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Azithromycin Causes a Novel Proarrhythmic Syndrome.
Yang Z, Prinsen JK, Bersell KR, Shen W, Yermalitskaya L, Sidorova T, Luis PB, Hall L, Zhang W, Du L, Milne G, Tucker P, George AL, Campbell CM, Pickett RA, Shaffer CM, Chopra N, Yang T, Knollmann BC, Roden DM, Murray KT
(2017) Circ Arrhythm Electrophysiol 10:
MeSH Terms: Action Potentials, Animals, Anti-Bacterial Agents, Arrhythmias, Cardiac, Azithromycin, CHO Cells, Calcium Channel Blockers, Calcium Channels, L-Type, Cricetulus, Dose-Response Relationship, Drug, Electrocardiography, Ambulatory, Female, HEK293 Cells, Heart Rate, Humans, KCNQ1 Potassium Channel, Mice, Inbred C57BL, Myocytes, Cardiac, NAV1.5 Voltage-Gated Sodium Channel, Potassium Channel Blockers, Potassium Channels, Inwardly Rectifying, Potassium Channels, Voltage-Gated, Rabbits, Sodium Channel Blockers, Telemetry, Time Factors, Transfection, Young Adult
Show Abstract · Added July 6, 2017
BACKGROUND - The widely used macrolide antibiotic azithromycin increases risk of cardiovascular and sudden cardiac death, although the underlying mechanisms are unclear. Case reports, including the one we document here, demonstrate that azithromycin can cause rapid, polymorphic ventricular tachycardia in the absence of QT prolongation, indicating a novel proarrhythmic syndrome. We investigated the electrophysiological effects of azithromycin in vivo and in vitro using mice, cardiomyocytes, and human ion channels heterologously expressed in human embryonic kidney (HEK 293) and Chinese hamster ovary (CHO) cells.
METHODS AND RESULTS - In conscious telemetered mice, acute intraperitoneal and oral administration of azithromycin caused effects consistent with multi-ion channel block, with significant sinus slowing and increased PR, QRS, QT, and QTc intervals, as seen with azithromycin overdose. Similarly, in HL-1 cardiomyocytes, the drug slowed sinus automaticity, reduced phase 0 upstroke slope, and prolonged action potential duration. Acute exposure to azithromycin reduced peak SCN5A currents in HEK cells (IC=110±3 μmol/L) and Na current in mouse ventricular myocytes. However, with chronic (24 hour) exposure, azithromycin caused a ≈2-fold increase in both peak and late SCN5A currents, with findings confirmed for I in cardiomyocytes. Mild block occurred for K currents representing I (CHO cells expressing hERG; IC=219±21 μmol/L) and I (CHO cells expressing KCNQ1+KCNE1; IC=184±12 μmol/L), whereas azithromycin suppressed L-type Ca currents (rabbit ventricular myocytes, IC=66.5±4 μmol/L) and I (HEK cells expressing Kir2.1, IC=44±3 μmol/L).
CONCLUSIONS - Chronic exposure to azithromycin increases cardiac Na current to promote intracellular Na loading, providing a potential mechanistic basis for the novel form of proarrhythmia seen with this macrolide antibiotic.
© 2017 American Heart Association, Inc.
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28 MeSH Terms
Kidney-specific transposon-mediated gene transfer in vivo.
Woodard LE, Cheng J, Welch RC, Williams FM, Luo W, Gewin LS, Wilson MH
(2017) Sci Rep 7: 44904
MeSH Terms: Acute Kidney Injury, Animals, DNA Transposable Elements, Erythropoietin, Gene Expression, Gene Expression Regulation, Gene Transfer Techniques, Genes, Reporter, Genetic Vectors, Hydrodynamics, Immunosuppressive Agents, Kidney, Male, Mice, Organ Specificity, Promoter Regions, Genetic, Transfection
Show Abstract · Added September 11, 2017
Methods enabling kidney-specific gene transfer in adult mice are needed to develop new therapies for kidney disease. We attempted kidney-specific gene transfer following hydrodynamic tail vein injection using the kidney-specific podocin and gamma-glutamyl transferase promoters, but found expression primarily in the liver. In order to achieve kidney-specific transgene expression, we tested direct hydrodynamic injection of a DNA solution into the renal pelvis and found that luciferase expression was strong in the kidney and absent from extra-renal tissues. We observed heterogeneous, low-level transfection of the collecting duct, proximal tubule, distal tubule, interstitial cells, and rarely glomerular cells following injection. To assess renal injury, we performed the renal pelvis injections on uninephrectomised mice and found that their blood urea nitrogen was elevated at two days post-transfer but resolved within two weeks. Although luciferase expression quickly decreased following renal pelvis injection, the use of the piggyBac transposon system improved long-term expression. Immunosuppression with cyclophosphamide stabilised luciferase expression, suggesting immune clearance of the transfected cells occurs in immunocompetent animals. Injection of a transposon expressing erythropoietin raised the haematocrit, indicating that the developed injection technique can elicit a biologic effect in vivo. Hydrodynamic renal pelvis injection enables transposon mediated-kidney specific gene transfer in adult mice.
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17 MeSH Terms
Clostridium difficile Toxin A Undergoes Clathrin-Independent, PACSIN2-Dependent Endocytosis.
Chandrasekaran R, Kenworthy AK, Lacy DB
(2016) PLoS Pathog 12: e1006070
MeSH Terms: Adaptor Proteins, Signal Transducing, Animals, Bacterial Toxins, Blotting, Western, Caco-2 Cells, Clathrin, Clostridium Infections, Clostridium difficile, Endocytosis, Enterotoxins, Fluorescent Antibody Technique, Gene Knockdown Techniques, HEK293 Cells, Humans, Image Processing, Computer-Assisted, Mice, Microscopy, Confocal, Protein Transport, Reverse Transcriptase Polymerase Chain Reaction, Transfection, Virulence Factors
Show Abstract · Added April 26, 2017
Clostridium difficile infection affects a significant number of hospitalized patients in the United States. Two homologous exotoxins, TcdA and TcdB, are the major virulence factors in C. difficile pathogenesis. The toxins are glucosyltransferases that inactivate Rho family-GTPases to disrupt host cellular function and cause fluid secretion, inflammation, and cell death. Toxicity depends on receptor binding and subsequent endocytosis. TcdB has been shown to enter cells by clathrin-dependent endocytosis, but the mechanism of TcdA uptake is still unclear. Here, we utilize a combination of RNAi-based knockdown, pharmacological inhibition, and cell imaging approaches to investigate the endocytic mechanism(s) that contribute to TcdA uptake and subsequent cytopathic and cytotoxic effects. We show that TcdA uptake and cellular intoxication is dynamin-dependent but does not involve clathrin- or caveolae-mediated endocytosis. Confocal microscopy using fluorescently labeled TcdA shows significant colocalization of the toxin with PACSIN2-positive structures in cells during entry. Disruption of PACSIN2 function by RNAi-based knockdown approaches inhibits TcdA uptake and toxin-induced downstream effects in cells indicating that TcdA entry is PACSIN2-dependent. We conclude that TcdA and TcdB utilize distinct endocytic mechanisms to intoxicate host cells.
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21 MeSH Terms
The L-Arginine Transporter Solute Carrier Family 7 Member 2 Mediates the Immunopathogenesis of Attaching and Effacing Bacteria.
Singh K, Al-Greene NT, Verriere TG, Coburn LA, Asim M, Barry DP, Allaman MM, Hardbower DM, Delgado AG, Piazuelo MB, Vallance BA, Gobert AP, Wilson KT
(2016) PLoS Pathog 12: e1005984
MeSH Terms: Animals, Blotting, Western, Cationic Amino Acid Transporter 2, Cell Line, Citrobacter rodentium, Disease Models, Animal, Enterobacteriaceae Infections, Host-Parasite Interactions, Humans, Immunophenotyping, Mice, Mice, Inbred C57BL, Mice, Knockout, Transfection
Show Abstract · Added October 27, 2016
Solute carrier family 7 member 2 (SLC7A2) is an inducible transporter of the semi-essential amino acid L-arginine (L-Arg), which has been implicated in immune responses to pathogens. We assessed the role of SLC7A2 in murine infection with Citrobacter rodentium, an attaching and effacing enteric pathogen that causes colitis. Induction of SLC7A2 was upregulated in colitis tissues, and localized predominantly to colonic epithelial cells. Compared to wild-type mice, Slc7a2-/-mice infected with C. rodentium had improved survival and decreased weight loss, colon weight, and histologic injury; this was associated with decreased colonic macrophages, dendritic cells, granulocytes, and Th1 and Th17 cells. In infected Slc7a2-/-mice, there were decreased levels of the proinflammatory cytokines G-CSF, TNF-α, IL-1α, IL-1β, and the chemokines CXCL1, CCL2, CCL3, CCL4, CXCL2, and CCL5. In bone marrow chimeras, the recipient genotype drove the colitis phenotype, indicative of the importance of epithelial, rather than myeloid SLC7A2. Mice lacking Slc7a2 exhibited reduced adherence of C. rodentium to the colonic epithelium and decreased expression of Talin-1, a focal adhesion protein involved in the attachment of the bacterium. The importance of SLC7A2 and Talin-1 in the intimate attachment of C. rodentium and induction of inflammatory response was confirmed in vitro, using conditionally-immortalized young adult mouse colon (YAMC) cells with shRNA knockdown of Slc7a2 or Tln1. Inhibition of L-Arg uptake with the competitive inhibitor, L-lysine (L-Lys), also prevented attachment of C. rodentium and chemokine expression. L-Lys and siRNA knockdown confirmed the role of L-Arg and SLC7A2 in human Caco-2 cells co-cultured with enteropathogenic Escherichia coli. Overexpression of SLC7A2 in human embryonic kidney cells increased bacterial adherence and chemokine expression. Taken together, our data indicate that C. rodentium enhances its own pathogenicity by inducing the expression of SLC7A2 to favor its attachment to the epithelium and thus create its ecological niche.
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14 MeSH Terms
Contribution of Organic Anion-Transporting Polypeptides 1A/1B to Doxorubicin Uptake and Clearance.
Lee HH, Leake BF, Kim RB, Ho RH
(2017) Mol Pharmacol 91: 14-24
MeSH Terms: ATP Binding Cassette Transporter, Subfamily B, Member 1, Animals, Biological Transport, Cell Membrane, Dogs, Doxorubicin, HeLa Cells, Humans, Kinetics, Liver, Liver-Specific Organic Anion Transporter 1, Madin Darby Canine Kidney Cells, Male, Mice, Models, Biological, Mutant Proteins, Organic Anion Transporters, Organic Anion Transporters, Sodium-Independent, Organic Cation Transport Proteins, Rats, Transfection
Show Abstract · Added November 10, 2016
The organic anion-transporting polypeptides represent an important family of drug uptake transporters that mediate the cellular uptake of a broad range of substrates including numerous drugs. Doxorubicin is a highly efficacious and well-established anthracycline chemotherapeutic agent commonly used in the treatment of a wide range of cancers. Although doxorubicin is a known substrate for efflux transporters such as P-glycoprotein (P-gp; MDR1, ABCB1), significantly less is known regarding its interactions with drug uptake transporters. Here, we investigated the role of organic anion transporting polypeptide (OATP) transporters to the disposition of doxorubicin. A recombinant vaccinia-based method for expressing uptake transporters in HeLa cells revealed that OATP1A2, but not OATP1B1 or OATP1B3, and the rat ortholog Oatp1a4 were capable of significant doxorubicin uptake. Interestingly, transwell assays using Madin-Darby canine kidney II cell line cells stably expressing specific uptake and/or efflux transporters revealed that OATP1B1, OATP1B3, and OATP1A2, either alone or in combination with MDR1, significantly transported doxorubicin. An assessment of polymorphisms in SLCO1A2 revealed that four variants were associated with significantly impaired doxorubicin transport in vitro. In vivo doxorubicin disposition studies revealed that doxorubicin plasma area under the curve was significantly higher (1.7-fold) in Slco1a/1b versus wild-type mice. The liver-to-plasma ratio of doxorubicin was significantly decreased (2.3-fold) in Slco1a/1b2 mice and clearance was reduced by 40% compared with wild-type mice, suggesting Oatp1b transporters are important for doxorubicin hepatic uptake. In conclusion, we demonstrate important roles for OATP1A/1B in transporter-mediated uptake and disposition of doxorubicin.
Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.
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21 MeSH Terms
Development and Antiparkinsonian Activity of VU0418506, a Selective Positive Allosteric Modulator of Metabotropic Glutamate Receptor 4 Homomers without Activity at mGlu2/4 Heteromers.
Niswender CM, Jones CK, Lin X, Bubser M, Thompson Gray A, Blobaum AL, Engers DW, Rodriguez AL, Loch MT, Daniels JS, Lindsley CW, Hopkins CR, Javitch JA, Conn PJ
(2016) ACS Chem Neurosci 7: 1201-11
MeSH Terms: Allosteric Regulation, Animals, Antiparkinson Agents, Antipsychotic Agents, Apomorphine, Brain, Catalepsy, Disease Models, Animal, Dopamine Agonists, Forelimb, Glutamic Acid, HEK293 Cells, Haloperidol, Humans, Oxidopamine, Parkinson Disease, Rats, Rats, Sprague-Dawley, Receptors, Metabotropic Glutamate, Transfection
Show Abstract · Added March 3, 2020
Metabotropic glutamate receptor 4 (mGlu4) is emerging as a potential therapeutic target for numerous central nervous system indications, including Parkinson's disease (PD). As the glutamate binding sites among the eight mGlu receptors are highly conserved, modulation of receptor activity via allosteric sites within the receptor transmembrane domains using positive and negative allosteric modulators (PAMs and NAMs, respectively) has become a common strategy. We and others have used PAMs targeting mGlu4 to show that potentiation of receptor signaling induces antiparkinsonian activity in a variety of PD animal models, including haloperidol-induced catalepsy and 6-hydroxydopamine-induced lesion. Recently, mGlu4 has been reported to form heteromeric complexes with other mGlu receptor subtypes, such as mGlu2, and the resulting heteromer exhibits a distinct pharmacological profile in response to allosteric modulators. For example, some mGlu4 PAMs do not appear to potentiate glutamate activity when mGlu2 and mGlu4 are coexpressed, whereas other compounds potentiate mGlu4 responses regardless of mGlu2 coexpression. We report here the discovery and characterization of VU0418506, a novel mGlu4 PAM with activity in rodent PD models. Using pharmacological approaches and Complemented Donor-Acceptor resonance energy transfer (CODA-RET) technology, we find that VU0418506 does not potentiate agonist-induced activity when mGlu2 and mGlu4 are heterodimerized, suggesting that the antiparkinsonian action of mGlu4 PAMs can be induced by compounds without activity at mGlu2/4 heteromers.
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Laminin-111 peptide C16 regulates invadopodia activity of malignant cells through β1 integrin, Src and ERK 1/2.
Siqueira AS, Pinto MP, Cruz MC, Smuczek B, Cruz KS, Barbuto JA, Hoshino D, Weaver AM, Freitas VM, Jaeger RG
(2016) Oncotarget 7: 47904-47917
MeSH Terms: Carcinoma, Squamous Cell, Cell Line, Tumor, Fibrosarcoma, Head and Neck Neoplasms, Humans, Integrin beta1, Laminin, MAP Kinase Signaling System, Mouth Neoplasms, Peptide Fragments, Podosomes, Squamous Cell Carcinoma of Head and Neck, Transfection, src-Family Kinases
Show Abstract · Added April 26, 2017
Laminin peptides influence tumor behavior. In this study, we addressed whether laminin peptide C16 (KAFDITYVRLKF, γ1 chain) would increase invadopodia activity of cells from squamous cell carcinoma (CAL27) and fibrosarcoma (HT1080). We found that C16 stimulates invadopodia activity over time in both cell lines. Rhodamine-conjugated C16 decorates the edge of cells, suggesting a possible binding to membrane receptors. Flow cytometry showed that C16 increases activated β1 integrin, and β1 integrin miRNA-mediated depletion diminishes C16-induced invadopodia activity in both cell lines. C16 stimulates Src and ERK 1/2 phosphorylation, and ERK 1/2 inhibition decreases peptide-induced invadopodia activity. C16 also increases cortactin phosphorylation in both cells lines. Based on our findings, we propose that C16 regulates invadopodia activity over time of squamous carcinoma and fibrosarcoma cells, probably through β1 integrin, Src and ERK 1/2 signaling pathways.
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14 MeSH Terms
ML418: The First Selective, Sub-Micromolar Pore Blocker of Kir7.1 Potassium Channels.
Swale DR, Kurata H, Kharade SV, Sheehan J, Raphemot R, Voigtritter KR, Figueroa EE, Meiler J, Blobaum AL, Lindsley CW, Hopkins CR, Denton JS
(2016) ACS Chem Neurosci 7: 1013-23
MeSH Terms: Animals, Dose-Response Relationship, Drug, HEK293 Cells, Humans, Membrane Potentials, Models, Molecular, Mutagenesis, Site-Directed, Mutation, Patch-Clamp Techniques, Potassium, Potassium Channel Blockers, Potassium Channels, Inwardly Rectifying, Structure-Activity Relationship, Time Factors, Transfection
Show Abstract · Added April 8, 2017
The inward rectifier potassium (Kir) channel Kir7.1 (KCNJ13) has recently emerged as a key regulator of melanocortin signaling in the brain, electrolyte homeostasis in the eye, and uterine muscle contractility during pregnancy. The pharmacological tools available for exploring the physiology and therapeutic potential of Kir7.1 have been limited to relatively weak and nonselective small-molecule inhibitors. Here, we report the discovery in a fluorescence-based high-throughput screen of a novel Kir7.1 channel inhibitor, VU714. Site-directed mutagenesis of pore-lining amino acid residues identified glutamate 149 and alanine 150 as essential determinants of VU714 activity. Lead optimization with medicinal chemistry generated ML418, which exhibits sub-micromolar activity (IC50 = 310 nM) and superior selectivity over other Kir channels (at least 17-fold selective over Kir1.1, Kir2.1, Kir2.2, Kir2.3, Kir3.1/3.2, and Kir4.1) except for Kir6.2/SUR1 (equally potent). Evaluation in the EuroFins Lead Profiling panel of 64 GPCRs, ion-channels, and transporters for off-target activity of ML418 revealed a relatively clean ancillary pharmacology. While ML418 exhibited low CLHEP in human microsomes which could be modulated with lipophilicity adjustments, it showed high CLHEP in rat microsomes regardless of lipophilicity. A subsequent in vivo PK study of ML418 by intraperitoneal (IP) administration (30 mg/kg dosage) revealed a suitable PK profile (Cmax = 0.20 μM and Tmax = 3 h) and favorable CNS distribution (mouse brain/plasma Kp of 10.9 to support in vivo studies. ML418, which represents the current state-of-the-art in Kir7.1 inhibitors, should be useful for exploring the physiology of Kir7.1 in vitro and in vivo.
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15 MeSH Terms
The Ste20 kinases SPAK and OSR1 travel between cells through exosomes.
Koumangoye R, Delpire E
(2016) Am J Physiol Cell Physiol 311: C43-53
MeSH Terms: Cell Communication, Cell Membrane, Coculture Techniques, Culture Media, Conditioned, Exosomes, HEK293 Cells, HeLa Cells, Humans, Luminescent Proteins, Microscopy, Fluorescence, Particle Size, Phosphorylation, Protein Transport, Protein-Serine-Threonine Kinases, Recombinant Proteins, Solute Carrier Family 12, Member 2, Tetraspanin 30, Time Factors, Transfection
Show Abstract · Added May 3, 2017
Proteomics studies have identified Ste20-related proline/alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1) in exosomes isolated from body fluids such as blood, saliva, and urine. Because proteomics studies likely overestimate the number of exosome proteins, we sought to confirm and extend this observation using traditional biochemical and cell biology methods. We utilized HEK293 cells in culture to verify the packaging of these Ste20 kinases in exosomes. Using a series of centrifugation and filtration steps of conditioned culture medium isolated from HEK293 cells, we isolated nanovesicles in the range of 40-100 nm. We show that these small vesicles express the tetraspanin protein CD63 and lack endoplasmic reticulum and Golgi markers, consistent with these being exosomes. We show by Western blot and immunogold analyses that these exosomes express SPAK, OSR1, and Na-K-Cl cotransporter 1 (NKCC1). We show that exosomes are not only secreted by cells, but also accumulated by adjacent cells. Indeed, exposing cultured cells to exosomes produced by other cells expressing a fluorescently labeled kinase resulted in the kinase finding its way into the cytoplasm of these cells, consistent with the idea of exosomes serving as cell-to-cell communication vessels. Similarly, coculturing cells expressing different fluorescently tagged proteins resulted in the exchange of proteins between cells. In addition, we show that both SPAK and OSR1 kinases entering cells through exosomes are preferentially expressed at the plasma membrane and that the kinases in exosomes are functional and maintain NKCC1 in a phosphorylated state.
Copyright © 2016 the American Physiological Society.
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19 MeSH Terms
Mammalian retinal Müller cells have circadian clock function.
Xu L, Ruan G, Dai H, Liu AC, Penn J, McMahon DG
(2016) Mol Vis 22: 275-83
MeSH Terms: Animals, CLOCK Proteins, Cells, Cultured, Circadian Clocks, Circadian Rhythm, Ependymoglial Cells, Female, Fluorescent Antibody Technique, Indirect, Genetic Vectors, Humans, Lentivirus, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, RNA, Small Interfering, Transfection
Show Abstract · Added March 18, 2020
PURPOSE - To test whether Müller glia of the mammalian retina have circadian rhythms.
METHODS - We used Müller glia cultures isolated from mouse lines or from humans and bioluminescent reporters of circadian clock genes to monitor molecular circadian rhythms. The clock gene dependence of the Müller cell rhythms was tested using clock gene knockout mouse lines or with siRNA for specific clock genes.
RESULTS - We demonstrated that retinal Müller glia express canonical circadian clock genes, are capable of sustained circadian oscillations in isolation from other cell types, and exhibit unique features of their molecular circadian clock compared to the retina as a whole. Mouse and human Müller cells demonstrated circadian clock function; however, they exhibited species-specific differences in the gene dependence of their clocks.
CONCLUSIONS - Müller cells are the first mammalian retinal cell type in which sustained circadian rhythms have been demonstrated in isolation from other retinal cells.
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