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Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney.
Woodard LE, Welch RC, Williams FM, Luo W, Cheng J, Wilson MH
(2018) J Vis Exp :
MeSH Terms: Animals, DNA, Hydrodynamics, Injections, Kidney, Kidney Pelvis, Male, Mice, Plasmids, Protein Biosynthesis, Transfection, Transgenes
Show Abstract · Added March 14, 2018
Hydrodynamic injection creates a local, high-pressure environment to transfect various tissues with plasmid DNA and other substances. Hydrodynamic tail vein injection, for example, is a well-established method by which the liver can be transfected. This manuscript describes an application of hydrodynamic principles by injection of the mouse kidney directly with plasmid DNA for kidney-specific gene expression. Mice are anesthetized and the kidney is exposed by a flank incision followed by a fast injection of a plasmid DNA-containing solution directly into the renal pelvis. The needle is kept in place for ten seconds and the incision site is sutured. The following day, live animal imaging, Western blot, or immunohistochemistry may be used to assay gene expression, or other assays suited to the transgene of choice are used for detection of the protein of interest. Published methods to prolong gene expression include transposon-mediated transgene integration and cyclophosphamide treatment to inhibit the immune response to the transgene.
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12 MeSH Terms
The Dihydroxy Metabolite of the Teratogen Thalidomide Causes Oxidative DNA Damage.
Wani TH, Chakrabarty A, Shibata N, Yamazaki H, Guengerich FP, Chowdhury G
(2017) Chem Res Toxicol 30: 1622-1628
MeSH Terms: Catalase, DNA Cleavage, DNA Damage, Free Radical Scavengers, HEK293 Cells, Hep G2 Cells, Human Umbilical Vein Endothelial Cells, Humans, Microscopy, Fluorescence, Plasmids, Poly(ADP-ribose) Polymerases, Reactive Oxygen Species, Teratogens, Thalidomide
Show Abstract · Added March 14, 2018
Thalidomide [α-(N-phthalimido)glutarimide] (1) is a sedative and antiemetic drug originally introduced into the clinic in the 1950s for the treatment of morning sickness. Although marketed as entirely safe, more than 10 000 babies were born with severe birth defects. Thalidomide was banned and subsequently approved for the treatment of multiple myeloma and complications associated with leprosy. Although known for more than 5 decades, the mechanism of teratogenicity remains to be conclusively understood. Various theories have been proposed in the literature including DNA damage and ROS and inhibition of angiogenesis and cereblon. All of the theories have their merits and limitations. Although the recently proposed cereblon theory has gained wide acceptance, it fails to explain the metabolism and low-dose requirement reported by a number of groups. Recently, we have provided convincing structural evidence in support of the presence of arene oxide and the quinone-reactive intermediates. However, the ability of these reactive intermediates to impart toxicity/teratogenicity needs investigation. Herein we report that the oxidative metabolite of thalidomide, dihydroxythalidomide, is responsible for generating ROS and causing DNA damage. We show, using cell lines, the formation of comet (DNA damage) and ROS. Using DNA-cleavage assays, we also show that catalase, radical scavengers, and desferal are capable of inhibiting DNA damage. A mechanism of teratogenicity is proposed that not only explains the DNA-damaging property but also the metabolism, low concentration, and species-specificity requirements of thalidomide.
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14 MeSH Terms
Evolution of a Heavy Metal Homeostasis/Resistance Island Reflects Increasing Copper Stress in Enterobacteria.
Staehlin BM, Gibbons JG, Rokas A, O'Halloran TV, Slot JC
(2016) Genome Biol Evol 8: 811-26
MeSH Terms: Chromosomes, Bacterial, Copper, Enterobacteriaceae, Gene Transfer, Horizontal, Homeostasis, Humans, Metals, Heavy, Phylogeny, Plasmids, Shewanella
Show Abstract · Added February 22, 2016
Copper homeostasis in bacteria is challenged by periodic elevation of copper levels in the environment, arising from both natural sources and human inputs. Several mechanisms have evolved to efflux copper from bacterial cells, including thecus(copper sensing copper efflux system), andpco(plasmid-borne copper resistance system) systems. The genes belonging to these two systems can be physically clustered in a Copper Homeostasis and Silver Resistance Island (CHASRI) on both plasmids and chromosomes in Enterobacteria. Increasing use of copper in agricultural and industrial applications raises questions about the role of human activity in the evolution of novel copper resistance mechanisms. Here we present evidence that CHASRI emerged and diversified in response to copper deposition across aerobic and anaerobic environments. An analysis of diversification rates and a molecular clock model suggest that CHASRI experienced repeated episodes of elevated diversification that could correspond to peaks in human copper production. Phylogenetic analyses suggest that CHASRI originated in a relative ofEnterobacter cloacaeas the ultimate product of sequential assembly of several pre-existing two-gene modules. Once assembled, CHASRI dispersed via horizontal gene transfer within Enterobacteriaceae and also to certain members of Shewanellaceae, where the originalpcomodule was replaced by a divergentpcohomolog. Analyses of copper stress mitigation suggest that CHASRI confers increased resistance aerobically, anaerobically, and during shifts between aerobic and anaerobic environments, which could explain its persistence in facultative anaerobes and emergent enteric pathogens.
© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
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10 MeSH Terms
Acute blockade of the Caenorhabditis elegans dopamine transporter DAT-1 by the mammalian norepinephrine transporter inhibitor nisoxetine reveals the influence of genetic modifications of dopamine signaling in vivo.
Bermingham DP, Hardaway JA, Snarrenberg CL, Robinson SB, Folkes OM, Salimando GJ, Jinnah H, Blakely RD
(2016) Neurochem Int 98: 122-8
MeSH Terms: Animals, Caenorhabditis elegans, Caenorhabditis elegans Proteins, Dopamine, Dopamine Plasma Membrane Transport Proteins, Dopamine Uptake Inhibitors, Fluoxetine, Gene Deletion, Mutation, Norepinephrine Plasma Membrane Transport Proteins, Paralysis, Plasmids, Signal Transduction, Swimming
Show Abstract · Added February 15, 2016
Modulation of neurotransmission by the catecholamine dopamine (DA) is conserved across phylogeny. In the nematode Caenorhabditis elegans, excess DA signaling triggers Swimming-Induced Paralysis (Swip), a phenotype first described in animals with loss of function mutations in the presynaptic DA transporter (dat-1). Swip has proven to be a phenotype suitable for the identification of novel dat-1 mutations as well as the identification of novel genes that impact DA signaling. Pharmacological manipulations can also induce Swip, though the reagents employed to date lack specificity and potency, limiting their use in evaluation of dat-1 expression and function. Our lab previously established the mammalian norepinephrine transporter (NET) inhibitor nisoxetine to be a potent antagonist of DA uptake conferred by DAT-1 following heterologous expression. Here we demonstrate the ability of low (μM) concentrations of nisoxetine to trigger Swip within minutes of incubation, with paralysis dependent on DA release and signaling, and non-additive with Swip triggered by dat-1 deletion. Using nisoxetine in combination with genetic mutations that impact DA release, we further demonstrate the utility of the drug for demonstrating contributions of presynaptic DA receptors and ion channels to Swip. Together, these findings reveal nisoxetine as a powerful reagent for monitoring multiple dimensions of DA signaling in vivo, thus providing a new resource that can be used to evaluate contributions of dat-1 and other genes linked to DA signaling without the potential for compensations that attend constitutive genetic mutations.
Copyright © 2016 Elsevier Ltd. All rights reserved.
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14 MeSH Terms
A Novel Saccharomyces cerevisiae FG Nucleoporin Mutant Collection for Use in Nuclear Pore Complex Functional Experiments.
Adams RL, Terry LJ, Wente SR
(2015) G3 (Bethesda) 6: 51-8
MeSH Terms: Gene Expression, Genetic Association Studies, Mutation, Nuclear Pore Complex Proteins, Phenotype, Plasmids, Protein Interaction Domains and Motifs, Saccharomyces cerevisiae, Sequence Deletion
Show Abstract · Added February 15, 2016
FG nucleoporins (Nups) are the class of proteins that both generate the permeability barrier and mediate selective transport through the nuclear pore complex (NPC). The FG Nup family has 11 members in Saccharomyces cerevisiae, and the study of mutants lacking different FG domains has been instrumental in testing transport models. To continue analyzing the distinct functional roles of FG Nups in vivo, additional robust genetic tools are required. Here, we describe a novel collection of S. cerevisiae mutant strains in which the FG domains of different groups of Nups are absent (Δ) in the greatest number documented to date. Using this plasmid-based ΔFG strategy, we find that a GLFG domain-only pore is sufficient for viability. The resulting extensive plasmid and strain resources are available to the scientific community for future in-depth in vivo studies of NPC transport.
Copyright © 2016 Adams et al.
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9 MeSH Terms
Evaluating the potential for undesired genomic effects of the piggyBac transposon system in human cells.
Saha S, Woodard LE, Charron EM, Welch RC, Rooney CM, Wilson MH
(2015) Nucleic Acids Res 43: 1770-82
MeSH Terms: 5' Untranslated Regions, Chromosomes, Artificial, Bacterial, DNA Damage, DNA Transposable Elements, Genome, Human, Green Fluorescent Proteins, HEK293 Cells, Humans, Plasmids, Polymerase Chain Reaction, Transgenes, Transposases
Show Abstract · Added January 30, 2015
Non-viral transposons have been used successfully for genetic modification of clinically relevant cells including embryonic stem, induced pluripotent stem, hematopoietic stem and primary human T cell types. However, there has been limited evaluation of undesired genomic effects when using transposons for human genome modification. The prevalence of piggyBac(PB)-like terminal repeat (TR) elements in the human genome raises concerns. We evaluated if there were undesired genomic effects of the PB transposon system to modify human cells. Expression of the transposase alone revealed no mobilization of endogenous PB-like sequences in the human genome and no increase in DNA double-strand breaks. The use of PB in a plasmid containing both transposase and transposon greatly increased the probability of transposase integration; however, using transposon and transposase from separate vectors circumvented this. Placing a eGFP transgene within transposon vector backbone allowed isolation of cells free from vector backbone DNA. We confirmed observable directional promoter activity within the 5'TR element of PB but found no significant enhancer effects from the transposon DNA sequence. Long-term culture of primary human cells modified with eGFP-transposons revealed no selective growth advantage of transposon-harboring cells. PB represents a promising vector system for genetic modification of human cells with limited undesired genomic effects.
Published by Oxford University Press on behalf of Nucleic Acids Research 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.
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12 MeSH Terms
Frequency of disinfectant resistance genes in pediatric strains of methicillin-resistant Staphylococcus aureus.
Johnson JG, Saye EJ, Jimenez-Truque N, Soper N, Thomsen I, Talbot TR, Creech CB
(2013) Infect Control Hosp Epidemiol 34: 1326-7
MeSH Terms: Anti-Infective Agents, Local, Antiporters, Bacterial Proteins, Chlorhexidine, DNA, Bacterial, Disinfectants, Drug Resistance, Bacterial, Membrane Transport Proteins, Methicillin-Resistant Staphylococcus aureus, Plasmids, Quaternary Ammonium Compounds
Added February 3, 2014
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11 MeSH Terms
Structure and expression of a novel compact myelin protein - small VCP-interacting protein (SVIP).
Wu J, Peng D, Voehler M, Sanders CR, Li J
(2013) Biochem Biophys Res Commun 440: 173-8
MeSH Terms: Amino Acid Sequence, Animals, Anions, Carrier Proteins, DNA, Complementary, Escherichia coli, Gene Expression, Humans, Membrane Proteins, Mice, Molecular Sequence Data, Nuclear Proteins, Plasmids, Protein Structure, Secondary, Rats, Sciatic Nerve
Show Abstract · Added May 20, 2014
SVIP (small p97/VCP-interacting protein) was initially identified as one of many cofactors regulating the valosin containing protein (VCP), an AAA+ ATPase involved in endoplasmic-reticulum-associated protein degradation (ERAD). Our previous study showed that SVIP is expressed exclusively in the nervous system. In the present study, SVIP and VCP were seen to be co-localized in neuronal cell bodies. Interestingly, we also observed that SVIP co-localizes with myelin basic protein (MBP) in compact myelin, where VCP was absent. Furthermore, using nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopic measurements, we determined that SVIP is an intrinsically disordered protein (IDP). However, upon binding to the surface of membranes containing a net negative charge, the helical content of SVIP increases dramatically. These findings provide structural insight into interactions between SVIP and myelin membranes.
Published by Elsevier Inc.
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16 MeSH Terms
Selective targeting of gain-of-function KCNQ1 mutations predisposing to atrial fibrillation.
Campbell CM, Campbell JD, Thompson CH, Galimberti ES, Darbar D, Vanoye CG, George AL
(2013) Circ Arrhythm Electrophysiol 6: 960-6
MeSH Terms: Animals, Atrial Fibrillation, Cells, Cultured, Chromans, Cricetulus, Genetic Predisposition to Disease, KCNQ1 Potassium Channel, Mutation, Myocytes, Cardiac, Patch-Clamp Techniques, Plasmids, Potassium Channels, Voltage-Gated, Rabbits, Risk Factors, Sulfonamides
Show Abstract · Added March 7, 2014
BACKGROUND - Atrial fibrillation is the most common sustained cardiac arrhythmia in adults. We hypothesized that gain-of-function KCNQ1 mutations previously associated with familial atrial fibrillation have distinct pharmacological properties that may enable targeted inhibition.
METHODS AND RESULTS - Wild-type (WT) KCNQ1 or the familial atrial fibrillation mutation KCNQ1-S140G was heterologously coexpressed with KCNE1 to enable electrophysiological recording of the slow delayed rectifier current (IKs) and investigation of pharmacological effects of the IKs selective blocker HMR-1556. Coexpression of KCNQ1-S140G with KCNE1 generated potassium currents (S140G-IKs) that exhibited greater sensitivity to HMR-1556 than WT-IKs. Enhanced HMR-1556 sensitivity was also observed for another gain-of-function atrial fibrillation mutation, KCNQ1-V141M. Heteromeric expression of KCNE1 with both KCNQ1-WT and KCNQ1-S140G generated currents (HET-IKs) with gain-of-function features, including larger amplitude, a constitutively active component, hyperpolarized voltage dependence of activation, and extremely slow deactivation. A low concentration of HMR-1556, which had little effect on WT-IKs but was capable of inhibiting the mutant channel, reduced both instantaneous and steady state HET-IKs to levels that were not significantly different from WT-IKs and attenuated use-dependent accumulation of the current. In cultured adult rabbit left atrial myocytes, expression of S140G-IKs shortened action potential duration compared with WT-IKs. Application of HMR-1556 mitigated S140G-IKs-induced action potential duration shortening and did not alter action potential duration in cells expressing WT-IKs.
CONCLUSIONS - The enhanced sensitivity of KCNQ1 gain-of-function mutations for HMR-1556 suggests the possibility of selective therapeutic targeting, and, therefore, our data illustrate a potential proof of principle for genotype-specific treatment of this heritable arrhythmia.
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15 MeSH Terms
Interaction of members of the heat shock protein-70 family with the macrophage mannose receptor.
Yang S, Vigerust DJ, Shepherd VL
(2013) J Leukoc Biol 93: 529-36
MeSH Terms: Amino Acid Sequence, Binding Sites, Blotting, Western, Catechin, Gene Expression, HEK293 Cells, HSP70 Heat-Shock Proteins, HeLa Cells, Humans, Immunoprecipitation, Lectins, C-Type, Macrophages, Mannose-Binding Lectins, Mass Spectrometry, Microscopy, Confocal, Molecular Sequence Data, Plasmids, Protein Binding, Protein Interaction Domains and Motifs, Protein Transport, Receptors, Cell Surface, Transfection
Show Abstract · Added March 22, 2016
The macrophage MR has been the subject of investigation for over 20 years, and several important physiological functions have been described. However, the molecular mechanisms that regulate MR signaling and trafficking during these processes still remain elusive. The focus of the current paper was to identify potential cellular MR-interacting proteins. An initial screen of binding proteins in MR-expressing cells was performed using coimmunoprecipitation, followed by identification of matching peptide sequences using proteomics and MS. The major class of binding proteins identified belonged to the heat shock family of proteins. The specific interaction of the MR with HSP70 family members was validated by Western blot analysis, ligand binding assays, and intracellular colocalization using confocal microscopy. Additional studies indicated that inhibition of the HSP BiP by treatment of cells with EGCG reduced BiP interaction with and surface expression of the MR. Studies of possible motifs within the cytoplasmic tail of the receptor suggested that a juxtamembrane dibasic sequence may contribute to the interaction with BiP. These findings suggest that the molecular association of the MR with HSP70 family members via the receptor cytoplasmic tail may contribute to MR trafficking in macrophages.
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22 MeSH Terms