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Current Protocols in Pharmacology

Michael Williams
Many results reported in the biomedical research literature cannot be independently reproduced, undermining the basic foundations of science. This overview is intended for researchers who are committed to improving the quality and integrity of biomedical science by raising awareness of both the sources of irreproducibility, and activities specifically targeted to address the issue. The irreproducibility of biomedical research is due to a variety of factors, known and unknown, that markedly influence experimental outcomes...
June 2018: Current Protocols in Pharmacology
Gianluca Civenni, Giuseppina M Carbone, Carlo V Catapano
Prostate cancer (PCa) is the most common malignant visceral neoplasm in males in Western countries. Despite progress made in the early treatment of localized malignancies, there remains a need for therapies effective against advanced forms of the disease. Genetically engineered mouse (GEM) models are valuable tools for addressing this issue, particularly in defining the cellular and molecular mechanisms responsible for tumor initiation and progression. While cell and tissue culture systems are important models for this purpose as well, they cannot recapitulate the complex interactions within heterotypic cells and the tumor microenvironment that are crucial in the initiation and progression of prostate tumors...
June 2018: Current Protocols in Pharmacology
Dan A Klaerke, Maria de Los Angeles Tejada, Vibeke Grøsfjeld Christensen, Mette Lassen, Per Amstrup Pedersen, Kirstine Calloe
Detergent-solubilized purified ion channels can be reconstituted into lipid bilayers for electrophysiological analysis. Traditionally, ion channels were inserted into vesicles and subsequently fused with planar "black lipid membranes" formed from lipids dissolved in a hydrophobic solvent such as decane. Provided in this article is a step-by-step guide to reconstitute purified ion channel proteins into giant unilamellar vesicles (GUVs). This procedure results in the formation of proteoliposomes that can be used for planar bilayer formation and electrophysiological characterization of single-channel currents...
June 2018: Current Protocols in Pharmacology
Doodipala Samba Reddy, Xin Wu, Victoria M Golub, W Mohaiza Dashwood, Roderick H Dashwood
Histone deacetylases (HDACs) represent a family of enzymes that are targets for epigenetic modulation of genomic activity and may be beneficial in the treatment of many diseases, including cancer and central nervous system disorders. In animal models, HDAC inhibitors have neuroprotective, antiepileptogenic, and antidepressant effects. Assaying HDAC activity provides a robust method for identifying HDAC inhibitors and for assessing their effects under various physiological conditions or after pathological insults...
June 2018: Current Protocols in Pharmacology
Matthew R Durk
Intracerebral microdialysis is a powerful technique for quantifying unbound brain extracellular fluid concentrations of drugs and drug candidates over time in live, conscious, freely moving animals. The results provide crucial information on the brain penetrance of the administered agent. This unit details the surgical procedures, study planning and execution, and data analysis required for a brain microdialysis study following intravenous infusion of a test substance to steady state. The accumulated data makes possible the determination of the area under the concentration-time curve ratio and steady-state concentration ratio for unbound compound in rat brain extracellular fluid as compared to that in plasma (Kpu,u )...
March 2018: Current Protocols in Pharmacology
Shubha Gururaja Rao, Devasena Ponnalagu, Neel J Patel, Harpreet Singh
Intracellular organelles are membranous structures central for maintaining cellular physiology and the overall health of the cell. To maintain cellular function, intracellular organelles are required to tightly regulate their ionic homeostasis. Any imbalance in ionic concentrations can disrupt energy production (mitochondria), protein degradation (lysosomes), DNA replication (nucleus), or cellular signaling (endoplasmic reticulum). Ionic homeostasis is also important for volume regulation of intracellular organelles and is maintained by cation and anion channels as well as transporters...
March 2018: Current Protocols in Pharmacology
Nina I Zolotarjova, Richard Wynn
Bromodomains are protein domains that recognize acetylated lysine residues and are important for recruiting a large number of protein and multiprotein complexes to sites of lysine acetylation. They play an important role in chromatin biology and are popular targets for drug discovery. Compound screening in this area requires the use of biochemical assays to assess the binding potency of potential drug candidates. Foremost among the efforts to target bromodomains are those aimed at identifying compounds that interact with the bromodomain and extra-terminal domain (BET) family of bromodomain-containing proteins (BRD2, BRD3, BRD4, and BRDT)...
March 2018: Current Protocols in Pharmacology
Hanna Tarhonskaya, Anthony Tumber, Akane Kawamura, Christopher J Schofield
Histone modifications, including lysine methylation marks on histone tails, modulate the accessibility of genes for transcription. Changes in histone tail methylation patterns can cause transcriptional activation or repression. The dynamic regulation of lysine methylation patterns is enabled by two distinct groups of enzymes: histone methyltransferases (KMTs) and demethylases (KDMs). The Jumonji C (JmjC) domain-containing lysine histone demethylases (JmjC-KDMs) alter the methylation levels of histone tails by removing tri-, di-, or mono-methylation marks...
March 2018: Current Protocols in Pharmacology
James A L Brown
Acetylation is a core cellular process involved in maintaining genomic integrity, gene regulation, and metabolism. Histone acetyltransferases (HATs) are an enzyme family that regulates these processes by catalyzing the transfer of an acetyl moiety onto target proteins. Perturbations of cellular acetylation profiles have been associated with a variety of disease states, including cancer. Changes in acetylation profiles can be achieved by mechanisms associated with acetyltransferases, such as gene down-regulation or alterations in the activity of key acetyltransferase enzymes...
December 20, 2017: Current Protocols in Pharmacology
Shaili Aggarwal, Ole V Mortensen
The dopamine (DAT), serotonin (SERT), and norepinephrine (NET) transporters, which are collectively referred to as monoamine transporters (MATs), play significant roles in regulating the neuronal response to these neurotransmitters. MATs terminate the action of these neurotransmitters by translocating them from the synaptic space into the presynaptic neurons. These three transmitters are responsible for controlling a number of physiological, emotional, and behavioral functions, with their transporters being the site of action of drugs employed for the treatment of a variety of conditions, including depression, anxiety, ADHD, schizophrenia, and psychostimulant abuse...
December 20, 2017: Current Protocols in Pharmacology
Sujay Ramanathan, Botros B Shenoda, Seena K Ajit
MicroRNA(miRNA)-mediated gene regulation underlies cellular processes, playing an important role in homeostasis and diseases. The expression and function of miRNAs are altered by various pharmacological agents, with differences in the endogenous levels of miRNAs influencing drug efficacy and toxicity. Thus, miRNA levels could be a biomarker for predicting treatment response, efficacy, and safety. In addition, elucidating the mechanistic significance of miRNA alterations can aid in the identification of therapeutic targets and patient selection, and guide personalized therapy...
December 20, 2017: Current Protocols in Pharmacology
Shaili Aggarwal, Ole V Mortensen
Detailed in this unit are protocols for studying the in vitro uptake of dopamine (DA) as a means for defining the functional characteristics of dopamine transporters. All assays are performed using commercially available cell lines that transiently express the transporter under investigation. The three main assays provided are: a kinetic assay to calculate the affinity (KM ) and maximal velocity (Vmax ) of radiolabeled DA uptake into cells; concentration-response assays to measure the potencies (IC50 /Ki values) of test compounds as transport inhibitors; and an efflux assay to assess the ability and potency (EC50 ) of a ligand to elicit reverse transport of DA accumulated in the cell...
December 20, 2017: Current Protocols in Pharmacology
Botros B Shenoda, Sujay Ramanathan, Seena K Ajit
Pharmacogenomic approaches used to investigate how genes affect drug responses are critical for designing personalized therapies aimed at maximizing efficacy and minimizing adverse effects. Drug efficacy is often dependent on the sequence and expression levels of drug target genes or those involved in the metabolism and transport of the therapeutic agent. Expression of these genes, in turn, is negatively regulated by small noncoding miRNAs. The levels of miRNAs in bodily fluids have been studied extensively as potential diagnostic and prognostic biomarkers...
December 20, 2017: Current Protocols in Pharmacology
Matthew C Stubbs, Andrei V Krivtsov
MLL-rearranged leukemia represents approximately 5% to 10% of adult acute myelogenous leukemia (AML) and nearly half of all infant/pediatric acute leukemia cases. These leukemias have a poor prognosis, and there are no approved therapeutic options. The rearrangement in the MLL gene leads to aberrant expression of MLL-fusion proteins. These are transforming in murine bone marrow and, in particular, on stem cells and myeloid progenitors derived from bone marrow or fetal liver. The commonality of the MLL fusions is the in-frame fusion of 8 to 11 N-terminal exons of MLL1 (KMT2a) with the C-terminus of a partner fusion gene...
September 11, 2017: Current Protocols in Pharmacology
Christopher A Werley, Ted Brookings, Hansini Upadhyay, Luis A Williams, Owen B McManus, Graham T Dempsey
A key challenge for establishing a phenotypic screen for neuronal excitability is measurement of membrane potential changes with high throughput and accuracy. Most approaches for probing excitability rely on low-throughput, invasive methods or lack cell-specific information. These limitations stimulated the development of novel strategies for characterizing the electrical properties of cultured neurons. Among these was the development of optogenetic technologies (Optopatch) that allow for stimulation and recording of membrane voltage signals from cultured neurons with single-cell sensitivity and millisecond temporal resolution...
September 11, 2017: Current Protocols in Pharmacology
Bhavna Verma, Michael Ritchie, Maria Mancini
With the recent approval of four novel immune oncology agents for the treatment of various cancers, the emerging power of this drug class has been substantiated. However, the full potential of such agents is yet to be realized, with only a fraction of the patient population responding to these drugs. A more advanced pre-clinical and translational research platform may increase our understanding of the mechanisms associated with immune-mediated cancer cell death, thereby facilitating the design and development of more generally efficacious agents and drug regimens...
September 11, 2017: Current Protocols in Pharmacology
Guy A Higgins, Leo B Silenieks
The 5-choice serial reaction time task (5-CSRTT) is employed extensively to measure attention in rodents. The assay involves animals trained to respond to a brief, unpredictable visual stimulus presented in one of five locations. The effects of experimental manipulations on response speed and choice accuracy are measured, and each related to attentional performance. The 5-CSRTT is also used to measure motor impulsivity. Adapted from a human task, the 5-CSRTT can be employed with rodents or primates, highlighting its translational value...
September 11, 2017: Current Protocols in Pharmacology
Beatrice Kisser, Eva Mangelsen, Caroline Wingolf, Lars Ivo Partecke, Claus-Dieter Heidecke, Christer Tannergren, Stefan Oswald, Markus Keiser
The Ussing chamber is an old but still powerful technique originally designed to study the vectorial transport of ions through frog skin. This technique is also used to investigate the transport of chemical agents through the intestinal barrier as well as drug metabolism in enterocytes, both of which are key determinants for the bioavailability of orally administered drugs. More contemporary model systems, such as Caco-2 cell monolayers or stably transfected cells, are more limited in their use compared to the Ussing chamber because of differences in expression rates of transporter proteins and/or metabolizing enzymes...
June 22, 2017: Current Protocols in Pharmacology
Andrew Dunbar, Abbas Nazir, Ross Levine
Myeloproliferative neoplasms (MPNs) are a class of hematologic diseases characterized by aberrant proliferation of one or more myeloid lineages and progressive bone marrow fibrosis. In 2005, seminal work by multiple groups identified the JAK2V617F mutation in a significant fraction of MPN patients. Since that time, murine models of JAK2V617F have greatly enhanced the understanding of the role of aberrant JAK-STAT signaling in MPN pathogenesis and have provided an in vivo pre-clinical platform that can be used to develop novel therapies...
June 22, 2017: Current Protocols in Pharmacology
Manuel Grundmann
Label-free biosensors are increasingly employed in drug discovery. Cell-based biosensors provide valuable insights into the biological consequences of exposing cells and tissues to chemical agents and the underlying molecular mechanisms associated with these effects. Optical biosensors based on the detection of dynamic mass redistribution (DMR) and impedance biosensors using cellular dielectric spectroscopy (CDS) capture changes of the cytoskeleton of living cells in real time. Because signal transduction correlates with changes in cell morphology, DMR and CDS biosensors are exquisitely suited for recording integrated cell responses in an unbiased, yet pathway-specific manner without the use of labels that may interfere with cell function...
June 22, 2017: Current Protocols in Pharmacology
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