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Results: 1 to 10 of 16

Publication Record


Selective Small Molecule Activators of TREK-2 Channels Stimulate Dorsal Root Ganglion c-Fiber Nociceptor Two-Pore-Domain Potassium Channel Currents and Limit Calcium Influx.
Dadi PK, Vierra NC, Days E, Dickerson MT, Vinson PN, Weaver CD, Jacobson DA
(2017) ACS Chem Neurosci 8: 558-568
MeSH Terms: Action Potentials, Animals, Antibodies, Calcium, Dinoprostone, Electric Stimulation, Fluoxetine, Ganglia, Spinal, HEK293 Cells, Humans, Lectins, Mice, Mice, Inbred C57BL, Mutation, Nociceptors, Potassium Channel Blockers, Potassium Channels, Tandem Pore Domain, Protein Synthesis Inhibitors, Tetracycline
Show Abstract · Added November 13, 2017
The two-pore-domain potassium (K2P) channel TREK-2 serves to modulate plasma membrane potential in dorsal root ganglia c-fiber nociceptors, which tunes electrical excitability and nociception. Thus, TREK-2 channels are considered a potential therapeutic target for treating pain; however, there are currently no selective pharmacological tools for TREK-2 channels. Here we report the identification of the first TREK-2 selective activators using a high-throughput fluorescence-based thallium (Tl) flux screen (HTS). An initial pilot screen with a bioactive lipid library identified 11-deoxy prostaglandin F2α as a potent activator of TREK-2 channels (EC ≈ 0.294 μM), which was utilized to optimize the TREK-2 Tl flux assay (Z' = 0.752). A HTS was then performed with 76 575 structurally diverse small molecules. Many small molecules that selectively activate TREK-2 were discovered. As these molecules were able to activate single TREK-2 channels in excised membrane patches, they are likely direct TREK-2 activators. Furthermore, TREK-2 activators reduced primary dorsal root ganglion (DRG) c-fiber Ca influx. Interestingly, some of the selective TREK-2 activators such as 11-deoxy prostaglandin F2α were found to inhibit the K2P channel TREK-1. Utilizing chimeric channels containing portions of TREK-1 and TREK-2, the region of the TREK channels that allows for either small molecule activation or inhibition was identified. This region lies within the second pore domain containing extracellular loop and is predicted to play an important role in modulating TREK channel activity. Moreover, the selective TREK-2 activators identified in this HTS provide important tools for assessing human TREK-2 channel function and investigating their therapeutic potential for treating chronic pain.
0 Communities
1 Members
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19 MeSH Terms
Control of gene expression in Helicobacter pylori using the Tet repressor.
McClain MS, Duncan SS, Gaddy JA, Cover TL
(2013) J Microbiol Methods 95: 336-41
MeSH Terms: Bacterial Proteins, Blotting, Western, Gene Expression Regulation, Bacterial, Genetics, Microbial, Helicobacter pylori, Molecular Biology, Operator Regions, Genetic, Recombination, Genetic, Repressor Proteins, Tetracyclines, Transcriptional Activation
Show Abstract · Added January 13, 2014
The lack of a versatile system to control gene expression in Helicobacter pylori has hampered efforts to study H. pylori physiology and pathogenesis. To overcome these limitations, we evaluated the utility of an inducible system based on the well-characterized Tet repressor (TetR) and Tet operator (tetO). As validation of this system, we introduced three copies of tetO into the promoter region upstream of the cagUT operon (encoding two virulence factors required for function of the H. pylori Cag type IV secretion system) and expressed tetR by introducing a codon-optimized gene into the chromosomal ureA locus. Introduction of the tetO copies upstream of cagUT did not disrupt promoter activity, as determined by immunoblotting for CagT. The subsequent introduction of tetR, however, did repress CagT synthesis. Production of CagT was restored when strains were cultured in the presence of the inducer, anhydrotetracycline. To demonstrate one potential application of this new tool, we analyzed the function of the Cag type IV secretion system. When the modified H. pylori strains were co-cultured with AGS cells, activity of the Cag type IV secretion system was dependent on the presence of anhydrotetracycline as evidenced by inducer-dependent induction of IL-8 secretion, CagA translocation, and appearance of type IV secretion system pili at the bacteria-host interface. These studies demonstrate the effectiveness of the tetR-tetO system to control gene expression in H. pylori and provide an improved system for studying H. pylori physiology and pathogenesis.
© 2013.
1 Communities
3 Members
0 Resources
11 MeSH Terms
Minocycline- and tetracycline-class antibiotics are protective against partial seizures in vivo.
Wang DD, Englot DJ, Garcia PA, Lawton MT, Young WL
(2012) Epilepsy Behav 24: 314-8
MeSH Terms: Animals, Anti-Bacterial Agents, Anticonvulsants, Dose-Response Relationship, Drug, Doxycycline, Epilepsies, Partial, Male, Mice, Minocycline, Neurons, Rats, Rats, Sprague-Dawley, Seizures, Tetracycline
Show Abstract · Added August 12, 2016
INTRODUCTION - Increasing evidence suggests the role of inflammation in enhancing neuronal excitability and contributing to epileptogenesis. Tetracycline-class antibiotics minocycline, doxycycline and tetracycline have been shown to have anti-apoptotic and anti-inflammatory effects.
METHODS - We investigated the anti-seizure effects of tetracycline-class antibiotics minocycline, doxycycline and tetracycline in vivo by using the maximal electric shock (MES), 6-Hz (minimal clonic seizure) test and subcutaneous Metrazol (scMET) models of epilepsy.
RESULTS - Minocycline, doxycycline and tetracycline showed anticonvulsant effects in abolishing partial seizures in the mouse 6-Hz seizure test. A dose-dependent effect was found, with ED(50) of 170 mg/kg for minocycline, 157 mg/kg for doxycycline, and 255 mg/kg for tetracycline with peak onset at 0.5h. At high doses, minocycline (250 mg/kg) and doxycycline (150 mg/kg) also had toxic effects, from motor impairments to respiratory failure and death. These drugs had no effects on the MES and scMET tests.
CONCLUSIONS - In the three tests of anti-seizure activity, minocycline, doxycycline, and tetracycline were found to be protective in one: the 6-Hz seizure model. Our data suggest that minocycline and other tetracycline-class drugs may offer some degree of anticonvulsant effect in the setting of CNS disease trials.
Published by Elsevier Inc.
0 Communities
1 Members
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14 MeSH Terms
DKK1 mediated inhibition of Wnt signaling in postnatal mice leads to loss of TEC progenitors and thymic degeneration.
Osada M, Jardine L, Misir R, Andl T, Millar SE, Pezzano M
(2010) PLoS One 5: e9062
MeSH Terms: Animals, Apoptosis, Cell Count, Cell Proliferation, Doxycycline, Epithelial Cells, Female, Gene Expression Regulation, In Situ Hybridization, Intercellular Signaling Peptides and Proteins, Keratins, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Microscopy, Confocal, Reverse Transcriptase Polymerase Chain Reaction, Signal Transduction, Stem Cells, Tetracycline, Thymus Gland, Trans-Activators, Wnt Proteins
Show Abstract · Added January 30, 2013
BACKGROUND - Thymic epithelial cell (TEC) microenvironments are essential for the recruitment of T cell precursors from the bone marrow, as well as the subsequent expansion and selection of thymocytes resulting in a mature self-tolerant T cell repertoire. The molecular mechanisms, which control both the initial development and subsequent maintenance of these critical microenvironments, are poorly defined. Wnt signaling has been shown to be important to the development of several epithelial tissues and organs. Regulation of Wnt signaling has also been shown to impact both early thymocyte and thymic epithelial development. However, early blocks in thymic organogenesis or death of the mice have prevented analysis of a role of canonical Wnt signaling in the maintenance of TECs in the postnatal thymus.
METHODOLOGY/PRINCIPAL FINDINGS - Here we demonstrate that tetracycline-regulated expression of the canonical Wnt inhibitor DKK1 in TECs localized in both the cortex and medulla of adult mice, results in rapid thymic degeneration characterized by a loss of DeltaNP63(+) Foxn1(+) and Aire(+) TECs, loss of K5K8DP TECs thought to represent or contain an immature TEC progenitor, decreased TEC proliferation and the development of cystic structures, similar to an aged thymus. Removal of DKK1 from DKK1-involuted mice results in full recovery, suggesting that canonical Wnt signaling is required for the differentiation or proliferation of TEC populations needed for maintenance of properly organized adult thymic epithelial microenvironments.
CONCLUSIONS/SIGNIFICANCE - Taken together, the results of this study demonstrate that canonical Wnt signaling within TECs is required for the maintenance of epithelial microenvironments in the postnatal thymus, possibly through effects on TEC progenitor/stem cell populations. Downstream targets of Wnt signaling, which are responsible for maintenance of these TEC progenitors may provide useful targets for therapies aimed at counteracting age associated thymic involution or the premature thymic degeneration associated with cancer therapy and bone marrow transplants.
0 Communities
1 Members
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23 MeSH Terms
Focal adhesion kinase modulates cell adhesion strengthening via integrin activation.
Michael KE, Dumbauld DW, Burns KL, Hanks SK, García AJ
(2009) Mol Biol Cell 20: 2508-19
MeSH Terms: Animals, Biomechanical Phenomena, Cell Adhesion, Fibroblasts, Fibronectins, Focal Adhesion Protein-Tyrosine Kinases, Gene Knockdown Techniques, Humans, Integrin alpha5beta1, Integrins, Kinetics, Mice, Phosphorylation, Phosphotyrosine, Protein Binding, Solubility, Talin, Tetracycline, Vinculin
Show Abstract · Added January 20, 2015
Focal adhesion kinase (FAK) is an essential nonreceptor tyrosine kinase regulating cell migration, adhesive signaling, and mechanosensing. Using FAK-null cells expressing FAK under an inducible promoter, we demonstrate that FAK regulates the time-dependent generation of adhesive forces. During the early stages of adhesion, FAK expression in FAK-null cells enhances integrin activation to promote integrin binding and, hence, the adhesion strengthening rate. Importantly, FAK expression regulated integrin activation, and talin was required for the FAK-dependent effects. A role for FAK in integrin activation was confirmed in human fibroblasts with knocked-down FAK expression. The FAK autophosphorylation Y397 site was required for the enhancements in adhesion strengthening and integrin-binding responses. This work demonstrates a novel role for FAK in integrin activation and the time-dependent generation of cell-ECM forces.
0 Communities
1 Members
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19 MeSH Terms
Positive regulation of Raf1-MEK1/2-ERK1/2 signaling by protein serine/threonine phosphatase 2A holoenzymes.
Adams DG, Coffee RL, Zhang H, Pelech S, Strack S, Wadzinski BE
(2005) J Biol Chem 280: 42644-54
MeSH Terms: Blotting, Western, Catalytic Domain, Cell Line, Dimerization, Enzyme Activation, Enzyme Inhibitors, Epitopes, Gene Expression Regulation, Enzymologic, Humans, Immunoblotting, Immunoprecipitation, Luciferases, MAP Kinase Kinase 1, MAP Kinase Kinase 2, MAP Kinase Signaling System, Mitogen-Activated Protein Kinase 1, Mitogen-Activated Protein Kinase 3, Phosphoprotein Phosphatases, Phosphorylation, Plasmids, Protein Phosphatase 2, Proto-Oncogene Proteins c-raf, RNA Interference, Reverse Transcriptase Polymerase Chain Reaction, Serine, Signal Transduction, Tetracycline, Transfection
Show Abstract · Added December 10, 2013
Protein serine/threonine phosphatase 2A (PP2A) regulates a wide variety of cellular signal transduction pathways. The predominant form of PP2A in cells is a heterotrimeric holoenzyme consisting of a scaffolding (A) subunit, a regulatory (B) subunit, and a catalytic (C) subunit. Although PP2A is known to regulate Raf1-MEK1/2-ERK1/2 signaling at multiple steps in this pathway, the specific PP2A holoenzymes involved remain unclear. To address this question, we established tetracycline-inducible human embryonic kidney 293 cell lines for overexpression of FLAG-tagged Balpha/delta regulatory subunits by approximately 3-fold or knock-down of Balpha by greater than 70% compared with endogenous levels. The expression of functional epitope-tagged B subunits was confirmed by the detection of A and C subunits as well as phosphatase activity in FLAG immune complexes from extracts of cells overexpressing the FLAG-Balpha/delta subunit. Western analysis of the cell extracts using phosphospecific antibodies for MEK1/2 and ERK1/2 demonstrated that activation of these kinases in response to epidermal growth factor was markedly diminished in Balpha knock-down cells but elevated in Balpha- and Bdelta-overexpressing cells as compared with control cells. In parallel with the activation of MEK1/2 and ERK1/2, the inhibitory phosphorylation site of Raf1 (Ser-259) was dephosphorylated in cells overexpressing Balpha or Bdelta. Pharmacological inhibitor studies as well as reporter assays for ERK-dependent activation of the transcription factor Elk1 revealed that the PP2A holoenzymes ABalphaC and ABdeltaC act downstream of Ras and upstream of MEK1 to promote activation of this MAPK signaling cascade. Furthermore both PP2A holoenzymes were found to associate with Raf1 and catalyze dephosphorylation of inhibitory phospho-Ser-259. Together these findings indicate that PP2A ABalphaC and ABdeltaC holoenzymes function as positive regulators of Raf1-MEK1/2-ERK1/2 signaling by targeting Raf1.
0 Communities
1 Members
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28 MeSH Terms
Tetracycline protects myocardium against ischemic injury.
Kagawa N, Senbonmatsu TA, Satoh K, Ichihara K, Yamagata N, Hatano O, Saito T, Nguyen VQ, Waterman MR, Price E, Atkinson JB, Inagami T
(2005) Front Biosci 10: 608-19
MeSH Terms: Animals, Dogs, HeLa Cells, Heart, Hemodynamics, Humans, Ischemia, Mice, Myocardial Ischemia, Myocardium, Reperfusion, Tetracycline
Show Abstract · Added February 12, 2015
Stress pretreatments protect myocardium from ischemic injury. We hypothesized that tetracycline, an antibiotic, may induce a stress response via the inhibition of mitochondrial translation as it induces the cold stress response by translational inhibition in E. coli. If so, tetracycline may protect myocardium from ischemic injury as stress pretreatments do. Thus, we investigated the effects of tetracycline on myocardial ischemia and its association with stress response. In a dog model of acute ischemia, 4mg/kg tetracycline injected 30 min prior to the occlusion improved the functional recovery from stunning of myocardium caused by ischemia. The same dosage of tetracycline dramatically reduced the size of infarct area in murine hearts analyzed by tetrazolium staining. In HeLa cells, tetracycline induced molecules that were increased by cold stress, which suggests that tetracycline may induce a cold stress-like response in mammalian cells. These molecules were also induced by ischemic stress in murine hearts, suggesting that the stress response caused by translational inhibition in mitochondria may be associated with the cardioprotection by tetracycline. Our results suggest that a subclinical dosage of tetracycline may protect heart from ischemic injury. Therefore, tetracycline may be of great use in suppressing the development of infarction caused by myocardial ischemia. This study is also important for providing new insights into the non-antimicrobial effects of tetracycline and its derivatives.
0 Communities
1 Members
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12 MeSH Terms
Development of a tightly regulated U6 promoter for shRNA expression.
Lin X, Yang J, Chen J, Gunasekera A, Fesik SW, Shen Y
(2004) FEBS Lett 577: 376-80
MeSH Terms: Blotting, Western, Cell Line, Tumor, Cloning, Molecular, Gene Expression Regulation, Genes, Reporter, Genetic Variation, HeLa Cells, Humans, Luciferases, Molecular Sequence Data, Promoter Regions, Genetic, RNA, Small Interfering, Sequence Homology, Nucleic Acid, TATA Box, Tetracycline, Transcription, Genetic
Show Abstract · Added March 5, 2014
Short hairpin RNAs (shRNAs) have been used to achieve stable target knockdown in a variety of biological systems. Here, we report the development of a tightly regulated tetracycline-responsive human U6 promoter for shRNA expression. By engineering two copies of the tet operators flanking the TATA box of the human U6 promoter, we created a U6 promoter derivative (2O2) that exhibited much lower basal transcriptional activity compared with recently reported inducible pol III dependent promoters. As a consequence of its tighter regulation, the 2O2 system greatly improved the success rate in making inducible knockdown cell lines.
0 Communities
1 Members
0 Resources
16 MeSH Terms
CMT-3. CollaGenex.
Fingleton B
(2003) Curr Opin Investig Drugs 4: 1460-7
MeSH Terms: Animals, Clinical Trials as Topic, Drugs, Investigational, Enzyme Inhibitors, Humans, Matrix Metalloproteinase Inhibitors, Matrix Metalloproteinases, Neoplasms, Technology, Pharmaceutical, Tetracyclines
Show Abstract · Added March 5, 2014
CMT-3 is an orally active matrix metalloprotease inhibitor, and one of a series of inhibitors of multiple proteases and cytokines, under development by CollaGenex. The compound is currently undergoing phase II trials for the potential treatment of cancer, in particular metastatic cancer and HIV-related Kaposi's sarcoma.
1 Communities
1 Members
0 Resources
10 MeSH Terms
Inducible podocyte-specific gene expression in transgenic mice.
Shigehara T, Zaragoza C, Kitiyakara C, Takahashi H, Lu H, Moeller M, Holzman LB, Kopp JB
(2003) J Am Soc Nephrol 14: 1998-2003
MeSH Terms: Animals, Cytomegalovirus, DNA, Dose-Response Relationship, Drug, Doxycycline, Gene Expression Regulation, Genes, Reporter, Humans, Kidney, Kidney Glomerulus, Lac Operon, Mice, Mice, Transgenic, Microscopy, Fluorescence, Plasmids, Polymerase Chain Reaction, Promoter Regions, Genetic, Protein Structure, Tertiary, Tetracycline, Time Factors, Tissue Distribution, Transcription, Genetic, Transcriptional Activation, Transgenes, beta-Galactosidase
Show Abstract · Added September 9, 2013
The podocyte plays a key role in glomerular function and glomerular disease. To facilitate studies of podocyte function, we have developed a transgenic mouse model with inducible expression in the podocyte. The tetracycline-inducible transgenic system facilitates gene expression with restricted cellular distribution and tight temporal control. Recently, Bujard and colleagues have developed a functionally improved reverse tetracycline-controlled transcriptional activator (rtTA) with substantially lower background in the off state (the absence of tetracycline) and greater inducibility in the on state (the presence of tetracycline). We used the human podocin (NPHS2) gene promoter to control expression of the rtTA cassette and bred these mice with a reporter mouse line that contains the cytomegalovirus minimal promoter and tetO promoter elements together with LacZ, encoding beta-galactosidase. Dual transgenic mice, bearing both podocin-rtTA and tetO-LacZ transgenes, had no detectable expression in kidney or other organs in the absence of tetracycline. Administration of tetracycline in the drinking water was associated with podocyte expression of beta-galactosidase, in a fashion that was time dependent (maximal at 1 wk) and dose-dependent (maximal at 2 mg/ml). Podocyte expression was confirmed in two ways: histochemical staining for beta-galactosidase and double-immunostaining using the podocyte marker WT-1 and beta-galactosidase. This transgenic system should aid future investigations of podocyte function.
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25 MeSH Terms