre , Guillaume Philippe 
Published: June 2004

AbstractArrhythmias arise as the result of either disorders of cardiac impulse formation or disorders of impulse conduction, or a combination of both. Screening methods have been developed to produce experimental arrhythmias and to select compounds on the basis of their ability to prevent or to reverse these arrhythmias. In this unit, cardiac glycosides are used produce arrhythmias (premature ventricular contractions, then sustained ventricular tachycardia and ventricular fibrillation) and can be used to detect the arrhythmogenic/antiarrhythmic potential of compounds in the guinea pig. The antihypertensive effects of compounds may be assessed using relatively simple in vivo techniques which are adapted to primary screening. The spontaneously hypertensive rat (SHR) model requires no surgery or pharmacological agent to induce hypertension, and it is sensitive to most of the clinically active antihypertensive drugs. The SHR method described in this unit can be used to detect the antihypertensive potential of compounds
Models of Affective Illness: Chronic Mild Stress
Authors: Papp Mariusz 
Published: June 2004

AbstractAntidepressants can elevate the mood of depressed patients, but have no effect in normal (i.e., non‐depressed) individuals. This characteristic makes predicting the clinical action of an antidepressant, based on the effects it has in normal laboratory animals, difficult. The best strategy for studying the psychobiological causes and treatment of depression involves the use of appropriate animal models. Of the few models that meet the criteria required for a valid animal model of depression, the chronic mild stress paradigm is the most valid and the most extensively investigated. In addition to the protocol describing generation of the model, data supporting the model's validation are presented along with a discussion of technical aspects of the procedure.
Models of Anxiety: Ultrasonic Vocalizations of Isolated Rat Pups
Authors: van Oorschot Ruud , Mos Jan , Olivier Berend 
Published: June 2004

AbstractWhen rat pups of 9 to 11 days are separated from their mother and littermates, they emit ultrasonic calls in the range of 30 to 50 kHz. This ultrasonic vocalization can be used as a measure of distress or anxiety. Measurement of the number of vocalizations during a short testing period (5 min) can be used to quantify the amount of distress (anxiety). While the ultrasonic cries are sensitive to drugs with anxiolytic properties, the paradigm also distinguishes other drug classes. Described in this unit are the basic methods for measurement and quantification of ultrasonic calls in rat pups under two different stress conditions: a cold plate (18°C) and a warm plate (37°C) condition. The utility of this protocol in psychopharmacologic testing and a method for determining drug side‐effects is described.
Models for Environmentally Induced Eating Disorders: Dietary Hyperphagia and Anorexia Nervosa
Authors: Pelleymounter Mary Ann , Kant R.H. , Aravich Paul 
Published: June 2004

AbstractThe two protocols in this unit provide suggestions for constructing models of eating disorders that are at the opposite ends of the spectrum: dietary hyperphagia and anorexia nervosa. The greatest degree of dietary hyperphagia is induced by giving rats or mice access to a daily choice of highly palatable foods (e.g., chocolate or bread) in addition to their regular chow. Like humans, rats overeat and actually develop physiological requirements for these foods. This model can be used to test the effects of putative anorectic agents on both acute and chronic administration regimens. The second protocol describes a model of compulsive behavior that results in profound weight loss, which is produced by moderate food deprivation along with continuous access to exercise wheels.
Models of Renal Insufficiency: The Anti‐Thy‐1.1 Model of Acute Proliferative Glomerulonephritis
Authors: Reinhart Glenn A. , Cox Bryan F. 
Published: June 2004

AbstractThis unit describes the rat anti‐Thy‐1.1 model of acute proliferative glomerulonephritis for the study of chronic renal insufficiency. A procedure is detailed for the induction of glomerulonephritis in rats as well as measurement of daily urinary excretion of protein, which is a convenient, primary screening tool. The unit also provides methods for assessment of glomerular filtration rate and effective renal plasma flow in anesthetized rats with anti‐Thy‐1.1‐induced renal insufficiency.
Models of Cardiac Ischemia‐Reperfusion Injury in Dogs and Rats
Authors: Gross Garrett J. 
Published: June 2004

AbstractA marked reduction in coronary blood flow produces a reduction in myocardial function, electrocardiographic abnormalities and eventually a myocardial infarction if the ischemic episode were to persist for more than 20 to 30 min. There have been numerous studies in animal models in search of a magic bullet or drug that can ameliorate these symptoms and result in a reduction in infarct size, improvement in the recovery of contractile function, and abrogation of malignant ventricular arrhythmias in humans. This unit describes two animal models of myocardial ischemia/reperfusion injury which are used to evaluate pharmacological agents that may eventually demonstrate cardioprotective activity in a clinical setting.
Tissue Expression Profiling Using Real‐Time PCR
Authors: Strum Jay C. , Carrick Kevin M. , Stuart Joan S. , Martensen Shelby A. 
Published: June 2004

AbstractThe recent development of real‐time PCR allows for the rapid and accurate quantitation of gene expression in cells and tissues. Real‐time PCR instrumentation is designed for the simultaneous quantitation of gene expression from a few samples up to 384 samples. The normal tissue expression profile of a gene can provide useful insights into its potential role in normal physiological processes. When combined with the tissue expression profile of the gene in diseased tissues, information concerning the potential role in pathological processes can be determined. This unit describes a protocol to determine the relative abundance of mRNA in a panel of human tissues using real‐time PCR.
In Vivo Measurement of Blood‐Brain Barrier Permeability
Authors: Boje Kathleen M.K. 
Published: June 2004

AbstractThis unit describes various protocols for the in vivo quantitation of drug permeability across the rodent blood‐brain barrier. Methods for the measurement of drug influx and efflux are described, and support protocols are provided for determining intravascular capillary volume and cerebral perfusion flow. An in situ perfusion technique is also provided for assessing whether transport of a test compound occurs by carrier‐mediated or saturable transport.
Determination of the Stability of Drugs in Plasma
Authors: Hartman David A. 
Published: June 2004

AbstractIn order for a drug to be effective, enough of the active form must reach the target to elicit the desired effect. The first‐pass effect for drugs taken orally is well known but the metabolism of drugs in plasma must also be considered during the drug discovery and development process. For instance, hydrolysis of a compound by plasma enzymes can significantly alter the bioavailability of the active compound. This unit describes methods for the determination of the stability of compounds in plasma.
Screening in Constitutively Active Receptor Systems
Authors: Kenakin Terry 
Published: June 2004

AbstractThis unit discusses the use of constitutive G protein‐coupled receptor (GPCR) activity for the screening of new drug entities. It begins with an introduction to constitutive GPCR activity and then centers on the three basic components of a constitutive screening system, namely the receptor, the receptor coupling components (G protein) and the response reporting system. Then, the influence of ligands and particular application of such systems to drug screening, with reference to optimal conditions, is discussed. Finally, the relative advantages and disadvantages of constitutive screening are considered. This unit discusses the use of constitutive G protein‐coupled receptor (GPCR) activity for the screening of new drug entities
The Action Potential of the Purkinje Fiber: An In Vitro Model for Evaluation of the Proarrhythmic Potential of Cardiac and Noncardiac Drugs
Authors: Rouet René , Ducroq Joffrey , Picard Sandra 
Published: June 2004

AbstractThe proarrhythmic potential of new drugs can be investigated using in vitro electrophysiological techniques measuring the cardiac action potential in isolated Purkinje fibers. Different types of arrhythmias may occur, as early after‐depolarizations (EADs), which are favored by action potential duration lengthening and bradycardia, or delayed after‐depolarizations (DADs), which are facilitated by tachycardia. The effects of a test compound on the occurrence of these arrhythmias, known to induce in vivo torsades de pointes (or triggered activities), can be studied following the experimental protocols described in this unit.
Electrophysiological Analysis of ATP‐Sensitive Potassium Channels in Mammalian Cells and Xenopus Oocytes
Authors: Shieh Char‐Chang , Gopalakrishnan Murali 
Published: June 2004

AbstractThis unit describes general methodologies for the characterization of ATP‐sensitive K+ channels and the study of ligand‐channel interactions in native tissues and clonal cell lines by electrophysiological techniques. Detailed protocols on how to establish patch‐clamp single‐channel and whole‐cell current recording are presented. Two‐electrode voltage clamp techniques for studying ATP‐sensitive K+ channels expressed in Xenopus oocytes are also included.
Overview of Electrophysiological Characterization of Neuronal Nicotinic Acetylcholine Receptors
Authors: Bertrand Sonia , Bertrand Daniel 
Published: June 2004

AbstractElectrophysiology is one of the best tools to characterize ligand‐gated channels such as neuronal nicotinic acetylcholine receptors. In this unit, the properties of these receptors are discussed along with approaches for how they can be characterized. Special emphasis is given to agonist and antagonist profiles as well as allosteric effectors that offer alternative possibilities in drug discovery.
Chondrocyte Culture and Assay
Authors: Liebman Jeffrey , Goldberg Ronald L. 
Published: June 2004

AbstractChondrocytes constitute the sole cell type found within cartilage, and control the formation and composition of cartilage. Cellular, biochemical and pharmacological studies of arthritis and other cartilage disorders have increasingly focused on chondrocyte function. Three methods are presented in this unit for culturing chondrocytes, and two assays are described that characterize proteoglycan synthesis, a key measure of chondrocyte function.Chondrocytes constitute the sole cell type found within cartilage, and control the formation and composition of cartilage
Immunohistochemical Localization of Proteins in the Nervous System
Authors: Volpicelli‐Daley Laura A. , Levey Allan 
Published: June 2004

AbstractThe immunohistological methods described in this unit can be used to determine the precise localization of neurochemicals, receptors, and proteins throughout the nervous system. Determining the localization of a protein within defined brain nuclei and neuronal cell populations can provide important clues regarding its potential function. Immunoperoxidase reactions and light microscopy are commonly used to visualize the distribution of a single primary antibody directed to an antigen of interest. Double‐labeling immunofluorescence and confocal microscopy techniques detect the localization of one protein relative to another protein and allow analysis of colocalization at a cellular and subcellular level. The colocalization of two proteins can also be quantified, allowing analysis of the extent of overlap between two labeled markers and measurements of changes in the localization of one protein relative to another following drug treatment or in animals that have been genetically modified. The theoretical limit of resolution of confocal microscopy is 0.1 to 0.2 µm.
Transneuronal Circuit Analysis With Pseudorabies Viruses
Authors: Card J. Patrick , Enquist Lynn W. 
Published: June 2004

AbstractOver the past decade there has been a dramatic increase in the use of viruses as transneuronal tracers of neuronal circuitry. The method exploits the propensity of neurotropic viruses to invade neurons and then produce infectious progeny that cross synapses to infect other neurons within a circuit. The protocols and commentaries included in this unit focus upon the use of the swine alpha herpesvirus known as pseudorabies virus (PRV) for polysynaptic analysis. Here, the aspects of experimental design that have the greatest import for successful use of viruses in circuit definition are presented. Accordingly, the protocols included in this unit can be applied in concert with methods in which the use of classical tract tracers has been detailed. A procedure for retrograde infection of CNS circuits in the rat CNS by peripheral injection of virus is detailed, while transneuronal analysis by intracerebral injection is also described. A variant of these procedures, transneuronal analysis with multiple recombinant strains, is also described along with methods for growing and titering viral stocks, and procedures for single and dual immunohistochemical localization of viral antigens in fixed brain tissue.