bluuebunny/crossref_metadata_2025_split huggingface dataset:
In Vitro Enzymatic Assays of Protein Tyrosine Phosphatase 1B
Authors: Lubben Thomas , Clampit Jill , Stashko Michael , Trevillyan James , Jirousek Michael R. 
Published: June 2004

AbstractMany hormone or growth factor receptors signal via the activation of protein‐tyrosine kinases and phosphatases. Alteration of the phosphorylation state of tyrosine residues in certain proteins can directly regulate enzyme activity or cause formation of protein complexes necessary for transducing intracellular signals. Genetic and biochemical evidence also implicates protein‐tyrosine phosphatases in several disease processes, including negative regulation of insulin receptor signaling at the level of the insulin receptor and perhaps in signaling at the IRS‐1 level. The expression of protein tyrosine phosphatase‐1B (PTP1B) is elevated in muscle and adipose tissue in insulin‐resistant states both in man and rodents suggesting that PTP1B may play a role in the insulin‐resistant state associated with diabetes and obesity. As described in this unit, PTP1B activity can be determined with the small molecule substrate, p‐nitrophenyl phosphate (pNPP), in which the cleavage of the phosphate results in production of p‐nitrophenol (pNP) and an increase in absorbance at 405 nm. Alternatively, PTP1B activity can be measured as described using model phosphotyrosyl‐containing peptide substrates in which the release of free phosphate from the peptide is determined using a malachite green colorimetric assay.
Measurement of Glutamate Carboxypeptidase II (NAALADase) Enzyme Activity by the Hydrolysis of [3H]‐N‐Acetylaspartylglutamate (NAAG)
Authors: Tiffany Carol W. , Slusher Barbara S. 
Published: June 2004

AbstractThe peptide N‐Acetylaspartylglutamate (NAAG) is hydrolyzed by N‐Acetylated‐alpha‐linked‐acidic dipeptidase (NAALADase, glutamate carboxypeptidase II) into N‐Acetylated aspartate (NAA) and glutamate. Hydrolysis can be measured as described in this unit by employing radiolabeled NAAG (NAA‐[3H]glu) as the substrate. The occurrence of NAALADase activity in a wide range of tissues has implications for a variety of physiological purposes. The assay described here is useful for the analysis of NAALADase activity and its inhibition in brain synaptosomal preparations, tissue homogenates and tissue culture cell pellets.
Overview of Receptor Interactions of Agonists and Antagonists
Authors: Kenakin Terry 
Published: June 2004

AbstractExperiments with isolated tissues have been crucial in defining interactions between drugs and receptors, and in formulating drug receptor theory. Described in this unit are procedures which can be used to define properties of drugs and receptors that transcend tissue hosts. This allows the extrapolation of such data to other tissue hosts and also to human therapeutic potential. The common currency of such studies is the dose‐response curve. In this unit, two views of agonism are discussed: classical receptor occupancy theory and the operational model. The models yield similar equations for the calculation of drug receptor constants in isolated tissues. The techniques for estimating molecular constants for drug/receptor pairs are identical within the context of either the occupancy or the operational theory; only the interpretation differs. Both theoretical frameworks are widely used in current pharmacological literature, and therefore techniques for measuring drug receptor constants utilize both models in this unit. Experiments with isolated tissues have been crucial in defining interactions between drugs and receptors, and in formulating drug receptor‐theory.
Opioid Receptor Assays
Authors: Berzetei‐Gurske Ilona 
Published: June 2004

AbstractOpioid receptors mediate the pharmacologic actions of morphine‐like analgesics, a class of drugs that are important clinical agents which are also widely abused socially as a result of their euphorogenic and addictive properties. Cloning of the opioid receptors has confirmed the original postulate that there are only three classes of opioid receptors: µ, δ, and κ. The most commonly used isolated tissue preparations for measuring activity at these receptors are the guinea pig ileum and the mouse vas deferens. This unit describes preparation of these tissues to measure agonist and antagonist properties of test compounds at the µ‐ and κ‐opioid receptors in guinea pig ileum and the d‐opioid receptor in mouse vas deferens.
Tachykinin Receptor Assays
Authors: Patacchini Riccardo , Maggi Carlo Alberto 
Published: June 2004

AbstractThe tachykinins are a family of peptides (including Substance P, neurokinin A and neurokinin B) that mediate a wide variety of biological activities, and smooth muscle contraction following stimulation of the tachykinin receptors has been well characterized, making isolated smooth muscle is a preferred tissue in which to study tachykinin receptors and their ligands. Three receptor subtypes have been isolated and cloned, and the protocols in this unit provide precise instruction on ligand‐binding assays using isolated tissues bearing the various subtypes (NK1, NK2 or NK3). A plethora of representative data illustrate expected results, so the investigator can compare and validate data obtained from test compounds.
Dopamine 2 (DA‐2) Receptor Assays Using Sympathetic Nerve Terminals
Authors: Long John P. 
Published: June 2004

AbstractIn vitro experiments using isolated atria with and without superfusion can be used to quantify the dopamine‐2 (DA‐2) receptor stimulating activity of compounds. This receptor mechanism decreases the amount of norepinephrine released from sympathetic nerve terminals by neuronal impulses. DA‐2 receptor agonists (stimulants) are very active and induce several physiological changes, such as bradycardia, hypotension, lower intraocular pressure, etc. In this unit, tissue preparation and dopamine receptor assays are described for isolated cat atria with field stimulation or with superfusion, and isolated guinea pig atria with field stimulation.
5‐Hydroxytryptamine Receptor Assays
Authors: Figueroa Katherine W. , Morton Magda F. , Prendergast Clodagh E. , Shankley Nigel P. , Martin Graeme R. 
Published: June 2004

AbstractThis unit describes in vitro bioassays for studying 5‐hydroxytryptamine receptors in isolated intact animal tissues. In total, nine in vitro bioassays are described for 5‐HT1A, 5‐HT1B, 5‐HT1D, 5‐HT2A, 5‐HT2B, 5‐HT2C, 5‐HT3, 5‐HT4, and 5‐HT7 receptors in isolated intact guinea pig, rat and rabbit tissues. The 5‐HT1A, 5‐HT3, and 5‐HT7 assays use ileum preparations, whereas, the 5‐HT1B, 5‐HT1D, 5‐HT2A and 5‐HT2C receptor assays are all based on vascular preparations. The 5‐HT2B receptor is best studied in stomach fundic muscle and the 5‐HT4 receptor in esophageal tissue. To date, no functional isolated organ bioassays are available for the 5‐ht1e, 5‐ht1f, 5‐ht5 or 5‐HT6 receptors. This unit describes in vitro bioassays for studying 5‐hydroxytryptamine receptors in isolated intact animal tissues
Models of Pulmonary Disease: Acute and Chronic Allergic Asthma in the Monkey and Acute and Chronic Viral Pulmonitis in the Mouse
Authors: Wegner Craig D. , Shaughnessy Thomas , Mussatto Donna 
Published: June 2004

AbstractThree models/protocols designed to mimic an inflammation process characteristic of a specific lung disease are described in this unit. Included are a single allergen inhalation in monkeys to model acute asthmatic episodes, repeated allergen inhalations in monkeys to model chronic asthma, and a lower‐respiratory viral infection in mice to model acute and chronic viral alveolitis and bronchitis. In each case, alterations in lung functions believed to correlate with symptoms of the respective disease are measured along with pulmonary inflammation.
Gastrointestinal Models: Intestinal Transit and Ulcerogenic Activity in the Rat
Authors: Lacroix Pierre , Guillaume Philippe 
Published: June 2004

AbstractSlowing or acceleration of intestinal transit is readily observed in laboratory animals following administration of a number of drugs such as those interfering with the autonomic nervous system. Because serious side effects of these drugs include constipation or diarrhea, it is important to assess such effects when developing new drugs. Additionally, acute mucosal erosions are readily observed in the stomachs of laboratory animals following oral administration of ulcerogens such as nonsteroidal anti‐inflammatory drugs (NSAIDs). A serious side‐effect of these drugs in man is development or reactivation of gastroduodenal ulcers. This unit presents protocols to detect the effect of compounds on intestinal transit in the small intestine in the rats and mice, and to detect the ulcerogenic potential of compounds in the rat.
Models of Inflammation: Carrageenan Air Pouch in the Rat
Authors: Whiteley Phyllis E. , Dalrymple Stacie A. 
Published: June 2004

AbstractThe rat air pouch is an in vivo model used to study acute inflammation. The air pouch is formed by subcutaneous injection of air into the intracapsular area of the back of the rat. The pouch is composed of a lining of cells that consists primarily of macrophages and fibroblasts. Injection of a carrageenan solution into the pouch produces an inflammatory reaction that is characterized by an infiltration of cells, an increase in exudate, and a marked production of biochemical mediators such as prostaglandins, leukotrienes, and cytokines. This unit describes a method to elicit a cellular influx and inflammatory exudate in the six‐day‐old air pouch in the rat. Potential anti‐inflammatory drugs can be tested by measuring the reduction in carrageenan‐induced inflammation.
Models of Pain: Hot‐Plate and Formalin Test in Rodents
Authors: Bannon Anthony W. 
Published: June 2004

AbstractExperimental models of pain can be separated into three basic categories: acute, persistent, and neuropathic. Acute pain is modeled by the hot‐plate test, persistent pain by the formalin test, and neuropathic pain by nerve ligation. The neurotransmitters and neuromodulators that mediate the pain response in each of these models can differ. As a result, different drugs may be effective in attenuating pain for each model. A basic understanding of the mechanisms involved in the pain response can help investigators choose the model that would be most appropriate for testing the analgesic properties of specific compounds or classes of compounds. This unit presents protocols for both an acute model of pain using the hot‐plate test, and a persistent model of pain using the formalin test. Each protocol describes the preparation of animals (mice or rats) for the test, administration of a compound being tested for its analgesic properties, and data collection.
Models of Affective Illness: Forced Swimming and Tail Suspension Tests in Rodents
Authors: Porsolt Roger D. , Brossard Geneviève , Hautbois Carine , Roux Sylvain 
Published: June 2004

AbstractRodents forced to swim in a narrow space from which there is no escape adopt, after an initial period of vigorous activity, a characteristic immobile posture, moving only when necessary to keep their heads above the water. The animals immobility was hypothesized to show they had learned that escape was impossible and had given up hope. Immobility was therefore given the name behavioral despair. It was subsequently found that immobility could be reduced by a wide range of clinically active antidepressant drugs. This simple behavioral procedure has since become a widely used test for screening novel antidepressants. Rodents forced to swim in a narrow space from which there is no escape adopt, after an initial period of vigorous activity.
Models of Urogenital Dysfunction: Assessment of Volume‐Evoked Micturition Reflex (VEMR) in the Conscious Rat by Cystometry
Authors: Nunn Philip , Williamson Ian , Newgreen Donald , Bushfield Mark 
Published: June 2004

AbstractThis unit describes a conscious rat model that allows the investigation of compounds administered by various routes (e.g., oral, intravenous and subcutaneous). This procedure can be used to examine the effects of agents on supraspinal, spinal and peripheral control of the micturition reflex, in addition to effects on parasympathetic neurotransmission and detrusor relaxation. One advantage to this procedure over anesthetized dog and rat models is the ability to determine pharmacological effects in the absence of possible interfering effects of anesthetics.
Models of Dementia: Delayed Alternation in Aged Rats
Authors: Roux Sylvian , Hubert Isabelle , Porsolt Roger D. 
Published: June 2004

AbstractOperant delayed alternation tasks are useful tools for assessing age‐related memory deficits in rats. Decreased accuracy in these tasks seems to reflect mainly an inability to remember and utilize recent events (short‐term memory deficit). The task described in this unit requires the rat to learn a delayed alternation for food reward in a standard Skinner box, so it must retain spatial information over a short period of time. Aged rats exhibit clear short‐term memory impairments (decrease in response accuracy) in the task compared to young rats during learning and later, after extended training, also exhibit performance deficits. An advantage of studying these two phases is the possibility to examine either chronic effects (repeated drug administrations during acquisition) or acute effects (single treatments given before the test session during the stabilized performance phase). Operant delayed alternation tasks are useful tools for assessing age‐related memory deficits in rats
Models of Anxiety: Stress‐Induced Hyperthermia (SIH) in Group‐Housed Mice
Authors: Zethof Theo J.J. , van der Heyden Jan A.M. , Olivier Berend 
Published: June 2004

AbstractThis unit describes a procedure for quantifying anxiety in group‐housed mice by measuring their rectal temperature at 1‐min intervals. When rectal temperature is measured in this fashion, the temperature of the last mouse (T10) is higher than that of the first (T0). This difference in temperature is termed stress‐induced hyperthermia (SIH), and is thought to reflect an increased anxiety level in the animal. It is a robust, stable, and reproducible parameter. Under these conditions, the rectal temperature of the last mouse is 1(to 1.5(C higher than that of the first mouse. When mice are pretreated with anxiolytics (benzodiazepines, 5‐HT1A receptor agonists, ethanol), SIH decreases, making this a relatively simple procedure for screening drugs of this class. Moreover, the test reveals the intrinsic effects of a test compound on basal body temperature. An advantage of this assay is that it does not require time‐consuming training of the animals.
Overview of the Use of Transgenic Animals in CNS Drug Discovery
Authors: Nestler Eric J. , Olivier Berend 
Published: June 2004

AbstractThis overview highlights how transgenic mice, and related genetic manipulations, can contribute in important ways to the goal of developing novel treatments for neuropsychiatric disorders. A brief background of the field of drug discovery is covered and then new animal models of CNS disorders are discussed along with the topics of target validation, identification of novel targets, and functional genomics.
Models of Epilepsy: Electroshock and Chemical Induced Convulsions in the Mouse
Authors: Giardina William J. 
Published: June 2004

AbstractEpilepsy is a disorder of the central nervous system that is characterized by seizures. This unit describes protocols for identifying and differentiating the antiseizure or anticonvulsant activity of novel test substances. Variations of two basic test methods using mice are described: blockade of electroshock‐induced convulsions (the maximal electroshock test) and blockade of chemical‐induced convulsions (the pentylenetetrazole test). This unit also describes variations of the same methods that can be used to assess potential proconvulsant activity of test substances in safety evaluation studies.
Classic In Vivo Cancer Models: Three Examples of Mouse Models Used in Experimental Therapeutics
Authors: Kruczynski Anna , Hill Bridget T. 
Published: June 2004

AbstractTransplantable animal tumors have been associated with the discovery of most clinically active anticancer agents. They are still useful today in conducting detailed evaluations of new candidate anticancer drugs. Three protocols relating to transplantable experimental tumors are described in this unit. Included are the intravenously‐implanted murine P388 leukemia, the subcutaneously‐implanted murine B16 melanoma and two examples of subcutaneously‐implanted human tumor xenografts, the LX‐1 (lung) and MX‐1 (breast) tumors.
Social Recognition Task in the Rat
Authors: Lemaire Martine 
Published: June 2004

AbstractAmong the numerous tasks designed for assessing distinct memory processes, the social recognition task in the rat offers the opportunity to evaluate a form of short‐term working memory in the domain of social cognition, and its modification by pharmacological agents or physiopathological states, such as aging. Social cognition in humans is obviously of great importance and its deficits, e.g., during aging and Alzheimer's dementia, often have dramatic consequences for the patient and their environment. Two protocols are described in this unit that permit evaluation of positive and negative drug effects on social recognition memory in adult male rats and beneficial drug effects on age‐related social recognition amnesia in aged male rats.
Emetic Liability Testing in Ferrets
Authors: Osinski Mark A. , Seifert Terese R. , Shaughnessy Thomas K. , Gintant Gary A. , Cox Bryan F. 
Published: June 2004

AbstractEvidence of a candidate drug's efficacy and safety is mandatory for successful drug registration by regulatory authorities. However, a third property, tolerability, often determines a drug's acceptance by the patient population. Gastrointestinal events often determine the maximum tolerated dose in Phase I clinical trials. If the plasma concentrations achieved at the maximum tolerable dose are below those required for efficacy, the drug will certainly fail. The identification of a compound's emetic/nauseogenic liability early in the discovery process can be critical to the ultimate success of the drug discovery project. Ferrets are small carnivores (∼1 kg) of the Mustelidae family that vomit in response to many pharmacological classes of drugs as well as to cytotoxic chemotherapeutics and radiation. This unit describes a simple method for evaluating the emetic and nauseogenic potential of drug candidates in ferrets.
Overview of Receptor Cloning
Authors: Nash Norman 
Published: June 2004

AbstractReceptors are generally categorized into four major superfamilies according to their structural and genetic similarities: steroid receptors, growth factor receptors, ionotropic receptors (i.e., ligand‐gated ion channels), and metabotropic receptors (i.e., G protein‐coupled receptors). This unit describes the methodology and critical issues for cloning receptors by several methods. Although cloning by protein purification was the first major method, it is now less frequently employed. The early, more tedious procedures broadened knowledge about receptors and established a strong foundation that has enabled the utilization of the newer approaches discussed in this unit, including: homology cloning by screening with oligonucleotides, DNA fragments or the products of direct polymerase chain reaction (PCR); expression cloning using function, ligand binding, and antibody recognition; and differential display techniques.
Receptor Reporter Systems
Authors: Burns David J. 
Published: June 2004

AbstractReceptor‐linked reporter systems allow the study of pharmacological properties of known receptors, identification of new receptor subtypes, examination of the specific signal transduction pathways linked to receptor activation, and development of functional assays. Reporter systems provide high‐throughput screening methods that allow researchers to expeditiously identify and characterize compounds that modulate the activities of therapeutically relevant receptors or influence events downstream of receptor activation. This unit provides two representative protocols for coupling cell‐surface receptors to reporter molecules that signal receptor activation. The first is a luciferase‐based assay and the second uses green fluorescent protein (GFP). This unit provides a comparison of several reporter assays as well as reporter systems that rely on chloramphenicol acetyltransferase activity.
Expression of Cloned Receptors in Mammalian Cell Lines
Authors: Gopalakrishnan Murali , Molinari Eduardo J. 
Published: June 2004

AbstractThe expression of cloned receptor and ion‐channel proteins is a very useful tool for studying the physiological and pharmacological properties of receptors. The process of introducing a gene of interest into eukaryotic cells by biochemical or physical methods is termed transfection. After transfection, some of the DNA is transported into the nucleus, where it is transcribed. The resulting mRNA is transferred into the cytoplasm and translated into protein. The transfection method described in this unit is a versatile, plasmid‐based, liposome‐mediated gene‐transfer technique known as lipofection. This technique has gained wide acceptance because of its simplicity, suitability for a wide range of cell types, reasonable transfection efficiencies, and reproducibility. This unit also includes a procedure for assessing transfection efficiency via cotransfection with a reporter gene (lacZ) and assay for coexpression.
Overview of Pharmacogenetics
Authors: Katz David A. 
Published: June 2004

AbstractPharmacogenetics is the study of relationships between genetic variation and inter‐individual differences of drug response. As the field has matured over the past 15 years, a remarkable diversity of pathways, variation types, and mechanisms have been found to be relevant pharmacogenetic factors. Today, pharmacogenetics is becoming more important in pharmacology for target validation, lead optimization, and understanding of idiosyncratic toxicity. This unit provides an overview of the history of pharmacogenetics and current research applications in drug discovery, as well as a discussion of research quality issues relevant for human subjects research in the pharmacogenetics laboratory.
Estimating Intestinal Mucosal Permeation of Compounds Using Caco‐2 Cell Monolayers
Authors: Gao Jinnian , Hugger Erin D. , Beck‐Westermeyer Melissa S. , Borchardt Ronald T. 
Published: June 2004

AbstractStep‐by‐step protocols are provided in this unit for the measurement of apparent permeability coefficients of compounds using Caco‐2 cell monolayers as an in vitro model of the intestinal mucosa. Procedures for culturing the cells and transmonolayer transport studies are also included. Critical issues for successfully estimating intestinal mucosal permeation of drugs are discussed. Step‐by‐step protocols are provided in this unit for the measurement of apparent permeability coefficients of compounds using
Uptake Studies for Evaluating Activity of Efflux Transporters in a Cell Line Representative of the Blood‐Brain Barrier
Authors: Silverstein Peter S. , Karunaratne D. Nedra , Audus Kenneth L. 
Published: June 2004

AbstractIn this unit, protocols are presented for performing uptake studies with bovine brain microvessel endothelial cells (BBMECs) using either radiolabeled compounds, or rhodamine 123 as a marker for a P‐glycoprotein substrate. The protocols can easily be modified for the investigation of substrate uptake in other cell lines.