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Genomic Instability Associated with p53 Knockdown in the Generation of Huntington's Disease Human Induced Pluripotent Stem Cells.
Tidball AM, Neely MD, Chamberlin R, Aboud AA, Kumar KK, Han B, Bryan MR, Aschner M, Ess KC, Bowman AB
(2016) PLoS One 11: e0150372
MeSH Terms: Adult, Aged, Cells, Cultured, DNA Damage, Gene Knockdown Techniques, Genomic Instability, Humans, Huntington Disease, Induced Pluripotent Stem Cells, Karyotyping, Middle Aged, Nucleic Acid Synthesis Inhibitors, Signal Transduction, Tumor Suppressor Protein p53, Young Adult, Zinostatin
Show Abstract · Added August 18, 2016
Alterations in DNA damage response and repair have been observed in Huntington's disease (HD). We generated induced pluripotent stem cells (iPSC) from primary dermal fibroblasts of 5 patients with HD and 5 control subjects. A significant fraction of the HD iPSC lines had genomic abnormalities as assessed by karyotype analysis, while none of our control lines had detectable genomic abnormalities. We demonstrate a statistically significant increase in genomic instability in HD cells during reprogramming. We also report a significant association with repeat length and severity of this instability. Our karyotypically normal HD iPSCs also have elevated ATM-p53 signaling as shown by elevated levels of phosphorylated p53 and H2AX, indicating either elevated DNA damage or hypersensitive DNA damage signaling in HD iPSCs. Thus, increased DNA damage responses in the HD genotype is coincidental with the observed chromosomal aberrations. We conclude that the disease causing mutation in HD increases the propensity of chromosomal instability relative to control fibroblasts specifically during reprogramming to a pluripotent state by a commonly used episomal-based method that includes p53 knockdown.
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16 MeSH Terms
Efficacy and safety of rifampicin for multiple system atrophy: a randomised, double-blind, placebo-controlled trial.
Low PA, Robertson D, Gilman S, Kaufmann H, Singer W, Biaggioni I, Freeman R, Perlman S, Hauser RA, Cheshire W, Lessig S, Vernino S, Mandrekar J, Dupont WD, Chelimsky T, Galpern WR
(2014) Lancet Neurol 13: 268-75
MeSH Terms: Aged, Cohort Studies, Double-Blind Method, Female, Follow-Up Studies, Humans, Male, Middle Aged, Multiple System Atrophy, Nucleic Acid Synthesis Inhibitors, Rifampin, Treatment Outcome
Show Abstract · Added March 21, 2014
BACKGROUND - No available treatments slow or halt progression of multiple system atrophy, which is a rare, progressive, fatal neurological disorder. In a mouse model of multiple system atrophy, rifampicin inhibited formation of α-synuclein fibrils, the neuropathological hallmark of the disease. We aimed to assess the safety and efficacy of rifampicin in patients with multiple system atrophy.
METHODS - In this randomised, double-blind, placebo-controlled trial we recruited participants aged 30-80 years with possible or probable multiple system atrophy from ten US medical centres. Eligible participants were randomly assigned (1:1) via computer-generated permuted block randomisation to rifampicin 300 mg twice daily or matching placebo (50 mg riboflavin capsules), stratified by subtype (parkinsonian vs cerebellar), with a block size of four. The primary outcome was rate of change (slope analysis) from baseline to 12 months in Unified Multiple System Atrophy Rating Scale (UMSARS) I score, analysed in all participants with at least one post-baseline measurement. This study is registered with ClinicalTrials.gov, number NCT01287221.
FINDINGS - Between April 22, 2011, and April 19, 2012, we randomly assigned 100 participants (50 to rifampicin and 50 to placebo). Four participants in the rifampicin group and five in the placebo group withdrew from study prematurely. Results of the preplanned interim analysis (n=15 in each group) of the primary endpoint showed that futility criteria had been met, and the trial was stopped (the mean rate of change [slope analysis] of UMSARS I score was 0.62 points [SD 0.85] per month in the rifampicin group vs 0.47 points [0.48] per month in the placebo group; futility p=0.032; efficacy p=0.76). At the time of study termination, 49 participants in the rifampicin group and 50 in the placebo group had follow-up data and were included in the final analysis. The primary endpoint was 0.5 points (SD 0.7) per month for rifampicin and 0.5 points (0.5) per month for placebo (difference 0.0, 95% CI -0.24 to 0.24; p=0.82). Three (6%) of 50 participants in the rifampicin group and 12 (24%) of 50 in the placebo group had one or more serious adverse events; none was thought to be related to treatment.
INTERPRETATION - Our results show that rifampicin does not slow or halt progression of multiple system atrophy. Despite the negative result, the trial does provide information that could be useful in the design of future studies assessing potential disease modifying therapies in patients with multiple system atrophy.
FUNDING - National Institutes of Health, Mayo Clinic Center for Translational Science Activities, and Mayo Funds.
Copyright © 2014 Elsevier Ltd. All rights reserved.
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12 MeSH Terms
Leukotriene biosynthesis inhibitor MK886 impedes DNA polymerase activity.
Ketkar A, Zafar MK, Maddukuri L, Yamanaka K, Banerjee S, Egli M, Choi JY, Lloyd RS, Eoff RL
(2013) Chem Res Toxicol 26: 221-32
MeSH Terms: Binding Sites, DNA-Directed DNA Polymerase, Humans, Indoles, Kinetics, Leukotrienes, Lipoxygenase Inhibitors, Molecular Docking Simulation, Nucleic Acid Synthesis Inhibitors
Show Abstract · Added May 27, 2014
Specialized DNA polymerases participate in replication stress responses and in DNA repair pathways that function as barriers against cellular senescence and genomic instability. These events can be co-opted by tumor cells as a mechanism to survive chemotherapeutic and ionizing radiation treatments and as such, represent potential targets for adjuvant therapies. Previously, a high-throughput screen of ∼16,000 compounds identified several first generation proof-of-principle inhibitors of human DNA polymerase kappa (hpol κ). The indole-derived inhibitor of 5-lipoxygenase activating protein (FLAP), MK886, was one of the most potent inhibitors of hpol κ discovered in that screen. However, the specificity and mechanism of inhibition remained largely undefined. In the current study, the specificity of MK886 against human Y-family DNA polymerases and a model B-family DNA polymerase was investigated. MK886 was found to inhibit the activity of all DNA polymerases tested with similar IC(50) values, the exception being a 6- to 8-fold increase in the potency of inhibition against human DNA polymerase iota (hpol ι), a highly error-prone enzyme that uses Hoogsteen base-pairing modes during catalysis. The specificity against hpol ι was partially abrogated by inclusion of the recently annotated 25 a.a. N-terminal extension. On the basis of Michaelis-Menten kinetic analyses and DNA binding assays, the mechanism of inhibition by MK886 appears to be mixed. In silico docking studies were used to produce a series of models for MK886 binding to Y-family members. The docking results indicate that two binding pockets are conserved between Y-family polymerases, while a third pocket near the thumb domain appears to be unique to hpol ι. Overall, these results provide insight into the general mechanism of DNA polymerase inhibition by MK886.
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9 MeSH Terms
A comprehensive strategy to discover inhibitors of the translesion synthesis DNA polymerase κ.
Yamanaka K, Dorjsuren D, Eoff RL, Egli M, Maloney DJ, Jadhav A, Simeonov A, Lloyd RS
(2012) PLoS One 7: e45032
MeSH Terms: Acrolein, Benzimidazoles, Biphenyl Compounds, Cell Line, Transformed, Cell Survival, DNA, DNA Adducts, DNA Damage, DNA Repair, DNA-Directed DNA Polymerase, Deoxyguanosine, Dose-Response Relationship, Drug, Drug Evaluation, Preclinical, Enzyme Inhibitors, Humans, Indoles, Nucleic Acid Synthesis Inhibitors, Small Molecule Libraries, Terpenes, Tetrazoles, Ultraviolet Rays
Show Abstract · Added May 27, 2014
Human DNA polymerase kappa (pol κ) is a translesion synthesis (TLS) polymerase that catalyzes TLS past various minor groove lesions including N(2)-dG linked acrolein- and polycyclic aromatic hydrocarbon-derived adducts, as well as N(2)-dG DNA-DNA interstrand cross-links introduced by the chemotherapeutic agent mitomycin C. It also processes ultraviolet light-induced DNA lesions. Since pol κ TLS activity can reduce the cellular toxicity of chemotherapeutic agents and since gliomas overexpress pol κ, small molecule library screens targeting pol κ were conducted to initiate the first step in the development of new adjunct cancer therapeutics. A high-throughput, fluorescence-based DNA strand displacement assay was utilized to screen ∼16,000 bioactive compounds, and the 60 top hits were validated by primer extension assays using non-damaged DNAs. Candesartan cilexetil, manoalide, and MK-886 were selected as proof-of-principle compounds and further characterized for their specificity toward pol κ by primer extension assays using DNAs containing a site-specific acrolein-derived, ring-opened reduced form of γ-HOPdG. Furthermore, candesartan cilexetil could enhance ultraviolet light-induced cytotoxicity in xeroderma pigmentosum variant cells, suggesting its inhibitory effect against intracellular pol κ. In summary, this investigation represents the first high-throughput screening designed to identify inhibitors of pol κ, with the characterization of biochemical and biologically relevant endpoints as a consequence of pol κ inhibition. These approaches lay the foundation for the future discovery of compounds that can be applied to combination chemotherapy.
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21 MeSH Terms
Single molecule analysis of serotonin transporter regulation using antagonist-conjugated quantum dots reveals restricted, p38 MAPK-dependent mobilization underlying uptake activation.
Chang JC, Tomlinson ID, Warnement MR, Ustione A, Carneiro AM, Piston DW, Blakely RD, Rosenthal SJ
(2012) J Neurosci 32: 8919-29
MeSH Terms: Actins, Animals, Cell Line, Transformed, Cholera Toxin, Cholesterol, Cyclic GMP, Cytochalasin D, Enzyme Inhibitors, Gangliosidosis, GM1, Imidazoles, Ligands, Membrane Microdomains, Microscopy, Confocal, Neurons, Normal Distribution, Nucleic Acid Synthesis Inhibitors, Protein Transport, Pyridines, Quantum Dots, Rats, Serotonin, Serotonin Plasma Membrane Transport Proteins, Thionucleotides, beta-Cyclodextrins, p38 Mitogen-Activated Protein Kinases
Show Abstract · Added November 8, 2013
The presynaptic serotonin (5-HT) transporter (SERT) is targeted by widely prescribed antidepressant medications. Altered SERT expression or regulation has been implicated in multiple neuropsychiatric disorders, including anxiety, depression and autism. Here, we implement a generalizable strategy that exploits antagonist-conjugated quantum dots (Qdots) to monitor, for the first time, single SERT proteins on the surface of serotonergic cells. We document two pools of SERT proteins defined by lateral mobility, one that exhibits relatively free diffusion, and a second, localized to cholesterol and GM1 ganglioside-enriched microdomains, that displays restricted mobility. Receptor-linked signaling pathways that enhance SERT activity mobilize transporters that, nonetheless, remain confined to membrane microdomains. Mobilization of transporters arises from a p38 MAPK-dependent untethering of the SERT C terminus from the juxtamembrane actin cytoskeleton. Our studies establish the utility of ligand-conjugated Qdots for analysis of the behavior of single membrane proteins and reveal a physical basis for signaling-mediated SERT regulation.
1 Communities
5 Members
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25 MeSH Terms
POLG mutations cause decreased mitochondrial DNA repopulation rates following induced depletion in human fibroblasts.
Stewart JD, Schoeler S, Sitarz KS, Horvath R, Hallmann K, Pyle A, Yu-Wai-Man P, Taylor RW, Samuels DC, Kunz WS, Chinnery PF
(2011) Biochim Biophys Acta 1812: 321-5
MeSH Terms: Adult, Amino Acid Substitution, Case-Control Studies, DNA Polymerase gamma, DNA Replication, DNA, Mitochondrial, DNA-Directed DNA Polymerase, Diffuse Cerebral Sclerosis of Schilder, Enzyme Inhibitors, Epilepsy, Ethidium, Female, Fibroblasts, Heterozygote, Homozygote, Humans, Infant, Male, Mitochondria, Muscular Diseases, Mutation, Nucleic Acid Synthesis Inhibitors, Thymidine Kinase
Show Abstract · Added December 12, 2013
Disorders of mitochondrial DNA (mtDNA) maintenance have emerged as an important cause of human genetic disease, but demonstrating the functional consequences of de novo mutations remains a major challenge. We studied the rate of depletion and repopulation of mtDNA in human fibroblasts exposed to ethidium bromide in patients with heterozygous POLG mutations, POLG2 and TK2 mutations. Ethidium bromide induced mtDNA depletion occurred at the same rate in human fibroblasts from patients and healthy controls. By contrast, the restoration of mtDNA levels was markedly delayed in fibroblasts from patients with compound heterozygous POLG mutations. Specific POLG2 and TK2 mutations did not delay mtDNA repopulation rates. These observations are consistent with the hypothesis that mutations in POLG impair mtDNA repopulation within intact cells, and provide a potential method of demonstrating the functional consequences of putative pathogenic alleles causing a defect of mtDNA synthesis.
Copyright © 2010 Elsevier B.V. All rights reserved.
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23 MeSH Terms
An analysis of enzyme kinetics data for mitochondrial DNA strand termination by nucleoside reverse transcription inhibitors.
Wendelsdorf KV, Song Z, Cao Y, Samuels DC
(2009) PLoS Comput Biol 5: e1000261
MeSH Terms: Base Sequence, DNA Replication, DNA, Mitochondrial, DNA-Directed DNA Polymerase, Deoxyribonucleotides, Dose-Response Relationship, Drug, Humans, Kinetics, Models, Biological, Nucleic Acid Synthesis Inhibitors, Nucleosides, Reverse Transcriptase Inhibitors, Zidovudine
Show Abstract · Added December 12, 2013
Nucleoside analogs used in antiretroviral treatment have been associated with mitochondrial toxicity. The polymerase-gamma hypothesis states that this toxicity stems from the analogs' inhibition of the mitochondrial DNA polymerase (polymerase-gamma) leading to mitochondrial DNA (mtDNA) depletion. We have constructed a computational model of the interaction of polymerase-gamma with activated nucleoside and nucleotide analog drugs, based on experimentally measured reaction rates and base excision rates, together with the mtDNA genome size, the human mtDNA sequence, and mitochondrial dNTP concentrations. The model predicts an approximately 1000-fold difference in the activated drug concentration required for a 50% probability of mtDNA strand termination between the activated di-deoxy analogs d4T, ddC, and ddI (activated to ddA) and the activated forms of the analogs 3TC, TDF, AZT, FTC, and ABC. These predictions are supported by experimental and clinical data showing significantly greater mtDNA depletion in cell culture and patient samples caused by the di-deoxy analog drugs. For zidovudine (AZT) we calculated a very low mtDNA replication termination probability, in contrast to its reported mitochondrial toxicity in vitro and clinically. Therefore AZT mitochondrial toxicity is likely due to a mechanism that does not involve strand termination of mtDNA replication.
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1 Members
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13 MeSH Terms
Dpb11 activates the Mec1-Ddc2 complex.
Mordes DA, Nam EA, Cortez D
(2008) Proc Natl Acad Sci U S A 105: 18730-4
MeSH Terms: Adaptor Proteins, Signal Transducing, Cell Cycle Proteins, DNA Damage, DNA Repair, Enzyme Activation, Humans, Hydroxyurea, Intracellular Signaling Peptides and Proteins, Nucleic Acid Synthesis Inhibitors, Phosphoproteins, Protein-Serine-Threonine Kinases, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins
Show Abstract · Added March 5, 2014
The Saccharomyces cerevisiae Mec1-Ddc2 checkpoint kinase complex (the ortholog to human ATR-ATRIP) is an essential regulator of genomic integrity. The S. cerevisiae BRCT repeat protein Dpb11 functions in the initiation of both DNA replication and cell cycle checkpoints. Here, we report a genetic and physical interaction between Dpb11 and Mec1-Ddc2. A C-terminal domain of Dpb11 is sufficient to associate with Mec1-Ddc2 and strongly stimulates the kinase activity of Mec1 in a Ddc2-dependent manner. Furthermore, Mec1 phosphorylates Dpb11 and thereby amplifies the stimulating effect of Dpb11 on Mec1-Ddc2 kinase activity. Thus, Dpb11 is a functional ortholog of human TopBP1, and the Mec1/ATR activation mechanism is conserved from yeast to humans.
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1 Members
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13 MeSH Terms
Activation of the DNA-dependent protein kinase by drug-induced and radiation-induced DNA strand breaks.
Mårtensson S, Nygren J, Osheroff N, Hammarsten O
(2003) Radiat Res 160: 291-301
MeSH Terms: Aminoglycosides, Anti-Bacterial Agents, Antibiotics, Antineoplastic, Antigens, Nuclear, Antimetabolites, Antineoplastic, Bleomycin, DNA, DNA Damage, DNA Helicases, DNA Repair, DNA Topoisomerases, Type II, DNA-Activated Protein Kinase, DNA-Binding Proteins, Dose-Response Relationship, Drug, Enediynes, Energy Transfer, Enzyme Activation, Etoposide, Ferritins, Ku Autoantigen, Models, Biological, Nucleic Acid Synthesis Inhibitors, Plasmids, Protein Binding, Protein-Serine-Threonine Kinases, Radiation, Ionizing
Show Abstract · Added March 5, 2014
The DNA-dependent protein kinase (DNA-PK) is a DNA-end activated protein kinase that is required for efficient repair of DNA double-strand breaks (DSBs) and for normal resistance to ionizing radiation. DNA-PK is composed of a DNA-binding subunit, Ku, and a catalytic subunit, DNA-PKcs (PRKDC). We have previously shown that PRKDC is activated when the enzyme interacts with the terminal nucleotides of a DSB. These nucleotides are often damaged when DSBs are introduced by anticancer agents and could therefore prevent recognition by DNA-PK. To determine whether DNA-PK could recognize DNA strand breaks generated by agents used in the treatment of cancer, we damaged plasmid DNA with anticancer drugs and ionizing radiation. The DNA breaks were tested for the ability to activate purified DNA-PK. The data indicate that DSBs produced by bleomycin, calicheamicin and two types of ionizing radiation ((137)Cs gamma rays and N(7+) ions: high and low linear energy transfer, respectively) activate DNA-PK to levels matching the kinase activation obtained with simple restriction endonuclease-induced DSBs. In contrast, the protein-linked DSBs produced by etoposide and topoisomerase II failed to bind and activate DNA-PK. Our findings indicate that DNA-PK recognizes DSBs regardless of chemical complexity but cannot recognize the protein-linked DSBs produced by etoposide and topoisomerase II.
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26 MeSH Terms
Cyclooxygenase-2 expression in cultured cortical thick ascending limb of Henle increases in response to decreased extracellular ionic content by both transcriptional and post-transcriptional mechanisms. Role of p38-mediated pathways.
Cheng HF, Harris RC
(2002) J Biol Chem 277: 45638-43
MeSH Terms: Animals, Binding Sites, Cells, Cultured, Cyclooxygenase 2, Dactinomycin, Enzyme Inhibitors, Genes, Reporter, Humans, I-kappa B Kinase, Isoenzymes, Loop of Henle, Membrane Proteins, Mice, Mitogen-Activated Protein Kinases, Mutation, NF-kappa B, Nucleic Acid Synthesis Inhibitors, Promoter Regions, Genetic, Prostaglandin-Endoperoxide Synthases, Protein-Serine-Threonine Kinases, RNA Stability, Rabbits, Sodium Chloride, Transcription, Genetic, p38 Mitogen-Activated Protein Kinases
Show Abstract · Added August 21, 2013
We showed previously that decreased extracellular salt or chloride up-regulates the cortical thick ascending limb of Henle (cTALH) COX-2 expression via a p38-dependent pathway. The present studies determined that low salt medium increased COX-2 mRNA expression 3.9-fold control by 6 h in cultured cTALH, which was blocked by actinomycin D pretreatment, suggesting transcriptional regulation. Luciferase activity (normalized to beta-galactosidase activity) of the full-length (-3400) COX-2 promoter in cTALH increased from 1.8 +/- 0.3 in control media to 5.8 +/- 0.7 in low salt (n = 9; p < 0.01). Low chloride medium had similar effects as low salt has on COX-2 promoter activity. Deletion constructs -815, -512, and -410 were similarly stimulated, but -385 could not be stimulated significantly by low salt (1.8 +/- 0.3 versus 2.4 +/- 0.5, n = 10). This suggested involvement of an NF-kappaB cis-element located in this region, which was confirmed by utilizing a construct with a point mutation of this NF-kappaB-binding site that was not stimulated by low salt medium. Co-incubation of the specific p38 inhibitor, SB203580 or PD169316, inhibited a low salt-induced increase in luciferase activity of the intact COX-2 promoter (5.8 +/- 0.7 versus 1.1 +/- 0.2, n = 8 and 1.4 +/- 0.4, n = 4 respectively, p < 0.01). Mobility shift assays indicated that the low salt medium stimulated NF-kappaB binding activity, and this stimulation was inhibited by p38 inhibitors. To test whether p38 also increased COX-2 expression by increasing mRNA stability, cTALH were incubated in low salt for 2 h, and actinomycin was then added with or without SB203580. p38 inhibition led to a decreased half-life of COX-2 mRNA (from 68 to 18 min, n = 4-7, p < 0.05). Therefore, these studies indicate that p38 stimulates COX-2 expression in cTALH and macula densa by transcriptional regulation predominantly via a NF-kappaB-dependent pathway and by post-transcriptional increases in mRNA stability.
1 Communities
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25 MeSH Terms