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Results: 1 to 10 of 26

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Discovery of human cell selective effector molecules using single cell multiplexed activity metabolomics.
Earl DC, Ferrell PB, Leelatian N, Froese JT, Reisman BJ, Irish JM, Bachmann BO
(2018) Nat Commun 9: 39
MeSH Terms: Aged, Bone Marrow, Cell Extracts, Chromatography, Liquid, DNA Damage, Female, Flow Cytometry, Humans, Leukemia, Leukemia, Myeloid, Acute, Lymphocytes, Male, Mass Spectrometry, Metabolome, Metabolomics, Monocytes, Signal Transduction, Streptomyces, Tumor Cells, Cultured, Young Adult
Show Abstract · Added January 4, 2018
Discovering bioactive metabolites within a metabolome is challenging because there is generally little foreknowledge of metabolite molecular and cell-targeting activities. Here, single-cell response profiles and primary human tissue comprise a response platform used to discover novel microbial metabolites with cell-type-selective effector properties in untargeted metabolomic inventories. Metabolites display diverse effector mechanisms, including targeting protein synthesis, cell cycle status, DNA damage repair, necrosis, apoptosis, or phosphoprotein signaling. Arrayed metabolites are tested against acute myeloid leukemia patient bone marrow and molecules that specifically targeted blast cells or nonleukemic immune cell subsets within the same tissue biopsy are revealed. Cell-targeting polyketides are identified in extracts from biosynthetically prolific bacteria, including a previously unreported leukemia blast-targeting anthracycline and a polyene macrolactam that alternates between targeting blasts or nonmalignant cells by way of light-triggered photochemical isomerization. High-resolution cell profiling with mass cytometry confirms response mechanisms and is used to validate initial observations.
3 Communities
1 Members
0 Resources
20 MeSH Terms
Identification of a Paralog-Specific Notch1 Intracellular Domain Degron.
Broadus MR, Chen TW, Neitzel LR, Ng VH, Jodoin JN, Lee LA, Salic A, Robbins DJ, Capobianco AJ, Patton JG, Huppert SS, Lee E
(2016) Cell Rep 15: 1920-9
MeSH Terms: Amino Acid Sequence, Animals, Cell Extracts, Embryo, Nonmammalian, F-Box Proteins, HEK293 Cells, Humans, Muscle Proteins, Mutation, Protein Binding, Protein Domains, Protein Stability, Proteolysis, Receptor, Notch1, Regulatory Sequences, Nucleic Acid, Sequence Homology, Amino Acid, Transcription, Genetic, Ubiquitin-Protein Ligases, Xenopus, Zebrafish
Show Abstract · Added February 13, 2017
Upon Notch pathway activation, the receptor is cleaved to release the Notch intracellular domain (NICD), which translocates to the nucleus to activate gene transcription. Using Xenopus egg extracts, we have identified a Notch1-specific destruction signal (N1-Box). We show that mutations in the N1-Box inhibit NICD1 degradation and that the N1-Box is transferable for the promotion of degradation of heterologous proteins in Xenopus egg extracts and in cultured human cells. Mutation of the N1-Box enhances Notch1 activity in cultured human cells and zebrafish embryos. Human cancer mutations within the N1-Box enhance Notch1 signaling in transgenic zebrafish, highlighting the physiological relevance of this destruction signal. We find that binding of the Notch nuclear factor, CSL, to the N1-Box blocks NICD1 turnover. Our studies reveal a mechanism by which degradation of NICD1 is regulated by the N1-Box to minimize stochastic flux and to establish a threshold for Notch1 pathway activation.
Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.
0 Communities
2 Members
0 Resources
20 MeSH Terms
Toward a Broader View of Ube3a in a Mouse Model of Angelman Syndrome: Expression in Brain, Spinal Cord, Sciatic Nerve and Glial Cells.
Grier MD, Carson RP, Lagrange AH
(2015) PLoS One 10: e0124649
MeSH Terms: Alleles, Angelman Syndrome, Animals, Astrocytes, Brain, Cell Extracts, Cells, Cultured, Disease Models, Animal, Female, Gene Silencing, Genomic Imprinting, Humans, Male, Mice, Mice, Inbred C57BL, Neuroglia, Oligodendroglia, Sciatic Nerve, Spinal Cord, Time Factors, Ubiquitin-Protein Ligases
Show Abstract · Added March 14, 2018
Angelman Syndrome (AS) is a devastating neurodevelopmental disorder characterized by developmental delay, speech impairment, movement disorder, sleep disorders and refractory epilepsy. AS is caused by loss of the Ube3a protein encoded for by the imprinted Ube3a gene. Ube3a is expressed nearly exclusively from the maternal chromosome in mature neurons. While imprinting in neurons of the brain has been well described, the imprinting and expression of Ube3a in other neural tissues remains relatively unexplored. Moreover, given the overwhelming deficits in brain function in AS patients, the possibility of disrupted Ube3a expression in the infratentorial nervous system and its consequent disability have been largely ignored. We evaluated the imprinting status of Ube3a in the spinal cord and sciatic nerve and show that it is also imprinted in these neural tissues. Furthermore, a growing body of clinical and radiological evidence has suggested that myelin dysfunction may contribute to morbidity in many neurodevelopmental syndromes. However, findings regarding Ube3a expression in non-neuronal cells of the brain have varied. Utilizing enriched primary cultures of oligodendrocytes and astrocytes, we show that Ube3a is expressed, but not imprinted in these cell types. Unlike many other neurodevelopmental disorders, AS symptoms do not become apparent until roughly 6 to 12 months of age. To determine the temporal expression pattern and silencing, we analyzed Ube3a expression in AS mice at several time points. We confirm relaxed imprinting of Ube3a in neurons of the postnatal developing cortex, but not in structures in which neurogenesis and migration are more complete. This furthers the hypothesis that the apparently normal window of development in AS patients is supported by an incompletely silenced paternal allele in developing neurons, resulting in a relative preservation of Ube3a expression during this crucial epoch of early development.
0 Communities
1 Members
0 Resources
21 MeSH Terms
Biotinylated probes for the analysis of protein modification by electrophiles.
Codreanu SG, Kim HY, Porter NA, Liebler DC
(2012) Methods Mol Biol 803: 77-95
MeSH Terms: Aldehydes, Amino Acid Sequence, Biochemistry, Biotin, Biotinylation, Blood Proteins, Blotting, Western, Cell Extracts, Cell Line, Tumor, Databases, Protein, Humans, Immunoblotting, Indicators and Reagents, Mass Spectrometry, Molecular Probes, Molecular Sequence Data, Peptides, Protein Processing, Post-Translational, Proteins, Streptavidin, Trypsin
Show Abstract · Added March 7, 2014
Formation of covalent protein adducts by lipid electrophiles contributes to diseases and toxicities linked to oxidative stress, but analysis of the adducts presents a challenging analytical problem. We describe selective adduct capture using biotin affinity probes to enrich protein and peptide adducts for analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). One approach employs biotinamidohexanoic acid hydrazide to covalently label residual carbonyl groups on adducts. The other employs alkynyl analogs of lipid electrophiles, which form adducts that can be postlabeled with azidobiotin tags by Cu(+)-catalyzed cycloaddition (Click chemistry). To enhance the selectivity of adduct capture, we use an azidobiotin reagent with a photocleavable linker, which allows recovery of adducted proteins and peptides under mild conditions. This approach allows both the identification of protein targets of lipid electrophiles and sequence mapping of the adducts.
0 Communities
2 Members
0 Resources
21 MeSH Terms
Assessing neuronal bioenergetic status.
Zeiger SL, Stankowski JN, McLaughlin B
(2011) Methods Mol Biol 758: 215-35
MeSH Terms: Adenosine Triphosphate, Animals, Brain, Cell Extracts, Cell Hypoxia, Cell Line, Tumor, Cell Survival, Embryo, Mammalian, Energy Metabolism, Female, Galactose, Glucose, Humans, L-Lactate Dehydrogenase, Lactic Acid, Neurons, Pregnancy, Pyruvic Acid, Rats
Show Abstract · Added January 26, 2015
Drug discovery and therapeutic development for disorders of the central nervous system (CNS) represents one of the largest unmet markets in modern medicine. We have increasingly recognized that the lack of stringent assessment of mitochondrial function during the discovery process has resulted in drug recalls, black box warnings, and an urgent need to understand the metabolic liability of small molecules in neural systems. Given that the brain is the most energetically demanding organ, even modest perturbations in neuronal energetic pathways have been shown to impact growth, signaling, connectivity, and the restorative capacity of the CNS. In this work, we describe several tools to assess metabolic activity of primary neuronal cultures and neural cell lines using an acute model of injury induced by oxygen glucose deprivation. Methods include the measurement of total ATP and NADH, enzymatic assessment of lactate production by anaerobic respiration, as well as viability assays. We also present a modified screening method for assessing aerobic respiration of immortalized cell lines using galactose challenge.
1 Communities
1 Members
0 Resources
19 MeSH Terms
Trichloroacetic acid (TCA) precipitation of proteins.
Link AJ, LaBaer J
(2011) Cold Spring Harb Protoc 2011: 993-4
MeSH Terms: Cell Extracts, Chemical Fractionation, Chemical Precipitation, Proteins, Trichloroacetic Acid
Added February 20, 2015
0 Communities
1 Members
0 Resources
5 MeSH Terms
Detection of respiratory syncytial virus using nanoparticle amplified immuno-polymerase chain reaction.
Perez JW, Vargis EA, Russ PK, Haselton FR, Wright DW
(2011) Anal Biochem 410: 141-8
MeSH Terms: Animals, Antibodies, Antigens, Base Sequence, Biosensing Techniques, Cell Extracts, Cell Line, DNA, Viral, Gold, Immunoassay, Limit of Detection, Metal Nanoparticles, Polymerase Chain Reaction, Quartz Crystal Microbalance Techniques, Reproducibility of Results, Respiratory Syncytial Viruses
Show Abstract · Added May 27, 2014
In traditional immuno-polymerase chain reaction (immuno-PCR), a single antibody recognition event is associated with one to three DNA tags, which are subsequently amplified by PCR. Here we describe a nanoparticle-amplified immuno-PCR (NPA-IPCR) assay that combines antibody recognition of enzyme-linked immunosorbent assay (ELISA) with a 50-fold nanoparticle valence amplification step prior to tag amplification by PCR. The assay detects a respiratory syncytial virus (RSV) surface protein using an antibody bound to a 15-nm gold nanoparticle cofunctionalized with thiolated DNA complementary to a hybridized 76-base tag DNA with a tag DNA/antibody ratio of 50:1. The presence of virus particles triggers the formation of a "sandwich" complex composed of the gold nanoparticle construct, virus, and an antibody-functionalized magnetic particle used for extraction. After extraction, DNA tags are released by heating to 95°C and detected via real-time PCR. The limit of detection of the assay was compared with ELISA and reversion transcription (RT) PCR using RSV-infected HEp-2 cell extracts. NPA-IPCR showed an approximately 4000-fold improvement in the limit of detection compared with ELISA and a 4-fold improvement compared with viral RNA extraction followed by traditional RT-PCR. NPA-IPCR offers a viable platform for the development of early-stage diagnostics requiring an exceptionally low limit of detection.
2010 Elsevier Inc. All rights reserved.
0 Communities
1 Members
0 Resources
16 MeSH Terms
Small-molecule inhibition of Wnt signaling through activation of casein kinase 1α.
Thorne CA, Hanson AJ, Schneider J, Tahinci E, Orton D, Cselenyi CS, Jernigan KK, Meyers KC, Hang BI, Waterson AG, Kim K, Melancon B, Ghidu VP, Sulikowski GA, LaFleur B, Salic A, Lee LA, Miller DM, Lee E
(2010) Nat Chem Biol 6: 829-36
MeSH Terms: Adaptor Proteins, Signal Transducing, Adenomatous Polyposis Coli, Animals, Axin Protein, Casein Kinase I, Casein Kinase Ialpha, Cell Extracts, Cell Line, Tumor, Cell Proliferation, Colonic Neoplasms, Dose-Response Relationship, Drug, Enzyme Activation, Enzyme Inhibitors, Humans, Intracellular Signaling Peptides and Proteins, Oocytes, Pyrvinium Compounds, Repressor Proteins, Signal Transduction, Wnt Proteins, Xenopus Proteins, Xenopus laevis, beta Catenin
Show Abstract · Added February 3, 2014
Wnt/β-catenin signaling is critically involved in metazoan development, stem cell maintenance and human disease. Using Xenopus laevis egg extract to screen for compounds that both stabilize Axin and promote β-catenin turnover, we identified an FDA-approved drug, pyrvinium, as a potent inhibitor of Wnt signaling (EC(50) of ∼10 nM). We show pyrvinium binds all casein kinase 1 (CK1) family members in vitro at low nanomolar concentrations and pyrvinium selectively potentiates casein kinase 1α (CK1α) kinase activity. CK1α knockdown abrogates the effects of pyrvinium on the Wnt pathway. In addition to its effects on Axin and β-catenin levels, pyrvinium promotes degradation of Pygopus, a Wnt transcriptional component. Pyrvinium treatment of colon cancer cells with mutation of the gene for adenomatous polyposis coli (APC) or β-catenin inhibits both Wnt signaling and proliferation. Our findings reveal allosteric activation of CK1α as an effective mechanism to inhibit Wnt signaling and highlight a new strategy for targeted therapeutics directed against the Wnt pathway.
0 Communities
5 Members
0 Resources
23 MeSH Terms
A novel in vitro assay to assess phosphorylation of 3'-[(18)F]fluoro-3'-deoxythymidine.
Guo N, Xie J, Manning HC, Deane NG, Ansari MS, Coffey RJ, Gore J, Price RR, Baldwin RM, McIntyre JO
(2011) Mol Imaging Biol 13: 257-64
MeSH Terms: Biological Transport, Cell Extracts, Cell Line, Tumor, Cell Membrane, Dideoxynucleosides, Enzyme Assays, Enzyme Inhibitors, Humans, Kinetics, Nucleosides, Phosphorylation, Thymidine Kinase, Time Factors
Show Abstract · Added May 10, 2013
PURPOSE - 3'-[(18)F]fluoro-3'-deoxythymidine ([(18)F]FLT) is phosphorylated by thymidine kinase 1 (TK-1), a cell cycle regulated enzyme. Appropriate use of [(18)F]FLT tracer requires validation of the TK-1 activity. Here, we report development of a novel phosphoryl-transfer assay to assess phosphorylation of [(18)F]FLT both in tumor cell lysates and tumor cells.
PROCEDURES - The intrinsic F-18 radioactivity was used to quantify both substrate and phosphorylated products using a rapid thin layer chromatography method. Phosphorylation kinetics of [(18)F]FLT in SW480 and DiFi tumor cell lysates and cellular uptake were measured.
RESULTS - The apparent Michaelis-Menten kinetic parameters for [(18)F]FLT are K(m) = 4.8 ± 0.3 μM and V(max) = 7.4 pmol min(-1) per 1 × 10(6) cells with ~2-fold higher TK-1 activity in DiFi versus SW480 lysates.
CONCLUSIONS - The apparent K (m) of [(18)F]FLT was comparable to the value reported with purified recombinant TK-1. The uptake of [(18)F]FLT by SW480 cells is inhibited by nitrobenzylthioinosine or dipyridamole indicating that uptake is mediated predominantly by the equilibrative nucleoside transporters in these tumor cells.
2 Communities
4 Members
0 Resources
13 MeSH Terms
Binding of NIR-conPK and NIR-6T to astrocytomas and microglial cells: evidence for a protein related to TSPO.
Sexton M, Woodruff G, Cudaback E, Kreitzer FR, Xu C, Lin YH, Möller T, Bai M, Manning HC, Bornhop D, Stella N
(2009) PLoS One 4: e8271
MeSH Terms: Animals, Astrocytoma, Cell Extracts, Chemokines, Fluorescent Dyes, Gene Expression Regulation, Indoles, Kinetics, Mice, Microglia, Polymerase Chain Reaction, RNA, Messenger, Receptors, GABA-A
Show Abstract · Added March 5, 2014
PK 11195 and DAA1106 bind with high-affinity to the translocator protein (TSPO, formerly known as the peripheral benzodiazepine receptor). TSPO expression in glial cells increases in response to cytokines and pathological stimuli. Accordingly, [(11)C]-PK 11195 and [(11)C]-DAA1106 are recognized molecular imaging (MI) agents capable of monitoring changes in TSPO expression occurring in vivo and in response to various neuropathologies.Here we tested the pharmacological characteristics and TSPO-monitoring potential of two novel MI agents: NIR-conPK and NIR-6T. NIR-conPK is an analogue of PK 11195 conjugated to the near-infrared (NIR) emitting fluorophore: IRDye 800CW. NIR-6T is a DAA1106 analogue also conjugated to IRDye 800CW.We found that NIR-6T competed for [(3)H]-PK 11195 binding in astrocytoma cell homogenates with nanomolar affinity, but did not exhibit specific binding in intact astrocytoma cells in culture, indicating that NIR-6T is unlikely to constitute a useful MI agent for monitoring TSPO expression in intact cells. Conversely, we found that NIR-conPK did not compete for [(3)H]-PK 11195 binding in astrocytoma cell homogenate, but exhibited specific binding in intact astrocytoma cells in culture with nanomolar affinity, suggesting that NIR-conPK binds to a protein distinct, but related to, TSPO. Accordingly, treating intact astrocytoma cells and microglia in culture with cytokines led to significant changes in the amount of NIR-conPK specific binding without corresponding change in TSPO expression. Remarkably, the cytokine-induced changes in the protein targeted by NIR-conPK in intact microglia were selective, since IFN-gamma (but not TNFalpha and TGFbeta) increased the amount of NIR-conPK specific binding in these cells.Together these results suggest that NIR-conPK binds to a protein that is related to TSPO, and expressed by astrocytomas and microglia. Our results also suggest that the expression of this protein is increased by specific cytokines, and thus allows for the monitoring of a particular subtype of microglia activation.
0 Communities
2 Members
0 Resources
13 MeSH Terms