Current Protocols in Pharmacology

Current Protocols in Pharmacology

Online ISBN: 9780471141754

DOI: 10.1002/0471141755

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  1. Preface
  2. Foreword
  3. Selected Suppliers of Reagents and Equipment
  4. Chapter 1 Receptor Binding
    1. Introduction
    2. UNIT 1.1 Receptors as Drug Targets
    3. UNIT 1.2 Receptor Theory
    4. UNIT 1.3 Practical Aspects of Radioligand Binding
    5. UNIT 1.4 Characterization of Opioid and ORL1 Receptors
    6. UNIT 1.5 Characterization of Adrenoceptors
    7. UNIT 1.6 Characterization of Dopamine Receptors
    8. UNIT 1.7 Characterization of GABA Receptors
    9. UNIT 1.8 Radioligand Binding Characterization of Neuronal Nicotinic Acetylcholine Receptors
    10. UNIT 1.9 Characterization of P1 (Adenosine) Purinoceptors
    11. UNIT 1.10 Characterization of Angiotensin II Receptors
    12. UNIT 1.11 Characterization of Neuropeptide Y (NPY) Receptors
    13. UNIT 1.12 Characterization of Cholecystokinin (CCK) Receptors
    14. UNIT 1.13 Characterization of Corticotropin-Releasing Factor (CRF) Receptors
    15. UNIT 1.14 Characterization of Wild-Type Excitatory Amino Acid Ion Channel Receptors
    16. UNIT 1.15 Characterization of Tachykinin Receptors
    17. UNIT 1.16 Benzodiazepine Binding to GABAA Receptors
    18. UNIT 1.17 Characterization of Potassium Channel Binding
    19. UNIT 1.18 Characterization of the Picrotoxin Site of GABAA Receptors
    20. UNIT 1.19 Characterization of Histaminergic Receptors
    21. UNIT 1.20 [3H](+)MK801 Radioligand Binding Assay at the N-Methyl-D-Aspartate Receptor
    22. UNIT 1.21 Overview of Receptor Allosterism
    23. UNIT 1.22 Quantification of Allosteric Interactions at G Protein–Coupled Receptors Using Radioligand Binding Assays
    24. UNIT 1.23 Characterization of 5-HT1A,B and 5-HT2A,C Serotonin Receptor Binding
    25. UNIT 1.24 Characterization of Chemokine Receptors
    26. UNIT 1.25 Characterization of Calcium Channel Binding
    27. UNIT 1.26 Characterization of Cannabinoid Receptors
    28. UNIT 1.27 Radioligand Binding Assays for the Glycine Site on N-Methyl-D-Aspartate Receptors
    29. UNIT 1.28 Characterization of Melanocortin Receptors
    30. UNIT 1.29 Characterization of Neurotensin Receptors
    31. UNIT 1.30 Characterization of CGRP Receptor Binding
    32. UNIT 1.31 Overview of Approaches to the Identification of Inhibitors of Cytokine Action
    33. UNIT 1.32 A Homogeneous Assay to Assess GABA Transporter Activity
    34. UNIT 1.33 In Vitro Muscarinic Receptor Radioligand-Binding Assays
    35. UNIT 1.34 Sigma Receptor Binding Assays
    36. UNIT 1.35 In Vitro Binding of [3H]PSB-0413 to P2Y12 Receptors
  5. Chapter 2 Signal Transduction
    1. Introduction
    2. UNIT 2.1 Overview of Signal Transduction
    3. UNIT 2.2 Adenylyl and Guanylyl Cyclase Assays
    4. UNIT 2.3 Overview of Phosphoinositide Hydrolysis
    5. UNIT 2.4 Nitric Oxide Synthase Assays
    6. UNIT 2.5 Analysis of Early Gene Responses
    7. UNIT 2.6 Agonist-Stimulated [35S]GTPγS Binding
    8. UNIT 2.7 Assay of Receptor-Stimulated Phosphoinositide Turnover
    9. UNIT 2.8 Evaluating Modulators of “Regulator of G-protein Signaling” (RGS) Proteins
    10. UNIT 2.9 The Human Kinome and Kinase Inhibition
    11. UNIT 2.10 Overview of Different Mechanisms of Arrestin-Mediated Signaling
    12. UNIT 2.11 Arrestin Expression in E. coli and Purification
    13. UNIT 2.12 Arrestin-3-Dependent Activation of c-Jun N-Terminal Kinases (JNKs)
    14. UNIT 2.13 Determination of GPCR Phosphorylation Status: Establishing a Phosphorylation Barcode
    15. UNIT 2.14 Using Bioluminescence Resonance Energy Transfer (BRET) to Characterize Agonist-Induced Arrestin Recruitment to Modified and Unmodified G Protein-Coupled Receptors
  6. Chapter 3 Enzyme Assays
    1. Introduction
    2. UNIT 3.1 Cyclooxygenase Assays
    3. UNIT 3.2 HIV Protease Assays
    4. UNIT 3.3 Topoisomerase Assays
    5. UNIT 3.4 Protein Farnesyltransferase Assays
    6. UNIT 3.5 Protein Tyrosine Kinase Activity Assays
    7. UNIT 3.6 Monoamine Oxidase Assays
    8. UNIT 3.7 Characterization of Matrix Metalloproteinase Inhibitors: Enzymatic Assays
    9. UNIT 3.8 In Vitro Enzymatic Assays of Protein Tyrosine Phosphatase 1B
    10. UNIT 3.9 Cytochrome P450 Assays
    11. UNIT 3.10 Measurement of Glutamate Carboxypeptidase II (NAALADase) Enzyme Activity by the Hydrolysis of [3H]-N-Acetylaspartylglutamate (NAAG)
    12. UNIT 3.11 In Vitro Enzymatic Assays for Ser/Thr-Selective Protein Kinases
    13. UNIT 3.12 Cyclic Nucleotide Phosphodiesterase Assay Technology
    14. UNIT 3.13 Assessing Sensitivity to Antibacterial Topoisomerase II Inhibitors
  7. Chapter 4 Isolated Tissues
    1. Introduction
    2. UNIT 4.1 Overview of Receptor Interactions of Agonists and Antagonists
    3. UNIT 4.2 Overview of Length-Tension Relationships in Isolated Tissue
    4. UNIT 4.3 Isolated Cardiac Muscle Assays
    5. UNIT 4.4 Isolated Blood Vessel Assays
    6. UNIT 4.5 α-Adrenoceptor Assays
    7. UNIT 4.6 β-Adrenoceptor Assays
    8. UNIT 4.7 P1 (Adenosine) Purinoceptor Assays
    9. UNIT 4.8 In Vitro Opioid Receptor Assays
    10. UNIT 4.9 Angiotensin II Receptor Assays
    11. UNIT 4.10 Tachykinin Receptor Assays
    12. UNIT 4.11 Neuropeptide Y (NPY) Receptor Assays
    13. UNIT 4.12 Nicotinic Acetylcholine Receptor Assays
    14. UNIT 4.13 Cholecystokinin (CCK) Assays
    15. UNIT 4.14 Dopamine 2 (DA-2) Receptor Assays Using Sympathetic Nerve Terminals
    16. UNIT 4.15 In Vitro Isolated Tissue Functional Muscarinic Receptor Assays
    17. UNIT 4.16 Cysteinyl Leukotriene Receptor Assays
    18. UNIT 4.17 Histamine Receptor Assays
    19. UNIT 4.18 Prostanoid Receptor Assays
    20. UNIT 4.19 5-Hydroxytryptamine Receptor Assays
  8. Chapter 5 Animal Models of Disease
    1. Introduction
    2. UNIT 5.1 Models of Cardiovascular Disease: Measurement of Arrhythmogenic/Antiarrhythmic Activity in the Guinea Pig
    3. UNIT 5.2 Models of Pulmonary Disease: Acute and Chronic Allergic Asthma in the Monkey and Acute and Chronic Viral Pulmonitis in the Mouse
    4. UNIT 5.3 Gastrointestinal Models: Intestinal Transit, Gastric Emptying, and Ulcerogenic Activity in the Rat
    5. UNIT 5.4 Models of Inflammation: Carrageenan- or Complete Freund's Adjuvant (CFA)–Induced Edema and Hypersensitivity in the Rat
    6. UNIT 5.5 Models of Inflammation: Adjuvant-Induced Arthritis in the Rat
    7. UNIT 5.6 Models of Inflammation: Carrageenan Air Pouch
    8. UNIT 5.7 Models of Pain: Hot-Plate and Formalin Test in Rodents
    9. UNIT 5.8 Rodent Models of Depression: Forced Swim and Tail Suspension Behavioral Despair Tests in Rats and Mice
    10. UNIT 5.9 Models of Affective Illness: Chronic Mild Stress in the Rat
    11. UNIT 5.10 Models of Urogenital Dysfunction: Benign Prostatic Hyperplasia (BPH)
    12. UNIT 5.11 Models of Urogenital Dysfunction: Urinary Urge Incontinence (UUI) in Anesthetized Dog and Rat Models
    13. UNIT 5.12 Models of Urogenital Dysfunction: Assessment of Volume-Evoked Micturition Reflex (VEMR) in the Conscious Rat by Cystometry
    14. UNIT 5.13 Models of Dementia: Delayed Alternation in Aged Rats
    15. UNIT 5.14 Models of Neurological Disease (Alzheimer's Disease): Spatial Discrimination Water Maze Test in Septal-Lesioned Rats
    16. UNIT 5.15 Models of Schizophrenia: Phencyclidine Disruption of Prepulse Inhibition (PPI) of Startle in Rats
    17. UNIT 5.16 Models of Anxiety: Stress-Induced Hyperthermia (SIH) in Singly Housed Mice
    18. UNIT 5.17 Models for Assessing Antipsychotics: Antagonism of Amphetamine-Induced Hyperactivity and Stereotypies in Mice
    19. UNIT 5.18 Models of Anxiety: Ultrasonic Vocalizations of Isolated Rat Pups
    20. UNIT 5.19 Models for Environmentally Induced Eating Disorders: Dietary Hyperphagia and Anorexia Nervosa
    21. UNIT 5.20 Overview of the Use of Transgenic Animals in CNS Drug Discovery
    22. UNIT 5.21 Models of Renal Insufficiency: The Anti-Thy-1.1 Model of Acute Proliferative Glomerulonephritis
    23. UNIT 5.22 Acute Seizure Tests in Epilepsy Research: Electroshock- and Chemical-Induced Convulsions in the Mouse
    24. UNIT 5.23 Characterization of Matrix Metalloproteinase Inhibitors: Angiogenesis and Tumor Models
    25. UNIT 5.24 Classic In Vivo Cancer Models: Three Examples of Mouse Models Used in Experimental Therapeutics
    26. UNIT 5.25 Overview of Animal Models of Asthma
    27. UNIT 5.26 Guinea Pig Models of Asthma
    28. UNIT 5.27 Models of Cardiac Ischemia-Reperfusion Injury in Dogs and Rats
    29. UNIT 5.28 Intraperitoneal and Subcutaneous Tumor Models for Assessing Anti-Neoplastic Agents in Rodents
    30. UNIT 5.29 Electrocardiographic Toxicity in the Guinea Pig
    31. UNIT 5.30 Social Recognition Task in the Rat
    32. UNIT 5.31 Emetic Liability Testing in Ferrets
    33. UNIT 5.32 Models of Neuropathic Pain in the Rat
    34. UNIT 5.33 Electrical Amygdala Kindling
    35. UNIT 5.34 A Rat Model of Postoperative Pain
    36. UNIT 5.35 Models of Muscle Pain: Carrageenan Model and Acidic Saline Model
    37. UNIT 5.36 Models of Visceral Pain: Colorectal Distension (CRD)
    38. UNIT 5.37 Model of Attention Deficit Hyperactivity Disorder: Five-Trial, Repeated Acquisition Inhibitory Avoidance in Spontaneously Hypertensive Rat Pups
    39. UNIT 5.38 Animal Tests of Anxiety
    40. UNIT 5.39 Measurement and Characterization of Energy Expenditure as a Tool in the Development of Drugs for Metabolic Diseases, such as Obesity and Diabetes
    41. UNIT 5.40 Measurement and Characterization of Energy Intake in the Mouse
    42. UNIT 5.41 Animal Models of Erectile Dysfunction
    43. UNIT 5.42 MPTP-Induced Models of Parkinson's Disease in Mice and Non-Human Primates
    44. UNIT 5.43 Dog EEG for Wake-Promotion Studies
    45. UNIT 5.44 Rat Model for Evaluation of Therapeutics on Peripheral Vascular Resistance
    46. UNIT 5.45 Orthostatic Hypotension Induced by Postural Change in the Rat (Tilt Test)
    47. UNIT 5.46 Mouse Models of Bleomycin-Induced Pulmonary Fibrosis
    48. UNIT 5.47 Streptozotocin-Induced Diabetic Models in Mice and Rats
    49. UNIT 5.48 Fear-Potentiated Startle and Light-Enhanced Startle Models in Drug Discovery
    50. UNIT 5.49 Rodent Model of Attention: The 5-Choice Serial Reaction Time Task
    51. UNIT 5.50 Cardiomyopathic Syrian Hamster as a Model of Congestive Heart Failure
    52. UNIT 5.51 Animal Models of Collagen-Induced Arthritis
    53. UNIT 5.52 A Model of Cystitis Pain in the Mouse
    54. UNIT 5.53 Models of Cardiovascular Disease: Measurement of Antihypertensive Activity in the Conscious Rat (SHR, DOCA-Salt, and Goldblatt Hypertension Models)
    55. UNIT 5.54 Models of Aspects of Schizophrenia: Behavioral Sensitization Induced by Subchronic Phencyclidine Administration
    56. UNIT 5.55 Chemically Induced Mouse Models of Colitis
    57. UNIT 5.56 Monocrotaline-Induced Pulmonary Hypertension in Wistar Rats
    58. UNIT 5.57 Overview of Mouse Models of Inflammatory Bowel Disease and Their Use in Drug Discovery
    59. UNIT 5.58 Methods of Inducing Inflammatory Bowel Disease in Mice
    60. UNIT 5.59 Novel Object Recognition in the Rat: A Facile Assay for Cognitive Function
    61. UNIT 5.60 Animal Models of Systemic Lupus Erythematosus (SLE) and Ex Vivo Assay Design for Drug Discovery
    62. UNIT 5.61 Overview of Animal Models of Obesity
    63. UNIT 5.62 Assessment of Substance Abuse Liability in Rodents: Self-Administration, Drug Discrimination, and Locomotor Sensitization
    64. UNIT 5.63 Water Maze Testing to Identify Compounds for Cognitive Enhancement
    65. UNIT 5.64 Cigarette Smoke Exposure as a Model of Inflammation Associated with COPD
    66. UNIT 5.65 Models of Depression: Unpredictable Chronic Mild Stress in Mice
    67. UNIT 5.66 Overview of Mouse Models of Autism Spectrum Disorders
    68. UNIT 5.67 Rodent Models of Amyotrophic Lateral Sclerosis
  9. Chapter 6 Molecular Biology
    1. Introduction
    2. UNIT 6.1 Overview of Receptor Cloning
    3. UNIT 6.2 Receptor Reporter Systems
    4. UNIT 6.3 Expression of Cloned Receptors in Mammalian Cell Lines
    5. UNIT 6.4 Isotopic Assays for Reporter Gene Activity
    6. UNIT 6.5 Nonisotopic Assays for Reporter Gene Activity
    7. UNIT 6.6 Use of the A. Victoria Green Fluorescent Protein to Study Protein Dynamics In Vivo
    8. UNIT 6.7 Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization
    9. UNIT 6.8 Mobility Shift DNA-Binding Assay Using Gel Electrophoresis
    10. UNIT 6.9 Tissue Expression Profiling Using Real-Time PCR
    11. UNIT 6.10 Overview of Pharmacogenetics
    12. UNIT 6.11 Pharmacological Applications of Baculovirus-Mediated Protein Expression in Mammalian Cells
  10. Chapter 7 Pharmacokinetics
    1. Introduction
    2. UNIT 7.1 Overview of Pharmacokinetics
    3. UNIT 7.2 Estimating Intestinal Mucosal Permeation of Compounds Using Caco-2 Cell Monolayers
    4. UNIT 7.3 Overview of Drug Product Development
    5. UNIT 7.4 In Vivo Measurement of Blood-Brain Barrier Permeability
    6. UNIT 7.5 Determination of Compound Binding to Plasma Proteins
    7. UNIT 7.6 Determination of the Stability of Drugs in Plasma
    8. UNIT 7.7 Uptake Studies for Evaluating Activity of Efflux Transporters in a Cell Line Representative of the Blood-Brain Barrier
    9. UNIT 7.8 In Vitro Drug Metabolism Using Liver Microsomes
    10. UNIT 7.9 Hepatic Clearance and Drug Metabolism Using Isolated Perfused Rat Liver
    11. UNIT 7.10 In Vitro Species Comparisons and Metabolite Identification
    12. UNIT 7.11 Cytochrome P450 Inhibition Assays Using Traditional and Fluorescent Substrates
    13. UNIT 7.12 The Ussing Chamber and Measurement of Drug Actions on Mucosal Ion Transport
    14. UNIT 7.13 Assessment of P-Glycoprotein Substrate and Inhibition Potential of Test Compounds in MDR1-Transfected MDCK Cells
    15. UNIT 7.14 Assessment of the Time-Dependent Inhibition (TDI) Potential of Test Compounds with Human Liver Microsomes by IC50 Shift Method Using a Nondilution Approach
    16. UNIT 7.15 Overview of Experimental Models of the Blood-Brain Barrier in CNS Drug Discovery
  11. Chapter 8 Receptor/Enzyme Localization
    1. Introduction
    2. UNIT 8.1 In Vitro Autoradiography
    3. UNIT 8.2 Autoradiographic Localization of Growth Factor Receptors in Neuronal Tissues
  12. Chapter 9 Drug Discovery Technologies
    1. Introduction
    2. UNIT 9.1 Overview of Chemical Diversity
    3. UNIT 9.2 Cell-Based Assays Using the Fluorometric Imaging Plate Reader (FLIPR)
    4. UNIT 9.3 Overview of Combinatorial Chemistry
    5. UNIT 9.4 Overview of High-Throughput Screening
    6. UNIT 9.5 Testing for Inverse Agonism with Constitutive Receptor Systems
    7. UNIT 9.6 Overview of Microarrays in Drug Discovery and Development
    8. UNIT 9.7 Overview on the Use of Therapeutic Antibodies in Drug Discovery
    9. UNIT 9.8 Approaches to the Molecular Modeling of 7-Transmembrane Helical Receptors
    10. UNIT 9.9 Overview of Drug Discovery and Development
    11. UNIT 9.10 The IND Application
    12. UNIT 9.11 Natural Products as a Foundation for Drug Discovery
    13. UNIT 9.12 Overview on the Rule of Five
    14. UNIT 9.13 Label-Free Imaging and Temporal Signature in Phenotypic Cellular Assays: A New Approach to High-Content Screening
    15. UNIT 9.14 Measuring Receptor Target Coverage: A Radioligand Competition Binding Protocol for Assessing the Association and Dissociation Rates of Unlabeled Compounds
    16. UNIT 9.15 High Throughput Functional Assays for P2X Receptors
    17. UNIT 9.16 Parallel Chemistry in the 21st Century
    18. UNIT 9.17 Overview of Determination of Biopharmaceutical Properties for Development Candidate Selection
    19. UNIT 9.18 A High-Throughput (HTS) Assay for Enzyme Reaction Phenotyping in Human Recombinant P450 Enzymes Using LC-MS/MS
    20. UNIT 9.19 Drug Combinations: Tests and Analysis with Isoboles
  13. Chapter 10 Safety Pharmacology/Toxicology
    1. Introduction
    2. UNIT 10.1 Overview of Safety Pharmacology
    3. UNIT 10.2 Models of Inflammation: Measuring Gastrointestinal Ulceration in the Rat
    4. UNIT 10.3 Toxicology in the Drug Discovery and Development Process
    5. UNIT 10.4 Place Preference Test in Rodents
    6. UNIT 10.5 Models of Neurological Disease (Substance Abuse): Self-Administration in Monkeys
    7. UNIT 10.6 Vigilance-Controlled Quantified EEG in Safety Pharmacology
    8. UNIT 10.7 An Overview of QT Interval Assessment in Safety Pharmacology
    9. UNIT 10.8 Patch-Clamp Studies of Human Cardiac Ion Channels in the Evaluation of Cardiac Electrophysiological Effects of Compounds
    10. UNIT 10.9 Respiratory Function Assessment in Safety Pharmacology
    11. UNIT 10.10 Primary Observation (Irwin) Test in Rodents for Assessing Acute Toxicity of a Test Agent and its Effects on Behavior and Physiological Function
    12. UNIT 10.11 Head-Out Plethysmography in Safety Pharmacology Assessment
    13. UNIT 10.12 Evaluation of Neurotoxicity Potential in Rats: The Functional Observational Battery
    14. UNIT 10.13 The Fentanyl/Etomidate-Anesthetized Beagle (FEAB) Model in Safety Pharmacology Assessment
    15. UNIT 10.14 Measurement of Action Potential Generation in Isolated Canine Left Ventricular Midmyocardial Myocytes
    16. UNIT 10.15 Whole-Cell Configuration of the Patch-Clamp Technique in the hERG Channel Assay to Predict the Ability of a Compound to Prolong QT Interval
    17. UNIT 10.16 Predicting Drug-Induced QT Prolongation and Torsades de Pointes: A Review of Preclinical Endpoint Measures
  14. Chapter 11 Electrophysiological Techniques
    1. UNIT 11.0 Electrophysiological Techniques
    2. UNIT 11.1 Overview of Electrophysiological Techniques
    3. UNIT 11.2 Electrophysiological Analysis of G Protein–Coupled Receptors in Mammalian Neurons
    4. UNIT 11.3 The Action Potential of the Purkinje Fiber: An In Vitro Model for Evaluation of the Proarrhythmic Potential of Cardiac and Noncardiac Drugs
    5. UNIT 11.4 Use of Electrophysiological Methods in the Study of Recombinant and Native Neuronal Ligand-Gated Ion Channels
    6. UNIT 11.5 Electrophysiological Analysis of Heterologously Expressed Kv and SK/IK Potassium Channels
    7. UNIT 11.6 Electrophysiological Analysis of ATP-Sensitive Potassium Channels in Mammalian Cells and Xenopus Oocytes
    8. UNIT 11.7 Overview of Electrophysiological Characterization of Neuronal Nicotinic Acetylcholine Receptors
    9. UNIT 11.8 Electrophysiological Analysis of Tetrodotoxin-Resistant Sodium Channel Pharmacology
    10. UNIT 11.9 Electrophysiological Characterization of Recombinant and Native P2X Receptors
    11. UNIT 11.10 Electrophysiology of Airway Nerves
    12. UNIT 11.11 Electrophysiological Techniques for Studying Synaptic Activity In Vivo
    13. UNIT 11.12 Functional Characterization of Human Stem Cell–Derived Cardiomyocytes
    14. UNIT 11.13 Automated Patch Clamp Analysis of nAChα7 and NaV1.7 Channels
    15. UNIT 11.14 Electrophysiological Studies of Voltage-Gated Sodium Channels Using QPatch HT, an Automated Patch-Clamp System
    16. UNIT 11.15 In Vivo Electrophysiological Recording Techniques for the Study of Neuropathic Pain in Rodent Models
    17. UNIT 11.16 Single-Channel Recording of Glutamate Receptors
    18. UNIT 11.17 Single-Channel Analysis of Glutamate Receptors
    19. UNIT 11.18 A Human Induced Pluripotent Stem Cell−Derived Cardiomyocyte (hiPSC-CM) Multielectrode Array Assay for Preclinical Cardiac Electrophysiology Safety Screening
  15. Chapter 12 In Vitro Cellular Assays
    1. Introduction
    2. UNIT 12.1 Overview of Cell and Tissue Culture Techniques
    3. UNIT 12.2 Chondrocyte Culture and Assay
    4. UNIT 12.3 Preparation of Nuclear and Cytoplasmic Extracts from Mammalian Cells
    5. UNIT 12.4 Cellular Assays of Chemokine Receptor Activation
    6. UNIT 12.5 Production and Use of HIV-1 Luciferase Reporter Viruses
    7. UNIT 12.6 Methods of Measuring Internalization of G Protein–Coupled Receptors
    8. UNIT 12.7 Measurement of VLA-4/CS-1 and VLA-4/VCAM Adhesion Inhibition
    9. UNIT 12.8 Measurement of Cell Death in Mammalian Cells
    10. UNIT 12.9 Functional Screening in the Melanophore Bioassay
    11. UNIT 12.10 Detection of Nitros(yl)ated Metabolites of Nitric Oxide (NO) In Vivo by Gas-Phase Chemiluminescence Assay
    12. UNIT 12.11 Drug Testing in Cellular Chemotaxis Assays
    13. UNIT 12.12 In Vitro Assay of Angiogenesis: Inhibition of Capillary Tube Formation
    14. UNIT 12.13 In Vivo Pharmacodynamic Assays for M2 and M3 Muscarinic Receptors
    15. UNIT 12.14 Measurement of Glucose Uptake in Cultured Cells
    16. UNIT 12.15 Harvesting, Isolation, and Functional Assessment of Primary Vagal Ganglia Cells
  16. Chapter 13 Anti-Infectives
    1. Introduction
    2. Section A Antibacterials
      1. UNIT 13A.1 Overview of Anti-Infective Drug Development
      2. UNIT 13A.2 Overview of Antibacterial Target Selection
      3. UNIT 13A.3 Basic Microbiological Techniques Used in Antibacterial Drug Discovery
      4. UNIT 13A.4 Primary Rodent Infection Models for Testing Antibacterial Compound Efficacy In Vivo
      5. UNIT 13A.5 In Vivo Animal Models: Quantitative Models Used for Identifying Antibacterial Agents
      6. UNIT 13A.6 In Vitro Antibacterial Resistance Selection and Quantitation
      7. UNIT 13A.7 Macromolecular Synthesis and Membrane Perturbation Assays for Mechanisms of Action Studies of Antimicrobial Agents
      8. UNIT 13A.8 Static Biofilm Cultures of Gram-Positive Pathogens Grown in a Microtiter Format Used for Anti-Biofilm Drug Discovery
    3. Section B Antivirals
      1. UNIT 13B.1 Evaluation of Compound Activity Against Hepatitis C Virus in Replicon Systems
      2. UNIT 13B.2 Studies on Hepatitis C Virus Resistance to Inhibitors in Replicon Systems
      3. UNIT 13B.3 High-Throughput Screening of Viral Entry Inhibitors Using Pseudotyped Virus
      4. UNIT 13B.4 Generation and Quantitation of Infectious Hepatitis C Virus Derived from Cell Culture (HCVcc)
      5. UNIT 13B.5 In Vitro Anti-Hepatitis C Virus (HCV) Resistance Selection and Characterization
      6. UNIT 13B.6 Preparation of HCV NS3 and NS5B Proteins to Support Small-Molecule Drug Discovery
      7. UNIT 13B.7 Biochemical Evaluation of HCV NS3 Protease Inhibitors
  17. Chapter 14 Cellular and Animal Models in Oncology and Tumor Biology
    1. Introduction
    2. UNIT 14.1 Murine and Canine Models of Appendicular Osteosarcoma
    3. UNIT 14.2 Carcinogen-Induced Animal Models of Head and Neck Squamous Cell Carcinoma
    4. UNIT 14.3 Orthotopic Model of Human Pancreatic Ductal Adenocarcinoma and Cancer Cachexia in Nude Mice
    5. UNIT 14.4 Orthotopic Models of Human Gastric Carcinoma in Nude Mice: Applications for Study of Tumor Growth and Progression
    6. UNIT 14.5 Metastatic Model of Colon Carcinoma in Mice: Utility in the Study of Tumor Growth and Progression
    7. UNIT 14.6 Models of Melanoma Metastasis: Using a Transient siRNA-Based Protein Inhibition Strategy in Mice to Validate the Functional Relevance of Pharmacological Agents
    8. UNIT 14.7 Application of Radiotherapy and Chemotherapy Protocols to Pre-Clinical Tumor Models
    9. UNIT 14.8 Preclinical Chemotherapeutic Tumor Models of Common Childhood Cancers: Solid Tumors, Acute Lymphoblastic Leukemia, and Disseminated Neuroblastoma
    10. UNIT 14.9 Animal Models of Multiple Myeloma and Their Utility in Drug Discovery
    11. UNIT 14.10 Murine Retroviral Bone Marrow Transplantation Models for the Study of Human Myeloproliferative Disorders
    12. UNIT 14.11 Genetically Engineered Mouse Models of Ovarian Cancer and Their Utility in Drug Discovery
    13. UNIT 14.12 Xenograft and Transgenic Mouse Models of Epithelial Ovarian Cancer and Non-Invasive Imaging Modalities to Monitor Ovarian Tumor Growth In Situ: Applications in Evaluating Novel Therapeutic Agents
    14. UNIT 14.13 Pre-Clinical Models of Renal Carcinoma and Their Utility in Drug Development
    15. UNIT 14.14 Mouse Models of Human Bladder Cancer as a Tool for Drug Discovery
    16. UNIT 14.15 Preclinical Mouse Models of Human Prostate Cancer and Their Utility in Drug Discovery
    17. UNIT 14.16 Establishment, Maintenance, and In Vitro and In Vivo Applications of Primary Human Glioblastoma Multiforme (GBM) Xenograft Models for Translational Biology Studies and Drug Discovery
    18. UNIT 14.17 Preclinical Models of Pediatric Solid Tumors (Neuroblastoma) and Their Use in Drug Discovery
    19. UNIT 14.18 Preclinical Orthotopic and Intracardiac Injection Models of Human Breast Cancer Metastasis to Bone and Their Use in Drug Discovery
    20. UNIT 14.19 Orthotopic Xenograft Model of Cervical Cancer for Studying Microenvironmental Effects on Metastasis Formation and Response to Drug Treatment
    21. UNIT 14.20 Orthotopic Models of Esophageal Carcinoma and Their Use in Drug Discovery
    22. UNIT 14.21 Pre-Clinical Mouse Models of Primary and Metastatic Pleural Cancers of the Lung and Breast and the Use of Bioluminescent Imaging to Monitor Pleural Tumor Burden
    23. UNIT 14.22 Overview of Human Primary Tumorgraft Models: Comparisons with Traditional Oncology Preclinical Models and the Clinical Relevance and Utility of Primary Tumorgrafts in Basic and Translational Oncology Research
    24. UNIT 14.23 Patient-Derived Models of Human Breast Cancer: Protocols for In Vitro and In Vivo Applications in Tumor Biology and Translational Medicine
    25. UNIT 14.24 Discovery of Biologically Active Oncologic and Immunologic Small Molecule Therapies using Zebrafish: Overview and Example of Modulation of T Cell Activation
    26. UNIT 14.25 Overview of Cancer Stem Cells (CSCs) and Mechanisms of Their Regulation: Implications for Cancer Therapy
    27. UNIT 14.26 Primary Human Non-Small Cell Lung and Pancreatic Tumorgraft Models—Utility and Applications in Drug Discovery and Tumor Biology
    28. UNIT 14.27 Utility and Applications of Orthotopic Models of Human Non-Small Cell Lung Cancer (NSCLC) for the Evaluation of Novel and Emerging Cancer Therapeutics
    29. UNIT 14.28 Isolation and Characterization of Potential Cancer Stem Cells from Solid Human Tumors—Potential Applications
    30. UNIT 14.29 Overview of Genetically Engineered Mouse Models of Colorectal Carcinoma to Enable Translational Biology and Drug Development
    31. UNIT 14.30 The DEN and CCl4-Induced Mouse Model of Fibrosis and Inflammation-Associated Hepatocellular Carcinoma
    32. UNIT 14.31 Modeling Human Liver Cancer Heterogeneity: Virally Induced Transgenic Models and Mouse Genetic Models of Chronic Liver Inflammation
    33. UNIT 14.32 Generation of Human Acute Lymphoblastic Leukemia Xenografts for Use in Oncology Drug Discovery
    34. UNIT 14.33 Overview of Genetically Engineered Mouse Models of Papillary and Anaplastic Thyroid Cancers: Enabling Translational Biology for Patient Care Improvement
    35. UNIT 14.34 Murine Models of Helicobacter (pylori or felis)-associated Gastric Cancer
    36. UNIT 14.35 Overview of KRAS-Driven Genetically Engineered Mouse Models of Non-Small Cell Lung Cancer
    37. UNIT 14.36 Overview of Genetically Engineered Mouse Models of Breast Cancer Used in Translational Biology and Drug Development
    38. UNIT 14.37 Overview of Transgenic Glioblastoma and Oligoastrocytoma CNS Models and Their Utility in Drug Discovery
    39. UNIT 14.38 Overview of Genetically Engineered Mouse Models of Distinct Breast Cancer Subtypes
  18. Appendix 1 Nomenclature and Useful Data
    1. APPENDIX 1A Enzyme Nomenclature
    2. APPENDIX 1B Receptor Nomenclature Guidelines
  19. Appendix 2 Stock Solutions, Equipment, and Laboratory Guidelines
    1. APPENDIX 2A Common Stock Solutions, Buffers, and Media
    2. APPENDIX 2B Standard Laboratory Equipment and Supplies
  20. Appendix 3 Standard Techniques
    1. APPENDIX 3A Assays for Determination of Protein Concentration
    2. APPENDIX 3B Protein Analysis by SDS-PAGE and Detection by Coomassie Blue or Silver Staining
    3. APPENDIX 3C Purification and Concentration of DNA from Aqueous Solutions
    4. APPENDIX 3D Fixation, Embedding, and Sectioning of Tissues, Embryos, and Single Cells
    5. APPENDIX 3E Cryosectioning
    6. APPENDIX 3F In Situ Hybridization to Cellular RNA
  21. Appendix 4 Animal Care Guidelines
    1. APPENDIX 4 Guidelines for Animal Care and Use in Biomedical Research
    2. APPENDIX 4B Guidelines for the Care and Use of Laboratory Animals in Biomedical Research