Springer protocols feed by Pharmacology/Toxicology 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Virus-Mediated Expression of DREADDs for In Vivo Metabolic Studies
During the past few years, CNO-sensitive designer G protein-coupled receptors (GPCRs) known as DREADDs (designer receptors exclusively activated by designer drugs) have emerged as powerful new tools for the study of GPCR physiology. In this chapter, we present protocols employing adeno-associated viruses (AAVs) to express a Gq-coupled DREADD (Dq) in two metabolically important cell types, AgRP neurons of the hypothalamus and hepatocytes of the liver. We also provide examples dealing with the metabolic analysis of the Dq mutant mice after administration of CNO in vivo. The approaches described in this chapter can be applied...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news
Bioluminescence Resonance Energy Transfer Approaches to Discover Bias in GPCR Signaling
Bioluminescence resonance energy transfer (BRET) is a well-established technique for investigating G protein-coupled receptor (GPCR) pharmacology. BRET enables the monitoring of molecular proximity through the use of heterologously expressed proteins of interest and/or fluorophore-labeled ligands. Fusion to a donor luciferase enzyme or an acceptor fluorophore and subsequent detection of resonance energy transfer indicate the close proximity of the molecules of interest. As BRET is readily applied to the study of numerous GPCR signaling and regulatory paths, it is an ideal technique for investigating the pharmacology of bia...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news
Approaches to Assess Functional Selectivity in GPCRs: Evaluating G Protein Signaling in an Endogenous Environment
This report presents our method and offers tips for evaluating G protein signaling in endogenous tissues. Predominately, brain tissues are discussed here; optimization points that can be applied to any tissues are highlighted. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news
The Measurement of Receptor Signaling Bias
This chapter describes a method to quantify biased signaling effects of agonists; this approach can furnish a scale for medicinal chemists to optimize biased profiles. Biased ligands have different pharmacological properties on a molecular level (stabilization of different receptor active states) and thus can have different pharmacological profiles therapeutically. The calculation of transduction ratios (ΔΔlog(τ/K
A)) values (where τ is efficacy and K
A a measure of affinity) allows the identification of agonists that demonstrate unique signaling either for different signaling pathways linked to the rec...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news
Detection and Quantification of Intracellular Signaling Using FRET-Based Biosensors and High Content Imaging
Förster resonance energy transfer (FRET) biosensors represent invaluable tools to detect the spatiotemporal context of second messenger production and intracellular signaling that cannot be attained using traditional methods. Here, we describe a detailed protocol for the use of high content imaging in combination with FRET biosensors to assess second messenger production and intracellular signaling in a time-effective manner. We use four different FRET biosensors to measure cAMP levels, kinase (ERK and PKC), and GTPase activity. Importantly, we provide the protocols to express and measure these sensors in a variety of...
Source: Springer protocols feed by Pharmacology/Toxicology - August 11, 2015 Category: Drugs & Pharmacology Source Type: news
A Systems Toxicology Approach to Investigating the Cardiovascular Effects of Cigarette Smoke and Environmental Pollutants in ApoE-Deficient Mice
Epidemiological evidence indicates that exposure to combustion-derived particles is linked to an increased risk of cardiovascular disease. Despite this strong association, there remains a lack of data that can be used to identify the molecular mechanisms through which exposure to particulate matter (PM) leads to adverse cardiovascular events. The biological complexity of the responses generated by exposure to PM is compounded by the inherent multidimensional nature of the chemical mixtures associated with PM emitted from a range of sources, including diesel and gasoline exhausts, cigarette smoke, and ambient particles. The...
Source: Springer protocols feed by Pharmacology/Toxicology - July 23, 2015 Category: Drugs & Pharmacology Source Type: news
High-Content Screening: Understanding and Managing Mechanistic Data to Better Predict Toxicity
An increased understanding of the cellular pathways involved in toxicity responses, coupled with a simultaneous advance in technology, has allowed for a shift in the way that the safety assessment of novel chemicals is performed. The development of assays that offer a high-throughput and low-cost option in comparison to more traditional approaches has been a focus of recent years. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - July 23, 2015 Category: Drugs & Pharmacology Source Type: news
Xenobiotic Metabolism Activation as a Biomarker of Cigarette Smoke Exposure Response
The recent advances in “omics” technologies have generated various in silico approaches for toxicity assessment. In silico-based toxicity predictions can overcome certain major drawbacks of laboratory experiments, including the limitation of conducting experiments in a chemical-by-chemical basis that can be expensive. This chapter discusses some recent applications of in silico approaches utilizing xenobiotic metabolism that can be used to assess the impact of cigarette smoke (CS). We first outline recent studies using quantum mechanics/molecular modeling and quantitative structure–activity relationships ...
Source: Springer protocols feed by Pharmacology/Toxicology - July 23, 2015 Category: Drugs & Pharmacology Source Type: news
Analysis of Proteomic Data for Toxicological Applications
Toward a comprehensive characterization of toxicant responses, systems toxicology requires the integration of different data modalities. Proteomics approaches measure changes in the levels of proteins and their posttranslational modifications, which can closely reflect the biological effects of a toxicant. With a focus on mass spectrometry-based proteomics, we describe the isobaric tag-based approach for quantitative proteomics (iTRAQ® and TMT™) and describe computational approaches to derive biological/mechanistic insights. Specifically, we describe the generation and quantification of mass-spectrometry data and...
Source: Springer protocols feed by Pharmacology/Toxicology - July 23, 2015 Category: Drugs & Pharmacology Source Type: news
Quantifying the Biological Impact of Active Substances Using Causal Network Models
In this chapter a five-step strategy is described that provides comparative evaluations of the effects of biologically active substances. These evaluations constitute an integral part of the determination of the risks for the human population to exposure to these substances. The strategy is based on the concept of biological impact quantification for which novel computational methodologies have been developed in the past few years; these methodologies are reviewed in this chapter. The effects of the active substances are then described in terms of networks containing the biological mechanisms involved in the response to th...
Source: Springer protocols feed by Pharmacology/Toxicology - July 23, 2015 Category: Drugs & Pharmacology Source Type: news
Mitochondrial Targeting of Catalytic RNAs
We describe here mitochondrial targeting of trans-cleaving ribozymes destined to knockdown organelle RNAs for regulation studies and inverse genetics and biotechnological purposes. The design and functional assessment of chimeric RNAs combining the ribozyme and the mitochondrial shuttle are detailed, followed by all procedures to prepare constructs for in vivo expression, generate stable plant transformants, and establish target RNA knockdown in mitochondria. (Source: Springer protocols feed by Pharmacology/Toxicology)
Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news
Mitochondrial Targeting of Recombinant RNA
Mitochondrial import of small noncoding RNA is found in a large variety of species. In mammalian cells, this pathway can be used for therapeutic purpose, to restore the mitochondrial functions affected by pathogenic mutations. Recently, we developed mitochondrial RNA vectors able to address therapeutic oligoribonucleotides into human mitochondria. Here we provide the protocol for transfection of cultured human cells with small recombinant RNA molecules and describe two approaches useful to demonstrate their import into mitochondria: (1) isolation of RNA from purified mitochondria and quantitative hybridization analysis and...
Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news
Allotopic Expression of ATP6 in the Mouse as a Transgenic Model of Mitochondrial Disease
Progress in animal modeling of polymorphisms and mutations in mitochondrial DNA (mtDNA) is not as developed as nuclear transgenesis due to a host of cellular and physiological distinctions. mtDNA mutation modeling is of critical importance as mutations in the mitochondrial genome give rise to a variety of pathological conditions and play a contributing role in many others. Nuclear localization and transcription of mtDNA genes followed by cytoplasmic translation and transport into mitochondria (allotopic expression, AE) provide an opportunity to create in vivo modeling of a targeted mutation in mitochondrial genes and has b...
Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news
Analysis of Pollutant-Induced Changes in Mitochondrial DNA Methylation
There is increasing evidence that exposure to air pollutants is associated with human disease and may act through epigenetic modification of the nuclear genome, but there have been few publications describing their impact upon the mitochondrial genome. Mitochondrial DNA may be more susceptible to pollutant-induced changes via increased oxidative stress in the cell, and therefore this field of research is of growing interest. Many techniques employed to study DNA methylation of the nuclear genome are also applicable to mitochondrial epigenetic studies. In this chapter, we describe a protocol for the isolation of mitochondri...
Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news
ETS and DEAS Studies of the Reduction of Xenobiotics in Mitochondrial Intermembrane Space
This chapter describes the complementary experimental techniques electron transmission spectroscopy (ETS) and dissociative electron attachment spectroscopy (DEAS), two of the most suitable means for investigating interactions between electrons and gas-phase molecules, resonance formation of temporary molecular negative ions, and their possible decay through the dissociative electron attachment (DEA) mechanism. The latter can be seen as the gas-phase counterpart of the transfer of a solvated electron in solution, accompanied by dissociation of the molecular anion, referred to as dissociative electron transfer (DET). DET tak...
Source: Springer protocols feed by Pharmacology/Toxicology - February 5, 2015 Category: Drugs & Pharmacology Source Type: news
