Eicosapentaenoic acid-enriched phosphatidylcholine isolated from Cucumaria frondosa exhibits anti-hyperglycemic effects via activating phosphoinositide 3-kinase/protein kinase B signal pathway.

Science.gov (United States)

Hu, Shiwei; Xu, Leilei; Shi, Di; Wang, Jingfeng; Wang, Yuming; Lou, Qiaoming; Xue, Changhu

2014-04-01

Eicosapentaenoic acid-enriched phosphatidylcholine was isolated from the sea cucumber Cucumaria frondosa (Cucumaria-PC) and its effects on streptozotocin (STZ)-induced hyperglycemic rats were investigated. Male Sprague-Dawley rats were randomly divided into normal control, model control (STZ), low- and high-dose Cucumaria-PC groups (STZ + Cucumaria-PC at 25 and 75 mg/Kg·b·wt, intragastrically, respectively). Blood glucose, insulin, glycogen in liver and gastrocnemius were determined over 60 days. Insulin signaling in the rats' gastrocnemius was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. The results showed that Cucumaria-PC significantly decreased blood glucose level, increased insulin secretion and glycogen synthesis in diabetic rats. RT-PCR analysis revealed that Cucumaria-PC significantly promoted the expressions of glycometabolism-related genes of insulin receptor (IR), insulin receptor substrate-1 (IRS-1), phosphoinositide 3-kinase (PI3K), protein kinase B (PKB), and glucose transporter 4 (GLUT4) in gastrocnemius. Western blotting assay demonstrated that Cucumaria-PC remarkably enhanced the proteins abundance of IR-β, PI3K, PKB, GLUT4, as well as phosphorylation of Tyr-IR-β, p85-PI3K, Ser473-PKB (P insulin. Nutritional supplementation with Cucumaria-PC, if validated for human studies, may offer an adjunctive therapy for diabetes mellitus. Copyright © 2013 The Society for Biotechnology, Japan. All rights reserved.

Selected nondestructive assay instrumentation for an international safeguards system at uranium enrichment plants

International Nuclear Information System (INIS)

Tape, J.W.; Baker, M.P.; Strittmatter, R.; Jain, M.; Evans, M.L.

1979-01-01

A selected set of nondestructive assay instruments for an international safeguards system at uranium enrichment plants is currently under development. These instruments are of three types: in-line enrichment meters for feed, product, and tails streams; area radiation monitors for direct detection of high-enriched uranium production, and an enrichment meter for spent alumina trap material. The current status of the development of each of these instruments is discussed, with supporting data, as well as the role each would play in a total international safeguards system. 5 figures

A Discovery Strategy for Selective Inhibitors of c-Src in Complex with the Focal Adhesion Kinase SH3/SH2-binding Region.

Science.gov (United States)

Moroco, Jamie A; Baumgartner, Matthew P; Rust, Heather L; Choi, Hwan Geun; Hur, Wooyoung; Gray, Nathanael S; Camacho, Carlos J; Smithgall, Thomas E

2015-08-01

The c-Src tyrosine kinase co-operates with the focal adhesion kinase to regulate cell adhesion and motility. Focal adhesion kinase engages the regulatory SH3 and SH2 domains of c-Src, resulting in localized kinase activation that contributes to tumor cell metastasis. Using assay conditions where c-Src kinase activity required binding to a tyrosine phosphopeptide based on the focal adhesion kinase SH3-SH2 docking sequence, we screened a kinase-biased library for selective inhibitors of the Src/focal adhesion kinase peptide complex versus c-Src alone. This approach identified an aminopyrimidinyl carbamate compound, WH-4-124-2, with nanomolar inhibitory potency and fivefold selectivity for c-Src when bound to the phospho-focal adhesion kinase peptide. Molecular docking studies indicate that WH-4-124-2 may preferentially inhibit the 'DFG-out' conformation of the kinase active site. These findings suggest that interaction of c-Src with focal adhesion kinase induces a unique kinase domain conformation amenable to selective inhibition. © 2014 John Wiley & Sons A/S.

Selective elimination of neuroblastoma cells by synergistic effect of Akt kinase inhibitor and tetrathiomolybdate.

Science.gov (United States)

Navrátilová, Jarmila; Karasová, Martina; Kohutková Lánová, Martina; Jiráková, Ludmila; Budková, Zuzana; Pacherník, Jiří; Šmarda, Jan; Beneš, Petr

2017-09-01

Neuroblastoma is the most common extracranial solid tumour of infancy. Pathological activation of glucose consumption, glycolysis and glycolysis-activating Akt kinase occur frequently in neuroblastoma cells, and these changes correlate with poor prognosis of patients. Therefore, several inhibitors of glucose utilization and the Akt kinase activity are in preclinical trials as potential anti-cancer drugs. However, metabolic plasticity of cancer cells might undermine efficacy of this approach. In this work, we identified oxidative phosphorylation as compensatory mechanism preserving viability of neuroblastoma cells with inhibited glucose uptake/Akt kinase. It was oxidative phosphorylation that maintained intracellular level of ATP and proliferative capacity of these cells. The oxidative phosphorylation inhibitors (rotenone, tetrathiomolybdate) synergized with inhibitor of the Akt kinase/glucose uptake in down-regulation of both viability of neuroblastoma cells and clonogenic potential of cells forming neuroblastoma spheroids. Interestingly, tetrathiomolybdate acted as highly specific inhibitor of oxygen consumption and activator of lactate production in neuroblastoma cells, but not in normal fibroblasts and neuronal cells. Moreover, the reducing effect of tetrathiomolybdate on cell viability and the level of ATP in the cells with inhibited Akt kinase/glucose uptake was also selective for neuroblastoma cells. Therefore, efficient elimination of neuroblastoma cells requires inhibition of both glucose uptake/Akt kinase and oxidative phosphorylation activities. The use of tetrathiomolybdate as a mitochondrial inhibitor contributes to selectivity of this combined treatment, preferentially targeting neuroblastoma cells. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Structural elucidation of the DFG-Asp in and DFG-Asp out states of TAM kinases and insight into the selectivity of their inhibitors.

Science.gov (United States)

Messoussi, Abdellah; Peyronnet, Lucile; Feneyrolles, Clémence; Chevé, Gwénaël; Bougrin, Khalid; Yasri, Aziz

2014-10-10

Structural elucidation of the active (DFG-Asp in) and inactive (DFG-Asp out) states of the TAM family of receptor tyrosine kinases is required for future development of TAM inhibitors as drugs. Herein we report a computational study on each of the three TAM members Tyro-3, Axl and Mer. DFG-Asp in and DFG-Asp out homology models of each one were built based on the X-ray structure of c-Met kinase, an enzyme with a closely related sequence. Structural validation and in silico screening enabled identification of critical amino acids for ligand binding within the active site of each DFG-Asp in and DFG-Asp out model. The position and nature of amino acids that differ among Tyro-3, Axl and Mer, and the potential role of these residues in the design of selective TAM ligands, are discussed.

Structure-Based Design of a Novel Series of Potent, Selective Inhibitors of the Class I Phosphatidylinositol 3-Kinases

Energy Technology Data Exchange (ETDEWEB)

Smith, Adrian L.; D’Angelo, Noel D.; Bo, Yunxin Y.; Booker, Shon K.; Cee, Victor J.; Herberich, Brad; Hong, Fang-Tsao; Jackson, Claire L.M.; Lanman, Brian A.; Liu, Longbin; Nishimura, Nobuko; Pettus, Liping H.; Reed, Anthony B.; Tadesse, Seifu; Tamayo, Nuria A.; Wurz, Ryan P.; Yang, Kevin; Andrews, Kristin L.; Whittington, Douglas A.; McCarter, John D.; Miguel, Tisha San; Zalameda, Leeanne; Jiang, Jian; Subramanian, Raju; Mullady, Erin L.; Caenepeel, Sean; Freeman, Daniel J.; Wang, Ling; Zhang, Nancy; Wu, Tian; Hughes, Paul E.; Norman, Mark H. (Amgen)

2012-09-17

A highly selective series of inhibitors of the class I phosphatidylinositol 3-kinases (PI3Ks) has been designed and synthesized. Starting from the dual PI3K/mTOR inhibitor 5, a structure-based approach was used to improve potency and selectivity, resulting in the identification of 54 as a potent inhibitor of the class I PI3Ks with excellent selectivity over mTOR, related phosphatidylinositol kinases, and a broad panel of protein kinases. Compound 54 demonstrated a robust PD-PK relationship inhibiting the PI3K/Akt pathway in vivo in a mouse model, and it potently inhibited tumor growth in a U-87 MG xenograft model with an activated PI3K/Akt pathway.

Identification and Structure-Function Analysis of Subfamily Selective G Protein-Coupled Receptor Kinase Inhibitors

Energy Technology Data Exchange (ETDEWEB)

Homan, Kristoff T.; Larimore, Kelly M.; Elkins, Jonathan M.; Szklarz, Marta; Knapp, Stefan; Tesmer, John J.G. [Michigan; (Oxford)

2015-02-13

Selective inhibitors of individual subfamilies of G protein-coupled receptor kinases (GRKs) would serve as useful chemical probes as well as leads for therapeutic applications ranging from heart failure to Parkinson’s disease. To identify such inhibitors, differential scanning fluorimetry was used to screen a collection of known protein kinase inhibitors that could increase the melting points of the two most ubiquitously expressed GRKs: GRK2 and GRK5. Enzymatic assays on 14 of the most stabilizing hits revealed that three exhibit nanomolar potency of inhibition for individual GRKs, some of which exhibiting orders of magnitude selectivity. Most of the identified compounds can be clustered into two chemical classes: indazole/dihydropyrimidine-containing compounds that are selective for GRK2 and pyrrolopyrimidine-containing compounds that potently inhibit GRK1 and GRK5 but with more modest selectivity. The two most potent inhibitors representing each class, GSK180736A and GSK2163632A, were cocrystallized with GRK2 and GRK1, and their atomic structures were determined to 2.6 and 1.85 Å spacings, respectively. GSK180736A, developed as a Rho-associated, coiled-coil-containing protein kinase inhibitor, binds to GRK2 in a manner analogous to that of paroxetine, whereas GSK2163632A, developed as an insulin-like growth factor 1 receptor inhibitor, occupies a novel region of the GRK active site cleft that could likely be exploited to achieve more selectivity. However, neither compound inhibits GRKs more potently than their initial targets. This data provides the foundation for future efforts to rationally design even more potent and selective GRK inhibitors.

Leveraging the Pre-DFG Residue Thr-406 To Obtain High Kinase Selectivity in an Aminopyrazole-Type PAK1 Inhibitor Series.

Science.gov (United States)

Rudolph, Joachim; Aliagas, Ignacio; Crawford, James J; Mathieu, Simon; Lee, Wendy; Chao, Qi; Dong, Ping; Rouge, Lionel; Wang, Weiru; Heise, Christopher; Murray, Lesley J; La, Hank; Liu, Yanzhou; Manning, Gerard; Diederich, François; Hoeflich, Klaus P

2015-06-11

To increase kinase selectivity in an aminopyrazole-based PAK1 inhibitor series, analogues were designed to interact with the PAK1 deep-front pocket pre-DFG residue Thr-406, a residue that is hydrophobic in most kinases. This goal was achieved by installing lactam head groups to the aminopyrazole hinge binding moiety. The corresponding analogues represent the most kinase selective ATP-competitive Group I PAK inhibitors described to date. Hydrogen bonding with the Thr-406 side chain was demonstrated by X-ray crystallography, and inhibitory activities, particularly against kinases with hydrophobic pre-DFG residues, were mitigated. Leveraging hydrogen bonding side chain interactions with polar pre-DFG residues is unprecedented, and similar strategies should be applicable to other appropriate kinases.

Selectivity analysis of protein kinase CK2 inhibitors DMAT, TBB and resorufin in cisplatin-induced stress responses

DEFF Research Database (Denmark)

Fritz, Gerhard; Issinger, Olaf-Georg; Olsen, Birgitte Brinkmann

2009-01-01

Targeting protein kinases as a therapeutic approach to treat various diseases, especially cancer is currently a fast growing business. Although many inhibitors are available, exhibiting remarkable potency, the major challenge is their selectivity. Here we show that the protein kinase CK2 inhibito...

Structural Elucidation of the DFG-Asp in and DFG-Asp out States of TAM Kinases and Insight into the Selectivity of Their Inhibitors

Directory of Open Access Journals (Sweden)

Abdellah Messoussi

2014-10-01

Full Text Available Structural elucidation of the active (DFG-Asp in and inactive (DFG-Asp out states of the TAM family of receptor tyrosine kinases is required for future development of TAM inhibitors as drugs. Herein we report a computational study on each of the three TAM members Tyro-3, Axl and Mer. DFG-Asp in and DFG-Asp out homology models of each one were built based on the X-ray structure of c-Met kinase, an enzyme with a closely related sequence. Structural validation and in silico screening enabled identification of critical amino acids for ligand binding within the active site of each DFG-Asp in and DFG-Asp out model. The position and nature of amino acids that differ among Tyro-3, Axl and Mer, and the potential role of these residues in the design of selective TAM ligands, are discussed.

Identification, Selection, and Enrichment of Cardiomyocyte Precursors

Directory of Open Access Journals (Sweden)

Bianca Ferrarini Zanetti

2013-01-01

Full Text Available The large-scale production of cardiomyocytes is a key step in the development of cell therapy and tissue engineering to treat cardiovascular diseases, particularly those caused by ischemia. The main objective of this study was to establish a procedure for the efficient production of cardiomyocytes by reprogramming mesenchymal stem cells from adipose tissue. First, lentiviral vectors expressing neoR and GFP under the control of promoters expressed specifically during cardiomyogenesis were constructed to monitor cell reprogramming into precardiomyocytes and to select cells for amplification and characterization. Cellular reprogramming was performed using 5′-azacytidine followed by electroporation with plasmid pOKS2a, which expressed Oct4, Sox2, and Klf4. Under these conditions, GFP expression began only after transfection with pOKS2a, and less than 0.015% of cells were GFP+. These GFP+ cells were selected for G418 resistance to find molecular markers of cardiomyocytes by RT-PCR and immunocytochemistry. Both genetic and protein markers of cardiomyocytes were present in the selected cells, with some variations among them. Cell doubling time did not change after selection. Together, these results indicate that enrichment with vectors expressing GFP and neoR under cardiomyocyte-specific promoters can produce large numbers of cardiomyocyte precursors (CMPs, which can then be differentiated terminally for cell therapy and tissue engineering.

Selective enrichment of a methanol-utilizing consortium using pulp & paper mill waste streams

Energy Technology Data Exchange (ETDEWEB)

Gregory R. Mockos; William A. Smith; Frank J. Loge; David N. Thompson

2007-04-01

Efficient utilization of carbon inputs is critical to the economic viability of the current forest products sector. Input carbon losses occur in various locations within a pulp mill, including losses as volatile organics and wastewater . Opportunities exist to capture this carbon in the form of value-added products such as biodegradable polymers. Waste activated sludge from a pulp mill wastewater facility was enriched for 80 days for a methanol-utilizing consortium with the goal of using this consortium to produce biopolymers from methanol-rich pulp mill waste streams. Five enrichment conditions were utilized: three high-methanol streams from the kraft mill foul condensate system, one methanol-amended stream from the mill wastewater plant, and one methanol-only enrichment. Enrichment reactors were operated aerobically in sequencing batch mode at neutral pH and 25°C with a hydraulic residence time and a solids retention time of four days. Non-enriched waste activated sludge did not consume methanol or reduce chemical oxygen demand. With enrichment, however, the chemical oxygen demand reduction over 24 hour feed/decant cycles ranged from 79 to 89 %, and methanol concentrations dropped below method detection limits. Neither the non-enriched waste activated sludge nor any of the enrichment cultures accumulated polyhydroxyalkanoates (PHAs) under conditions of nitrogen sufficiency. Similarly, the non-enriched waste activated sludge did not accumulate PHAs under nitrogen limited conditions. By contrast, enriched cultures accumulated PHAs to nearly 14% on a dry weight basis under nitrogen limited conditions. This indicates that selectively-enriched pulp mill waste activated sludge can serve as an inoculum for PHA production from methanol-rich pulp mill effluents.

The selectivity of receptor tyrosine kinase signaling is controlled by a secondary SH2 domain binding site.

Science.gov (United States)

Bae, Jae Hyun; Lew, Erin Denise; Yuzawa, Satoru; Tomé, Francisco; Lax, Irit; Schlessinger, Joseph

2009-08-07

SH2 domain-mediated interactions represent a crucial step in transmembrane signaling by receptor tyrosine kinases. SH2 domains recognize phosphotyrosine (pY) in the context of particular sequence motifs in receptor phosphorylation sites. However, the modest binding affinity of SH2 domains to pY containing peptides may not account for and likely represents an oversimplified mechanism for regulation of selectivity of signaling pathways in living cells. Here we describe the crystal structure of the activated tyrosine kinase domain of FGFR1 in complex with a phospholipase Cgamma fragment. The structural and biochemical data and experiments with cultured cells show that the selectivity of phospholipase Cgamma binding and signaling via activated FGFR1 are determined by interactions between a secondary binding site on an SH2 domain and a region in FGFR1 kinase domain in a phosphorylation independent manner. These experiments reveal a mechanism for how SH2 domain selectivity is regulated in vivo to mediate a specific cellular process.

Development of dihydropyridone indazole amides as selective Rho-kinase inhibitors.

Science.gov (United States)

Goodman, Krista B; Cui, Haifeng; Dowdell, Sarah E; Gaitanopoulos, Dimitri E; Ivy, Robert L; Sehon, Clark A; Stavenger, Robert A; Wang, Gren Z; Viet, Andrew Q; Xu, Weiwei; Ye, Guosen; Semus, Simon F; Evans, Christopher; Fries, Harvey E; Jolivette, Larry J; Kirkpatrick, Robert B; Dul, Edward; Khandekar, Sanjay S; Yi, Tracey; Jung, David K; Wright, Lois L; Smith, Gary K; Behm, David J; Bentley, Ross; Doe, Christopher P; Hu, Erding; Lee, Dennis

2007-01-11

Rho kinase (ROCK1) mediates vascular smooth muscle contraction and is a potential target for the treatment of hypertension and related disorders. Indazole amide 3 was identified as a potent and selective ROCK1 inhibitor but possessed poor oral bioavailability. Optimization of this lead resulted in the discovery of a series of dihydropyridones, exemplified by 13, with improved pharmacokinetic parameters relative to the initial lead. Indazole substitution played a critical role in decreasing clearance and improving oral bioavailability.

Selectivity Profiling and Biological Activity of Novel β-Carbolines as Potent and Selective DYRK1 Kinase Inhibitors.

Directory of Open Access Journals (Sweden)

Katharina Rüben

Full Text Available DYRK1A is a pleiotropic protein kinase with diverse functions in cellular regulation, including cell cycle control, neuronal differentiation, and synaptic transmission. Enhanced activity and overexpression of DYRK1A have been linked to altered brain development and function in Down syndrome and neurodegenerative diseases such as Alzheimer's disease. The β-carboline alkaloid harmine is a high affinity inhibitor of DYRK1A but suffers from the drawback of inhibiting monoamine oxidase A (MAO-A with even higher potency. Here we characterized a series of novel harmine analogs with minimal or absent MAO-A inhibitory activity. We identified several inhibitors with submicromolar potencies for DYRK1A and selectivity for DYRK1A and DYRK1B over the related kinases DYRK2 and HIPK2. An optimized inhibitor, AnnH75, inhibited CLK1, CLK4, and haspin/GSG2 as the only off-targets in a panel of 300 protein kinases. In cellular assays, AnnH75 dose-dependently reduced the phosphorylation of three known DYRK1A substrates (SF3B1, SEPT4, and tau without negative effects on cell viability. AnnH75 inhibited the cotranslational tyrosine autophosphorylation of DYRK1A and threonine phosphorylation of an exogenous substrate protein with similar potency. In conclusion, we have characterized an optimized β-carboline inhibitor as a highly selective chemical probe that complies with desirable properties of drug-like molecules and is suitable to interrogate the function of DYRK1A in biological studies.

A proteomic approach for comprehensively screening substrates of protein kinases such as Rho-kinase.

Directory of Open Access Journals (Sweden)

Mutsuki Amano

Full Text Available BACKGROUND: Protein kinases are major components of signal transduction pathways in multiple cellular processes. Kinases directly interact with and phosphorylate downstream substrates, thus modulating their functions. Despite the importance of identifying substrates in order to more fully understand the signaling network of respective kinases, efficient methods to search for substrates remain poorly explored. METHODOLOGY/PRINCIPAL FINDINGS: We combined mass spectrometry and affinity column chromatography of the catalytic domain of protein kinases to screen potential substrates. Using the active catalytic fragment of Rho-kinase/ROCK/ROK as the model bait, we obtained about 300 interacting proteins from the rat brain cytosol fraction, which included the proteins previously reported as Rho-kinase substrates. Several novel interacting proteins, including doublecortin, were phosphorylated by Rho-kinase both in vitro and in vivo. CONCLUSIONS/SIGNIFICANCE: This method would enable identification of novel specific substrates for kinases such as Rho-kinase with high sensitivity.

Novel HIV-1 knockdown targets identified by an enriched kinases/phosphatases shRNA library using a long-term iterative screen in Jurkat T-cells.

Directory of Open Access Journals (Sweden)

Sylvie Rato

2010-02-01

Full Text Available HIV-1 is a complex retrovirus that uses host machinery to promote its replication. Understanding cellular proteins involved in the multistep process of HIV-1 infection may result in the discovery of more adapted and effective therapeutic targets. Kinases and phosphatases are a druggable class of proteins critically involved in regulation of signal pathways of eukaryotic cells. Here, we focused on the discovery of kinases and phosphatases that are essential for HIV-1 replication but dispensable for cell viability. We performed an iterative screen in Jurkat T-cells with a short-hairpin-RNA (shRNA library highly enriched for human kinases and phosphatases. We identified 14 new proteins essential for HIV-1 replication that do not affect cell viability. These proteins are described to be involved in MAPK, JNK and ERK pathways, vesicular traffic and DNA repair. Moreover, we show that the proteins under study are important in an early step of HIV-1 infection before viral integration, whereas some of them affect viral transcription/translation. This study brings new insights for the complex interplay of HIV-1/host cell and opens new possibilities for antiviral strategies.

Involvement of glycosphingolipid-enriched lipid rafts in inflammatory responses.

Science.gov (United States)

Iwabuchi, Kazuhisa

2015-01-01

Glycosphingolipids (GSLs) are membrane components consisting of hydrophobic ceramide and hydrophilic sugar moieties. GSLs cluster with cholesterol in cell membranes to form GSL-enriched lipid rafts. Biochemical analyses have demonstrated that GSL-enriched lipid rafts contain several kinds of transducer molecules, including Src family kinases. Among the GSLs, lactosylceramide (LacCer, CDw17) can bind to various microorganisms, is highly expressed on the plasma membranes of human phagocytes, and forms lipid rafts containing the Src family tyrosine kinase Lyn. LacCer-enriched lipid rafts mediate immunological and inflammatory reactions, including superoxide generation, chemotaxis, and non-opsonic phagocytosis. Therefore, LacCer-enriched membrane microdomains are thought to function as pattern recognition receptors (PRRs), which recognize pathogen-associated molecular patterns (PAMPs) expressed on microorganisms. LacCer also serves as a signal transduction molecule for functions mediated by CD11b/CD18-integrin (αM/β2-integrin, CR3, Mac-1), as well as being associated with several key cellular processes. LacCer recruits PCKα/ε and phospholipase A2 to stimulate PECAM-1 expression in human monocytes and their adhesion to endothelial cells, as well as regulating β1-integrin clustering and endocytosis on cell surfaces. This review describes the organizational and inflammation-related functions of LacCer-enriched lipid rafts.

Structure-Based Design of Potent and Selective 3-Phosphoinositide-Dependent Kinase-1 (PDK1) Inhibitors

Energy Technology Data Exchange (ETDEWEB)

Medina, Jesus R.; Becker, Christopher J.; Blackledge, Charles W.; Duquenne, Celine; Feng, Yanhong; Grant, Seth W.; Heerding, Dirk; Li, William H.; Miller, William H.; Romeril, Stuart P.; Scherzer, Daryl; Shu, Arthur; Bobko, Mark A.; Chadderton, Antony R.; Dumble, Melissa; Gardiner, Christine M.; Gilbert, Seth; Liu, Qi; Rabindran, Sridhar K.; Sudakin, Valery; Xiang, Hong; Brady, Pat G.; Campobasso, Nino; Ward, Paris; Axten, Jeffrey M. (GSKPA)

2014-10-02

Phosphoinositide-dependent protein kinase-1(PDK1) is a master regulator of the AGC family of kinases and an integral component of the PI3K/AKT/mTOR pathway. As this pathway is among the most commonly deregulated across all cancers, a selective inhibitor of PDK1 might have utility as an anticancer agent. Herein we describe our lead optimization of compound 1 toward highly potent and selective PDK1 inhibitors via a structure-based design strategy. The most potent and selective inhibitors demonstrated submicromolar activity as measured by inhibition of phosphorylation of PDK1 substrates as well as antiproliferative activity against a subset of AML cell lines. In addition, reduction of phosphorylation of PDK1 substrates was demonstrated in vivo in mice bearing OCl-AML2 xenografts. These observations demonstrate the utility of these molecules as tools to further delineate the biology of PDK1 and the potential pharmacological uses of a PDK1 inhibitor.

Phosphopeptide Enrichment by Covalent Chromatography after Derivatization of Protein Digests Immobilized on Reversed-Phase Supports

Science.gov (United States)

Nika, Heinz; Nieves, Edward; Hawke, David H.; Angeletti, Ruth Hogue

2013-01-01

A rugged sample-preparation method for comprehensive affinity enrichment of phosphopeptides from protein digests has been developed. The method uses a series of chemical reactions to incorporate efficiently and specifically a thiol-functionalized affinity tag into the analyte by barium hydroxide catalyzed β-elimination with Michael addition using 2-aminoethanethiol as nucleophile and subsequent thiolation of the resulting amino group with sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-dithiopropionate. Gentle oxidation of cysteine residues, followed by acetylation of α- and ε-amino groups before these reactions, ensured selectivity of reversible capture of the modified phosphopeptides by covalent chromatography on activated thiol sepharose. The use of C18 reversed-phase supports as a miniaturized reaction bed facilitated optimization of the individual modification steps for throughput and completeness of derivatization. Reagents were exchanged directly on the supports, eliminating sample transfer between the reaction steps and thus, allowing the immobilized analyte to be carried through the multistep reaction scheme with minimal sample loss. The use of this sample-preparation method for phosphopeptide enrichment was demonstrated with low-level amounts of in-gel-digested protein. As applied to tryptic digests of α-S1- and β-casein, the method enabled the enrichment and detection of the phosphorylated peptides contained in the mixture, including the tetraphosphorylated species of β-casein, which has escaped chemical procedures reported previously. The isolates proved highly suitable for mapping the sites of phosphorylation by collisionally induced dissociation. β-Elimination, with consecutive Michael addition, expanded the use of the solid-phase-based enrichment strategy to phosphothreonyl peptides and to phosphoseryl/phosphothreonyl peptides derived from proline-directed kinase substrates and to their O-sulfono- and O-linked β-N-acetylglucosamine (O

The use of titanium dioxide for selective enrichment of phosphorylated peptides

DEFF Research Database (Denmark)

Thingholm, Tine E.; Larsen, Martin R.

2016-01-01

acid (DHB), phthalic acid, lactic acid, or glycolic acid has been shown to improve selectivity significantly by reducing unspecific binding of non-phosphorylated peptides. The phosphopeptides bound to the TiO2 are subsequently eluted from the chromatographic material using an alkaline buffer. TiO2......Titanium dioxide (TiO2) has very high affinity for phosphopeptides and in recent years it has become one of the most popular methods for phosphopeptide enrichment from complex biological samples. Peptide loading onto TiO2 resin in a highly acidic environment in the presence of 2,5-dihydroxybenzoic...... chromatography is extremely tolerant towards most buffers used in biological experiments, highly robust and as such it has become the method of choice in large-scale phosphoproteomics. Here we describe a batch mode protocol for phosphopeptide enrichment using TiO2 chromatographic material followed by desalting...

An in silico high-throughput screen identifies potential selective inhibitors for the non-receptor tyrosine kinase Pyk2

Directory of Open Access Journals (Sweden)

Meirson T

2017-05-01

Full Text Available Tomer Meirson, Abraham O Samson, Hava Gil-Henn Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, Israel Abstract: The non-receptor tyrosine kinase proline-rich tyrosine kinase 2 (Pyk2 is a critical mediator of signaling from cell surface growth factor and adhesion receptors to cell migration, proliferation, and survival. Emerging evidence indicates that signaling by Pyk2 regulates hematopoietic cell response, bone density, neuronal degeneration, angiogenesis, and cancer. These physiological and pathological roles of Pyk2 warrant it as a valuable therapeutic target for invasive cancers, osteoporosis, Alzheimer’s disease, and inflammatory cellular response. Despite its potential as a therapeutic target, no potent and selective inhibitor of Pyk2 is available at present. As a first step toward discovering specific potential inhibitors of Pyk2, we used an in silico high-throughput screening approach. A virtual library of six million lead-like compounds was docked against four different high-resolution Pyk2 kinase domain crystal structures and further selected for predicted potency and ligand efficiency. Ligand selectivity for Pyk2 over focal adhesion kinase (FAK was evaluated by comparative docking of ligands and measurement of binding free energy so as to obtain 40 potential candidates. Finally, the structural flexibility of a subset of the docking complexes was evaluated by molecular dynamics simulation, followed by intermolecular interaction analysis. These compounds may be considered as promising leads for further development of highly selective Pyk2 inhibitors. Keywords: virtual screen, efficiency metrics, MM-GBSA, molecular dynamics

The SH2 domain of Abl kinases regulates kinase autophosphorylation by controlling activation loop accessibility

Science.gov (United States)

Lamontanara, Allan Joaquim; Georgeon, Sandrine; Tria, Giancarlo; Svergun, Dmitri I.; Hantschel, Oliver

2014-11-01

The activity of protein kinases is regulated by multiple molecular mechanisms, and their disruption is a common driver of oncogenesis. A central and almost universal control element of protein kinase activity is the activation loop that utilizes both conformation and phosphorylation status to determine substrate access. In this study, we use recombinant Abl tyrosine kinases and conformation-specific kinase inhibitors to quantitatively analyse structural changes that occur after Abl activation. Allosteric SH2-kinase domain interactions were previously shown to be essential for the leukemogenesis caused by the Bcr-Abl oncoprotein. We find that these allosteric interactions switch the Abl activation loop from a closed to a fully open conformation. This enables the trans-autophosphorylation of the activation loop and requires prior phosphorylation of the SH2-kinase linker. Disruption of the SH2-kinase interaction abolishes activation loop phosphorylation. Our analysis provides a molecular mechanism for the SH2 domain-dependent activation of Abl that may also regulate other tyrosine kinases.

Selective C(sp3 )-H Aerobic Oxidation Enabled by Decatungstate Photocatalysis in Flow.

Science.gov (United States)

Laudadio, Gabriele; Govaerts, Sebastian; Wang, Ying; Ravelli, Davide; Koolman, Hannes F; Fagnoni, Maurizio; Djuric, Stevan W; Noël, Timothy

2018-04-03

A mild and selective C(sp 3 )-H aerobic oxidation enabled by decatungstate photocatalysis has been developed. The reaction can be significantly improved in a microflow reactor enabling the safe use of oxygen and enhanced irradiation of the reaction mixture. Our method allows for the oxidation of both activated and unactivated C-H bonds (30 examples). The ability to selectively oxidize natural scaffolds, such as (-)-ambroxide, pregnenolone acetate, (+)-sclareolide, and artemisinin, exemplifies the utility of this new method. © 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

The Roles of Protein Kinases in Learning and Memory

Science.gov (United States)

Giese, Karl Peter; Mizuno, Keiko

2013-01-01

In the adult mammalian brain, more than 250 protein kinases are expressed, but only a few of these kinases are currently known to enable learning and memory. Based on this information it appears that learning and memory-related kinases either impact on synaptic transmission by altering ion channel properties or ion channel density, or regulate…

Induction of osteoblast differentiation by selective activation of kinase-mediated actions of the estrogen receptor.

Science.gov (United States)

Kousteni, Stavroula; Almeida, Maria; Han, Li; Bellido, Teresita; Jilka, Robert L; Manolagas, Stavros C

2007-02-01

Estrogens control gene transcription by cis or trans interactions of the estrogen receptor (ER) with target DNA or via the activation of cytoplasmic kinases. We report that selective activation of kinase-mediated actions of the ER with 4-estren-3alpha,17beta-diol (estren) or an estradiol-dendrimer conjugate, each a synthetic compound that stimulates kinase-mediated ER actions 1,000 to 10,000 times more potently than direct DNA interactions, induced osteoblastic differentiation in established cell lines of uncommitted osteoblast precursors and primary cultures of osteoblast progenitors by stimulating Wnt and BMP-2 signaling in a kinase-dependent manner. In sharp contrast, 17beta-estradiol (E(2)) suppressed BMP-2-induced osteoblast progenitor commitment and differentiation. Consistent with the in vitro findings, estren, but not E(2), stimulated Wnt/beta-catenin-mediated transcription in T-cell factor-lacZ transgenic mice. Moreover, E(2) stimulated BMP signaling in mice in which ERalpha lacks DNA binding activity and classical estrogen response element-mediated transcription (ERalpha(NERKI/-)) but not in wild-type controls. This evidence reveals for the first time the existence of a large signalosome in which inputs from the ER, kinases, bone morphogenetic proteins, and Wnt signaling converge to induce differentiation of osteoblast precursors. ER can either induce it or repress it, depending on whether the activating ligand (and presumably the resulting conformation of the receptor protein) precludes or accommodates ERE-mediated transcription.

Enabling Interoperable and Selective Data Sharing among Social Networking Sites

Science.gov (United States)

Shin, Dongwan; Lopes, Rodrigo

With the widespread use of social networking (SN) sites and even introduction of a social component in non-social oriented services, there is a growing concern over user privacy in general, how to handle and share user profiles across SN sites in particular. Although there have been several proprietary or open source-based approaches to unifying the creation of third party applications, the availability and retrieval of user profile information are still limited to the site where the third party application is run, mostly devoid of the support for data interoperability. In this paper we propose an approach to enabling interopearable and selective data sharing among SN sites. To support selective data sharing, we discuss an authenticated dictionary (ADT)-based credential which enables a user to share only a subset of her information certified by external SN sites with applications running on an SN site. For interoperable data sharing, we propose an extension to the OpenSocial API so that it can provide an open source-based framework for allowing the ADT-based credential to be used seamlessly among different SN sites.

A DUAL PLATFORM FOR SELECTIVE ANALYTE ENRICHMENT AND IONIZATION IN MASS SPECTROMETRY USING APTAMER-CONJUGATED GRAPHENE OXIDE

OpenAIRE

Gulbakan, Basri; Yasun, Emir; Shukoor, M. Ibrahim; Zhu, Zhi; You, Mingxu; Tan, Xiaohong; Sanchez, Hernan; Powell, David H.; Dai, Hongjie; Tan, Weihong

2010-01-01

This study demonstrates the use of aptamer-conjugated graphene oxide as an affinity extraction and detection platform for analytes from complex biological media. We have shown that cocaine and adenosine can be selectively enriched from plasma samples and that direct mass spectrometric readout can be obtained without a matrix and with greatly improved signal-to-noise ratios. The aptamer conjugated graphene oxide has clear advantages in target enrichment and in generating highly efficient ioniz...

Deuterium enrichment by selective photo-induced dissociation of an organic carbonyl compound

International Nuclear Information System (INIS)

Marling, J.B.

1981-01-01

A deuterium-enriched material is produced by selective photoinduced dissociation of a gas phase organic carbonyl compound containing at least one hydrogen atom bonded to an atom adjacent to a carbonyl group. Alkyl carbonyl compounds such as acetone, acetaldehyde, trifluoroacetic acid, cyclobutanone, cyclopentanone, methyl acetate, 3,3-dimethyl-2-butanone, 2,4-pentanedione, and 4-methyl-2-pentanone are preferred. The carbonyl compound is subjected to intense infrared radiation from one laser, or two lasers operating at different frequencies, to selectively dissociate the deuterated molecules into stable products. The undissociated compound may be redeuterated by direct aqueous liquid phase H/D exchange, or by indirect liquid phase exchange using an alkanol in an intermediate step

Chicken microsatellite markers isolated from libraries enriched for simple tandem repeats.

Science.gov (United States)

Gibbs, M; Dawson, D A; McCamley, C; Wardle, A F; Armour, J A; Burke, T

1997-12-01

The total number of microsatellite loci is considered to be at least 10-fold lower in avian species than in mammalian species. Therefore, efficient large-scale cloning of chicken microsatellites, as required for the construction of a high-resolution linkage map, is facilitated by the construction of libraries using an enrichment strategy. In this study, a plasmid library enriched for tandem repeats was constructed from chicken genomic DNA by hybridization selection. Using this technique the proportion of recombinant clones that cross-hybridized to probes containing simple tandem repeats was raised to 16%, compared with < 0.1% in a non-enriched library. Primers were designed from 121 different sequences. Polymerase chain reaction (PCR) analysis of two chicken reference pedigrees enabled 72 loci to be localized within the collaborative chicken genetic map, and at least 30 of the remaining loci have been shown to be informative in these or other crosses.

A screen for kinase inhibitors identifies antimicrobial imidazopyridine aminofurazans as specific inhibitors of the Listeria monocytogenes PASTA kinase PrkA.

Science.gov (United States)

Schaenzer, Adam J; Wlodarchak, Nathan; Drewry, David H; Zuercher, William J; Rose, Warren E; Striker, Rob; Sauer, John-Demian

2017-10-13

Bacterial signaling systems such as protein kinases and quorum sensing have become increasingly attractive targets for the development of novel antimicrobial agents in a time of rising antibiotic resistance. The family of bacterial P enicillin-binding-protein A nd S erine/ T hreonine kinase- A ssociated (PASTA) kinases is of particular interest due to the role of these kinases in regulating resistance to β-lactam antibiotics. As such, small-molecule kinase inhibitors that target PASTA kinases may prove beneficial as treatments adjunctive to β-lactam therapy. Despite this interest, only limited progress has been made in identifying functional inhibitors of the PASTA kinases that have both activity against the intact microbe and high kinase specificity. Here, we report the results of a small-molecule screen that identified GSK690693, an imidazopyridine aminofurazan-type kinase inhibitor that increases the sensitivity of the intracellular pathogen Listeria monocytogenes to various β-lactams by inhibiting the PASTA kinase PrkA. GSK690693 potently inhibited PrkA kinase activity biochemically and exhibited significant selectivity for PrkA relative to the Staphylococcus aureus PASTA kinase Stk1. Furthermore, other imidazopyridine aminofurazans could effectively inhibit PrkA and potentiate β-lactam antibiotic activity to varying degrees. The presence of the 2-methyl-3-butyn-2-ol (alkynol) moiety was important for both biochemical and antimicrobial activity. Finally, mutagenesis studies demonstrated residues in the back pocket of the active site are important for GSK690693 selectivity. These data suggest that targeted screens can successfully identify PASTA kinase inhibitors with both biochemical and antimicrobial specificity. Moreover, the imidazopyridine aminofurazans represent a family of PASTA kinase inhibitors that have the potential to be optimized for selective PASTA kinase inhibition.

The selective Aurora B kinase inhibitor AZD1152 is a potential new treatment for multiple myeloma.

Science.gov (United States)

Evans, Robert P; Naber, Claudia; Steffler, Tara; Checkland, Tamara; Maxwell, Christopher A; Keats, Jonathan J; Belch, Andrew R; Pilarski, Linda M; Lai, Raymond; Reiman, Tony

2008-02-01

Aurora kinases are potential targets for cancer therapy. Previous studies have validated Aurora kinase A as a therapeutic target in multiple myeloma (MM), and have demonstrated in vitro anti-myeloma effects of small molecule Aurora kinase inhibitors that inhibit both Aurora A and B. This study demonstrated that Aurora B kinase was strongly expressed in myeloma cell lines and primary plasma cells. The selective Aurora B inhibitor AZD1152-induced apoptotic death in myeloma cell lines at nanomolar concentrations, with a cell cycle phenotype consistent with that reported previously for Aurora B inhibition. In some cases, AZD1152 in combination with dexamethasone showed increased anti-myeloma activity compared with the use of either agent alone. AZD1152 was active against sorted CD138(+) BM plasma cells from myeloma patients but also, as expected, was toxic to CD138(-) marrow cells from the same patients. In a murine myeloma xenograft model, AZD1152-inhibited tumour growth at well-tolerated doses and induced cell death in established tumours, with associated mild, transient leucopenia. AZD1152 shows promise in these preclinical studies as a novel treatment for MM.

Dendrites Enable a Robust Mechanism for Neuronal Stimulus Selectivity.

Science.gov (United States)

Cazé, Romain D; Jarvis, Sarah; Foust, Amanda J; Schultz, Simon R

2017-09-01

Hearing, vision, touch: underlying all of these senses is stimulus selectivity, a robust information processing operation in which cortical neurons respond more to some stimuli than to others. Previous models assume that these neurons receive the highest weighted input from an ensemble encoding the preferred stimulus, but dendrites enable other possibilities. Nonlinear dendritic processing can produce stimulus selectivity based on the spatial distribution of synapses, even if the total preferred stimulus weight does not exceed that of nonpreferred stimuli. Using a multi-subunit nonlinear model, we demonstrate that stimulus selectivity can arise from the spatial distribution of synapses. We propose this as a general mechanism for information processing by neurons possessing dendritic trees. Moreover, we show that this implementation of stimulus selectivity increases the neuron's robustness to synaptic and dendritic failure. Importantly, our model can maintain stimulus selectivity for a larger range of loss of synapses or dendrites than an equivalent linear model. We then use a layer 2/3 biophysical neuron model to show that our implementation is consistent with two recent experimental observations: (1) one can observe a mixture of selectivities in dendrites that can differ from the somatic selectivity, and (2) hyperpolarization can broaden somatic tuning without affecting dendritic tuning. Our model predicts that an initially nonselective neuron can become selective when depolarized. In addition to motivating new experiments, the model's increased robustness to synapses and dendrites loss provides a starting point for fault-resistant neuromorphic chip development.

Selective inhibition of Sarcocystis neurona calcium-dependent protein kinase 1 for equine protozoal myeloencephalitis therapy.

Science.gov (United States)

Ojo, Kayode K; Dangoudoubiyam, Sriveny; Verma, Shiv K; Scheele, Suzanne; DeRocher, Amy E; Yeargan, Michelle; Choi, Ryan; Smith, Tess R; Rivas, Kasey L; Hulverson, Matthew A; Barrett, Lynn K; Fan, Erkang; Maly, Dustin J; Parsons, Marilyn; Dubey, Jitender P; Howe, Daniel K; Van Voorhis, Wesley C

2016-12-01

Sarcocystis neurona is the most frequent cause of equine protozoal myeloencephalitis, a debilitating neurological disease of horses that can be difficult to treat. We identified SnCDPK1, the S. neurona homologue of calcium-dependent protein kinase 1 (CDPK1), a validated drug target in Toxoplasma gondii. SnCDPK1 shares the glycine "gatekeeper" residue of the well-characterized T. gondii enzyme, which allows the latter to be targeted by bumped kinase inhibitors. This study presents detailed molecular and phenotypic evidence that SnCDPK1 can be targeted for rational drug development. Recombinant SnCDPK1 was tested against four bumped kinase inhibitors shown to potently inhibit both T. gondii (Tg) CDPK1 and T. gondii tachyzoite growth. SnCDPK1 was inhibited by low nanomolar concentrations of these BKIs and S. neurona growth was inhibited at 40-120nM concentrations. Thermal shift assays confirmed these bumped kinase inhibitors bind CDPK1 in S. neurona cell lysates. Treatment with bumped kinase inhibitors before or after invasion suggests that bumped kinase inhibitors interfere with S. neurona mammalian host cell invasion in the 0.5-2.5μM range but interfere with intracellular division at 2.5μM. In vivo proof-of-concept experiments were performed in a murine model of S. neurona infection. The experimental infected groups treated for 30days with compound BKI-1553 (n=10 mice) had no signs of disease, while the infected control group had severe signs and symptoms of infection. Elevated antibody responses were found in 100% of control infected animals, but only 20% of BKI-1553 treated infected animals. Parasites were found in brain tissues of 100% of the control infected animals, but only in 10% of the BKI-1553 treated animals. The bumped kinase inhibitors used in these assays have been chemically optimized for potency, selectivity and pharmacokinetic properties, and hence are good candidates for treatment of equine protozoal myeloencephalitis. Copyright © 2016

Crystal structure of human cyclin-dependent kinase-2 complex with MK2 inhibitor TEI-I01800: insight into the selectivity

Energy Technology Data Exchange (ETDEWEB)

Fujino, Aiko; Fukushima, Kei; Kubota, Takaharu; Kosugi, Tomomi; Takimoto-Kamimura, Midori, E-mail: m.kamimura@teijin.co.jp [Teijin Pharma Limited, 4-3-2 Asahigaoka, Hino-shi, Tokyo 191-8512 (Japan)

2013-11-01

The Gly-rich loop of cyclin-dependent kinase 2 (CDK2) bound to TEI-I01800 as an MK2 specific inhibitor forms a β-sheet which is a common structure in CDK2–ligand complexes. Here, the reason why TEI-I01800 does not become a strong inhibitor against CDK2 based on the conformation of TEI-I01800 is presented. Mitogen-activated protein kinase-activated protein kinase 2 (MK2 or MAPKAP-K2) is a Ser/Thr kinase from the p38 mitogen-activated protein kinase signalling pathway and plays an important role in inflammatory diseases. The crystal structure of the MK2–TEI-I01800 complex has been reported; its Gly-rich loop was found to form an α-helix, not a β-sheet as has been observed for other Ser/Thr kinases. TEI-I01800 is 177-fold selective against MK2 compared with CDK2; in order to understand the inhibitory mechanism of TEI-I01800, the cyclin-dependent kinase 2 (CDK2) complex structure with TEI-I01800 was determined at 2.0 Å resolution. Interestingly, the Gly-rich loop of CDK2 formed a β-sheet that was different from that of MK2. In MK2, TEI-I01800 changed the secondary structure of the Gly-rich loop from a β-sheet to an α-helix by collision between Leu70 and a p-ethoxyphenyl group at the 7-position and bound to MK2. However, for CDK2, TEI-I01800 bound to CDK2 without this structural change and lost the interaction with the substituent at the 7-position. In summary, the results of this study suggest that the reason for the selectivity of TEI-I01800 is the favourable conformation of TEI-I01800 itself, making it suitable for binding to the α-form MK2.

Selective C(sp3)−H aerobic oxidation enabled by decatungstate photocatalysis in flow

NARCIS (Netherlands)

Laudadio, G.; Govaerts, S.; Wang, Y.; Ravelli, D.; Koolman, H.; Fagnoni, M.; Djuric, S.; Noel, T.

2018-01-01

A mild and selective C(sp3)−H aerobic oxidation enabled by decatungstate photocatalysis has been developed. The reaction can be significantly improved in a microflow reactor enabling the safe use of oxygen and enhanced irradiation of the reaction mixture. Our method allows for the oxidation of both

The selective and inducible activation of endogenous PI 3-kinase in PC12 cells results in efficient NGF-mediated survival but defective neurite outgrowth.

Science.gov (United States)

Ashcroft, M; Stephens, R M; Hallberg, B; Downward, J; Kaplan, D R

1999-08-12

The Trk/Nerve Growth Factor receptor mediates the rapid activation of a number of intracellular signaling proteins, including phosphatidylinositol 3-kinase (PI 3-kinase). Here, we describe a novel, NGF-inducible system that we used to specifically address the signaling potential of endogenous PI 3-kinase in NGF-mediated neuronal survival and differentiation processes. This system utilizes a Trk receptor mutant (Trk(def)) lacking sequences Y490, Y785 and KFG important for the activation of the major Trk targets; SHC, PLC-gammal, Ras, PI 3-kinase and SNT. Trk(def) was kinase active but defective for NGF-induced responses when stably expressed in PC12nnr5 cells (which lack detectable levels of TrkA and are non-responsive to NGF). The PI 3-kinase consensus binding site, YxxM (YVPM), was introduced into the insert region within the kinase domain of Trk(def). NGF-stimulated tyrosine phosphorylation of the Trk(def)+PI 3-kinase addback receptor, resulted in the direct association and selective activation of PI 3-kinase in vitro and the production of PI(3,4)P2 and PI(3,4,5)P3 in vivo (comparable to wild-type). PC12nnr5 cells stably expressing Trk(def) + PI 3-kinase, initiated neurite outgrowth but failed to stably extend and maintain these neurites in response to NGF as compared to PC12 parental cells, or PC12nnr5 cells overexpressing wild-type Trk. However, Trk(def) + PI 3-kinase was fully competent in mediating NGF-induced survival processes. We propose that while endogenous PI 3-kinase can contribute in part to neurite initiation processes, its selective activation and subsequent signaling to downstream effectors such as Akt, functions mainly to promote cell survival in the PC12 system.

Selective enrichment of sialic acid-containing glycopeptides using titanium dioxide chromatography with analysis by HILIC and mass spectrometry

DEFF Research Database (Denmark)

Palmisano, Giuseppe; Lendal, Sara Eun; Engholm-Keller, Kasper

2010-01-01

-containing glycopeptides is achieved by using a low-pH buffer that contains a substituted acid such as glycolic acid to improve the binding efficiency and selectivity of SA-containing glycopeptides to the TiO(2) resin. By combining TiO(2) enrichment of sialylated glycopeptides with HILIC separation of deglycosylated...... of glycosylation sites and the characterization of glycan structures. In this paper, we describe a protocol for the selective enrichment of SA-containing glycopeptides using a combination of titanium dioxide (TiO(2)) and hydrophilic interaction liquid chromatography (HILIC). The selectivity of TiO(2) toward SA...... peptides, a more comprehensive analysis of formerly sialylated glycopeptides by MS can be achieved. Here we illustrate the efficiency of the method by the identification of 1,632 unique formerly sialylated glycopeptides from 817 sialylated glycoproteins. The TiO(2)/HILIC protocol requires 2 d...

Comparing Multi-Step IMAC and Multi-Step TiO2 Methods for Phosphopeptide Enrichment

Science.gov (United States)

Yue, Xiaoshan; Schunter, Alissa; Hummon, Amanda B.

2016-01-01

Phosphopeptide enrichment from complicated peptide mixtures is an essential step for mass spectrometry-based phosphoproteomic studies to reduce sample complexity and ionization suppression effects. Typical methods for enriching phosphopeptides include immobilized metal affinity chromatography (IMAC) or titanium dioxide (TiO2) beads, which have selective affinity and can interact with phosphopeptides. In this study, the IMAC enrichment method was compared with the TiO2 enrichment method, using a multi-step enrichment strategy from whole cell lysate, to evaluate their abilities to enrich for different types of phosphopeptides. The peptide-to-beads ratios were optimized for both IMAC and TiO2 beads. Both IMAC and TiO2 enrichments were performed for three rounds to enable the maximum extraction of phosphopeptides from the whole cell lysates. The phosphopeptides that are unique to IMAC enrichment, unique to TiO2 enrichment, and identified with both IMAC and TiO2 enrichment were analyzed for their characteristics. Both IMAC and TiO2 enriched similar amounts of phosphopeptides with comparable enrichment efficiency. However, phosphopeptides that are unique to IMAC enrichment showed a higher percentage of multi-phosphopeptides, as well as a higher percentage of longer, basic, and hydrophilic phosphopeptides. Also, the IMAC and TiO2 procedures clearly enriched phosphopeptides with different motifs. Finally, further enriching with two rounds of TiO2 from the supernatant after IMAC enrichment, or further enriching with two rounds of IMAC from the supernatant TiO2 enrichment does not fully recover the phosphopeptides that are not identified with the corresponding multi-step enrichment. PMID:26237447

A genome scan for positive selection in thoroughbred horses.

Directory of Open Access Journals (Sweden)

Jingjing Gu

2009-06-01

Full Text Available Thoroughbred horses have been selected for exceptional racing performance resulting in system-wide structural and functional adaptations contributing to elite athletic phenotypes. Because selection has been recent and intense in a closed population that stems from a small number of founder animals Thoroughbreds represent a unique population within which to identify genomic contributions to exercise-related traits. Employing a population genetics-based hitchhiking mapping approach we performed a genome scan using 394 autosomal and X chromosome microsatellite loci and identified positively selected loci in the extreme tail-ends of the empirical distributions for (1 deviations from expected heterozygosity (Ewens-Watterson test in Thoroughbred (n = 112 and (2 global differentiation among four geographically diverse horse populations (F(ST. We found positively selected genomic regions in Thoroughbred enriched for phosphoinositide-mediated signalling (3.2-fold enrichment; P<0.01, insulin receptor signalling (5.0-fold enrichment; P<0.01 and lipid transport (2.2-fold enrichment; P<0.05 genes. We found a significant overrepresentation of sarcoglycan complex (11.1-fold enrichment; P<0.05 and focal adhesion pathway (1.9-fold enrichment; P<0.01 genes highlighting the role for muscle strength and integrity in the Thoroughbred athletic phenotype. We report for the first time candidate athletic-performance genes within regions targeted by selection in Thoroughbred horses that are principally responsible for fatty acid oxidation, increased insulin sensitivity and muscle strength: ACSS1 (acyl-CoA synthetase short-chain family member 1, ACTA1 (actin, alpha 1, skeletal muscle, ACTN2 (actinin, alpha 2, ADHFE1 (alcohol dehydrogenase, iron containing, 1, MTFR1 (mitochondrial fission regulator 1, PDK4 (pyruvate dehydrogenase kinase, isozyme 4 and TNC (tenascin C. Understanding the genetic basis for exercise adaptation will be crucial for the identification of genes

Selective Enrichment of Omega-3 Fatty Acids in Oils by Phospholipase A1.

Directory of Open Access Journals (Sweden)

Tushar Ranjan Moharana

Full Text Available Omega fatty acids are recognized as key nutrients for healthier ageing. Lipases are used to release ω-3 fatty acids from oils for preparing enriched ω-3 fatty acid supplements. However, use of lipases in enrichment of ω-3 fatty acids is limited due to their insufficient specificity for ω-3 fatty acids. In this study use of phospholipase A1 (PLA1, which possesses both sn-1 specific activity on phospholipids and lipase activity, was explored for hydrolysis of ω-3 fatty acids from anchovy oil. Substrate specificity of PLA1 from Thermomyces lenuginosus was initially tested with synthetic p-nitrophenyl esters along with a lipase from Bacillus subtilis (BSL, as a lipase control. Gas chromatographic characterization of the hydrolysate obtained upon treatment of anchovy oil with these enzymes indicated a selective retention of ω-3 fatty acids in the triglyceride fraction by PLA1 and not by BSL. 13C NMR spectroscopy based position analysis of fatty acids in enzyme treated and untreated samples indicated that PLA1 preferably retained ω-3 fatty acids in oil, while saturated fatty acids were hydrolysed irrespective of their position. Hydrolysis of structured triglyceride,1,3-dioleoyl-2-palmitoylglycerol, suggested that both the enzymes hydrolyse the fatty acids at both the positions. The observed discrimination against ω-3 fatty acids by PLA1 appears to be due to its fatty acid selectivity rather than positional specificity. These studies suggest that PLA1 could be used as a potential enzyme for selective concentrationof ω-3 fatty acids.

Selective Enrichment of Omega-3 Fatty Acids in Oils by Phospholipase A1.

Science.gov (United States)

Ranjan Moharana, Tushar; Byreddy, Avinesh R; Puri, Munish; Barrow, Colin; Rao, Nalam Madhusudhana

2016-01-01

Omega fatty acids are recognized as key nutrients for healthier ageing. Lipases are used to release ω-3 fatty acids from oils for preparing enriched ω-3 fatty acid supplements. However, use of lipases in enrichment of ω-3 fatty acids is limited due to their insufficient specificity for ω-3 fatty acids. In this study use of phospholipase A1 (PLA1), which possesses both sn-1 specific activity on phospholipids and lipase activity, was explored for hydrolysis of ω-3 fatty acids from anchovy oil. Substrate specificity of PLA1 from Thermomyces lenuginosus was initially tested with synthetic p-nitrophenyl esters along with a lipase from Bacillus subtilis (BSL), as a lipase control. Gas chromatographic characterization of the hydrolysate obtained upon treatment of anchovy oil with these enzymes indicated a selective retention of ω-3 fatty acids in the triglyceride fraction by PLA1 and not by BSL. 13C NMR spectroscopy based position analysis of fatty acids in enzyme treated and untreated samples indicated that PLA1 preferably retained ω-3 fatty acids in oil, while saturated fatty acids were hydrolysed irrespective of their position. Hydrolysis of structured triglyceride,1,3-dioleoyl-2-palmitoylglycerol, suggested that both the enzymes hydrolyse the fatty acids at both the positions. The observed discrimination against ω-3 fatty acids by PLA1 appears to be due to its fatty acid selectivity rather than positional specificity. These studies suggest that PLA1 could be used as a potential enzyme for selective concentrationof ω-3 fatty acids.

Proteolytic Digestion and TiO2 Phosphopeptide Enrichment Microreactor for Fast MS Identification of Proteins

Science.gov (United States)

Deng, Jingren; Lazar, Iulia M.

2016-04-01

The characterization of phosphorylation state(s) of a protein is best accomplished by using isolated or enriched phosphoprotein samples or their corresponding phosphopeptides. The process is typically time-consuming as, often, a combination of analytical approaches must be used. To facilitate throughput in the study of phosphoproteins, a microreactor that enables a novel strategy for performing fast proteolytic digestion and selective phosphopeptide enrichment was developed. The microreactor was fabricated using 100 μm i.d. fused-silica capillaries packed with 1-2 mm beds of C18 and/or TiO2 particles. Proteolytic digestion-only, phosphopeptide enrichment-only, and sequential proteolytic digestion/phosphopeptide enrichment microreactors were developed and tested with standard protein mixtures. The protein samples were adsorbed on the C18 particles, quickly digested with a proteolytic enzyme infused over the adsorbed proteins, and further eluted onto the TiO2 microreactor for enrichment in phosphopeptides. A number of parameters were optimized to speed up the digestion and enrichments processes, including microreactor dimensions, sample concentrations, digestion time, flow rates, buffer compositions, and pH. The effective time for the steps of proteolytic digestion and enrichment was less than 5 min. For simple samples, such as standard protein mixtures, this approach provided equivalent or better results than conventional bench-top methods, in terms of both enzymatic digestion and selectivity. Analysis times and reagent costs were reduced ~10- to 15-fold. Preliminary analysis of cell extracts and recombinant proteins indicated the feasibility of integration of these microreactors in more advanced workflows amenable for handling real-world biological samples.

Non-degradative Ubiquitination of Protein Kinases.

Directory of Open Access Journals (Sweden)

K Aurelia Ball

2016-06-01

Full Text Available Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well.

Sn surface-enriched Pt-Sn bimetallic nanoparticles as a selective and stable catalyst for propane dehydrogenation

KAUST Repository

Zhu, Haibo

2014-12-01

A new one pot, surfactant-free, synthetic route based on the surface organometallic chemistry (SOMC) concept has been developed for the synthesis of Sn surface-enriched Pt-Sn nanoparticles. Bu3SnH selectively reacts with [Pt]-H formed in situ at the surface of Pt nanoparticles, Pt NPs, obtained by reduction of K2PtCl4 by LiB(C2H5)3H. Chemical analysis, 1H MAS and 13C CP/MAS solid-state NMR as well as two-dimensional double-quantum (DQ) and triple-quantum (TQ) experiments show that organo-tin moieties Sn(n-C4H9) are chemically linked to the surface of Pt NPs to produce, in fine, after removal of most of the n-butyl fragment, bimetallic Pt-Sn nanoparticles. The Sn(n-CH2CH2CH2CH3) groups remaining at the surface are believed to stabilize the as-synthesized Pt-Sn NPs, enabling the bimetallic NPs to be well dispersed in THF. Additionally, the Pt-Sn nanoparticles can be supported on MgAl2O4 during the synthesis of the nanoparticles. Some of the Pt-Sn/MgAl2O4 catalyst thus prepared exhibits high activity in PROX of CO and an extremely high selectivity and stability in propane dehydrogenation to propylene. The enhanced activity in propane dehydrogenation is associated with the high concentration of inactive Sn at the surface of Pt nanoparticles which ”isolates” the active Pt atoms. This conclusion is confirmed by XRD, NMR, TEM, and XPS analysis.

Uranium Enrichment, an overview

International Nuclear Information System (INIS)

Coates, J.H.

1994-01-01

This general presentation on uranium enrichment will be followed by lectures on more specific topics including descriptions of enrichment processes and assessments of the prevailing commercial and industrial situations. I shall therefore avoid as much as possible duplications with these other lectures, and rather dwell on: some theoretical aspects of enrichment in general, underlying the differences between statistical and selective processes, a review and comparison between enrichment processes, remarks of general order regarding applications, the proliferation potential of enrichment. It is noteworthy that enrichment: may occur twice in the LWR fuel cycle: first by enriching natural uranium, second by reenriching uranium recovered from reprocessing, must meet LWR requirements, and in particular higher assays required by high burn up fuel elements, bears on the structure of the entire front part of the fuel cycle, namely in the conversion/reconversion steps only involving UF 6 for the moment. (author). tabs., figs., 4 refs

Structure of Human G Protein-Coupled Receptor Kinase 2 in Complex with the Kinase Inhibitor Balanol

Energy Technology Data Exchange (ETDEWEB)

Tesmer, John J.G.; Tesmer, Valerie M.; Lodowski, David T.; Steinhagen, Henning; Huber, Jochen (Sanofi); (Michigan); (Texas)

2010-07-19

G protein-coupled receptor kinase 2 (GRK2) is a pharmaceutical target for the treatment of cardiovascular diseases such as congestive heart failure, myocardial infarction, and hypertension. To better understand how nanomolar inhibition and selectivity for GRK2 might be achieved, we have determined crystal structures of human GRK2 in complex with G{beta}{gamma} in the presence and absence of the AGC kinase inhibitor balanol. The selectivity of balanol among human GRKs is assessed.

Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase.

Science.gov (United States)

Jha, Sujata; Rollins, Madeline G; Fuchs, Gabriele; Procter, Dean J; Hall, Elizabeth A; Cozzolino, Kira; Sarnow, Peter; Savas, Jeffrey N; Walsh, Derek

2017-06-29

Ribosomes have the capacity to selectively control translation through changes in their composition that enable recognition of specific RNA elements. However, beyond differential subunit expression during development, evidence for regulated ribosome specification within individual cells has remained elusive. Here we report that a poxvirus kinase phosphorylates serine/threonine residues in the human small ribosomal subunit protein, receptor for activated C kinase (RACK1), that are not phosphorylated in uninfected cells or cells infected by other viruses. These modified residues cluster in an extended loop in RACK1, phosphorylation of which selects for translation of viral or reporter mRNAs with 5' untranslated regions that contain adenosine repeats, so-called polyA-leaders. Structural and phylogenetic analyses revealed that although RACK1 is highly conserved, this loop is variable and contains negatively charged amino acids in plants, in which these leaders act as translational enhancers. Phosphomimetics and inter-species chimaeras have shown that negative charge in the RACK1 loop dictates ribosome selectivity towards viral RNAs. By converting human RACK1 to a charged, plant-like state, poxviruses remodel host ribosomes so that adenosine repeats erroneously generated by slippage of the viral RNA polymerase confer a translational advantage. Our findings provide insight into ribosome customization through trans-kingdom mimicry and the mechanics of species-specific leader activity that underlie poxvirus polyA-leaders.

A dual platform for selective analyte enrichment and ionization in mass spectrometry using aptamer-conjugated graphene oxide.

Science.gov (United States)

Gulbakan, Basri; Yasun, Emir; Shukoor, M Ibrahim; Zhu, Zhi; You, Mingxu; Tan, Xiaohong; Sanchez, Hernan; Powell, David H; Dai, Hongjie; Tan, Weihong

2010-12-15

This study demonstrates the use of aptamer-conjugated graphene oxide as an affinity extraction and detection platform for analytes from complex biological media. We have shown that cocaine and adenosine can be selectively enriched from plasma samples and that direct mass spectrometric readouts can be obtained without a matrix and with greatly improved signal-to-noise ratios. Aptamer-conjugated graphene oxide has clear advantages in target enrichment and in generating highly efficient ionization of target molecules for mass spectrometry. These results demonstrate the utility of the approach for analysis of small molecules in real biological samples.

Phosphatidylinositol 3-Kinase (PI3K) and phosphatidylinositol 3-kinase-related kinase (PIKK) inhibitors: importance of the morpholine ring

Czech Academy of Sciences Publication Activity Database

Andrs, M.; Kobarecny, J.; Jun, D.; Hodný, Zdeněk; Bartek, Jiří; Kuca, K.

2015-01-01

Roč. 58, č. 1 (2015), s. 41-71 ISSN 0022-2623 R&D Projects: GA MŠk(CZ) CZ.1.07/2.3.00/30.0044 Grant - others:University Hospital Hradec Kralove(CZ) 00179906; Faculty of Military Health Sciences, University of Defence(CZ) SV/FVZ201402 Institutional support: RVO:68378050 Keywords : DEPENDENT PROTEIN-KINASE * STRAND BREAK REPAIR * SELECTIVE PI3K-BETA INHIBITORS * TELANGIECTASIA MUTATED KINASE Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.589, year: 2015

Optimization of Allosteric With-No-Lysine (WNK) Kinase Inhibitors and Efficacy in Rodent Hypertension Models

Energy Technology Data Exchange (ETDEWEB)

Yamada, Ken; Levell, Julian; Yoon, Taeyong; Kohls, Darcy; Yowe, David; Rigel, Dean F.; Imase, Hidetomo; Yuan, Jun; Yasoshima, Kayo; DiPetrillo, Keith; Monovich, Lauren; Xu, Lingfei; Zhu, Meicheng; Kato, Mitsunori; Jain, Monish; Idamakanti, Neeraja; Taslimi, Paul; Kawanami, Toshio; Argikar, Upendra A.; Kunjathoor, Vidya; Xie, Xiaoling; Yagi, Yukiko I.; Iwaki, Yuki; Robinson, Zachary; Park, Hyi-Man (Novartis)

2017-08-03

The observed structure–activity relationship of three distinct ATP noncompetitive With-No-Lysine (WNK) kinase inhibitor series, together with a crystal structure of a previously disclosed allosteric inhibitor bound to WNK1, led to an overlay hypothesis defining core and side-chain relationships across the different series. This in turn enabled an efficient optimization through scaffold morphing, resulting in compounds with a good balance of selectivity, cellular potency, and pharmacokinetic profile, which were suitable for in vivo proof-of-concept studies. When dosed orally, the optimized compound reduced blood pressure in mice overexpressing human WNK1, and induced diuresis, natriuresis and kaliuresis in spontaneously hypertensive rats (SHR), confirming that this mechanism of inhibition of WNK kinase activity is effective at regulating cardiovascular homeostasis.

Uranium enrichment: an overview

International Nuclear Information System (INIS)

Cazalet, J.

1995-01-01

This paper is a general presentation of uranium enrichment processes and assessments of the prevailing commercial and industrial situations. It gives first some theoretical aspects of enrichment in general and explains the differences between statistical and selective processes in particular. Then a review of the different processes is made with a comparison between them. Finally, some general remarks concerning applications are given and the risks of proliferation related to enrichment are mentioned. (J.S.). 4 refs., 5 figs., 8 tabs

New Variants of Tomato Thymidine Kinase 1 Selected for Increased Sensitivity of E. coli KY895 towards Azidothymidine

International Nuclear Information System (INIS)

Slot Christiansen, Louise; Egeblad, Louise; Munch-Petersen, Birgitte; Piškur, Jure; Knecht, Wolfgang

2015-01-01

Nucleoside analogues (NA) are prodrugs that are phosphorylated by deoxyribonucleoside kinases (dNKs) as the first step towards a compound toxic to the cell. During the last 20 years, research around dNKs has gone into new organisms other than mammals and viruses. Newly discovered dNKs have been tested as enzymes for suicide gene therapy. The tomato thymidine kinase 1 (ToTK1) is a dNK that has been selected for its in vitro kinetic properties and then successfully been tested in vivo for the treatment of malignant glioma. We present the selection of two improved variants of ToTK1 generated by random protein engineering for suicide gene therapy with the NA azidothymidine (AZT). We describe their selection, recombinant production and a subsequent kinetic and biochemical characterization. Their improved performance in killing of E. coli KY895 is accompanied by an increase in specificity for the NA AZT over the natural substrate thymidine as well as a decrease in inhibition by dTTP, the end product of the nucleoside salvage pathway for thymidine. The understanding of the enzymatic properties improving the variants efficacy is instrumental to further develop dNKs for use in suicide gene therapy

New Variants of Tomato Thymidine Kinase 1 Selected for Increased Sensitivity of E. coli KY895 towards Azidothymidine

Energy Technology Data Exchange (ETDEWEB)

Slot Christiansen, Louise [Department of Biology, Lund University, Lund 22362 (Sweden); Lund Protein Production Platform, Lund University, Lund 22362 (Sweden); Egeblad, Louise [Lund Protein Production Platform, Lund University, Lund 22362 (Sweden); Munch-Petersen, Birgitte [Department of Science, Systems and Models, Roskilde University, Roskilde 4000 (Denmark); Piškur, Jure [Department of Biology, Lund University, Lund 22362 (Sweden); Knecht, Wolfgang, E-mail: Louise.Slot_Christiansen@biol.lu.se [Department of Biology, Lund University, Lund 22362 (Sweden); Lund Protein Production Platform, Lund University, Lund 22362 (Sweden)

2015-06-08

Nucleoside analogues (NA) are prodrugs that are phosphorylated by deoxyribonucleoside kinases (dNKs) as the first step towards a compound toxic to the cell. During the last 20 years, research around dNKs has gone into new organisms other than mammals and viruses. Newly discovered dNKs have been tested as enzymes for suicide gene therapy. The tomato thymidine kinase 1 (ToTK1) is a dNK that has been selected for its in vitro kinetic properties and then successfully been tested in vivo for the treatment of malignant glioma. We present the selection of two improved variants of ToTK1 generated by random protein engineering for suicide gene therapy with the NA azidothymidine (AZT). We describe their selection, recombinant production and a subsequent kinetic and biochemical characterization. Their improved performance in killing of E. coli KY895 is accompanied by an increase in specificity for the NA AZT over the natural substrate thymidine as well as a decrease in inhibition by dTTP, the end product of the nucleoside salvage pathway for thymidine. The understanding of the enzymatic properties improving the variants efficacy is instrumental to further develop dNKs for use in suicide gene therapy.

Eukaryotic elongation factor 2 is a prognostic marker and its kinase a potential therapeutic target in HCC.

Science.gov (United States)

Pott, Leona L; Hagemann, Sascha; Reis, Henning; Lorenz, Kristina; Bracht, Thilo; Herold, Thomas; Skryabin, Boris V; Megger, Dominik A; Kälsch, Julia; Weber, Frank; Sitek, Barbara; Baba, Hideo A

2017-02-14

Hepatocellular carcinoma is a cancer with increasing incidence and largely refractory to current anticancer drugs. Since Sorafenib, a multikinase inhibitor has shown modest efficacy in advanced hepatocellular carcinoma additional treatments are highly needed. Protein phosphorylation via kinases is an important post-translational modification to regulate cell homeostasis including proliferation and apoptosis. Therefore kinases are valuable targets in cancer therapy. To this end we performed 2D differential gel electrophoresis and mass spectrometry analysis of phosphoprotein-enriched lysates of tumor and corresponding non-tumorous liver samples to detect differentially abundant phosphoproteins to screen for novel kinases as potential drug targets. We identified 34 differentially abundant proteins in phosphoprotein enriched lysates. Expression and distribution of the candidate protein eEF2 and its phosphorylated isoform was validated immunohistochemically on 78 hepatocellular carcinoma and non-tumorous tissue samples. Validation showed that total eEF2 and phosphorylated eEF2 at threonine 56 are prognostic markers for overall survival of HCC-patients. The activity of the regulating eEF2 kinase, compared between tumor and non-tumorous tissue lysates by in vitro kinase assays, is more than four times higher in tumor tissues. Functional analyzes regarding eEF2 kinase were performed in JHH5 cells with CRISPR/Cas9 mediated eEF2 kinase knock out. Proliferation and growth is decreased in eEF2 kinase knock out cells. eEF2 and phosphorylated eEF2 are prognostic markers for survival of hepatocellular carcinoma patients and the regulating eEF2 kinase is a potential drug target for tumor therapy.

Preferential Selectivity of Inhibitors with Human Tau Protein Kinase Gsk3 Elucidates Their Potential Roles for Off-Target Alzheimer’s Therapy

Directory of Open Access Journals (Sweden)

Jagadeesh Kumar Dasappa

2013-01-01

Full Text Available Alzheimer’s disease (AD is a neurodegenerative disorder characterized by the accumulation of amyloid beta peptides (A and neurofibrillary tangles (NFTs. The abnormal phosphorylation of tau leads to the formation of NFTs produced by the action of tau kinases, resulting in the loss of neurons and synapse, leading to dementia. Hence, tau kinases have become potential drug target candidates for small molecule inhibitors. With an aim to explore the identification of a common inhibitor, this investigation was undertaken towards analyzing all 10 tau kinases which are implicated in phosphorylation of AD. A set of 7 inhibitors with varied scaffolds were collected from the Protein Data Bank (PDB. The analysis, involving multiple sequence alignment, 3D structural alignment, catalytic active site overlap, and docking studies, has enabled elucidation of the pharmacophoric patterns for the class of 7 inhibitors. Our results divulge that tau protein kinases share a specific set of conserved structural elements for the binding of inhibitors and ATP, respectively. The scaffold of 3-aminopyrrolidine (inhibitor 6 exhibits high preferential affinity with GSK3. Surprisingly, the PDB does not contain the structural details of GSK3 with this specific inhibitor. Thus, our investigations provide vital clues towards design of novel off-target drugs for Alzheimer’s.

Centrifuge enrichment program

International Nuclear Information System (INIS)

Astley, E.R.

1976-01-01

Exxon Nuclear has been active in privately funded research and development of centrifuge enrichment technology since 1972. In October of 1975, Exxon Nuclear submitted a proposal to design, construct, and operate a 3000-MT SWU/yr centrifuge enrichment plant, under the provisions of the proposed Nuclear Fuel Assurance Act of 1975. The U.S. Energy Research and Development Administration (ERDA) accepted the proposal as a basis for negotiation. It was proposed to build a 1000-MT SWU/yr demonstration increment to be operational in 1982; and after successful operation for about one year, expand the facilities into a 3000-MT SWU/yr plant. As part of the overall centrifuge enrichment plant, a dedicated centrifuge manufacturing plant would be constructed; sized to support the full 3000-MT SWU/yr plant. The selection of the centrifuge process by Exxon Nuclear was based on an extremely thorough evaluation of current and projected enrichment technology; results show that the technology is mature and the process will be cost effective. The substantial savings in energy (about 93%) from utilization of the centrifuge option rather than gaseous diffusion is a compelling argument. As part of this program, Exxon Nuclear has a large hardware R and D program, plus a prototype centrifuge manufacturing capability in Malta, New York. To provide a full-scale machine and limited cascade test capability, Exxon Nuclear is constructing a $4,000,000 Centrifuge Test Facility in Richland, Washington. This facility was to initiate operations in the Fall of 1976. Exxon Nuclear is convinced that the centrifuge enrichment process is the rational selection for emergence of a commercial enrichment industry

Icotinib, a selective EGF receptor tyrosine kinase inhibitor, for the treatment of non-small-cell lung cancer.

Science.gov (United States)

Tan, Fenlai; Shi, Yuankai; Wang, Yinxiang; Ding, Lieming; Yuan, Xiaobin; Sun, Yan

2015-01-01

Advanced non-small-cell lung cancer (NSCLC) is the main cause for cancer-related mortality. Treatments for advanced NSCLC are largely palliative and a benefit plateau appears to have reached with the platinum-based chemotherapy regimens. EGF receptor (EGFR) tyrosine kinase inhibitors gefitinib, erlotinib and afatinib came up with prolonged progression-free survival and improved quality of life, especially in EGFR-mutated patients. Icotinib is an oral selective EGFR tyrosine kinase, which was approved by China Food and Drug administration in June 2011 for treating advanced NSCLC. Its approval was based on the registered Phase III trial (ICOGEN), which showed icotinib is noninferior to gefitinib. This review will discuss the role of icotinib in NSCLC, and its potential application and ongoing investigations.

Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns

DEFF Research Database (Denmark)

Larsen, Martin Røssel; Thingholm, Tine E; Jensen, Ole N

2005-01-01

based on TiO2microcolumns and peptide loading in 2,5-dihydroxybenzoic acid (DHB). The effect of DHB was a very efficient reduction in the binding of nonphosphorylated peptides to TiO2 while retaining its high binding affinity for phosphorylated peptides. Thus, inclusion of DHB dramatically increased...... the selectivity of the enrichment of phosphorylated peptides by TiO2. We demonstrated that this new procedure was more selective for binding phosphorylated peptides than IMAC using MALDI mass spectrometry. In addition, we showed that LC-ESI-MSMS was biased toward monophosphorylated peptides, whereas MALDI MS...... was not. Other substituted aromatic carboxylic acids were also capable of specifically reducing binding of nonphosphorylated peptides, whereas phosphoric acid reduced binding of both phosphorylated and nonphosphorylated peptides. A putative mechanism for this intriguing effect is presented....

Evolutionary diversification of plant shikimate kinase gene duplicates.

Directory of Open Access Journals (Sweden)

Geoffrey Fucile

2008-12-01

Full Text Available Shikimate kinase (SK; EC 2.7.1.71 catalyzes the fifth reaction of the shikimate pathway, which directs carbon from the central metabolism pool to a broad range of secondary metabolites involved in plant development, growth, and stress responses. In this study, we demonstrate the role of plant SK gene duplicate evolution in the diversification of metabolic regulation and the acquisition of novel and physiologically essential function. Phylogenetic analysis of plant SK homologs resolves an orthologous cluster of plant SKs and two functionally distinct orthologous clusters. These previously undescribed genes, shikimate kinase-like 1 (SKL1 and -2 (SKL2, do not encode SK activity, are present in all major plant lineages, and apparently evolved under positive selection following SK gene duplication over 400 MYA. This is supported by functional assays using recombinant SK, SKL1, and SKL2 from Arabidopsis thaliana (At and evolutionary analyses of the diversification of SK-catalytic and -substrate binding sites based on theoretical structure models. AtSKL1 mutants yield albino and novel variegated phenotypes, which indicate SKL1 is required for chloroplast biogenesis. Extant SKL2 sequences show a strong genetic signature of positive selection, which is enriched in a protein-protein interaction module not found in other SK homologs. We also report the first kinetic characterization of plant SKs and show that gene expression diversification among the AtSK inparalogs is correlated with developmental processes and stress responses. This study examines the functional diversification of ancient and recent plant SK gene duplicates and highlights the utility of SKs as scaffolds for functional innovation.

Stable acetate production in extreme-thermophilic (70ºC) mixed culture fermentation by selective enrichment of hydrogenotrophic methanogens

NARCIS (Netherlands)

Zhang, F.; Zhang, Y.; Ding, J.; Dai, K.; Van Loosdrecht, M.C.M.; Zeng, R.J.

2014-01-01

The control of metabolite production is difficult in mixed culture fermentation. This is particularly related to hydrogen inhibition. In this work, hydrogenotrophic methanogens were selectively enriched to reduce the hydrogen partial pressure and to realize efficient acetate production in

Tyrosine kinase inhibitors: Multi-targeted or single-targeted?

Science.gov (United States)

Broekman, Fleur; Giovannetti, Elisa; Peters, Godefridus J

2011-02-10

Since in most tumors multiple signaling pathways are involved, many of the inhibitors in clinical development are designed to affect a wide range of targeted kinases. The most important tyrosine kinase families in the development of tyrosine kinase inhibitors are the ABL, SCR, platelet derived growth factor, vascular endothelial growth factor receptor and epidermal growth factor receptor families. Both multi-kinase inhibitors and single-kinase inhibitors have advantages and disadvantages, which are related to potential resistance mechanisms, pharmacokinetics, selectivity and tumor environment. In different malignancies various tyrosine kinases are mutated or overexpressed and several resistance mechanisms exist. Pharmacokinetics is influenced by interindividual differences and differs for two single targeted inhibitors or between patients treated by the same tyrosine kinase inhibitor. Different tyrosine kinase inhibitors have various mechanisms to achieve selectivity, while differences in gene expression exist between tumor and stromal cells. Considering these aspects, one type of inhibitor can generally not be preferred above the other, but will depend on the specific genetic constitution of the patient and the tumor, allowing personalized therapy. The most effective way of cancer treatment by using tyrosine kinase inhibitors is to consider each patient/tumor individually and to determine the strategy that specifically targets the consequences of altered (epi)genetics of the tumor. This strategy might result in treatment by a single multi kinase inhibitor for one patient, but in treatment by a couple of single kinase inhibitors for other patients.

Enrichment: CRISLA [chemical reaction by isotope selective activation] aims to reduce costs

International Nuclear Information System (INIS)

Eerkens, J.W.

1989-01-01

Every year, more than $3 billion is spent on enriching uranium. CRISLA (Chemical Reaction by Isotope Selective Activation) uses a laser-catalyzed chemical reaction which, its proponents claim, could substantially reduce these costs. In CRISLA, an infrared CO laser illuminates the intracavity reaction cell (IC) at a frequency tuned to excite primarily UF 6 . When UF 6 and co-reactant RX are passed through the IC, the tuned laser photons preferentially enhance the reaction of UF 6 with RX ten-thousand-fold over the thermal reaction rate. Thus the laser serves as an activator and the chemical energy for separation is largely chemical. (author)

The selectivity and promiscuity of brain-neuroregenerative inhibitors between ROCK1 and ROCK2 isoforms: An integration of SB-QSSR modelling, QM/MM analysis and in vitro kinase assay.

Science.gov (United States)

Zhu, L; Yang, Y; Lu, X

2016-01-01

The Rho-associated kinases (ROCKs) have long been recognized as an attractive therapeutic target for various neurological diseases; selective inhibition of ROCK1 and ROCK2 isoforms would result in distinct biological effects on neurogenesis, neuroplasticity and neuroregeneration after brain surgery and traumatic brain injury. However, the discovery and design of isoform-selective inhibitors remain a great challenge due to the high conservation and similarity between the kinase domains of ROCK1 and ROCK2. Here, a structure-based quantitative structure-selectivity relationship (SB-QSSR) approach was used to correlate experimentally measured selectivity with the difference in inhibitor binding to the two kinase isoforms. The resulting regression models were examined rigorously through both internal cross-validation and external blind validation; a nonlinear predictor was found to have high fitting stability and strong generalization ability, which was then employed to perform virtual screening against a structurally diverse, drug-like compound library. Consequently, five and seven hits were identified as promising candidates of 1-o-2 and 2-o-1 selective inhibitors, respectively, from which seven purchasable compounds were tested in vitro using a standard kinase assay protocol to determine their inhibitory activity against and selectivity between ROCK1 and ROCK2. The structural basis, energetic property and biological implication underlying inhibitor selectivity and promiscuity were also investigated systematically using a hybrid quantum mechanics/molecular mechanics (QM/MM) scheme.

Alignment independent 3D-QSAR, quantum calculations and molecular docking of Mer specific tyrosine kinase inhibitors as anticancer drugs

Directory of Open Access Journals (Sweden)

Fereshteh Shiri

2016-03-01

Full Text Available Mer receptor tyrosine kinase is a promising novel cancer therapeutic target in many human cancers, because abnormal activation of Mer has been implicated in survival signaling and chemoresistance. 3D-QSAR analyses based on alignment independent descriptors were performed on a series of 81 Mer specific tyrosine kinase inhibitors. The fractional factorial design (FFD and the enhanced replacement method (ERM were applied and tested as variable selection algorithms for the selection of optimal subsets of molecular descriptors from a much greater pool of such regression variables. The data set was split into 65 molecules as the training set and 16 compounds as the test set. All descriptors were generated by using the GRid INdependent descriptors (GRIND approach. After variable selection, GRIND were correlated with activity values (pIC50 by PLS regression. Of the two applied variable selection methods, ERM had a noticeable improvement on the statistical parameters of PLS model, and yielded a q2 value of 0.77, an rpred2 of 0.94, and a low RMSEP value of 0.25. The GRIND information contents influencing the affinity on Mer specific tyrosine kinase were also confirmed by docking studies. In a quantum calculation study, the energy difference between HOMO and LUMO (gap implied the high interaction of the most active molecule in the active site of the protein. In addition, the molecular electrostatic potential energy at DFT level confirmed results obtained from the molecular docking. The identified key features obtained from the molecular modeling, enabled us to design novel kinase inhibitors.

Alignment independent 3D-QSAR, quantum calculations and molecular docking of Mer specific tyrosine kinase inhibitors as anticancer drugs.

Science.gov (United States)

Shiri, Fereshteh; Pirhadi, Somayeh; Ghasemi, Jahan B

2016-03-01

Mer receptor tyrosine kinase is a promising novel cancer therapeutic target in many human cancers, because abnormal activation of Mer has been implicated in survival signaling and chemoresistance. 3D-QSAR analyses based on alignment independent descriptors were performed on a series of 81 Mer specific tyrosine kinase inhibitors. The fractional factorial design (FFD) and the enhanced replacement method (ERM) were applied and tested as variable selection algorithms for the selection of optimal subsets of molecular descriptors from a much greater pool of such regression variables. The data set was split into 65 molecules as the training set and 16 compounds as the test set. All descriptors were generated by using the GRid INdependent descriptors (GRIND) approach. After variable selection, GRIND were correlated with activity values (pIC50) by PLS regression. Of the two applied variable selection methods, ERM had a noticeable improvement on the statistical parameters of PLS model, and yielded a q (2) value of 0.77, an [Formula: see text] of 0.94, and a low RMSEP value of 0.25. The GRIND information contents influencing the affinity on Mer specific tyrosine kinase were also confirmed by docking studies. In a quantum calculation study, the energy difference between HOMO and LUMO (gap) implied the high interaction of the most active molecule in the active site of the protein. In addition, the molecular electrostatic potential energy at DFT level confirmed results obtained from the molecular docking. The identified key features obtained from the molecular modeling, enabled us to design novel kinase inhibitors.

Rhodium(II) metallopeptide catalyst design enables fine control in selective functionalization of natural SH3 domains.

Science.gov (United States)

Vohidov, Farrukh; Coughlin, Jane M; Ball, Zachary T

2015-04-07

Chemically modified proteins are increasingly important for use in fundamental biophysical studies, chemical biology, therapeutic protein development, and biomaterials. However, chemical methods typically produce heterogeneous labeling and cannot approach the exquisite selectivity of enzymatic reactions. While bioengineered methods are sometimes an option, selective reactions of natural proteins remain an unsolved problem. Here we show that rhodium(II) metallopeptides combine molecular recognition with promiscuous catalytic activity to allow covalent decoration of natural SH3 domains, depending on choice of catalyst but independent of the specific residue present. A metallopeptide catalyst succeeds in modifying a single SH3-containing kinase at endogenous concentrations in prostate cancer (PC-3) cell lysate. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

L-Lactate-selective microbial sensor based on flavocytochrome b2-enriched yeast cells using recombinant and nanotechnology approaches.

Science.gov (United States)

Karkovska, Maria; Smutok, Oleh; Stasyuk, Nataliya; Gonchar, Mykhailo

2015-11-01

In the recent years, nanotechnology is the most developing branch due to a wide variety of potential applications in biomedical, biotechnological and agriculture fields. The binding nanoparticles with various biological molecules makes them attractive candidates for using in sensor technologies. The particularly actual is obtaining the bionanomembranes based on biocatalytic elements with improved sensing characteristics. The aim of this investigation is to study the properties of microbial L-lactate-selective sensor based on using the recombinant Hansenula polymorpha yeast cells overproducing flavocytochrome b2 (FC b2), as well as additionally enriched by the enzyme bound with gold nanoparticles (FC b2-nAu). Although, the high permeability of the living cells to nanoparticles is being intensively studied (mostly for delivery of drugs), the idea of using both recombinant technology and nanotechnology to increase the amount of the target enzyme in the biosensing layer is really novel. The FC b2-nAu-enriched living and permeabilized yeast cells were used for construction of a bioselective membrane of microbial L-lactate-selective amperometric biosensor. Phenazine methosulphate was served as a free defusing electron transfer mediator which provides effective electron transfer from the reduced enzyme to the electrode surface. It was shown that the output to L-lactate of FC b2-nAu-enriched permeabilized yeast cells is 2.5-fold higher when compared to the control cells. The obtained results confirm that additional enrichment of the recombinant yeast cell by the enzyme bound with nanoparticles improves the analytical parameters of microbial sensor. Copyright © 2015 Elsevier B.V. All rights reserved.

Bromovinyl-deoxyuridine: A selective substrate for mitochondrial thymidine kinase in cell extracts

International Nuclear Information System (INIS)

Franzolin, Elisa; Rampazzo, Chiara; Perez-Perez, Maria-Jesus; Hernandez, Ana-Isabel; Balzarini, Jan; Bianchi, Vera

2006-01-01

Cellular models of mitochondrial thymidine kinase (TK2) deficiency require a reliable method to measure TK2 activity in whole cell extracts containing two interfering deoxyribonucleoside kinases, thymidine kinase 1 (TK1) and deoxycytidine kinase. We tested the value of the thymidine analog (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) as a TK2-specific substrate. With extracts of OSTTK1 - cells containing TK2 as the only thymidine kinase and a highly specific TK2 inhibitor we established conditions to detect the low TK2 activity commonly present in cells. With extracts of TK1-proficient osteosarcoma cells and normal human fibroblasts we showed that BVDU, but not 1-(β-D-arabinofuranosyl)thymine (Ara-T), discriminates TK2 activity even in the presence of 100-fold excess TK1. A comparison with current procedures based on TK2 inhibition demonstrated the better performance of the new TK2 assay. When cultured human fibroblasts passed from proliferation to quiescence TK2 activity increased by 3-fold, stressing the importance of TK2 function in the absence of TK1

Bromovinyl-deoxyuridine: A selective substrate for mitochondrial thymidine kinase in cell extracts.

Science.gov (United States)

Franzolin, Elisa; Rampazzo, Chiara; Pérez-Pérez, María-Jesús; Hernández, Ana-Isabel; Balzarini, Jan; Bianchi, Vera

2006-05-26

Cellular models of mitochondrial thymidine kinase (TK2) deficiency require a reliable method to measure TK2 activity in whole cell extracts containing two interfering deoxyribonucleoside kinases, thymidine kinase 1 (TK1) and deoxycytidine kinase. We tested the value of the thymidine analog (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) as a TK2-specific substrate. With extracts of OSTTK1- cells containing TK2 as the only thymidine kinase and a highly specific TK2 inhibitor we established conditions to detect the low TK2 activity commonly present in cells. With extracts of TK1-proficient osteosarcoma cells and normal human fibroblasts we showed that BVDU, but not 1-(beta-d-arabinofuranosyl)thymine (Ara-T), discriminates TK2 activity even in the presence of 100-fold excess TK1. A comparison with current procedures based on TK2 inhibition demonstrated the better performance of the new TK2 assay. When cultured human fibroblasts passed from proliferation to quiescence TK2 activity increased by 3-fold, stressing the importance of TK2 function in the absence of TK1.

Furan-2-ylmethylene thiazolidinediones as novel, potent, and selective inhibitors of phosphoinositide 3-kinase gamma.

Science.gov (United States)

Pomel, Vincent; Klicic, Jasna; Covini, David; Church, Dennis D; Shaw, Jeffrey P; Roulin, Karen; Burgat-Charvillon, Fabienne; Valognes, Delphine; Camps, Montserrat; Chabert, Christian; Gillieron, Corinne; Françon, Bernard; Perrin, Dominique; Leroy, Didier; Gretener, Denise; Nichols, Anthony; Vitte, Pierre Alain; Carboni, Susanna; Rommel, Christian; Schwarz, Matthias K; Rückle, Thomas

2006-06-29

Class I phosphoinositide 3-kinases (PI3Ks), in particular PI3Kgamma, have become attractive drug targets for inflammatory and autoimmune diseases. Here, we disclose a novel series of furan-2-ylmethylene thiazolidinediones as selective, ATP-competitive PI3Kgamma inhibitors. Structure-based design and X-ray crystallography of complexes formed by inhibitors bound to PI3Kgamma identified key pharmacophore features for potency and selectivity. An acidic NH group on the thiazolidinedione moiety and a hydroxy group on the furan-2-yl-phenyl part of the molecule play crucial roles in binding to PI3K and contribute to class IB PI3K selectivity. Compound 26 (AS-252424), a potent and selective small-molecule PI3Kgamma inhibitor emerging from these efforts, was further profiled in three different cellular PI3K assays and shown to be selective for class IB PI3K-mediated cellular effects. Oral administration of 26 in a mouse model of acute peritonitis led to a significant reduction of leukocyte recruitment.

Isotope enrichment

International Nuclear Information System (INIS)

Garbuny, M.

1979-01-01

The invention discloses a method for deriving, from a starting material including an element having a plurality of isotopes, derived material enriched in one isotope of the element. The starting material is deposited on a substrate at less than a critical submonatomic surface density, typically less than 10 16 atoms per square centimeter. The deposit is then selectively irradiated by a laser (maser or electronic oscillator) beam with monochromatic coherent radiation resonant with the one isotope causing the material including the one istope to escape from the substrate. The escaping enriched material is then collected. Where the element has two isotopes, one of which is to be collected, the deposit may be irradiated with radiation resonant with the other isotope and the residual material enriched in the one isotope may be evaporated from the substrate and collected

Acute lymphoid and gastrointestinal toxicity induced by selective p38alpha map kinase and map kinase-activated protein kinase-2 (MK2) inhibitors in the dog.

Science.gov (United States)

Morris, Dale L; O'Neil, Shawn P; Devraj, Rajesh V; Portanova, Joseph P; Gilles, Richard W; Gross, Cindy J; Curtiss, Sandra W; Komocsar, Wendy J; Garner, Debra S; Happa, Fernando A; Kraus, Lori J; Nikula, Kristen J; Monahan, Joseph B; Selness, Shaun R; Galluppi, Gerald R; Shevlin, Kimberly M; Kramer, Jeffrey A; Walker, John K; Messing, Dean M; Anderson, David R; Mourey, Robert J; Whiteley, Laurence O; Daniels, John S; Yang, Jerry Z; Rowlands, Philip C; Alden, Carl L; Davis, John W; Sagartz, John E

2010-06-01

Exposure to moderately selective p38alpha mitogen-activated protein kinase (MAPK) inhibitors in the Beagle dog results in an acute toxicity consisting of mild clinical signs (decreased activity, diarrhea, and fever), lymphoid necrosis and depletion in the gut-associated lymphoid tissue (GALT), mesenteric lymph nodes and spleen, and linear colonic and cecal mucosal hemorrhages. Lymphocyte apoptosis and necrosis in the GALT is the earliest and most prominent histopathologic change observed, followed temporally by neutrophilic infiltration and acute inflammation of the lymph nodes and spleen and multifocal mucosal epithelial necrosis and linear hemorrhages in the colon and cecum. These effects are not observed in the mouse, rat, or cynomolgus monkey. To further characterize the acute toxicity in the dog, a series of in vivo, in vitro, and immunohistochemical studies were conducted to determine the relationship between the lymphoid and gastrointestinal (GI) toxicity and p38 MAPK inhibition. Results of these studies demonstrate a direct correlation between p38alpha MAPK inhibition and the acute lymphoid and gastrointestinal toxicity in the dog. Similar effects were observed following exposure to inhibitors of MAPK-activated protein kinase-2 (MK2), further implicating the role of p38alpha MAPK signaling pathway inhibition in these effects. Based on these findings, the authors conclude that p38alpha MAPK inhibition results in acute lymphoid and GI toxicity in the dog and is unique among the species evaluated in these studies.

The phosphoinositide 3-kinase α selective inhibitor BYL719 enhances the effect of the protein kinase C inhibitor AEB071 in GNAQ/GNA11-mutant uveal melanoma cells.

Science.gov (United States)

Musi, Elgilda; Ambrosini, Grazia; de Stanchina, Elisa; Schwartz, Gary K

2014-05-01

G-protein mutations are one of the most common mutations occurring in uveal melanoma activating the protein kinase C (PKC)/mitogen-activated protein kinase and phosphoinositide 3-kinase (PI3K)/AKT pathways. In this study, we described the effect of dual pathway inhibition in uveal melanoma harboring GNAQ and GNA11 mutations via PKC inhibition with AEB071 (sotrastaurin) and PI3K/AKT inhibition with BYL719, a selective PI3Kα inhibitor. Growth inhibition was observed in GNAQ/GNA11-mutant cells with AEB071 versus no activity in wild-type cells. In the GNAQ-mutant cells, AEB071 decreased phosphorylation of myristoylated alanine-rich C-kinase substrate, a substrate of PKC, along with ERK1/2 and ribosomal S6, but persistent AKT activation was present. BYL719 had minimal antiproliferative activity in all uveal melanoma cell lines, and inhibited phosphorylation of AKT in most cell lines. In the GNA11-mutant cell line, similar effects were observed with ERK1/2 inhibition, mostly inhibited by BYL719. With the combination treatment, both GNAQ- and GNA11-mutant cell lines showed synergistic inhibition of cell proliferation and apoptotic cell death. In vivo studies correlated with in vitro findings showing reduced xenograft tumor growth with the combination therapy in a GNAQ-mutant model. These findings suggest a new therapy treatment option for G-protein-mutant uveal melanoma with a focus on specific targeting of multiple downstream pathways as part of combination therapy.

Mitogen activated protein kinases selectively regulate palytoxin-stimulated gene expression in mouse keratinocytes

International Nuclear Information System (INIS)

Zeliadt, Nicholette A.; Warmka, Janel K.; Wattenberg, Elizabeth V.

2003-01-01

We have been investigating how the novel skin tumor promoter palytoxin transmits signals through mitogen activated protein kinases (MAPKs). Palytoxin activates three major MAPKs, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, in a keratinocyte cell line derived from initiated mouse skin (308). We previously showed that palytoxin requires ERK to increase matrix metalloproteinase-13 (MMP-13) gene expression, an enzyme implicated in carcinogenesis. Diverse stimuli require JNK and p38 to increase MMP-13 gene expression, however. We therefore used the JNK and p38 inhibitors SP 600125 and SB 202190, respectively, to investigate the role of these MAPKs in palytoxin-induced MMP-13 gene expression. Surprisingly, palytoxin does not require JNK and p38 to increase MMP-13 gene expression. Accordingly, ERK activation, independent of palytoxin and in the absence of JNK and p38 activation, is sufficient to induce MMP-13 gene expression in 308 keratinocytes. Dexamethasone, a synthetic glucocorticoid that inhibits activator protein-1 (AP-1), blocked palytoxin-stimulated MMP-13 gene expression. Therefore, the AP-1 site present in the promoter of the MMP-13 gene appears to be functional and to play a key role in palytoxin-stimulated gene expression. Previous studies showed that palytoxin simulates an ERK-dependent selective increase in the c-Fos content of AP-1 complexes that bind to the promoter of the MMP-13 gene. JNK and p38 can also modulate c-Fos. Palytoxin does not require JNK or p38 to increase c-Fos binding, however. Altogether, these studies indicate that ERK plays a distinctly essential role in transmitting palytoxin-stimulated signals to specific nuclear targets in keratinocytes derived from initiated mouse skin

Multiplexed enrichment of rare DNA variants via sequence-selective and temperature-robust amplification

Science.gov (United States)

Wu, Lucia R.; Chen, Sherry X.; Wu, Yalei; Patel, Abhijit A.; Zhang, David Yu

2018-01-01

Rare DNA-sequence variants hold important clinical and biological information, but existing detection techniques are expensive, complex, allele-specific, or don’t allow for significant multiplexing. Here, we report a temperature-robust polymerase-chain-reaction method, which we term blocker displacement amplification (BDA), that selectively amplifies all sequence variants, including single-nucleotide variants (SNVs), within a roughly 20-nucleotide window by 1,000-fold over wild-type sequences. This allows for easy detection and quantitation of hundreds of potential variants originally at ≤0.1% in allele frequency. BDA is compatible with inexpensive thermocycler instrumentation and employs a rationally designed competitive hybridization reaction to achieve comparable enrichment performance across annealing temperatures ranging from 56 °C to 64 °C. To show the sequence generality of BDA, we demonstrate enrichment of 156 SNVs and the reliable detection of single-digit copies. We also show that the BDA detection of rare driver mutations in cell-free DNA samples extracted from the blood plasma of lung-cancer patients is highly consistent with deep sequencing using molecular lineage tags, with a receiver operator characteristic accuracy of 95%. PMID:29805844

Protein kinase activity of phosphoinositide 3-kinase regulates cytokine-dependent cell survival.

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Daniel Thomas

Full Text Available The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K, promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3 and granulocyte macrophage colony stimulating factor (GM-CSF receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting

GSL-enriched membrane microdomains in innate immune responses.

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Nakayama, Hitoshi; Ogawa, Hideoki; Takamori, Kenji; Iwabuchi, Kazuhisa

2013-06-01

Many pathogens target glycosphingolipids (GSLs), which, together with cholesterol, GPI-anchored proteins, and various signaling molecules, cluster on host cell membranes to form GSL-enriched membrane microdomains (lipid rafts). These GSL-enriched membrane microdomains may therefore be involved in host-pathogen interactions. Innate immune responses are triggered by the association of pathogens with phagocytes, such as neutrophils, macrophages and dendritic cells. Phagocytes express a diverse array of pattern-recognition receptors (PRRs), which sense invading microorganisms and trigger pathogen-specific signaling. PRRs can recognize highly conserved pathogen-associated molecular patterns expressed on microorganisms. The GSL lactosylceramide (LacCer, CDw17), which binds to various microorganisms, including Candida albicans, is expressed predominantly on the plasma membranes of human mature neutrophils and forms membrane microdomains together with the Src family tyrosine kinase Lyn. These LacCer-enriched membrane microdomains can mediate superoxide generation, migration, and phagocytosis, indicating that LacCer functions as a PRR in innate immunity. Moreover, the interactions of GSL-enriched membrane microdomains with membrane proteins, such as growth factor receptors, are important in mediating the physiological properties of these proteins. Similarly, we recently found that interactions between LacCer-enriched membrane microdomains and CD11b/CD18 (Mac-1, CR3, or αMβ2-integrin) are significant for neutrophil phagocytosis of non-opsonized microorganisms. This review describes the functional role of LacCer-enriched membrane microdomains and their interactions with CD11b/CD18.

GPS 2.1: enhanced prediction of kinase-specific phosphorylation sites with an algorithm of motif length selection.

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Xue, Yu; Liu, Zexian; Cao, Jun; Ma, Qian; Gao, Xinjiao; Wang, Qingqi; Jin, Changjiang; Zhou, Yanhong; Wen, Longping; Ren, Jian

2011-03-01

As the most important post-translational modification of proteins, phosphorylation plays essential roles in all aspects of biological processes. Besides experimental approaches, computational prediction of phosphorylated proteins with their kinase-specific phosphorylation sites has also emerged as a popular strategy, for its low-cost, fast-speed and convenience. In this work, we developed a kinase-specific phosphorylation sites predictor of GPS 2.1 (Group-based Prediction System), with a novel but simple approach of motif length selection (MLS). By this approach, the robustness of the prediction system was greatly improved. All algorithms in GPS old versions were also reserved and integrated in GPS 2.1. The online service and local packages of GPS 2.1 were implemented in JAVA 1.5 (J2SE 5.0) and freely available for academic researches at: http://gps.biocuckoo.org.

Thermal breeder fuel enrichment zoning

International Nuclear Information System (INIS)

Capossela, H.J.; Dwyer, J.R.; Luce, R.G.; McCoy, D.F.; Merriman, F.C.

1992-01-01

A method and apparatus for improving the performance of a thermal breeder reactor having regions of higher than average moderator concentration are disclosed. The fuel modules of the reactor core contain at least two different types of fuel elements, a high enrichment fuel element and a low enrichment fuel element. The two types of fuel elements are arranged in the fuel module with the low enrichment fuel elements located between the high moderator regions and the high enrichment fuel elements. Preferably, shim rods made of a fertile material are provided in selective regions for controlling the reactivity of the reactor by movement of the shim rods into and out of the reactor core. The moderation of neutrons adjacent the high enrichment fuel elements is preferably minimized as by reducing the spacing of the high enrichment fuel elements and/or using a moderator having a reduced moderating effect. 1 figure

In vivo conditions to identify Prkci phosphorylation targets using the analog-sensitive kinase method in zebrafish.

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Elena Cibrián Uhalte

Full Text Available Protein kinase C iota is required for various cell biological processes including epithelial tissue polarity and organ morphogenesis. To gain mechanistic insight into different roles of this kinase, it is essential to identify specific substrate proteins in their cellular context. The analog-sensitive kinase method provides a powerful tool for the identification of kinase substrates under in vivo conditions. However, it has remained a major challenge to establish screens based on this method in multicellular model organisms. Here, we report the methodology for in vivo conditions using the analog-sensitive kinase method in a genetically-tractable vertebrate model organism, the zebrafish. With this approach, kinase substrates can uniquely be labeled in the developing zebrafish embryo using bulky ATPγS analogs which results in the thiophosphorylation of substrates. The labeling of kinase substrates with a thiophosphoester epitope differs from phosphoesters that are generated by all other kinases and allows for an enrichment of thiophosphopeptides by immunoaffinity purification. This study provides the foundation for using the analog-sensitive kinase method in the context of complex vertebrate development, physiology, or disease.

Kinase impact assessment in the landscape of fusion genes that retain kinase domains: a pan-cancer study

Science.gov (United States)

Kim, Pora; Jia, Peilin; Zhao, Zhongming

2018-01-01

Abstract Assessing the impact of kinase in gene fusion is essential for both identifying driver fusion genes (FGs) and developing molecular targeted therapies. Kinase domain retention is a crucial factor in kinase fusion genes (KFGs), but such a systematic investigation has not been done yet. To this end, we analyzed kinase domain retention (KDR) status in chimeric protein sequences of 914 KFGs covering 312 kinases across 13 major cancer types. Based on 171 kinase domain-retained KFGs including 101 kinases, we studied their recurrence, kinase groups, fusion partners, exon-based expression depth, short DNA motifs around the break points and networks. Our results, such as more KDR than 5′-kinase fusion genes, combinatorial effects between 3′-KDR kinases and their 5′-partners and a signal transduction-specific DNA sequence motif in the break point intronic sequences, supported positive selection on 3′-kinase fusion genes in cancer. We introduced a degree-of-frequency (DoF) score to measure the possible number of KFGs of a kinase. Interestingly, kinases with high DoF scores tended to undergo strong gene expression alteration at the break points. Furthermore, our KDR gene fusion network analysis revealed six of the seven kinases with the highest DoF scores (ALK, BRAF, MET, NTRK1, NTRK3 and RET) were all observed in thyroid carcinoma. Finally, we summarized common features of ‘effective’ (highly recurrent) kinases in gene fusions such as expression alteration at break point, redundant usage in multiple cancer types and 3′-location tendency. Collectively, our findings are useful for prioritizing driver kinases and FGs and provided insights into KFGs’ clinical implications. PMID:28013235

Method of deuterium isotope separation and enrichment

International Nuclear Information System (INIS)

Benson, S.W.

1979-01-01

A method is described for separating and enriching deuterium containing molecules comprising the steps of: providing a source of organic molecules containing a normal abundance of deuterium atoms, the organic molecules having a structural formula RX, in which R is an organic radical selected from ethyl, isopropyl, t-butyl and 3-cyclopentenyl, and in which X is selected from F, Cl, Br and OH, and wherein R represents 3-cyclopentenyl, X may additionally represent H; exposing the molecules to the radiation of at least one pulsed infrared laser source which has been specifically tuned and focussed to selectively decompose RX molecules containing deuterium to form an enriched olefin specie containing deuterium, and HX; and separating the deuterium enriched olefin specie from the undecomposed deuterium depleted RX molecules and HX. (author)

Protein kinase C-related kinase 1 and 2 play an essential role in thromboxane-mediated neoplastic responses in prostate cancer

OpenAIRE

O'Sullivan, Aine G.; Mulvaney, Eamon P.; Hyland, Paula B.; Kinsella, B. Therese

2015-01-01

The prostanoid thromboxane (TX) A2 is increasingly implicated in neoplastic progression, including prostate cancer (PCa). Mechanistically, we recently identified protein kinase C-related kinase (PRK) 1 as a functional interactant of both the TP? and TP? isoforms of the human T prostanoid receptor (TP). The interaction with PRK1 was not only essential for TP?/TP?-induced PCa cell migration but also enabled the TXA2-TP axis to induce phosphorylation of histone H3 at Thr11 (H3Thr11), an epigenet...

Tyrosine kinase, aurora kinase and leucine aminopeptidase as attractive drug targets in anticancer therapy - characterisation of their inhibitors.

Science.gov (United States)

Ziemska, Joanna; Solecka, Jolanta

Cancers are the leading cause of deaths all over the world. Available anticancer agents used in clinics exhibit low therapeutic index and usually high toxicity. Wide spreading drug resistance of cancer cells induce a demanding need to search for new drug targets. Currently, many on-going studies on novel compounds with potent anticancer activity, high selectivity as well as new modes of action are conducted. In this work, we describe in details three enzyme groups, which are at present of extensive interest to medical researchers and pharmaceutical companies. These include receptor tyrosine kinases (e.g. EGFR enzymes) and non-receptor tyrosine kinases (Src enzymes), type A, B and C Aurora kinases and aminopeptidases, especially leucine aminopeptidase. We discuss classification of these enzymes, biochemistry as well as their role in the cell cycle under normal conditions and during cancerogenesis. Further on, the work describes enzyme inhibitors that are under in vitro, preclinical, clinical studies as well as drugs available on the market. Both, chemical structures of discovered inhibitors and the role of chemical moieties in novel drug design are discussed. Described enzymes play essential role in cell cycle, especially in mitosis (Aurora kinases), cell differentiation, growth and apoptosis (tyrosine kinases) as well as G1/S transition (leucine aminopeptidase). In cancer cells, they are overexpressed and only their inhibition may stop tumor progression. This review presents the clinical outcomes of selected inhibitors and argues the safety of drug usage in human volunteers. Clinical studies of EGFR and Src kinase inhibitors in different tumors clearly show the need for molecular selection of patients (to those with mutations in genes coding EGFR and Src) to achieve positive clinical response. Current data indicates the great necessity for new anticancer treatment and actions to limit off-target activity.

Identification of Phosphorylation Consensus Sequences and Endogenous Neuronal Substrates of the Psychiatric Risk Kinase TNIK.

Science.gov (United States)

Wang, Qi; Amato, Stephen P; Rubitski, David M; Hayward, Matthew M; Kormos, Bethany L; Verhoest, Patrick R; Xu, Lan; Brandon, Nicholas J; Ehlers, Michael D

2016-02-01

Traf2- and Nck-interacting kinase (TNIK) is a serine/threonine kinase highly expressed in the brain and enriched in the postsynaptic density of glutamatergic synapses in the mammalian brain. Accumulating genetic evidence and functional data have implicated TNIK as a risk factor for psychiatric disorders. However, the endogenous substrates of TNIK in neurons are unknown. Here, we describe a novel selective small molecule inhibitor of the TNIK kinase family. Using this inhibitor, we report the identification of endogenous neuronal TNIK substrates by immunoprecipitation with a phosphomotif antibody followed by mass spectrometry. Phosphorylation consensus sequences were defined by phosphopeptide sequence analysis. Among the identified substrates were members of the delta-catenin family including p120-catenin, δ-catenin, and armadillo repeat gene deleted in velo-cardio-facial syndrome (ARVCF), each of which is linked to psychiatric or neurologic disorders. Using p120-catenin as a representative substrate, we show TNIK-induced p120-catenin phosphorylation in cells requires intact kinase activity and phosphorylation of TNIK at T181 and T187 in the activation loop. Addition of the small molecule TNIK inhibitor or knocking down TNIK by two shRNAs reduced endogenous p120-catenin phosphorylation in cells. Together, using a TNIK inhibitor and phosphomotif antibody, we identify endogenous substrates of TNIK in neurons, define consensus sequences for TNIK, and suggest signaling pathways by which TNIK influences synaptic development and function linked to psychiatric and neurologic disorders. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

CYT387, a selective JAK1/JAK2 inhibitor: in vitro assessment of kinase selectivity and preclinical studies using cell lines and primary cells from polycythemia vera patients.

Science.gov (United States)

Pardanani, A; Lasho, T; Smith, G; Burns, C J; Fantino, E; Tefferi, A

2009-08-01

Somatic mutations in Janus kinase 2 (JAK2), including JAK2V617F, result in dysregulated JAK-signal transducer and activator transcription (STAT) signaling, which is implicated in myeloproliferative neoplasm (MPN) pathogenesis. CYT387 is an ATP-competitive small molecule that potently inhibits JAK1/JAK2 kinases (IC(50)=11 and 18 nM, respectively), with significantly less activity against other kinases, including JAK3 (IC(50)=155 nM). CYT387 inhibits growth of Ba/F3-JAK2V617F and human erythroleukemia (HEL) cells (IC(50) approximately 1500 nM) or Ba/F3-MPLW515L cells (IC(50)=200 nM), but has considerably less activity against BCR-ABL harboring K562 cells (IC=58 000 nM). Cell lines harboring mutated JAK2 alleles (CHRF-288-11 or Ba/F3-TEL-JAK2) were inhibited more potently than the corresponding pair harboring mutated JAK3 alleles (CMK or Ba/F3-TEL-JAK3), and STAT-5 phosphorylation was inhibited in HEL cells with an IC(50)=400 nM. Furthermore, CYT387 selectively suppressed the in vitro growth of erythroid colonies harboring JAK2V617F from polycythemia vera (PV) patients, an effect that was attenuated by exogenous erythropoietin. Overall, our data indicate that the JAK1/JAK2 selective inhibitor CYT387 has potential for efficacious treatment of MPN harboring mutated JAK2 and MPL alleles.

Hydrophilic Nb{sup 5+}-immobilized magnetic core–shell microsphere – A novel immobilized metal ion affinity chromatography material for highly selective enrichment of phosphopeptides

Energy Technology Data Exchange (ETDEWEB)

Sun, Xueni; Liu, Xiaodan; Feng, Jianan [Pharmaceutical Analysis Department, School of Pharmacy, Fudan University, Shanghai 201203 (China); Li, Yan, E-mail: yanli@fudan.edu.cn [Pharmaceutical Analysis Department, School of Pharmacy, Fudan University, Shanghai 201203 (China); Deng, Chunhui [Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433 (China); Duan, Gengli [Pharmaceutical Analysis Department, School of Pharmacy, Fudan University, Shanghai 201203 (China)

2015-06-23

Highlights: • A new IMAC material (Fe{sub 3}O{sub 4}@PD-Nb{sup 5+}) was synthesized. • The strong magnetic behaviors of the microspheres ensure fast and easy separation. • The enrichment ability was tested by human serum and nonfat milk. • The results were compared with other IMAC materials including the commercial kits. • All results proved the good enrichment ability, especially for multiphosphopeptides. - Abstract: Rapid and selective enrichment of phosphopeptides from complex biological samples is essential and challenging in phosphorylated proteomics. In this work, for the first time, niobium ions were directly immobilized on the surface of polydopamine-coated magnetic microspheres through a facile and effective synthetic route. The Fe{sub 3}O{sub 4}@polydopamine-Nb{sup 5+} (denoted as Fe{sub 3}O{sub 4}@PD-Nb{sup 5+}) microspheres possess merits of high hydrophilicity and good biological compatibility, and demonstrated low limit of detection (2 fmol). The selectivity was also basically satisfactory (β-casein:BSA = 1:500) to capture phosphopeptides. They were also successfully applied for enrichment of phosphopeptides from real biological samples such as human serum and nonfat milk. Compared with Fe{sub 3}O{sub 4}@PD-Ti{sup 4+} microspheres, the Fe{sub 3}O{sub 4}@PD-Nb{sup 5+} microspheres exhibit superior selectivity to multi-phosphorylated peptides, and thus may be complementary to the conventional IMAC materials.

A framework for classification of prokaryotic protein kinases.

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Nidhi Tyagi

Full Text Available BACKGROUND: Overwhelming majority of the Serine/Threonine protein kinases identified by gleaning archaeal and eubacterial genomes could not be classified into any of the well known Hanks and Hunter subfamilies of protein kinases. This is owing to the development of Hanks and Hunter classification scheme based on eukaryotic protein kinases which are highly divergent from their prokaryotic homologues. A large dataset of prokaryotic Serine/Threonine protein kinases recognized from genomes of prokaryotes have been used to develop a classification framework for prokaryotic Ser/Thr protein kinases. METHODOLOGY/PRINCIPAL FINDINGS: We have used traditional sequence alignment and phylogenetic approaches and clustered the prokaryotic kinases which represent 72 subfamilies with at least 4 members in each. Such a clustering enables classification of prokaryotic Ser/Thr kinases and it can be used as a framework to classify newly identified prokaryotic Ser/Thr kinases. After series of searches in a comprehensive sequence database we recognized that 38 subfamilies of prokaryotic protein kinases are associated to a specific taxonomic level. For example 4, 6 and 3 subfamilies have been identified that are currently specific to phylum proteobacteria, cyanobacteria and actinobacteria respectively. Similarly subfamilies which are specific to an order, sub-order, class, family and genus have also been identified. In addition to these, we also identify organism-diverse subfamilies. Members of these clusters are from organisms of different taxonomic levels, such as archaea, bacteria, eukaryotes and viruses. CONCLUSION/SIGNIFICANCE: Interestingly, occurrence of several taxonomic level specific subfamilies of prokaryotic kinases contrasts with classification of eukaryotic protein kinases in which most of the popular subfamilies of eukaryotic protein kinases occur diversely in several eukaryotes. Many prokaryotic Ser/Thr kinases exhibit a wide variety of modular

Metagenome enrichment approach used for selection of oil-degrading bacteria consortia for drill cutting residue bioremediation.

Science.gov (United States)

Guerra, Alaine B; Oliveira, Jorge S; Silva-Portela, Rita C B; Araújo, Wydemberg; Carlos, Aline C; Vasconcelos, Ana Tereza R; Freitas, Ana Teresa; Domingos, Yldeney Silva; de Farias, Mirna Ferreira; Fernandes, Glauber José Turolla; Agnez-Lima, Lucymara F

2018-04-01

Drill cuttings leave behind thousands of tons of residues without adequate treatment, generating a large environmental liability. Therefore knowledge about the microbial community of drilling residue may be useful for developing bioremediation strategies. In this work, samples of drilling residue were enriched in different culture media in the presence of petroleum, aiming to select potentially oil-degrading bacteria and biosurfactant producers. Total DNA was extracted directly from the drill cutting samples and from two enriched consortia and sequenced using the Ion Torrent platform. Taxonomic analysis revealed the predominance of Proteobacteria in the metagenome from the drill cuttings, while Firmicutes was enriched in consortia samples. Functional analysis using the Biosurfactants and Biodegradation Database (BioSurfDB) revealed a similar pattern among the three samples regarding hydrocarbon degradation and biosurfactants production pathways. However, some statistical differences were observed between samples. Namely, the pathways related to the degradation of fatty acids, chloroalkanes, and chloroalkanes were enriched in consortia samples. The degradation colorimetric assay using dichlorophenolindophenol as an indicator was positive for several hydrocarbon substrates. The consortia were also able to produce biosurfactants, with biosynthesis of iturin, lichnysin, and surfactin among the more abundant pathways. A microcosms assay followed by gas chromatography analysis showed the efficacy of the consortia in degrading alkanes, as we observed a reduction of around 66% and 30% for each consortium in total alkanes. These data suggest the potential use of these consortia in the bioremediation of drilling residue based on autochthonous bioaugmentation. Copyright © 2018 Elsevier Ltd. All rights reserved.

PSM/SH2-B distributes selected mitogenic receptor signals to distinct components in the PI3-kinase and MAP kinase signaling pathways.

Science.gov (United States)

Deng, Youping; Xu, Hu; Riedel, Heimo

2007-02-15

The Pro-rich, PH, and SH2 domain containing mitogenic signaling adapter PSM/SH2-B has been implicated as a cellular partner of various mitogenic receptor tyrosine kinases and related signaling mechanisms. Here, we report in a direct comparison of three peptide hormones, that PSM participates in the assembly of distinct mitogenic signaling complexes in response to insulin or IGF-I when compared to PDGF in cultured normal fibroblasts. The complex formed in response to insulin or IGF-I involves the respective peptide hormone receptor and presumably the established components leading to MAP kinase activation. However, our data suggest an alternative link from the PDGF receptor via PSM directly to MEK1/2 and consequently also to p44/42 activation, possibly through a scaffold protein. At least two PSM domains participate, the SH2 domain anticipated to link PSM to the respective receptor and the Pro-rich region in an association with an unidentified downstream component resulting in direct MEK1/2 and p44/42 regulation. The PDGF receptor signaling complex formed in response to PDGF involves PI 3-kinase in addition to the same components and interactions as described for insulin or IGF-I. PSM associates with PI 3-kinase via p85 and in addition the PSM PH domain participates in the regulation of PI 3-kinase activity, presumably through membrane interaction. In contrast, the PSM Pro-rich region appears to participate only in the MAP kinase signal. Both pathways contribute to the mitogenic response as shown by cell proliferation, survival, and focus formation. PSM regulates p38 MAP kinase activity in a pathway unrelated to the mitogenic response.

Enhanced production and isotope enrichment of recombinant glycoproteins produced in cultured mammalian cells

Energy Technology Data Exchange (ETDEWEB)

Skelton, David; Goodyear, Abbey [Florida State University, Department of Chemistry and Biochemistry (United States); Ni, DaQun; Walton, Wendy J.; Rolle, Myron; Hare, Joan T. [Florida State University, Institute of Molecular Biophysics (United States); Logan, Timothy M., E-mail: tlogan@fsu.ed [Florida State University, Department of Chemistry and Biochemistry (United States)

2010-10-15

NMR studies of post-translationally modified proteins are complicated by the lack of an efficient method to produce isotope enriched recombinant proteins in cultured mammalian cells. We show that reducing the glucose concentration and substituting glutamate for glutamine in serum-free medium increased cell viability while simultaneously increasing recombinant protein yield and the enrichment of non-essential amino acids compared to culture in unmodified, serum-free medium. Adding dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, further improves cell viability, recombinant protein yield, and isotope enrichment. We demonstrate the method by producing partially enriched recombinant Thy1 glycoprotein from Lec1 Chinese hamster ovary (CHO) cells using U-{sup 13}C-glucose and {sup 15}N-glutamate as labeled precursors. This study suggests that uniformly {sup 15}N,{sup 13}C-labeled recombinant proteins may be produced in cultured mammalian cells starting from a mixture of labeled essential amino acids, glucose, and glutamate.

Enhanced production and isotope enrichment of recombinant glycoproteins produced in cultured mammalian cells

International Nuclear Information System (INIS)

Skelton, David; Goodyear, Abbey; Ni, DaQun; Walton, Wendy J.; Rolle, Myron; Hare, Joan T.; Logan, Timothy M.

2010-01-01

NMR studies of post-translationally modified proteins are complicated by the lack of an efficient method to produce isotope enriched recombinant proteins in cultured mammalian cells. We show that reducing the glucose concentration and substituting glutamate for glutamine in serum-free medium increased cell viability while simultaneously increasing recombinant protein yield and the enrichment of non-essential amino acids compared to culture in unmodified, serum-free medium. Adding dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, further improves cell viability, recombinant protein yield, and isotope enrichment. We demonstrate the method by producing partially enriched recombinant Thy1 glycoprotein from Lec1 Chinese hamster ovary (CHO) cells using U- 13 C-glucose and 15 N-glutamate as labeled precursors. This study suggests that uniformly 15 N, 13 C-labeled recombinant proteins may be produced in cultured mammalian cells starting from a mixture of labeled essential amino acids, glucose, and glutamate.

Dendrite-free Li metal anode enabled by a 3D free-standing lithiophilic nitrogen-enriched carbon sponge

Science.gov (United States)

Hou, Guangmei; Ren, Xiaohua; Ma, Xiaoxin; Zhang, Le; Zhai, Wei; Ai, Qing; Xu, Xiaoyan; Zhang, Lin; Si, Pengchao; Feng, Jinkui; Ding, Fei; Ci, Lijie

2018-05-01

Lithium metal is considered as the ultimate anode material for high-energy Li battery systems. However, the commercial application of lithium anode is impeded by issues with safety and low coulombic efficiency induced by Li dendrite growth. Herein, a free-standing three-dimensional nitrogen-enriched graphitic carbon sponge with a high nitrogen content is proposed as a multifunctional current collect for Lithium accommodation. The abundant lithiophilic N-containing functional groups are served as preferred nucleation sites to guide a uniform Li deposition. In addition, the nitrogen-enriched graphitic carbon sponge with a high specific surface area can effectively reduce the local current density. As a result of the synergistic effect, the nitrogen-enriched graphitic carbon sponge electrode realizes a long-term stable cycling without dendrites formation. Notably, the as-obtained composite electrode can deliver an ultra-high specific capacity of ∼3175 mA h g-1. The nitrogen-enriched graphitic carbon sponge might provide innovative insights to design a superior matrix for dendrite-free Li anode.

Phosphorylation of the Transient Receptor Potential Ankyrin 1 by Cyclin-dependent Kinase 5 affects Chemo-nociception

OpenAIRE

Hall, Bradford E.; Prochazkova, Michaela; Sapio, Matthew R.; Minetos, Paul; Kurochkina, Natalya; Binukumar, B. K.; Amin, Niranjana D.; Terse, Anita; Joseph, John; Raithel, Stephen J.; Mannes, Andrew J.; Pant, Harish C.; Chung, Man-Kyo; Iadarola, Michael J.; Kulkarni, Ashok B.

2018-01-01

Cyclin-dependent kinase 5 (Cdk5) is a key neuronal kinase that is upregulated during inflammation, and can subsequently modulate sensitivity to nociceptive stimuli. We conducted an in silico screen for Cdk5 phosphorylation sites within proteins whose expression was enriched in nociceptors and identified the chemo-responsive ion channel Transient Receptor Potential Ankyrin 1 (TRPA1) as a possible Cdk5 substrate. Immunoprecipitated full length TRPA1 was shown to be phosphorylated by Cdk5 and th...

Stable isotope enrichment: Current and future potential

International Nuclear Information System (INIS)

Tracy, J.G.; Aaron, W.S.

1992-01-01

Oak Ridge National Laboratory (ORNL) operates the Isotope Enrichment Facility for the purpose of providing enriched stable isotopes, selected radioactive isotopes (including the actinides), and isotope-related materials and services for use in various research applications. ORNL is responsible for isotope enrichment and the distribution of approximately 225 nongaseous stable isotopes from 50 multi-isotopic elements. Many enriched isotope products are of prime importance in the fabrication of nuclear targets and the subsequent production of special radionuclides. State-of-the-art techniques to achieve special isotopic, chemical, and physical requirements are performed at ORNL This report describes the status and capabilities of the Isotope Enrichment Facility and the Isotope Research Materials Laboratory as well as emphasizing potential advancements in enrichment capabilities

Stable isotope enrichment - current and future potential

International Nuclear Information System (INIS)

Tracy, J.G.; Aaron, W.S.

1993-01-01

Oak Ridge National Laboratory (ORNL) operates the Isotope Enrichment Facility for the purpose of providing enriched stable isotopes, selected radioactive isotopes (including the actinides), and isotope-related materials and services for use in various research applications. ORNL is responsible for isotope enrichment and the distribution of approximately 225 nongaseous stable isotopes from 50 multi-isotopic elements. Many enriched isotope products are of prime importance in the fabrication of nuclear targets and the subsequent production of special radionuclides. State-of-the-art techniques to achieve special isotopic, chemical, and physical requirements are performed at ORNL. This report describes the status and capabilities of the Isotope Enrichment Facility and the Isotope Research Materials Laboratory as well as emphasizing potential advancements in enrichment capabilities. (orig.)

FAK dimerization controls its kinase-dependent functions at focal adhesions

KAUST Repository

Brami-Cherrier, Karen; Gervasi, Nicolas; Arsenieva, Diana A.; Walkiewicz, Katarzyna; Boutterin, Marie Claude; Ortega, Á lvaro Darí o; Leonard, Paul G.; Seantier, Bastien; Gasmi, Laï la; Bouceba, Tahar; Kadaré , Gress; Girault -, Jean Antoine; Arold, Stefan T.

2014-01-01

Focal adhesion kinase (FAK) controls adhesion-dependent cell motility, survival, and proliferation. FAK has kinase-dependent and kinase-independent functions, both of which play major roles in embryogenesis and tumor invasiveness. The precise mechanisms of FAK activation are not known. Using x-ray crystallography, small angle x-ray scattering, and biochemical and functional analyses, we show that the key step for activation of FAK's kinase-dependent functions-autophosphorylation of tyrosine-397-requires site-specific dimerization of FAK. The dimers form via the association of the N-terminal FERM domain of FAK and are stabilized by an interaction between FERM and the C-terminal FAT domain. FAT binds to a basic motif on FERM that regulates co-activation and nuclear localization. FAK dimerization requires local enrichment, which occurs specifically at focal adhesions. Paxillin plays a dual role, by recruiting FAK to focal adhesions and by reinforcing the FAT:FERM interaction. Our results provide a structural and mechanistic framework to explain how FAK combines multiple stimuli into a site-specific function. The dimer interfaces we describe are promising targets for blocking FAK activation. © 2014 The Authors.

FAK dimerization controls its kinase-dependent functions at focal adhesions

KAUST Repository

Brami-Cherrier, Karen

2014-01-30

Focal adhesion kinase (FAK) controls adhesion-dependent cell motility, survival, and proliferation. FAK has kinase-dependent and kinase-independent functions, both of which play major roles in embryogenesis and tumor invasiveness. The precise mechanisms of FAK activation are not known. Using x-ray crystallography, small angle x-ray scattering, and biochemical and functional analyses, we show that the key step for activation of FAK\\'s kinase-dependent functions-autophosphorylation of tyrosine-397-requires site-specific dimerization of FAK. The dimers form via the association of the N-terminal FERM domain of FAK and are stabilized by an interaction between FERM and the C-terminal FAT domain. FAT binds to a basic motif on FERM that regulates co-activation and nuclear localization. FAK dimerization requires local enrichment, which occurs specifically at focal adhesions. Paxillin plays a dual role, by recruiting FAK to focal adhesions and by reinforcing the FAT:FERM interaction. Our results provide a structural and mechanistic framework to explain how FAK combines multiple stimuli into a site-specific function. The dimer interfaces we describe are promising targets for blocking FAK activation. © 2014 The Authors.

Fragment-based approaches to the discovery of kinase inhibitors.

Science.gov (United States)

Mortenson, Paul N; Berdini, Valerio; O'Reilly, Marc

2014-01-01

Protein kinases are one of the most important families of drug targets, and aberrant kinase activity has been linked to a large number of disease areas. Although eminently targetable using small molecules, kinases present a number of challenges as drug targets, not least obtaining selectivity across such a large and relatively closely related target family. Fragment-based drug discovery involves screening simple, low-molecular weight compounds to generate initial hits against a target. These hits are then optimized to more potent compounds via medicinal chemistry, usually facilitated by structural biology. Here, we will present a number of recent examples of fragment-based approaches to the discovery of kinase inhibitors, detailing the construction of fragment-screening libraries, the identification and validation of fragment hits, and their optimization into potent and selective lead compounds. The advantages of fragment-based methodologies will be discussed, along with some of the challenges associated with using this route. Finally, we will present a number of key lessons derived both from our own experience running fragment screens against kinases and from a large number of published studies.

Diacylglycerol kinase ζ regulates RhoA activation via a kinase-independent scaffolding mechanism

DEFF Research Database (Denmark)

Ard, Ryan; Mulatz, Kirk; Abramovici, Hanan

2012-01-01

, but the underlying mechanisms are unclear. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, selectively dissociates Rac1 by stimulating PAK1-mediated phosphorylation of RhoGDI on Ser-101/174. Similarly, phosphorylation of RhoGDI on Ser-34 by protein kinase Cα (PKCα......GDI and was required for efficient interaction of PKCα and RhoA. DGKζ-null fibroblasts had condensed F-actin bundles and altered focal adhesion distribution, indicative of aberrant RhoA signaling. Two targets of the RhoA effector ROCK showed reduced phosphorylation in DGKζ-null cells. Collectively our findings suggest...

Motif enrichment tool.

Science.gov (United States)

Blatti, Charles; Sinha, Saurabh

2014-07-01

The Motif Enrichment Tool (MET) provides an online interface that enables users to find major transcriptional regulators of their gene sets of interest. MET searches the appropriate regulatory region around each gene and identifies which transcription factor DNA-binding specificities (motifs) are statistically overrepresented. Motif enrichment analysis is currently available for many metazoan species including human, mouse, fruit fly, planaria and flowering plants. MET also leverages high-throughput experimental data such as ChIP-seq and DNase-seq from ENCODE and ModENCODE to identify the regulatory targets of a transcription factor with greater precision. The results from MET are produced in real time and are linked to a genome browser for easy follow-up analysis. Use of the web tool is free and open to all, and there is no login requirement. ADDRESS: http://veda.cs.uiuc.edu/MET/. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia.

Science.gov (United States)

Dong, Qian; Ernst, Sarah E; Ostedgaard, Lynda S; Shah, Viral S; Ver Heul, Amanda R; Welsh, Michael J; Randak, Christoph O

2015-05-29

The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl(-) channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Accelerating the design of molecularly imprinted nanocomposite membranes modified by Au@polyaniline for selective enrichment and separation of ibuprofen

Science.gov (United States)

Wu, Xiuling; Wu, Yilin; Dong, Hongjun; Zhao, Juan; Wang, Chen; Zhou, Shi; Lu, Jian; Yan, Yongsheng; Li, He

2018-01-01

A novel system for harvesting molecularly imprinted nanocomposite membranes (MINcMs) with Au-modified polyaniline (Au@polyaniline) nanocomposite structure was developed for selective enrichment and separation of ibuprofen. This unique nanocomposite structure obviously enhanced the adsorption capacity, perm-selectivity performance, and regeneration ability of MINcMs. The as-prepared MINcMs showed outstanding adsorption capacity (22.02 mg g-1) of ibuprofen, which was four times higher than that of non-imprinted nanocomposite membranes (NINcMs). Furthermore, the selectivity factor of MINcMs for ibuprofen reached up to 4.67 and the perm-selectivity factor β was about 8.74, which indicated MINcMs had a good selective separation performance of ibuprofen. We envision that this novel synthesis method will open a new direction to manipulation of molecularly imprinted membrane materials and provide a simple yet convenient way to selective separation of ibuprofen.

The Axl kinase domain in complex with a macrocyclic inhibitor offers first structural insights into an active TAM receptor kinase.

Science.gov (United States)

Gajiwala, Ketan S; Grodsky, Neil; Bolaños, Ben; Feng, Junli; Ferre, RoseAnn; Timofeevski, Sergei; Xu, Meirong; Murray, Brion W; Johnson, Ted W; Stewart, Al

2017-09-22

The receptor tyrosine kinase family consisting of Tyro3, Axl, and Mer (TAM) is one of the most recently identified receptor tyrosine kinase families. TAM receptors are up-regulated postnatally and maintained at high levels in adults. They all play an important role in immunity, but Axl has also been implicated in cancer and therefore is a target in the discovery and development of novel therapeutics. However, of the three members of the TAM family, the Axl kinase domain is the only one that has so far eluded structure determination. To this end, using differential scanning fluorimetry and hydrogen-deuterium exchange mass spectrometry, we show here that a lower stability and greater dynamic nature of the Axl kinase domain may account for its poor crystallizability. We present the first structural characterization of the Axl kinase domain in complex with a small-molecule macrocyclic inhibitor. The Axl crystal structure revealed two distinct conformational states of the enzyme, providing a first glimpse of what an active TAM receptor kinase may look like and suggesting a potential role for the juxtamembrane region in enzyme activity. We noted that the ATP/inhibitor-binding sites of the TAM members closely resemble each other, posing a challenge for the design of a selective inhibitor. We propose that the differences in the conformational dynamics among the TAM family members could potentially be exploited to achieve inhibitor selectivity for targeted receptors. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Site-selective 13C labeling of proteins using erythrose

International Nuclear Information System (INIS)

Weininger, Ulrich

2017-01-01

NMR-spectroscopy enables unique experimental studies on protein dynamics at atomic resolution. In order to obtain a full atom view on protein dynamics, and to study specific local processes like ring-flips, proton-transfer, or tautomerization, one has to perform studies on amino-acid side chains. A key requirement for these studies is site-selective labeling with 13 C and/or 1 H, which is achieved in the most general way by using site-selectively 13 C-enriched glucose (1- and 2- 13 C) as the carbon source in bacterial expression systems. Using this strategy, multiple sites in side chains, including aromatics, become site-selectively labeled and suitable for relaxation studies. Here we systematically investigate the use of site-selectively 13 C-enriched erythrose (1-, 2-, 3- and 4- 13 C) as a suitable precursor for 13 C labeled aromatic side chains. We quantify 13 C incorporation in nearly all sites in all 20 amino acids and compare the results to glucose based labeling. In general the erythrose approach results in more selective labeling. While there is only a minor gain for phenylalanine and tyrosine side-chains, the 13 C incorporation level for tryptophan is at least doubled. Additionally, the Phe ζ and Trp η2 positions become labeled. In the aliphatic side chains, labeling using erythrose yields isolated 13 C labels for certain positions, like Ile β and His β, making these sites suitable for dynamics studies. Using erythrose instead of glucose as a source for site-selective 13 C labeling enables unique or superior labeling for certain positions and is thereby expanding the toolbox for customized isotope labeling of amino-acid side-chains.

Selective Cyclin-Dependent Kinase Inhibitors Discriminating between Cell Cycle and Transcriptional Kinases Future Reality or Utopia?

Czech Academy of Sciences Publication Activity Database

Wesierska-Gadek, J.; Kryštof, Vladimír

2009-01-01

Roč. 1171, - (2009), s. 228-241 ISSN 0077-8923 R&D Projects: GA ČR GA204/08/0511 Institutional research plan: CEZ:AV0Z50380511 Keywords : cell cycle * CYC202 * cyclin-dependent kinase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.670, year: 2009

The rational design of a novel potent analogue of the 5’-AMP-activated protein kinase inhibitor compound C with improved selectivity and cellular activity

Science.gov (United States)

Machrouhi, Fouzia; Ouhamou, Nouara; Laderoute, Keith; Calaoagan, Joy; Bukhtiyarova, Marina; Ehrlich, Paula J.; Klon, Anthony E.

2010-01-01

We have designed and synthesized analogues of compound C, a non-specific inhibitor of 5’-AMP-activated protein kinase (AMPK), using a computational fragment-based drug design (FBDD) approach. Synthesizing only twenty-seven analogues yielded a compound that was equipotent to compound C in the inhibition of the human AMPK (hAMPK) α2 subunit in the heterotrimeric complex in vitro, exhibited significantly improved selectivity against a subset of relevant kinases, and demonstrated enhanced cellular inhibition of AMPK. PMID:20932747

The 'retro-design' concept for novel kinase inhibitors.

Science.gov (United States)

Müller, Gerhard; Sennhenn, Peter C; Woodcock, Timothy; Neumann, Lars

2010-07-01

Protein kinases are among the most attractive therapeutic targets for a broad range of diseases. This feature review highlights and classifies the main design principles employed to generate active and selective kinase inhibitors. In particular, emphasis is focused on a fragment-based lead-generation approach, which constitutes a novel design method for developing type II kinase inhibitors with distinct binding kinetic attributes. This 'retro-design' strategy relies on a customized fragment library, and contrasts the traditional approach used in the design of type II inhibitors.

A comprehensive target selectivity survey of the BCR-ABL kinase inhibitor INNO-406 by kinase profiling and chemical proteomics in chronic myeloid leukemia cells.

Science.gov (United States)

Rix, U; Remsing Rix, L L; Terker, A S; Fernbach, N V; Hantschel, O; Planyavsky, M; Breitwieser, F P; Herrmann, H; Colinge, J; Bennett, K L; Augustin, M; Till, J H; Heinrich, M C; Valent, P; Superti-Furga, G

2010-01-01

Resistance to the BCR-ABL tyrosine kinase inhibitor imatinib poses a pressing challenge in treating chronic myeloid leukemia (CML). This resistance is often caused by point mutations in the ABL kinase domain or by overexpression of LYN. The second-generation BCR-ABL inhibitor INNO-406 is known to inhibit most BCR-ABL mutants and LYN efficiently. Knowledge of its full target spectrum would provide the molecular basis for potential side effects or suggest novel therapeutic applications and possible combination therapies. We have performed an unbiased chemical proteomics native target profile of INNO-406 in CML cells combined with functional assays using 272 recombinant kinases thereby identifying several new INNO-406 targets. These include the kinases ZAK, DDR1/2 and various ephrin receptors. The oxidoreductase NQO2, inhibited by both imatinib and nilotinib, is not a relevant target of INNO-406. Overall, INNO-406 has an improved activity over imatinib but a slightly broader target profile than both imatinib and nilotinib. In contrast to dasatinib and bosutinib, INNO-406 does not inhibit all SRC kinases and most TEC family kinases and is therefore expected to elicit fewer side effects. Altogether, these properties may make INNO-406 a valuable component in the drug arsenal against CML.

Neuron membrane trafficking and protein kinases involved in autism and ADHD.

Science.gov (United States)

Kitagishi, Yasuko; Minami, Akari; Nakanishi, Atsuko; Ogura, Yasunori; Matsuda, Satoru

2015-01-30

A brain-enriched multi-domain scaffolding protein, neurobeachin has been identified as a candidate gene for autism patients. Mutations in the synaptic adhesion protein cell adhesion molecule 1 (CADM1) are also associated with autism spectrum disorder, a neurodevelopmental disorder of uncertain molecular origin. Potential roles of neurobeachin and CADM1 have been suggested to a function of vesicle transport in endosomal trafficking. It seems that protein kinase B (AKT) and cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) have key roles in the neuron membrane trafficking involved in the pathogenesis of autism. Attention deficit hyperactivity disorder (ADHD) is documented to dopaminergic insufficiencies, which is attributed to synaptic dysfunction of dopamine transporter (DAT). AKT is also essential for the DAT cell-surface redistribution. In the present paper, we summarize and discuss the importance of several protein kinases that regulate the membrane trafficking involved in autism and ADHD, suggesting new targets for therapeutic intervention.

Neuron Membrane Trafficking and Protein Kinases Involved in Autism and ADHD

Directory of Open Access Journals (Sweden)

Yasuko Kitagishi

2015-01-01

Full Text Available A brain-enriched multi-domain scaffolding protein, neurobeachin has been identified as a candidate gene for autism patients. Mutations in the synaptic adhesion protein cell adhesion molecule 1 (CADM1 are also associated with autism spectrum disorder, a neurodevelopmental disorder of uncertain molecular origin. Potential roles of neurobeachin and CADM1 have been suggested to a function of vesicle transport in endosomal trafficking. It seems that protein kinase B (AKT and cyclic adenosine monophosphate (cAMP-dependent protein kinase A (PKA have key roles in the neuron membrane trafficking involved in the pathogenesis of autism. Attention deficit hyperactivity disorder (ADHD is documented to dopaminergic insufficiencies, which is attributed to synaptic dysfunction of dopamine transporter (DAT. AKT is also essential for the DAT cell-surface redistribution. In the present paper, we summarize and discuss the importance of several protein kinases that regulate the membrane trafficking involved in autism and ADHD, suggesting new targets for therapeutic intervention.

The Phosphoinositide 3-Kinaseα Selective Inhibitor, BYL719, Enhances the Effect of the Protein Kinase C Inhibitor, AEB071, in GNAQ/GNA11 Mutant Uveal Melanoma Cells

Science.gov (United States)

Musi, Elgilda; Ambrosini, Grazia; de Stanchina, Elisa; Schwartz, Gary K.

2014-01-01

G-protein mutations are one of the most common mutations occurring in uveal melanoma activating the protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-Kinase (PI3K)/AKT pathways. In this study, we described the effect of dual pathway inhibition in uveal melanoma harboring GNAQ and GNA11 mutations via PKC inhibition with AEB071 (Sotrastaurin) and PI3k/AKT inhibition with BYL719, a selective PI3Kα inhibitor. Growth inhibition was observed in GNAQ/GNA11 mutant cells with AEB071 versus no activity in WT cells. In the GNAQ-mutant cells, AEB071 decreased phosphorylation of MARCKS, a substrate of PKC, along with ERK1/2 and ribosomal S6, but persistent AKT activation was present. BYL719 had minimal anti-proliferative activity in all uveal melanoma cell lines, and inhibited phosphorylation of AKT in most cell lines. In the GNA11 mutant cell line, similar effects were observed with ERK1/2 inhibition, mostly inhibited by BYL719. With the combination treatment, both GNAQ and GNA11 mutant cell lines showed synergistic inhibition of cell proliferation and apoptotic cell death. In vivo studies correlated with in vitro findings showing reduced xenograft tumor growth with the combination therapy in a GNAQ mutant model. These findings suggest a new therapy treatment option for G-protein mutant uveal melanoma with a focus on specific targeting of multiple downstream pathways as part of combination therapy. PMID:24563540

Comparação da eficiência dos caldos de enriquecimento seletivo no isolamento de Salmonella Dublin Comparison of the efficiency of selective enrichment broths for Salmonella Dublin isolation

Directory of Open Access Journals (Sweden)

D.G. Silva

2008-06-01

Full Text Available The aim of this study was to compare three different selective enrichment broths: Rappaport-Vassiliadis (RV, selenite cystine (SC and Muller-Kauffmann tetrathionate (MKT for Salmonella Dublin isolation from faecal samples of calf experimentally infected. The bacteriological procedure involved pre-enrichment stages in Hajna-GN broth (only for the samples inoculated in RV broth, selective enrichment, culture in modified brilliant green agar (BGA, presumptive biochemistry tests (using triple-sugar-iron agar and lysine-agar and slide agglutination test with poli-O and poli-H Salmonella antiserum. The effects of enrichment temperatures using RV broth were also evaluated (37ºC and 42ºC. SC broth was significantly more efficient in the isolation of Salmonella Dublin (P<0,05, whereas RV broth incubated at 42ºC had a lower efficiency in the microbiological isolation.

Parkinson-Related LRRK2 Mutation R1628P Enables Cdk5 Phosphorylation of LRRK2 and Upregulates Its Kinase Activity.

Directory of Open Access Journals (Sweden)

Yang Shu

Full Text Available Recent studies have linked certain single nucleotide polymorphisms in the leucine-rich repeat kinase 2 (LRRK2 gene with Parkinson's disease (PD. Among the mutations, LRRK2 c.4883G>C (R1628P variant was identified to have a significant association with the risk of PD in ethnic Han-Chinese populations. But the molecular pathological mechanisms of R1628P mutation in PD is still unknown.Unlike other LRRK2 mutants in the Roc-COR-Kinase domain, the R1628P mutation didn't alter the LRRK2 kinase activity and promote neuronal death directly. LRRK2 R1628P mutation increased the binding affinity of LRRK2 with Cyclin-dependent kinase 5 (Cdk5. Interestingly, R1628P mutation turned its adjacent amino acid residue S1627 on LRRK2 protein to a novel phosphorylation site of Cdk5, which could be defined as a typical type II (+ phosphorylation-related single nucleotide polymorphism. Importantly, we showed that the phosphorylation of S1627 by Cdk5 could activate the LRRK2 kinase, and neurons ectopically expressing R1628P displayed a higher sensitivity to 1-methyl-4-phenylpyridinium, a bioactive metabolite of environmental toxin MPTP, in a Cdk5-dependent manner.Our data indicate that Parkinson-related LRRK2 mutation R1628P leads to Cdk5 phosphorylation of LRRK2 at S1627, which would upregulate the kinase activity of LRRK2 and consequently cause neuronal death.

Cocoa Enriched Diets Enhance Expression of Phosphatases and Decrease Expression of Inflammatory Molecules in Trigeminal Ganglion Neurons

Science.gov (United States)

Cady, Ryan J.; Durham, Paul L.

2010-01-01

Activation of trigeminal nerves and release of neuropeptides that promote inflammation are implicated in the underlying pathology of migraine and temporomandibular joint (TMJ) disorders. The overall response of trigeminal nerves to peripheral inflammatory stimuli involves a balance between enzymes that promote inflammation, kinases, and those that restore homeostasis, phosphatases. The goal of this study was to determine the effects of a cocoa-enriched diet on the expression of key inflammatory proteins in trigeminal ganglion neurons under basal and inflammatory conditions. Rats were fed a control diet or an isocaloric diet enriched in cocoa for 14 days prior to an injection of noxious stimuli to cause acute or chronic excitation of trigeminal neurons. In animals fed a cocoa-enriched diet, basal levels of the mitogen-activated kinase (MAP) phosphatases MKP-1 and MKP-3 were elevated in neurons. Importantly, the stimulatory effects of acute or chronic peripheral inflammation on neuronal expression of the MAPK p38 and extracellular signal-regulated kinases (ERK) were significantly repressed in response to cocoa. Similarly, dietary cocoa significantly suppressed basal neuronal expression of calcitonin gene-related peptide (CGRP) as well as stimulated levels of the inducible form of nitric oxide synthase (iNOS), proteins implicated in the underlying pathology of migraine and TMJ disorders. To our knowledge, this is first evidence that a dietary supplement can cause upregulation of MKP, and that cocoa can prevent inflammatory responses in trigeminal ganglion neurons. Furthermore, our data provide evidence that cocoa contains biologically active compounds that would be beneficial in the treatment of migraine and TMJ disorders. PMID:20138852

Improvement of mammalian cell culture performance through surfactant enabled concentrated feed media.

Science.gov (United States)

Hossler, Patrick; McDermott, Sean; Racicot, Christopher; Fann, John C H

2013-01-01

The design of basal and feed media in mammalian cell culture is paramount towards ensuring acceptable upstream process performance in various operation modes, especially fed-batch culture. Mammalian cell culture media designs have evolved from the classical formulations designed by Eagle and Ham, to today's formulations designed from continuous improvement and statistical frameworks. Feed media is especially important for ensuring robust cell growth, productivity, and ensuring the product quality of recombinant therapeutics are within acceptable ranges. Numerous studies have highlighted the benefit of various media designs, supplements, and feed addition strategies towards the resulting cell culture process. In this work we highlight the use of a top-down level approach towards feed media design enabled by the use of select surfactants for the targeted enrichment of a chemically defined feed media. The use of the enriched media was able to improve product titers at g/L levels, without adversely impacting the growth of multiple Chinese Hamster Ovary cell lines or the product quality of multiple recombinant antibodies. © 2013 American Institute of Chemical Engineers.

Multiplex Real-Time PCR for Detection of Staphylococcus aureus, mecA and Panton-Valentine Leukocidin (PVL) Genes from Selective Enrichments from Animals and Retail Meat

Science.gov (United States)

Velasco, Valeria; Sherwood, Julie S.; Rojas-García, Pedro P.; Logue, Catherine M.

2014-01-01

The aim of this study was to compare a real-time PCR assay, with a conventional culture/PCR method, to detect S. aureus, mecA and Panton-Valentine Leukocidin (PVL) genes in animals and retail meat, using a two-step selective enrichment protocol. A total of 234 samples were examined (77 animal nasal swabs, 112 retail raw meat, and 45 deli meat). The multiplex real-time PCR targeted the genes: nuc (identification of S. aureus), mecA (associated with methicillin resistance) and PVL (virulence factor), and the primary and secondary enrichment samples were assessed. The conventional culture/PCR method included the two-step selective enrichment, selective plating, biochemical testing, and multiplex PCR for confirmation. The conventional culture/PCR method recovered 95/234 positive S. aureus samples. Application of real-time PCR on samples following primary and secondary enrichment detected S. aureus in 111/234 and 120/234 samples respectively. For detection of S. aureus, the kappa statistic was 0.68–0.88 (from substantial to almost perfect agreement) and 0.29–0.77 (from fair to substantial agreement) for primary and secondary enrichments, using real-time PCR. For detection of mecA gene, the kappa statistic was 0–0.49 (from no agreement beyond that expected by chance to moderate agreement) for primary and secondary enrichment samples. Two pork samples were mecA gene positive by all methods. The real-time PCR assay detected the mecA gene in samples that were negative for S. aureus, but positive for Staphylococcus spp. The PVL gene was not detected in any sample by the conventional culture/PCR method or the real-time PCR assay. Among S. aureus isolated by conventional culture/PCR method, the sequence type ST398, and multi-drug resistant strains were found in animals and raw meat samples. The real-time PCR assay may be recommended as a rapid method for detection of S. aureus and the mecA gene, with further confirmation of methicillin-resistant S. aureus (MRSA) using

Multiplex real-time PCR for detection of Staphylococcus aureus, mecA and Panton-Valentine Leukocidin (PVL genes from selective enrichments from animals and retail meat.

Directory of Open Access Journals (Sweden)

Valeria Velasco

Full Text Available The aim of this study was to compare a real-time PCR assay, with a conventional culture/PCR method, to detect S. aureus, mecA and Panton-Valentine Leukocidin (PVL genes in animals and retail meat, using a two-step selective enrichment protocol. A total of 234 samples were examined (77 animal nasal swabs, 112 retail raw meat, and 45 deli meat. The multiplex real-time PCR targeted the genes: nuc (identification of S. aureus, mecA (associated with methicillin resistance and PVL (virulence factor, and the primary and secondary enrichment samples were assessed. The conventional culture/PCR method included the two-step selective enrichment, selective plating, biochemical testing, and multiplex PCR for confirmation. The conventional culture/PCR method recovered 95/234 positive S. aureus samples. Application of real-time PCR on samples following primary and secondary enrichment detected S. aureus in 111/234 and 120/234 samples respectively. For detection of S. aureus, the kappa statistic was 0.68-0.88 (from substantial to almost perfect agreement and 0.29-0.77 (from fair to substantial agreement for primary and secondary enrichments, using real-time PCR. For detection of mecA gene, the kappa statistic was 0-0.49 (from no agreement beyond that expected by chance to moderate agreement for primary and secondary enrichment samples. Two pork samples were mecA gene positive by all methods. The real-time PCR assay detected the mecA gene in samples that were negative for S. aureus, but positive for Staphylococcus spp. The PVL gene was not detected in any sample by the conventional culture/PCR method or the real-time PCR assay. Among S. aureus isolated by conventional culture/PCR method, the sequence type ST398, and multi-drug resistant strains were found in animals and raw meat samples. The real-time PCR assay may be recommended as a rapid method for detection of S. aureus and the mecA gene, with further confirmation of methicillin-resistant S. aureus (MRSA

Selective Audiovisual Semantic Integration Enabled by Feature-Selective Attention.

Science.gov (United States)

Li, Yuanqing; Long, Jinyi; Huang, Biao; Yu, Tianyou; Wu, Wei; Li, Peijun; Fang, Fang; Sun, Pei

2016-01-13

An audiovisual object may contain multiple semantic features, such as the gender and emotional features of the speaker. Feature-selective attention and audiovisual semantic integration are two brain functions involved in the recognition of audiovisual objects. Humans often selectively attend to one or several features while ignoring the other features of an audiovisual object. Meanwhile, the human brain integrates semantic information from the visual and auditory modalities. However, how these two brain functions correlate with each other remains to be elucidated. In this functional magnetic resonance imaging (fMRI) study, we explored the neural mechanism by which feature-selective attention modulates audiovisual semantic integration. During the fMRI experiment, the subjects were presented with visual-only, auditory-only, or audiovisual dynamical facial stimuli and performed several feature-selective attention tasks. Our results revealed that a distribution of areas, including heteromodal areas and brain areas encoding attended features, may be involved in audiovisual semantic integration. Through feature-selective attention, the human brain may selectively integrate audiovisual semantic information from attended features by enhancing functional connectivity and thus regulating information flows from heteromodal areas to brain areas encoding the attended features.

Quantitative and Dynamic Imaging of ATM Kinase Activity.

Science.gov (United States)

Nyati, Shyam; Young, Grant; Ross, Brian Dale; Rehemtulla, Alnawaz

2017-01-01

Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including DNA double-strand breaks (DSBs). ATM activation results in the initiation of a complex cascade of events facilitating DNA damage repair, cell cycle checkpoint control, and survival. Traditionally, protein kinases have been analyzed in vitro using biochemical methods (kinase assays using purified proteins or immunological assays) requiring a large number of cells and cell lysis. Genetically encoded biosensors based on optical molecular imaging such as fluorescence or bioluminescence have been developed to enable interrogation of kinase activities in live cells with a high signal to background. We have genetically engineered a hybrid protein whose bioluminescent activity is dependent on the ATM-mediated phosphorylation of a substrate. The engineered protein consists of the split luciferase-based protein complementation pair with a CHK2 (a substrate for ATM kinase activity) target sequence and a phospho-serine/threonine-binding domain, FHA2, derived from yeast Rad53. Phosphorylation of the serine residue within the target sequence by ATM would lead to its interaction with the phospho-serine-binding domain, thereby preventing complementation of the split luciferase pair and loss of reporter activity. Bioluminescence imaging of reporter expressing cells in cultured plates or as mouse xenografts provides a quantitative surrogate for ATM kinase activity and therefore the cellular DNA damage response in a noninvasive, dynamic fashion.

Practical enrichment technique for 33S (34S)

International Nuclear Information System (INIS)

McInteer, B.B.; Lyman, J.L.; Nilsson, A.C.; Quigley, G.P.

1982-01-01

The successful preparation of a macroscopic sample of enriched 33 S by laser-induced molecular dissociation is reported. Approach was to induce isotopically selective dissociation of SF 6 with CO 2 -laser pulses and to separate the remaining SF 6 from the sulfur-containing reaction products by cryogenic distillation. A 200 Hz, 0.75 J/pulse laser was used for photolysis of low-pressure (less than 1 torr) gas mixtures. The mixture of SF 6 and scavenger recirculated continuously throughout the irradiation chamber where the laser pulses selectively dissociated 32 SF 6 to give the final products: SF 4 or SOF 2 . The unreacted SF 6 was enriched in the heavier isotopes: 33 S, 34 S, and 36 S. A 1.3-g sample of SF 6 was collected with a 33 S enrichment factor of 1.96 and a 34 S enrichment factor of 2.25. A similar size sample of depleted ( 32 S) sulfur compounds was also collected. A scavenger was necessary to ensure high yield, and moist hydrogen was found to be best for our conditions. Removal of hydrogen fluoride was also necessary to prevent severe corrosion and to maintain high isotopic selectivity. 6 figures

Contribution of PIP-5 kinase Iα to raft-based FcγRIIA signaling

International Nuclear Information System (INIS)

Szymanska, Ewelina; Korzeniowski, Marek; Raynal, Patrick; Sobota, Andrzej; Kwiatkowska, Katarzyna

2009-01-01

Receptor FcγIIA (FcγRIIA) associates with plasma membrane rafts upon activation to trigger signaling cascades leading to actin polymerization. We examined whether compartmentalization of PI(4,5)P 2 and PI(4,5)P 2 -synthesizing PIP5-kinase Iα to rafts contributes to FcγRIIA signaling. A fraction of PIP5-kinase Iα was detected in raft-originating detergent-resistant membranes (DRM) isolated from U937 monocytes and other cells. The DRM of U937 monocytes contained also a major fraction of PI(4,5)P 2 . PIP5-kinase Iα bound PI(4,5)P 2 , and depletion of the lipid displaced PIP5-kinase Iα from the DRM. Activation of FcγRIIA in BHK transfectants led to recruitment of the kinase to the plasma membrane and enrichment of DRM in PI(4,5)P 2 . Immunofluorescence studies revealed that in resting cells the kinase was associated with the plasma membrane, cytoplasmic vesicles and the nucleus. After FcγRIIA activation, PIP5-kinase Iα and PI(4,5)P 2 co-localized transiently with the activated receptor at distinct cellular locations. Immunoelectron microscopy studies revealed that PIP5-kinase Iα and PI(4,5)P 2 were present at the edges of electron-dense assemblies containing activated FcγRIIA in their core. The data suggest that activation of FcγRIIA leads to membrane rafts coalescing into signaling platforms containing PIP5-kinase Iα and PI(4,5)P 2

Computational methods for analysis and inference of kinase/inhibitor relationships

Directory of Open Access Journals (Sweden)

Fabrizio eFerrè

2014-06-01

Full Text Available The central role of kinases in virtually all signal transduction networks is the driving motivation for the development of compounds modulating their activity. ATP-mimetic inhibitors are essential tools for elucidating signaling pathways and are emerging as promising therapeutic agents. However, off-target ligand binding and complex and sometimes unexpected kinase/inhibitor relationships can occur for seemingly unrelated kinases, stressing that computational approaches are needed for learning the interaction determinants and for the inference of the effect of small compounds on a given kinase. Recently published high-throughput profiling studies assessed the effects of thousands of small compound inhibitors, covering a substantial portion of the kinome. This wealth of data paved the road for computational resources and methods that can offer a major contribution in understanding the reasons of the inhibition, helping in the rational design of more specific molecules, in the in silico prediction of inhibition for those neglected kinases for which no systematic analysis has been carried yet, in the selection of novel inhibitors with desired selectivity, and offering novel avenues of personalized therapies.

Zirconium-doped magnetic microspheres for the selective enrichment of cis-diol-containing ribonucleosides.

Science.gov (United States)

Fan, Hua; Chen, Peihong; Wang, Chaozhan; Wei, Yinmao

2016-05-27

Zirconium-doped magnetic microspheres (Zr-Fe3O4) for the selective enrichment of cis-diol-containing biomolecules were easily synthesized via a one-step hydrothermal method. Characterization of the microspheres revealed that zirconium was successfully doped into the lattice of Fe3O4 at a doping level of 4.0 at%. Zr-Fe3O4 possessed good magnetic properties and high specificity towards cis-diol molecules, as shown using 28 compounds. For ribonucleosides, the adsorbent not only has favorable anti-interferential abilities but also has a high adsorption capacity up to 159.4μmol/g. As an example of a real application, four ribonucleosides in urine were efficiently enriched and detected via magnetic solid-phase extraction coupled with high-performance liquid chromatography. Under the optimized extraction conditions, the detection limits were determined to be between 0.005 and 0.017μg/mL, and the linearities ranged from 0.02 to 5.00μg/mL (R≥0.996) for these analytes. The accuracy of the analytical method was examined by studying the relative recoveries of the analytes in real urine samples, with recoveries varying from 77.8% to 119.6% (RSDs<10.6%, n=6). The results indicate that Zr-Fe3O4 is a suitable adsorbent for the analysis of cis-diol-containing biomolecules in practical applications. Copyright © 2016 Elsevier B.V. All rights reserved.

Utilizing ion-pairing hydrophilic interaction chromatography solid phase extraction for efficient glycopeptide enrichment in glycoproteomics

DEFF Research Database (Denmark)

Mysling, Simon; Palmisano, Giuseppe; Højrup, Peter

2010-01-01

Glycopeptide enrichment is a prerequisite to enable structural characterization of protein glycosylation in glycoproteomics. Here we present an improved method for glycopeptide enrichment based on zwitter-ionic hydrophilic interaction chromatography solid phase extraction (ZIC-HILIC SPE...

Training versus engagement as paths to cognitive enrichment with aging.

Science.gov (United States)

Stine-Morrow, Elizabeth A L; Payne, Brennan R; Roberts, Brent W; Kramer, Arthur F; Morrow, Daniel G; Payne, Laura; Hill, Patrick L; Jackson, Joshua J; Gao, Xuefei; Noh, Soo Rim; Janke, Megan C; Parisi, Jeanine M

2014-12-01

While a training model of cognitive intervention targets the improvement of particular skills through instruction and practice, an engagement model is based on the idea that being embedded in an intellectually and socially complex environment can impact cognition, perhaps even broadly, without explicit instruction. We contrasted these 2 models of cognitive enrichment by randomly assigning healthy older adults to a home-based inductive reasoning training program, a team-based competitive program in creative problem solving, or a wait-list control. As predicted, those in the training condition showed selective improvement in inductive reasoning. Those in the engagement condition, on the other hand, showed selective improvement in divergent thinking, a key ability exercised in creative problem solving. On average, then, both groups appeared to show ability-specific effects. However, moderators of change differed somewhat for those in the engagement and training interventions. Generally, those who started either intervention with a more positive cognitive profile showed more cognitive growth, suggesting that cognitive resources enabled individuals to take advantage of environmental enrichment. Only in the engagement condition did initial levels of openness and social network size moderate intervention effects on cognition, suggesting that comfort with novelty and an ability to manage social resources may be additional factors contributing to the capacity to take advantage of the environmental complexity associated with engagement. Collectively, these findings suggest that training and engagement models may offer alternative routes to cognitive resilience in late life. (PsycINFO Database Record (c) 2014 APA, all rights reserved).

Selecting an optimal number of binding site waters to improve virtual screening enrichments against the adenosine A2A receptor.

Science.gov (United States)

Lenselink, Eelke B; Beuming, Thijs; Sherman, Woody; van Vlijmen, Herman W T; IJzerman, Adriaan P

2014-06-23

A major challenge in structure-based virtual screening (VS) involves the treatment of explicit water molecules during docking in order to improve the enrichment of active compounds over decoys. Here we have investigated this in the context of the adenosine A2A receptor, where water molecules have previously been shown to be important for achieving high enrichment rates with docking, and where the positions of some binding site waters are known from a high-resolution crystal structure. The effect of these waters (both their presence and orientations) on VS enrichment was assessed using a carefully curated set of 299 high affinity A2A antagonists and 17,337 decoys. We show that including certain crystal waters greatly improves VS enrichment and that optimization of water hydrogen positions is needed in order to achieve the best results. We also show that waters derived from a molecular dynamics simulation - without any knowledge of crystallographic waters - can improve enrichments to a similar degree as the crystallographic waters, which makes this strategy applicable to structures without experimental knowledge of water positions. Finally, we used decision trees to select an ensemble of structures with different water molecule positions and orientations that outperforms any single structure with water molecules. The approach presented here is validated against independent test sets of A2A receptor antagonists and decoys from the literature. In general, this water optimization strategy could be applied to any target with waters-mediated protein-ligand interactions.

Application of enriched stable isotopes as tracers in biological systems

DEFF Research Database (Denmark)

Stürup, Stefan; Hansen, Helle Rüsz; Gammelgaard, Bente

2008-01-01

The application of enriched stable isotopes of minerals and trace elements as tracers in biological systems is a rapidly growing research field that benefits from the many new developments in inorganic mass spectrometric instrumentation, primarily within inductively coupled plasma mass spectrometry...... (ICP-MS) instrumentation, such as reaction/collision cell ICP-MS and multicollector ICP-MS with improved isotope ratio measurement and interference removal capabilities. Adaptation and refinement of radioisotope tracer experiment methodologies for enriched stable isotope experiments......, and the development of new methodologies coupled with more advanced compartmental and mathematical models for the distribution of elements in living organisms has enabled a broader use of enriched stable isotope experiments in the biological sciences. This review discusses the current and future uses of enriched...

Environmental Enrichment Mitigates Deficits after Repetitive Mild Traumatic Brain Injury.

Science.gov (United States)

Liu, Xixia; Qiu, Jianhua; Alcon, Sasha; Hashim, Jumana; Meehan, William P; Mannix, Rebekah

2017-08-15

Although environmental enrichment has been shown to improve functional and histologic outcomes in pre-clinical moderate-to-severe traumatic brain injury (TBI), there are a paucity of pre-clinical data regarding enrichment strategies in the setting of repetitive mild traumatic brain injury (rmTBI). Given the vast numbers of athletes and those in the military who sustain rmTBI, the mounting evidence of the long-term and progressive sequelae of rmTBI, and the lack of targeted therapies to mitigate these sequelae, successful enrichment interventions in rmTBI could have large public health significance. Here, we evaluated enrichment strategies in an established pre-clinical rmTBI model. Seventy-one male C57BL/6 mice were randomized to two different housing conditions, environmental enrichment (EE) or normal condition (NC), then subjected to rmTBI injury (seven injuries in 9 days) or sham injury (anesthesia only). Functional outcomes in all four groups (NC-TBI, EE-TBI, NC-sham, and EE-sham) were assessed by motor, exploratory/anxiety, and mnemonic behavioral tests. At the synaptic level, N-methyl d-aspartate receptor (NMDAR) subunit expression of phosphorylated glutamate receptor 1 (GluR1), phosphorylated Ca 2+ /calmodulin-dependent protein kinase II (CaMKII), and calpain were evaluated by western blot. Compared to injured NC-TBI mice, EE-TBI mice had improved memory and decreased anxiety and exploratory activity post-injury. Treatment with enrichment also corresponded to normal NMDAR subunit expression, decreased GluR1 phosphorylation, decreased phosphorylated CaMKII, and normal calpain expression post-rmTBI. These data suggest that enrichment strategies may improve functional outcomes and mitigate synaptic changes post-rmTBI. Given that enrichment strategies are feasible in the clinical setting, particularly for athletes and soldiers for whom the risk of repetitive injury is greatest, these data suggest that clinical trials may be warranted.

Band 3 tyrosine kinase in avian erythrocyte plasma membrane is immunologically related to pp60c-src

International Nuclear Information System (INIS)

Hillsgrove, D.; Shores, C.G.; Parker, J.C.; Maness, P.F.

1987-01-01

The authors have identified in the plasma membrane of the chicken erythrocyte a 60-kDa tyrosine-specific protein kinase immunologically related to the transforming protein pp60 v-src of Rous sarcoma virus. The erythrocyte protein kinase phosphorylated heavy chains of tumor-bearing rabbit (TBR) antibodies reactive with pp60 c-src at tyrosine in immune complex protein kinase assays. The kinase was identified as a 60-kDa protein by [ 35 S]methionine labeling of erythrocytes and by autophosphorylation in immune complexes. The kinase migrated on two-dimensional gel electrophoresis with an apparent pI and molecular mass similar to pp60 c-src . A plasma membrane-enriched fraction isolated from chicken red cells contained the majority of the kinase activity. Incubation of the plasma membrane fraction with [ 32 P]ATP resulted in tyrosine phosphorylation of the anion transport protein band 3. Band 3 phosphorylation was blocked by TBR antibodies, indicting that the kinase recognized by pp60 c-src antibodies was responsible for band 3 phosphorylation. These results demonstrate that the avian erythrocyte plasma membrane contains a tightly bound tyrosine-specific protein kinase identical or closely related to pp60 c-src and that this kinase is responsible for band 3 phosphorylation in vitro

Intramolecular Crosstalk between Catalytic Activities of Receptor Kinases

KAUST Repository

Kwezi, Lusisizwe

2018-01-22

Signal modulation is important for the growth and development of plants and this process is mediated by a number of factors including physiological growth regulators and their associated signal transduction pathways. Protein kinases play a central role in signaling, including those involving pathogen response mechanisms. We previously demonstrated an active guanylate cyclase (GC) catalytic center in the brassinosteroid insensitive receptor (AtBRI1) within an active intracellular kinase domain resulting in dual enzymatic activity. Here we propose a novel type of receptor architecture that is characterized by a functional GC catalytic center nested in the cytosolic kinase domain enabling intramolecular crosstalk. This may be through a cGMP-AtBRI1 complex forming that may induce a negative feedback mechanism leading to desensitisation of the receptor, regulated through the cGMP production pathway. We further argue that the comparatively low but highly localized cGMP generated by the GC in response to a ligand is sufficient to modulate the kinase activity. This type of receptor therefore provides a molecular switch that directly and/or indirectly affects ligand dependent phosphorylation of downstream signaling cascades and suggests that subsequent signal transduction and modulation works in conjunction with the kinase in downstream signaling.

Intramolecular Crosstalk between Catalytic Activities of Receptor Kinases

KAUST Repository

Kwezi, Lusisizwe; Wheeler, Janet I; Marondedze, Claudius; Gehring, Christoph A; Irving, Helen R

2018-01-01

Signal modulation is important for the growth and development of plants and this process is mediated by a number of factors including physiological growth regulators and their associated signal transduction pathways. Protein kinases play a central role in signaling, including those involving pathogen response mechanisms. We previously demonstrated an active guanylate cyclase (GC) catalytic center in the brassinosteroid insensitive receptor (AtBRI1) within an active intracellular kinase domain resulting in dual enzymatic activity. Here we propose a novel type of receptor architecture that is characterized by a functional GC catalytic center nested in the cytosolic kinase domain enabling intramolecular crosstalk. This may be through a cGMP-AtBRI1 complex forming that may induce a negative feedback mechanism leading to desensitisation of the receptor, regulated through the cGMP production pathway. We further argue that the comparatively low but highly localized cGMP generated by the GC in response to a ligand is sufficient to modulate the kinase activity. This type of receptor therefore provides a molecular switch that directly and/or indirectly affects ligand dependent phosphorylation of downstream signaling cascades and suggests that subsequent signal transduction and modulation works in conjunction with the kinase in downstream signaling.

Kinase-dead ATM protein is highly oncogenic and can be preferentially targeted by Topo-isomerase I inhibitors.

Science.gov (United States)

Yamamoto, Kenta; Wang, Jiguang; Sprinzen, Lisa; Xu, Jun; Haddock, Christopher J; Li, Chen; Lee, Brian J; Loredan, Denis G; Jiang, Wenxia; Vindigni, Alessandro; Wang, Dong; Rabadan, Raul; Zha, Shan

2016-06-15

Missense mutations in ATM kinase, a master regulator of DNA damage responses, are found in many cancers, but their impact on ATM function and implications for cancer therapy are largely unknown. Here we report that 72% of cancer-associated ATM mutations are missense mutations that are enriched around the kinase domain. Expression of kinase-dead ATM (Atm(KD/-)) is more oncogenic than loss of ATM (Atm(-/-)) in mouse models, leading to earlier and more frequent lymphomas with Pten deletions. Kinase-dead ATM protein (Atm-KD), but not loss of ATM (Atm-null), prevents replication-dependent removal of Topo-isomerase I-DNA adducts at the step of strand cleavage, leading to severe genomic instability and hypersensitivity to Topo-isomerase I inhibitors. Correspondingly, Topo-isomerase I inhibitors effectively and preferentially eliminate Atm(KD/-), but not Atm-proficientor Atm(-/-) leukemia in animal models. These findings identify ATM kinase-domain missense mutations as a potent oncogenic event and a biomarker for Topo-isomerase I inhibitor based therapy.

An Elementary Overview of the Selection of Materials for Service in Oxygen-Enriched Environments

Science.gov (United States)

Davis, Samuel Eddie

2012-01-01

The process for selecting materials for use in oxygen or oxygen-enriched environments is one that continues to be investigated by many industries due to the importance to those industries of oxygen systems. There are several excellent resources available to assist oxygen systems design engineers and end-users, with the most comprehensive being ASTM MNL-36, Safe Use of Oxygen and Oxygen Systems: Handbook for Design, Operation and Maintenance, 2nd Edition. ASTM also makes available several standards for oxygen systems. However, the ASTM publications are extremely detailed, and typically designed for professionals who already possess a working knowledge of oxygen systems. No notable resource exists, whether an ASTM or other organizational publication, which can be used to educate engineers or technicians who have no prior knowledge of the nuances of oxygen system design and safety. This paper will fill the void for information needed by organizations that design or operate oxygen systems. The information in this paper is not new information, but is a concise and easily understood summary of selecting materials for oxygen systems. This paper will serve well as an employee s first introduction to oxygen system materials selection, and probably the employee s first introduction to ASTM.

Laser-induced photochemical enrichment of boron isotopes

International Nuclear Information System (INIS)

Freund, S.M.; Ritter, J.J.

1976-01-01

A boron trichloride starting material containing both boron-10 isotopes and boron-11 isotopes is selectively enriched in one or the other of these isotopes by a laser-induced photochemical method involving the reaction of laser-excited boron trichloride with either H 2 S or D 2 S. The method is carried out by subjecting a low pressure gaseous mixture of boron trichloride starting material and the sulfide to infrared radiation from a carbon dioxide TE laser. The wave length of the radiation is selected so as to selectively excite one or the other of boron-10 BCl 3 molecules or boron-11 BCl 3 molecules, thereby making them preferentially more reactive with the sulfide. The laser-induced reaction produces both a boron-containing solid phase reaction product and a gaseous phase containing mostly unreacted BCl 3 and small amounts of sulfhydroboranes. Pure boron trichloride selectively enriched in one of the isotopes is recovered as the primary product of the method from the gaseous phase by a multi-step recovery procedure. Pure boron trichloride enriched in the other isotope is recovered as a secondary product of the method by the subsequent chlorination of the solid phase reaction product followed by separation of BCl 3 from the mixture of gaseous products resulting from the chlorination

Hsp90 inhibition differentially destabilises MAP kinase and TGF-beta signalling components in cancer cells revealed by kinase-targeted chemoproteomics

International Nuclear Information System (INIS)

Haupt, Armin; Dahl, Andreas; Lappe, Michael; Lehrach, Hans; Gonzalez, Cayetano; Drewes, Gerard; Lange, Bodo MH; Joberty, Gerard; Bantscheff, Marcus; Fröhlich, Holger; Stehr, Henning; Schweiger, Michal R; Fischer, Axel; Kerick, Martin; Boerno, Stefan T

2012-01-01

The heat shock protein 90 (Hsp90) is required for the stability of many signalling kinases. As a target for cancer therapy it allows the simultaneous inhibition of several signalling pathways. However, its inhibition in healthy cells could also lead to severe side effects. This is the first comprehensive analysis of the response to Hsp90 inhibition at the kinome level. We quantitatively profiled the effects of Hsp90 inhibition by geldanamycin on the kinome of one primary (Hs68) and three tumour cell lines (SW480, U2OS, A549) by affinity proteomics based on immobilized broad spectrum kinase inhibitors ('kinobeads'). To identify affected pathways we used the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway classification. We combined Hsp90 and proteasome inhibition to identify Hsp90 substrates in Hs68 and SW480 cells. The mutational status of kinases from the used cell lines was determined using next-generation sequencing. A mutation of Hsp90 candidate client RIPK2 was mapped onto its structure. We measured relative abundances of > 140 protein kinases from the four cell lines in response to geldanamycin treatment and identified many new potential Hsp90 substrates. These kinases represent diverse families and cellular functions, with a strong representation of pathways involved in tumour progression like the BMP, MAPK and TGF-beta signalling cascades. Co-treatment with the proteasome inhibitor MG132 enabled us to classify 64 kinases as true Hsp90 clients. Finally, mutations in 7 kinases correlate with an altered response to Hsp90 inhibition. Structural modelling of the candidate client RIPK2 suggests an impact of the mutation on a proposed Hsp90 binding domain. We propose a high confidence list of Hsp90 kinase clients, which provides new opportunities for targeted and combinatorial cancer treatment and diagnostic applications

Enrichment of Biologically Active Compounds from Selected Plants Using Adsorptive Bubble Separation

OpenAIRE

Thompson, Leonor

2006-01-01

In this research, foam fractionation a method belonging to the Adsorptive Bubble Separation Methods, was employed to enrich the following active principles contained in medically valuable plants: Faradiol esters from Calendula officinalis, catechins from Camellia sinensis, tryptanthrin from Isatis Tinctoria and cannabinoids from Cannabis sativa. The enrichment methods have been developed in the batch mode for these principles aqueous dilute solutions, based on their physicochemical nature and...

Template-directed ligation of tethered mononucleotides by t4 DNA ligase for kinase ribozyme selection.

Directory of Open Access Journals (Sweden)

David G Nickens

Full Text Available BACKGROUND: In vitro selection of kinase ribozymes for small molecule metabolites, such as free nucleosides, will require partition systems that discriminate active from inactive RNA species. While nucleic acid catalysis of phosphoryl transfer is well established for phosphorylation of 5' or 2' OH of oligonucleotide substrates, phosphorylation of diffusible small molecules has not been demonstrated. METHODOLOGY/PRINCIPAL FINDINGS: This study demonstrates the ability of T4 DNA ligase to capture RNA strands in which a tethered monodeoxynucleoside has acquired a 5' phosphate. The ligation reaction therefore mimics the partition step of a selection for nucleoside kinase (deoxyribozymes. Ligation with tethered substrates was considerably slower than with nicked, fully duplex DNA, even though the deoxynucleotides at the ligation junction were Watson-Crick base paired in the tethered substrate. Ligation increased markedly when the bridging template strand contained unpaired spacer nucleotides across from the flexible tether, according to the trends: A(2>A(1>A(3>A(4>A(0>A(6>A(8>A(10 and T(2>T(3>T(4>T(6 approximately T(1>T(8>T(10. Bridging T's generally gave higher yield of ligated product than bridging A's. ATP concentrations above 33 microM accumulated adenylated intermediate and decreased yields of the gap-sealed product, likely due to re-adenylation of dissociated enzyme. Under optimized conditions, T4 DNA ligase efficiently (>90% joined a correctly paired, or TratioG wobble-paired, substrate on the 3' side of the ligation junction while discriminating approximately 100-fold against most mispaired substrates. Tethered dC and dG gave the highest ligation rates and yields, followed by tethered deoxyinosine (dI and dT, with the slowest reactions for tethered dA. The same kinetic trends were observed in ligase-mediated capture in complex reaction mixtures with multiple substrates. The "universal" analog 5-nitroindole (dNI did not support ligation when

Allosteric small-molecule kinase inhibitors

DEFF Research Database (Denmark)

Wu, Peng; Clausen, Mads Hartvig; Nielsen, Thomas E.

2015-01-01

current barriers of kinase inhibitors, including poor selectivity and emergence of drug resistance. In spite of the small number of identified allosteric inhibitors in comparison with that of inhibitors targeting the ATP pocket, encouraging results, such as the FDA-approval of the first small...

Engineering and Functional Analysis of Mitotic Kinases Through Chemical Genetics.

Science.gov (United States)

Jones, Mathew J K; Jallepalli, Prasad V

2016-01-01

During mitosis, multiple protein kinases transform the cytoskeleton and chromosomes into new and highly dynamic structures that mediate the faithful transmission of genetic information and cell division. However, the large number and strong conservation of mammalian kinases in general pose significant obstacles to interrogating them with small molecules, due to the difficulty in identifying and validating those which are truly selective. To overcome this problem, a steric complementation strategy has been developed, in which a bulky "gatekeeper" residue within the active site of the kinase of interest is replaced with a smaller amino acid, such as glycine or alanine. The enlarged catalytic pocket can then be targeted in an allele-specific manner with bulky purine analogs. This strategy provides a general framework for dissecting kinase function with high selectivity, rapid kinetics, and reversibility. In this chapter we discuss the principles and techniques needed to implement this chemical genetic approach in mammalian cells.

Determination of reliability of express forecasting evaluation of radiometric enriching ability of non-ferrous ores

International Nuclear Information System (INIS)

Kirpishchikov, S.P.

1991-01-01

Use of the data of nuclear physical methods of sampling and logging enables to improve reliability of evaluation of radiometric enriching ability of ores, as well as to evaluate quantitatively this reliability. This problem may be solved by using some concepts of geostatistics. The presented results enable to conclude, that the data of nuclear-physical methods of sampling and logging can provide high reliability of evaluation of radiometric enriching ability of non-ferrous ores and their geometrization by technological types

Protein kinase substrate identification on functional protein arrays

Directory of Open Access Journals (Sweden)

Zhou Fang

2008-02-01

important new tool that enables multiplex analysis of protein phosphorylation, and thus can be utilized to identify novel kinase substrates. Integrating this technology with a systems biology approach to cell signalling will help uncover new layers in our understanding of this essential class of enzymes.

OSI-930: a novel selective inhibitor of Kit and kinase insert domain receptor tyrosine kinases with antitumor activity in mouse xenograft models.

Science.gov (United States)

Garton, Andrew J; Crew, Andrew P A; Franklin, Maryland; Cooke, Andrew R; Wynne, Graham M; Castaldo, Linda; Kahler, Jennifer; Winski, Shannon L; Franks, April; Brown, Eric N; Bittner, Mark A; Keily, John F; Briner, Paul; Hidden, Chris; Srebernak, Mary C; Pirrit, Carrie; O'Connor, Matthew; Chan, Anna; Vulevic, Bojana; Henninger, Dwight; Hart, Karen; Sennello, Regina; Li, An-Hu; Zhang, Tao; Richardson, Frank; Emerson, David L; Castelhano, Arlindo L; Arnold, Lee D; Gibson, Neil W

2006-01-15

OSI-930 is a novel inhibitor of the receptor tyrosine kinases Kit and kinase insert domain receptor (KDR), which is currently being evaluated in clinical studies. OSI-930 selectively inhibits Kit and KDR with similar potency in intact cells and also inhibits these targets in vivo following oral dosing. We have investigated the relationships between the potency observed in cell-based assays in vitro, the plasma exposure levels achieved following oral dosing, the time course of target inhibition in vivo, and antitumor activity of OSI-930 in tumor xenograft models. In the mutant Kit-expressing HMC-1 xenograft model, prolonged inhibition of Kit was achieved at oral doses between 10 and 50 mg/kg and this dose range was associated with antitumor activity. Similarly, prolonged inhibition of wild-type Kit in the NCI-H526 xenograft model was observed at oral doses of 100 to 200 mg/kg, which was the dose level associated with significant antitumor activity in this model as well as in the majority of other xenograft models tested. The data suggest that antitumor activity of OSI-930 in mouse xenograft models is observed at dose levels that maintain a significant level of inhibition of the molecular targets of OSI-930 for a prolonged period. Furthermore, pharmacokinetic evaluation of the plasma exposure levels of OSI-930 at these effective dose levels provides an estimate of the target plasma concentrations that may be required to achieve prolonged inhibition of Kit and KDR in humans and which would therefore be expected to yield a therapeutic benefit in future clinical evaluations of OSI-930.

A genome scan for positive selection in thoroughbred horses.

Science.gov (United States)

Gu, Jingjing; Orr, Nick; Park, Stephen D; Katz, Lisa M; Sulimova, Galina; MacHugh, David E; Hill, Emmeline W

2009-06-02

Thoroughbred horses have been selected for exceptional racing performance resulting in system-wide structural and functional adaptations contributing to elite athletic phenotypes. Because selection has been recent and intense in a closed population that stems from a small number of founder animals Thoroughbreds represent a unique population within which to identify genomic contributions to exercise-related traits. Employing a population genetics-based hitchhiking mapping approach we performed a genome scan using 394 autosomal and X chromosome microsatellite loci and identified positively selected loci in the extreme tail-ends of the empirical distributions for (1) deviations from expected heterozygosity (Ewens-Watterson test) in Thoroughbred (n = 112) and (2) global differentiation among four geographically diverse horse populations (F(ST)). We found positively selected genomic regions in Thoroughbred enriched for phosphoinositide-mediated signalling (3.2-fold enrichment; PThoroughbred athletic phenotype. We report for the first time candidate athletic-performance genes within regions targeted by selection in Thoroughbred horses that are principally responsible for fatty acid oxidation, increased insulin sensitivity and muscle strength: ACSS1 (acyl-CoA synthetase short-chain family member 1), ACTA1 (actin, alpha 1, skeletal muscle), ACTN2 (actinin, alpha 2), ADHFE1 (alcohol dehydrogenase, iron containing, 1), MTFR1 (mitochondrial fission regulator 1), PDK4 (pyruvate dehydrogenase kinase, isozyme 4) and TNC (tenascin C). Understanding the genetic basis for exercise adaptation will be crucial for the identification of genes within the complex molecular networks underlying obesity and its consequential pathologies, such as type 2 diabetes. Therefore, we propose Thoroughbred as a novel in vivo large animal model for understanding molecular protection against metabolic disease.

Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

Science.gov (United States)

Choi, Mal-Gi; Kurnov, Vladlen; Kersting, Michael C.; Sreenivas, Avula; Carman, George M.

2005-01-01

The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work, we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent, and dependent on the concentrations of choline kinase (Km = 27 μg/ml) and ATP (Km = 15 μM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSS25QRRHS (Vmax/Km = 17.5 mM-1 μmol min-1 mg-1) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo, the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Whereas the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHS30LTRQ) containing Ser30 was a substrate (Vmax/Km = 3.0 mM−1 μmol min−1 mg−1) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C. PMID:15919656

Applying ligands profiling using multiple extended electron distribution based field templates and feature trees similarity searching in the discovery of new generation of urea-based antineoplastic kinase inhibitors.

Directory of Open Access Journals (Sweden)

Eman M Dokla

Full Text Available This study provides a comprehensive computational procedure for the discovery of novel urea-based antineoplastic kinase inhibitors while focusing on diversification of both chemotype and selectivity pattern. It presents a systematic structural analysis of the different binding motifs of urea-based kinase inhibitors and the corresponding configurations of the kinase enzymes. The computational model depends on simultaneous application of two protocols. The first protocol applies multiple consecutive validated virtual screening filters including SMARTS, support vector-machine model (ROC = 0.98, Bayesian model (ROC = 0.86 and structure-based pharmacophore filters based on urea-based kinase inhibitors complexes retrieved from literature. This is followed by hits profiling against different extended electron distribution (XED based field templates representing different kinase targets. The second protocol enables cancericidal activity verification by using the algorithm of feature trees (Ftrees similarity searching against NCI database. Being a proof-of-concept study, this combined procedure was experimentally validated by its utilization in developing a novel series of urea-based derivatives of strong anticancer activity. This new series is based on 3-benzylbenzo[d]thiazol-2(3H-one scaffold which has interesting chemical feasibility and wide diversification capability. Antineoplastic activity of this series was assayed in vitro against NCI 60 tumor-cell lines showing very strong inhibition of GI(50 as low as 0.9 uM. Additionally, its mechanism was unleashed using KINEX™ protein kinase microarray-based small molecule inhibitor profiling platform and cell cycle analysis showing a peculiar selectivity pattern against Zap70, c-src, Mink1, csk and MeKK2 kinases. Interestingly, it showed activity on syk kinase confirming the recent studies finding of the high activity of diphenyl urea containing compounds against this kinase. Allover, the new series

Multi-lobulation of the nucleus in prolonged S phase by nuclear expression of Chk tyrosine kinase.

Science.gov (United States)

Nakayama, Yuji; Yamaguchi, Naoto

2005-04-01

Chk tyrosine kinase phosphorylates Src-family tyrosine kinases and suppresses their kinase activity. We recently showed that Chk localizes to the nucleus as well as the cytoplasm and inhibits cell proliferation. To investigate the role of nuclear Chk in proliferation, various Chk mutants were constructed and expressed. Nuclear localization of Chk-induced dynamic multi-lobulation of the nucleus and prolonged S phase of the cell cycle. The N-terminal domain of Chk and a portion of its kinase domain but not the kinase activity were responsible for induction of the multi-lobulation. Cell sorting analysis revealed that nuclear multi-lobulated cells were enriched in late S phase. Multi-lobulated nuclei were surrounded with lamin B1 that was particularly concentrated in concave regions of the nuclei. Furthermore, treatment with nocodazole or taxol disrupted multi-lobulation of the nucleus. These results suggest that nuclear multi-lobulation in late S phase, which is dependent on polymerization and depolymerization of microtubules, may be involved in nuclear Chk-induced inhibition of proliferation.

Multi-lobulation of the nucleus in prolonged S phase by nuclear expression of Chk tyrosine kinase

International Nuclear Information System (INIS)

Nakayama, Yuji; Yamaguchi, Naoto

2005-01-01

Chk tyrosine kinase phosphorylates Src-family tyrosine kinases and suppresses their kinase activity. We recently showed that Chk localizes to the nucleus as well as the cytoplasm and inhibits cell proliferation. To investigate the role of nuclear Chk in proliferation, various Chk mutants were constructed and expressed. Nuclear localization of Chk-induced dynamic multi-lobulation of the nucleus and prolonged S phase of the cell cycle. The N-terminal domain of Chk and a portion of its kinase domain but not the kinase activity were responsible for induction of the multi-lobulation. Cell sorting analysis revealed that nuclear multi-lobulated cells were enriched in late S phase. Multi-lobulated nuclei were surrounded with lamin B1 that was particularly concentrated in concave regions of the nuclei. Furthermore, treatment with nocodazole or taxol disrupted multi-lobulation of the nucleus. These results suggest that nuclear multi-lobulation in late S phase, which is dependent on polymerization and depolymerization of microtubules, may be involved in nuclear Chk-induced inhibition of proliferation

Visual Snapshots of Intracellular Kinase Activity At The Onset of Mitosis

Science.gov (United States)

Dai, Zhaohua; Dulyaninova, Natalya G.; Kumar, Sanjai; Bresnick, Anne R.; Lawrence, David S.

2007-01-01

Summary Visual snapshots of intracellular kinase activity can be acquired with exquisite temporal control using a light-activatable (caged) sensor, thereby providing a means to interrogate enzymatic activity at any point during the cell division cycle. Robust protein kinase activity transpires just prior to, but not immediately following, nuclear envelope breakdown (NEB). Furthermore, kinase activity is required for progression from prophase into metaphase. Finally, the application of selective protein kinase C (PKC) inhibitors, in combination with the caged sensor, correlates the action of the PKC β isoform with subsequent NEB. PMID:18022564

Enriched lithium collection from lithium plasma flow

International Nuclear Information System (INIS)

Karchevsky, A.I.; Laz'ko, V.S.; Muromkin, Y.A.; Pashkovsky, V.G.; Ustinov, A.L.; Dolgolenko, D.A.

1994-01-01

In order to understand the physical processes concerned with the selective heating by ion cyclotron resonance and with the subsequent collection of heated particles, experiments were carried out with the extraction of lithium samples, enriched with 6 Li isotopes. Probe and integral extractors allow to collect enriched Li at the end of the selective heating region. Surface density distribution on the collector and local isotopic content of lithium are measured, as a function of the screen height and the retarding potential. Dependence of the collected amount of lithium and of its isotopic content on the value of the magnetic field is also measured. 4 figs., 2 tabs., 5 refs

Small-molecule kinase inhibitors: an analysis of FDA-approved drugs

DEFF Research Database (Denmark)

Wu, Peng; Nielsen, Thomas Eiland; Clausen, Mads Hartvig

2016-01-01

Small-molecule kinase inhibitors (SMKIs), 28 of which are approved by the US Food and Drug Administration (FDA), have been actively pursued as promising targeted therapeutics. Here, we assess the key structural and physicochemical properties, target selectivity and mechanism of function, and ther......Small-molecule kinase inhibitors (SMKIs), 28 of which are approved by the US Food and Drug Administration (FDA), have been actively pursued as promising targeted therapeutics. Here, we assess the key structural and physicochemical properties, target selectivity and mechanism of function...

Selection based on indirect genetic effects for growth, environmental enrichment and coping style affect the immune status of pigs.

Science.gov (United States)

Reimert, Inonge; Rodenburg, T Bas; Ursinus, Winanda W; Kemp, Bas; Bolhuis, J Elizabeth

2014-01-01

Pigs living in intensive husbandry systems may experience both acute and chronic stress through standard management procedures and limitations in their physical and social environment, which may have implications for their immune status. Here, the effect of a new breeding method where pigs were selected on their heritable influence on their pen mates' growth, and environmental enrichment on the immune status of pigs was investigated. Hereto, 240 pigs with a relatively positive genetic effect on the growth of their pen mates (+SBV) and 240 pigs with a relatively negative genetic effect on the growth of their pen mates (-SBV) were housed in barren or straw-enriched pens from 4 to 23 weeks of age (n  =  80 pens in total). A blood sample was taken from the pigs before, three days after a 24 h regrouping test, and at week 22. In addition, effects of coping style, as assessed in a backtest, and gender were also investigated. Mainly, +SBV were found to have lower leukocyte, lymphocyte and haptoglobin concentrations than -SBV pigs. Enriched housed pigs had a lower neutrophil to lymphocyte (N:L) ratio and lower haptoglobin concentrations, but had higher antibody titers specific for Keyhole Limpet Hemocyanin (KLH) than barren housed pigs. No interactions were found between SBV class and housing. Furthermore, pigs with a proactive coping style had higher alternative complement activity and, in the enriched pens, higher antibody titers specific for KLH than pigs with a reactive coping style. Lastly, females tended to have lower leukocyte, but higher haptoglobin concentrations than castrated males. Overall, these results suggest that +SBV pigs and enriched housed pigs were less affected by stress than -SBV and barren housed pigs, respectively. Moreover, immune activation might be differently organized in individuals with different coping styles and to a lesser extent in individuals of opposite genders.

Selection based on indirect genetic effects for growth, environmental enrichment and coping style affect the immune status of pigs.

Directory of Open Access Journals (Sweden)

Inonge Reimert

Full Text Available Pigs living in intensive husbandry systems may experience both acute and chronic stress through standard management procedures and limitations in their physical and social environment, which may have implications for their immune status. Here, the effect of a new breeding method where pigs were selected on their heritable influence on their pen mates' growth, and environmental enrichment on the immune status of pigs was investigated. Hereto, 240 pigs with a relatively positive genetic effect on the growth of their pen mates (+SBV and 240 pigs with a relatively negative genetic effect on the growth of their pen mates (-SBV were housed in barren or straw-enriched pens from 4 to 23 weeks of age (n  =  80 pens in total. A blood sample was taken from the pigs before, three days after a 24 h regrouping test, and at week 22. In addition, effects of coping style, as assessed in a backtest, and gender were also investigated. Mainly, +SBV were found to have lower leukocyte, lymphocyte and haptoglobin concentrations than -SBV pigs. Enriched housed pigs had a lower neutrophil to lymphocyte (N:L ratio and lower haptoglobin concentrations, but had higher antibody titers specific for Keyhole Limpet Hemocyanin (KLH than barren housed pigs. No interactions were found between SBV class and housing. Furthermore, pigs with a proactive coping style had higher alternative complement activity and, in the enriched pens, higher antibody titers specific for KLH than pigs with a reactive coping style. Lastly, females tended to have lower leukocyte, but higher haptoglobin concentrations than castrated males. Overall, these results suggest that +SBV pigs and enriched housed pigs were less affected by stress than -SBV and barren housed pigs, respectively. Moreover, immune activation might be differently organized in individuals with different coping styles and to a lesser extent in individuals of opposite genders.

Site-selective {sup 13}C labeling of proteins using erythrose

Energy Technology Data Exchange (ETDEWEB)

Weininger, Ulrich, E-mail: ulrich.weininger@physik.uni-halle.de [Lund University, Department of Biophysical Chemistry, Center for Molecular Protein Science (Sweden)

2017-03-15

NMR-spectroscopy enables unique experimental studies on protein dynamics at atomic resolution. In order to obtain a full atom view on protein dynamics, and to study specific local processes like ring-flips, proton-transfer, or tautomerization, one has to perform studies on amino-acid side chains. A key requirement for these studies is site-selective labeling with {sup 13}C and/or {sup 1}H, which is achieved in the most general way by using site-selectively {sup 13}C-enriched glucose (1- and 2-{sup 13}C) as the carbon source in bacterial expression systems. Using this strategy, multiple sites in side chains, including aromatics, become site-selectively labeled and suitable for relaxation studies. Here we systematically investigate the use of site-selectively {sup 13}C-enriched erythrose (1-, 2-, 3- and 4-{sup 13}C) as a suitable precursor for {sup 13}C labeled aromatic side chains. We quantify {sup 13}C incorporation in nearly all sites in all 20 amino acids and compare the results to glucose based labeling. In general the erythrose approach results in more selective labeling. While there is only a minor gain for phenylalanine and tyrosine side-chains, the {sup 13}C incorporation level for tryptophan is at least doubled. Additionally, the Phe ζ and Trp η2 positions become labeled. In the aliphatic side chains, labeling using erythrose yields isolated {sup 13}C labels for certain positions, like Ile β and His β, making these sites suitable for dynamics studies. Using erythrose instead of glucose as a source for site-selective {sup 13}C labeling enables unique or superior labeling for certain positions and is thereby expanding the toolbox for customized isotope labeling of amino-acid side-chains.

Usage of adenovirus expressing thymidine kinase mediated hepatocellular damage for enabling mouse liver repopulation with allogenic or xenogenic hepatocytes.

Directory of Open Access Journals (Sweden)

Daniel Moreno

Full Text Available It has been shown that the liver of immunodeficient mice can be efficiently repopulated with human hepatocytes when subjected to chronic hepatocellular damage. Mice with such chimeric livers represent useful reagents for medical and clinical studies. However all previously reported models of humanized livers are difficult to implement as they involve cross-breeding of immunodeficient mice with mice exhibiting genetic alterations causing sustained hepatic injury. In this paper we attempted to create chimeric livers by inducing persistent hepatocellular damage in immunodeficient Rag2(-/- γc(-/- mice using an adenovirus encoding herpes virus thymidine kinase (AdTk and two consecutive doses of ganciclovir (GCV. We found that this treatment resulted in hepatocellular damage persisting for at least 10 weeks and enabled efficient engraftment and proliferation within the liver of either human or allogenic hepatocytes. Interestingly, while the nodules generated from the transplanted mouse hepatocytes were well vascularized, the human hepatocytes experienced progressive depolarization and exhibited reduced numbers of murine endothelial cells inside the nodules. In conclusion, AdTk/GCV-induced liver damage licenses the liver of immunodeficient mice for allogenic and xenogenic hepatocyte repopulation. This approach represents a simple alternative strategy for chimeric liver generation using immunodeficient mice without additional genetic manipulation of the germ line.

Reduction of Fibrogenesis by Selective Delivery of a Rho Kinase Inhibitor to Hepatic Stellate Cells in Mice

NARCIS (Netherlands)

van Beuge, M. M.; Prakash, J.; Lacombe, M.; Gosens, R.; Post, E.; Reker-Smit, C.; Beljaars, L.; Poelstra, K.

One of the pathways activated during liver fibrosis is the Rho kinase pathway, which regulates activation, migration, and contraction of hepatic stellate cells (HSC). Inhibition of this kinase by the Rho kinase inhibitor Y27632 [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide

Experimental study on enriching 12C by centrifuge method

International Nuclear Information System (INIS)

Xiao Huaxian

1994-07-01

The diamond made from the highly enriched 12 C, whose thermal conductivity and electric insulativity are much better than that of natural diamond, has widely uses in new and high technology. In many enriching 12 C methods, the gas centrifuge method is superior to others. After selecting the appropriate process gas and solving key problems, such as feed and extract, the separation experiments are performed by a single stage of centrifuge. To increase the separation capacity of single machine, various parameters in the centrifugal separation are optimized, and appropriate mechanical drive, thermal drive, hold-up and process parameters are selected. The optimal operating condition of single machine is also obtained in the cascade. Thus, highly enriched 12 C is produced in the centrifuge cascade

Autophagy suppresses RIP kinase-dependent necrosis enabling survival to mTOR inhibition.

Directory of Open Access Journals (Sweden)

Kevin Bray

Full Text Available mTOR inhibitors are used clinically to treat renal cancer but are not curative. Here we show that autophagy is a resistance mechanism of human renal cell carcinoma (RCC cell lines to mTOR inhibitors. RCC cell lines have high basal autophagy that is required for survival to mTOR inhibition. In RCC4 cells, inhibition of mTOR with CCI-779 stimulates autophagy and eliminates RIP kinases (RIPKs and this is blocked by autophagy inhibition, which induces RIPK- and ROS-dependent necroptosis in vitro and suppresses xenograft growth. Autophagy of mitochondria is required for cell survival since mTOR inhibition turns off Nrf2 antioxidant defense. Thus, coordinate mTOR and autophagy inhibition leads to an imbalance between ROS production and defense, causing necroptosis that may enhance cancer treatment efficacy.

Target-specific support vector machine scoring in structure-based virtual screening: computational validation, in vitro testing in kinases, and effects on lung cancer cell proliferation.

Science.gov (United States)

Li, Liwei; Khanna, May; Jo, Inha; Wang, Fang; Ashpole, Nicole M; Hudmon, Andy; Meroueh, Samy O

2011-04-25

We assess the performance of our previously reported structure-based support vector machine target-specific scoring function across 41 targets, 40 among them from the Directory of Useful Decoys (DUD). The area under the curve of receiver operating characteristic plots (ROC-AUC) revealed that scoring with SVM-SP resulted in consistently better enrichment over all target families, outperforming Glide and other scoring functions, most notably among kinases. In addition, SVM-SP performance showed little variation among protein classes, exhibited excellent performance in a test case using a homology model, and in some cases showed high enrichment even with few structures used to train a model. We put SVM-SP to the test by virtual screening 1125 compounds against two kinases, EGFR and CaMKII. Among the top 25 EGFR compounds, three compounds (1-3) inhibited kinase activity in vitro with IC₅₀ of 58, 2, and 10 μM. In cell cultures, compounds 1-3 inhibited nonsmall cell lung carcinoma (H1299) cancer cell proliferation with similar IC₅₀ values for compound 3. For CaMKII, one compound inhibited kinase activity in a dose-dependent manner among 20 tested with an IC₅₀ of 48 μM. These results are encouraging given that our in-house library consists of compounds that emerged from virtual screening of other targets with pockets that are different from typical ATP binding sites found in kinases. In light of the importance of kinases in chemical biology, these findings could have implications in future efforts to identify chemical probes of kinases within the human kinome.

Computational-experimental approach to drug-target interaction mapping: A case study on kinase inhibitors.

Directory of Open Access Journals (Sweden)

Anna Cichonska

2017-08-01

Full Text Available Due to relatively high costs and labor required for experimental profiling of the full target space of chemical compounds, various machine learning models have been proposed as cost-effective means to advance this process in terms of predicting the most potent compound-target interactions for subsequent verification. However, most of the model predictions lack direct experimental validation in the laboratory, making their practical benefits for drug discovery or repurposing applications largely unknown. Here, we therefore introduce and carefully test a systematic computational-experimental framework for the prediction and pre-clinical verification of drug-target interactions using a well-established kernel-based regression algorithm as the prediction model. To evaluate its performance, we first predicted unmeasured binding affinities in a large-scale kinase inhibitor profiling study, and then experimentally tested 100 compound-kinase pairs. The relatively high correlation of 0.77 (p < 0.0001 between the predicted and measured bioactivities supports the potential of the model for filling the experimental gaps in existing compound-target interaction maps. Further, we subjected the model to a more challenging task of predicting target interactions for such a new candidate drug compound that lacks prior binding profile information. As a specific case study, we used tivozanib, an investigational VEGF receptor inhibitor with currently unknown off-target profile. Among 7 kinases with high predicted affinity, we experimentally validated 4 new off-targets of tivozanib, namely the Src-family kinases FRK and FYN A, the non-receptor tyrosine kinase ABL1, and the serine/threonine kinase SLK. Our sub-sequent experimental validation protocol effectively avoids any possible information leakage between the training and validation data, and therefore enables rigorous model validation for practical applications. These results demonstrate that the kernel

Fluorescent Inhibitors as Tools To Characterize Enzymes: Case Study of the Lipid Kinase Phosphatidylinositol 4-Kinase IIIβ (PI4KB).

Science.gov (United States)

Humpolickova, Jana; Mejdrová, Ivana; Matousova, Marika; Nencka, Radim; Boura, Evzen

2017-01-12

The lipid kinase phosphatidylinositol 4-kinase IIIβ (PI4KB) is an essential host factor for many positive-sense single-stranded RNA (+RNA) viruses including human pathogens hepatitis C virus (HCV), Severe acute respiratory syndrome (SARS), coxsackie viruses, and rhinoviruses. Inhibitors of PI4KB are considered to be potential broad-spectrum virostatics, and it is therefore critical to develop a biochemical understanding of the kinase. Here, we present highly potent and selective fluorescent inhibitors that we show to be useful chemical biology tools especially in determination of dissociation constants. Moreover, we show that the coumarin-labeled inhibitor can be used to image PI4KB in cells using fluorescence-lifetime imaging microscopy (FLIM) microscopy.

Reconstructed ancestral enzymes reveal that negative selection drove the evolution of substrate specificity in ADP-dependent kinases.

Science.gov (United States)

Castro-Fernandez, Víctor; Herrera-Morande, Alejandra; Zamora, Ricardo; Merino, Felipe; Gonzalez-Ordenes, Felipe; Padilla-Salinas, Felipe; Pereira, Humberto M; Brandão-Neto, Jose; Garratt, Richard C; Guixe, Victoria

2017-09-22

One central goal in molecular evolution is to pinpoint the mechanisms and evolutionary forces that cause an enzyme to change its substrate specificity; however, these processes remain largely unexplored. Using the glycolytic ADP-dependent kinases of archaea, including the orders Thermococcales , Methanosarcinales , and Methanococcales , as a model and employing an approach involving paleoenzymology, evolutionary statistics, and protein structural analysis, we could track changes in substrate specificity during ADP-dependent kinase evolution along with the structural determinants of these changes. To do so, we studied five key resurrected ancestral enzymes as well as their extant counterparts. We found that a major shift in function from a bifunctional ancestor that could phosphorylate either glucose or fructose 6-phosphate (fructose-6-P) as a substrate to a fructose 6-P-specific enzyme was started by a single amino acid substitution resulting in negative selection with a ground-state mode against glucose and a subsequent 1,600-fold change in specificity of the ancestral protein. This change rendered the residual phosphorylation of glucose a promiscuous and physiologically irrelevant activity, highlighting how promiscuity may be an evolutionary vestige of ancestral enzyme activities, which have been eliminated over time. We also could reconstruct the evolutionary history of substrate utilization by using an evolutionary model of discrete binary characters, indicating that substrate uses can be discretely lost or acquired during enzyme evolution. These findings exemplify how negative selection and subtle enzyme changes can lead to major evolutionary shifts in function, which can subsequently generate important adaptive advantages, for example, in improving glycolytic efficiency in Thermococcales . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Progress in ultra-centrifuge enrichment technology

International Nuclear Information System (INIS)

Paul Dawson

2006-01-01

Urenco have undertaken a continuous development programme in centrifuge technology for over 35 years. This has seen development from sub-critical machines in the mid 1970's through to the company's world leading TC12 supercritical centrifuge, which has been deployed on a large-scale basis over the last decade. The latest centrifuge to emerge from this programme is Urenco's sixth generation centrifuge, the TC21, which will be commercially deployed from mid-2007 onwards. In recent times Urenco has vested its centrifuge technology in Enrichment Technology Company (ETC) as a vehicle to enable the use of this advanced technology by other operators for commercial purposes. This paper reviews why Urenco and ETC believe this technology represents the best choice for creating new global commercial enrichment capacity and its future development prospects. (author)

Discovery of a 1,2-bis(3-indolyl)ethane that selectively inhibits the pyruvate kinase of methicillin-resistant Staphylococcus aureus over human isoforms.

Science.gov (United States)

Zoraghi, Roya; Campbell, Sara; Kim, Catrina; Dullaghan, Edie M; Blair, Lachlan M; Gillard, Rachel M; Reiner, Neil E; Sperry, Jonathan

2014-11-01

Methicillin-resistant Staphylococcus aureus pyruvate kinase (MRSA PK) has recently been identified as a target for development of novel antibacterial agents. Testing a series of 1,2-bis(3-indolyl)ethanes against MRSA PK has led to the discovery of a potent inhibitor that is selective over human isoforms. Copyright © 2014 Elsevier Ltd. All rights reserved.

Detection of Salmonella spp. in veterinary samples by combining selective enrichment and real-time PCR.

Science.gov (United States)

Goodman, Laura B; McDonough, Patrick L; Anderson, Renee R; Franklin-Guild, Rebecca J; Ryan, James R; Perkins, Gillian A; Thachil, Anil J; Glaser, Amy L; Thompson, Belinda S

2017-11-01

Rapid screening for enteric bacterial pathogens in clinical environments is essential for biosecurity. Salmonella found in veterinary hospitals, particularly Salmonella enterica serovar Dublin, can pose unique challenges for culture and testing because of its poor growth. Multiple Salmonella serovars including Dublin are emerging threats to public health given increasing prevalence and antimicrobial resistance. We adapted an automated food testing method to veterinary samples and evaluated the performance of the method in a variety of matrices including environmental samples ( n = 81), tissues ( n = 52), feces ( n = 148), and feed ( n = 29). A commercial kit was chosen as the basis for this approach in view of extensive performance characterizations published by multiple independent organizations. A workflow was established for efficiently and accurately testing veterinary matrices and environmental samples by use of real-time PCR after selective enrichment in Rappaport-Vassiliadis soya (RVS) medium. Using this method, the detection limit for S. Dublin improved by 100-fold over subculture on selective agars (eosin-methylene blue, brilliant green, and xylose-lysine-deoxycholate). Overall, the procedure was effective in detecting Salmonella spp. and provided next-day results.

Selective enrichment and determination of monoamine neurotransmitters by CU(II) immobilized magnetic solid phase extraction coupled with high-performance liquid chromatography-fluorescence detection.

Science.gov (United States)

He, Maofang; Wang, Chaozhan; Wei, Yinmao

2016-01-15

In this paper, iminodiacetic acid-Cu(II) functionalized Fe3O4@SiO2 magnetic nanoparticles were prepared and used as new adsorbents for magnetic solid phase extraction (MSPE) of six monoamine neurotransmitters (MNTs) from rabbit plasma. The selective enrichment of MNTs at pH 5.0 was motivated by the specific coordination interaction between amino groups of MNTs and the immobilized Cu(II). The employed weak acidic extraction condition avoided the oxidation of MNTs, and thus facilitated operation and ensured higher recoveries. Under optimal conditions, the recoveries of six MNTs from rabbit plasma were in the range of 83.9-109.4%, with RSD of 2.0-10.0%. When coupled the Cu(II) immobilized MSPE with high-performance liquid chromatography-fluorescence detection, the method exhibited relatively lower detection limits than the previously reported methods, and the method was successfully used to determine the endogenous MNTs in rabbit plasma. The proposed method has potential application for the determination of MNTs in biological samples. Also, the utilization of coordination interaction to improve the selectivity might open another way to selectively enrich small alkaloids from complex samples. Copyright © 2015 Elsevier B.V. All rights reserved.

The Discovery of Aurora Kinase Inhibitor by Multi-Docking-Based Virtual Screening

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Jun-Tae Kim

2014-11-01

Full Text Available We report the discovery of aurora kinase inhibitor using the fragment-based virtual screening by multi-docking strategy. Among a number of fragments collected from eMololecules, we found four fragment molecules showing potent activity (>50% at 100 μM against aurora kinase. Based on the explored fragment scaffold, we selected two compounds in our synthesized library and validated the biological activity against Aurora kinase.

Physician communication via Internet-enabled technology: A systematic review.

Science.gov (United States)

Barr, Neil G; Randall, Glen E; Archer, Norman P; Musson, David M

2017-10-01

The use of Internet-enabled technology (information and communication technology such as smartphone applications) may enrich information exchange among providers and, consequently, improve health care delivery. The purpose of this systematic review was to gain a greater understanding of the role that Internet-enabled technology plays in enhancing communication among physicians. Studies were identified through a search in three electronic platforms: the Association for Computing Machinery Digital Library, ProQuest, and Web of Science. The search identified 5140 articles; of these, 21 met all inclusion criteria. In general, physicians were satisfied with Internet-enabled technology, but consensus was lacking regarding whether Internet-enabled technology improved efficiency or made a difference to clinical decision-making. Internet-enabled technology can play an important role in enhancing communication among physicians, but the extent of that benefit is influenced by (1) the impact of Internet-enabled technology on existing work practices, (2) the availability of adequate resources, and (3) the nature of institutional elements, such as privacy legislation.

Kinase inhibition by the Jamaican ball moss, Tillandsia recurvata L.

Science.gov (United States)

Lowe, Henry I C; Watson, Charah T; Badal, Simone; Toyang, Ngeh J; Bryant, Joseph

2012-10-01

This research was undertaken in order to investigate the inhibitory potential of the Jamaican ball moss, Tillandsia recurvata against several kinases. The inhibition of these kinases has emerged as a potential solution to restoring the tight regulation of normal cellular growth, the loss of which leads to cancer cell formation. Kinase inhibition was investigated using competition binding (to the ATP sites) assays, which have been previously established and authenticated. Four hundred and fifty one kinases were tested against the Jamaican ball moss extract and a dose-response was tested on 40 kinases, which were inhibited by more than 35% compared to the control. Out of the 40 kinases, the Jamaican ball moss selectively inhibited 5 (CSNK2A2, MEK5, GAK, FLT and DRAK1) and obtained Kd(50)s were below 20 μg/ml. Since MEK5 and GAK kinases have been associated with aggressive prostate cancer, the inhibitory properties of the ball moss against them, coupled with its previously found bioactivity towards the PC-3 cell line, makes it promising in the arena of drug discovery towards prostate cancer.

Isotopically enriched structural materials in nuclear devices

Energy Technology Data Exchange (ETDEWEB)

Morgan, L.W.G., E-mail: Lee.Morgan@ccfe.ac.uk [CCFE, Culham Science Centre, Abingdon, Oxfordshire OX14 3DB (United Kingdom); Shimwell, J. [CCFE, Culham Science Centre, Abingdon, Oxfordshire OX14 3DB (United Kingdom); Department of Physics and Astronomy, University of Sheffield, Hicks Building, Hounsfield Road, Sheffield S3 7RH (United Kingdom); Gilbert, M.R. [CCFE, Culham Science Centre, Abingdon, Oxfordshire OX14 3DB (United Kingdom)

2015-01-15

Highlights: • C-B analysis of isotopic enrichment of structural materials is presented. • Some, previously, prohibited elements could be used as alloying elements in LAM's. • Adding enriched molybdenum and nickel, to EUROFER, could increase availability. • Isotope enrichment for EUROFER could be cost-effective. • Isotopically enriching copper, in CuCrZr, can reduce helium production by 50%. - Abstract: A large number of materials exist which have been labeled as low activation structural materials (LAM). Most often, these materials have been designed in order to substitute-out or completely remove elements that become activated and contribute significantly to shut-down activity after being irradiated by neutrons in a reactor environment. To date, one of the fundamental principles from which LAMs have been developed is that natural elemental compositions are the building blocks of LAMs. Thus, elements such as Co, Al, Ni, Mo, Nb, N and Cu that produce long-lived decay products are significantly reduced or removed from the LAM composition. These elements have an important part to play in the composition of steels and the removal/substitution can have a negative impact on materials properties such as yield stress and fracture toughness. This paper looks in more detail at whether using isotopic selection of the more mechanically desirable, but prohibited due to activation, elements can improve matters. In particular, this paper focuses on the activation of Eurofer. Carefully chosen isotopically enriched elements, which are normally considered to be on the prohibited element list, are added to EUROFER steel as potential alloying elements. The EUROFER activation results show that some prohibited elements can be used as alloying elements in LAM steels, providing the selected isotopes do not have a significant impact on waste disposal rating or shut-down dose. The economic implications of isotopically enriching elements and the potential implications for

Urochordate ascidians possess a single isoform of Aurora kinase that localizes to the midbody via TPX2 in eggs and cleavage stage embryos.

Directory of Open Access Journals (Sweden)

Celine Hebras

Full Text Available Aurora kinases are key proteins found throughout the eukaryotes that control mitotic progression. Vertebrate Aurora-A and B kinases are thought to have evolved from a single Aurora-kinase isoform closest to that found in present day urochordates. In urochordate ascidians Aurora binds both TPX2 (a vertebrate AURKA partner and INCENP (a vertebrate AURKB partner and localizes to centrosomes and spindle microtubules as well as chromosomes and midbody during both meiosis and mitosis. Ascidian Aurora also displays this localization pattern during mitosis in echinoderms, strengthening the idea that non-vertebrate deuterostomes such as the urochordates and echinoderms possess a single form of Aurora kinase that has properties of vertebrate Aurora-kinase A and B. In the ascidian, TPX2 localizes to the centrosome and the spindle poles also as in vertebrates. However, we were surprised to find that TPX2 also localized strongly to the midbody in ascidian eggs and embryos. We thus examined more closely Aurora localization to the midbody by creating two separate point mutations of ascidian Aurora predicted to perturb binding to TPX2. Both forms of mutated Aurora behaved as predicted: neither localized to spindle poles where TPX2 is enriched. Interestingly, neither form of mutated Aurora localized to the midbody where TPX2 is also enriched, suggesting that ascidian Aurora midbody localization required TPX2 binding in ascidians. Functional analysis revealed that inhibition of Aurora kinase with a pharmacological inhibitor or with a dominant negative kinase dead form of Aurora caused cytokinesis failure and perturbed midbody formation during polar body extrusion. Our data support the view that vertebrate Aurora-A and B kinases evolved from a single non-vertebrate deuterostome ancestor. Moreover, since TPX2 localizes to the midbody in ascidian eggs and cleavage stage embryos it may be worthwhile re-assessing whether Aurora A kinase or TPX2 localize to the midbody

Enrichment of Acinetobacter spp. from food samples.

Science.gov (United States)

Carvalheira, Ana; Ferreira, Vânia; Silva, Joana; Teixeira, Paula

2016-05-01

Relatively little is known about the role of foods in the chain of transmission of acinetobacters and the occurrence of different Acinetobacter spp. in foods. Currently, there is no standard procedure to recover acinetobacters from food in order to gain insight into the food-related ecology and epidemiology of acinetobacters. This study aimed to assess whether enrichment in Dijkshoorn enrichment medium followed by plating in CHROMagar™ Acinetobacter medium is a useful method for the isolation of Acinetobacter spp. from foods. Recovery of six Acinetobacter species from food spiked with these organisms was compared for two selective enrichment media (Baumann's enrichment and Dijkshoorn's enrichment). Significantly (p enrichment. Next, the Dijkshoorn's enrichment followed by direct plating on CHROMagar™ Acinetobacter was applied to detect Acinetobacter spp. in different foods. Fourteen different presumptive acinetobacters were recovered and assumed to represent nine different strains on the basis of REP-PCR typing. Eight of these strains were identified by rpoB gene analysis as belonging to the species Acinetobacter johnsonii, Acinetobacter calcoaceticus, Acinetobacter guillouiae and Acinetobacter gandensis. It was not possible to identify the species level of one strain which may suggests that it represents a distinct species. Copyright © 2015 Elsevier Ltd. All rights reserved.

LOK is a major ERM kinase in resting lymphocytes and regulates cytoskeletal rearrangement through ERM phosphorylation.

Science.gov (United States)

Belkina, Natalya V; Liu, Yin; Hao, Jian-Jiang; Karasuyama, Hajime; Shaw, Stephen

2009-03-24

ERM (ezrin-radixin-moesin) proteins mediate linkage of actin cytoskeleton to plasma membrane in many cells. ERM activity is regulated in part by phosphorylation at a C-terminal threonine, but the identity of ERM kinases is unknown in lymphocytes and incompletely defined in other mammalian cells. Our studies show that lymphocyte-oriented kinase (LOK) is an ERM kinase in vitro and in vivo. Mass spectrometric analysis indicates LOK is abundant at the lymphocyte plasma membrane and immunofluorescence studies show LOK enrichment at the plasma membrane near ERM. In vitro peptide specificity analyses characterize LOK as a basophilic kinase whose optimal substrate sequence resembles the ERM site, including unusual preference for tyrosine at P-2. LOK's activity on moesin peptide and protein was comparable to reported ERM kinases ROCK and PKC but unlike them LOK displayed preferential specificity for moesin compared to traditional basophilic kinase substrates. Two genetic approaches demonstrate a role for LOK in ERM phosphorylation: cell transfection with LOK kinase domain augments ERM phosphorylation and lymphocytes from LOK knockout mice have >50% reduction in ERM phosphorylation. The findings on localization and specificity argue that LOK is a direct ERM kinase. The knockout mice have normal hematopoietic cell development but notably lymphocyte migration and polarization in response to chemokine are enhanced. These functional alterations fit the current understanding of the role of ERM phosphorylation in regulating cortical reorganization. Thus, these studies identify a new ERM kinase of importance in lymphocytes and confirm the role of ERM phosphorylation in regulating cell shape and motility.

Exploiting Chemical Libraries, Structure, and Genomics in the Search for Kinase Inhibitors

NARCIS (Netherlands)

Gray, Nathanael S.; Wodicka, Lisa; Thunnissen, Andy-Mark W.H.; Norman, Thea C.; Kwon, Soojin; Espinoza, F. Hernan; Morgan, David O.; Barnes, Georjana; LeClerc, Sophie; Meijer, Laurent; Kim, Sung-Hou; Lockhart, David J.; Schultz, Peter G.

1998-01-01

Selective protein kinase inhibitors were developed on the basis of the unexpected binding mode of 2,6,9-trisubstituted purines to the adenosine triphosphate-binding site of the human cyclin-dependent kinase 2 (CDK2). By iterating chemical library synthesis and biological screening, potent inhibitors

Enrichment allows identification of diverse, rare elements in metagenomic resistome-virulome sequencing.

Science.gov (United States)

Noyes, Noelle R; Weinroth, Maggie E; Parker, Jennifer K; Dean, Chris J; Lakin, Steven M; Raymond, Robert A; Rovira, Pablo; Doster, Enrique; Abdo, Zaid; Martin, Jennifer N; Jones, Kenneth L; Ruiz, Jaime; Boucher, Christina A; Belk, Keith E; Morley, Paul S

2017-10-17

Shotgun metagenomic sequencing is increasingly utilized as a tool to evaluate ecological-level dynamics of antimicrobial resistance and virulence, in conjunction with microbiome analysis. Interest in use of this method for environmental surveillance of antimicrobial resistance and pathogenic microorganisms is also increasing. In published metagenomic datasets, the total of all resistance- and virulence-related sequences accounts for enrichment system that incorporates unique molecular indices to count DNA molecules and correct for enrichment bias. The use of the bait-capture and enrichment system significantly increased on-target sequencing of the resistome-virulome, enabling detection of an additional 1441 gene accessions and revealing a low-abundance portion of the resistome-virulome that was more diverse and compositionally different than that detected by more traditional metagenomic assays. The low-abundance portion of the resistome-virulome also contained resistance genes with public health importance, such as extended-spectrum betalactamases, that were not detected using traditional shotgun metagenomic sequencing. In addition, the use of the bait-capture and enrichment system enabled identification of rare resistance gene haplotypes that were used to discriminate between sample origins. These results demonstrate that the rare resistome-virulome contains valuable and unique information that can be utilized for both surveillance and population genetic investigations of resistance. Access to the rare resistome-virulome using the bait-capture and enrichment system validated in this study can greatly advance our understanding of microbiome-resistome dynamics.

Selective vs. nonselective media and direct plating vs. enrichment technique in isolation of Vibrio cholerae: recommendations for clinical laboratories.

Science.gov (United States)

Rennels, M B; Levine, M M; Daya, V; Angle, P; Young, C

1980-09-01

The occurrence of human cholera along the Gulf of Mexico and the isolation of Vibrio cholerae O1 from the Gulf and Chesapeake Bay make it imperative that microbiology laboratories along estuaries develop the capabilities to culture for these pathogens. In attempts to devise a simplified but efficient culture procedure, a selective medium, thiosulfate-citrate-bile salts-sucrose (TCBS) agar, was compared with a nonselective medium, gelatin agar (GA), and the utility of enrichment was examined. TCBS agar detected 99% of the stools found to be positive by all techniques combined, whereas GA identified only 80%. Of acute diarrheal stools, 96% were positive on direct plating, whereas only 66% of formed stools containing V. cholerae were detected by direct plating. Stools from patients with acute diarrhea can be plated directly into TCBS agar alone; stools from persons shedding low numbers of organisms (such as contacts, carriers, or patients receiving antibiotics) should be incubated first in an enrichment broth and then on TCBS agar.

Identification of Mediator Kinase Substrates in Human Cells using Cortistatin A and Quantitative Phosphoproteomics.

Science.gov (United States)

Poss, Zachary C; Ebmeier, Christopher C; Odell, Aaron T; Tangpeerachaikul, Anupong; Lee, Thomas; Pelish, Henry E; Shair, Matthew D; Dowell, Robin D; Old, William M; Taatjes, Dylan J

2016-04-12

Cortistatin A (CA) is a highly selective inhibitor of the Mediator kinases CDK8 and CDK19. Using CA, we now report a large-scale identification of Mediator kinase substrates in human cells (HCT116). We identified over 16,000 quantified phosphosites including 78 high-confidence Mediator kinase targets within 64 proteins, including DNA-binding transcription factors and proteins associated with chromatin, DNA repair, and RNA polymerase II. Although RNA-seq data correlated with Mediator kinase targets, the effects of CA on gene expression were limited and distinct from CDK8 or CDK19 knockdown. Quantitative proteome analyses, tracking around 7,000 proteins across six time points (0-24 hr), revealed that CA selectively affected pathways implicated in inflammation, growth, and metabolic regulation. Contrary to expectations, increased turnover of Mediator kinase targets was not generally observed. Collectively, these data support Mediator kinases as regulators of chromatin and RNA polymerase II activity and suggest their roles extend beyond transcription to metabolism and DNA repair. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Resorufin: a lead for a new protein kinase CK2 inhibitor

DEFF Research Database (Denmark)

Sandholt, Iben Skjøth; Olsen, Birgitte Brinkmann; Guerra, Barbara

2009-01-01

Screening a natural compound library led to the identification of resorufin as a highly selective and potent inhibitor of protein kinase CK2. Out of 52 kinases tested, only CK2 was inhibited, in contrast to emodin, a structurally related, known CK2 inhibitor that, in addition to CK2, inhibited te...

Fluorescent Reporters and Biosensors for Probing the Dynamic Behavior of Protein Kinases

Directory of Open Access Journals (Sweden)

Juan A. González-Vera

2015-11-01

Full Text Available Probing the dynamic activities of protein kinases in real-time in living cells constitutes a major challenge that requires specific and sensitive tools tailored to meet the particular demands associated with cellular imaging. The development of genetically-encoded and synthetic fluorescent biosensors has provided means of monitoring protein kinase activities in a non-invasive fashion in their native cellular environment with high spatial and temporal resolution. Here, we review existing technologies to probe different dynamic features of protein kinases and discuss limitations where new developments are required to implement more performant tools, in particular with respect to infrared and near-infrared fluorescent probes and strategies which enable improved signal-to-noise ratio and controlled activation of probes.

Activation loop targeting strategy for design of receptor-interacting protein kinase 2 (RIPK2) inhibitors.

Science.gov (United States)

Suebsuwong, Chalada; Pinkas, Daniel M; Ray, Soumya S; Bufton, Joshua C; Dai, Bing; Bullock, Alex N; Degterev, Alexei; Cuny, Gregory D

2018-02-15

Development of selective kinase inhibitors remains a challenge due to considerable amino acid sequence similarity among family members particularly in the ATP binding site. Targeting the activation loop might offer improved inhibitor selectivity since this region of kinases is less conserved. However, the strategy presents difficulties due to activation loop flexibility. Herein, we report the design of receptor-interacting protein kinase 2 (RIPK2) inhibitors based on pan-kinase inhibitor regorafenib that aim to engage basic activation loop residues Lys169 or Arg171. We report development of CSR35 that displayed >10-fold selective inhibition of RIPK2 versus VEGFR2, the target of regorafenib. A co-crystal structure of CSR35 with RIPK2 revealed a resolved activation loop with an ionic interaction between the carboxylic acid installed in the inhibitor and the side-chain of Lys169. Our data provides principle feasibility of developing activation loop targeting type II inhibitors as a complementary strategy for achieving improved selectivity. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Selective inhibition reveals cyclin-dependent kinase 2 as another kinase that phosphorylates the androgen receptor at serine 81

Czech Academy of Sciences Publication Activity Database

Jorda, Radek; Bučková, Zuzana; Řezníčková, Eva; Bouchal, J.; Kryštof, Vladimír

2018-01-01

Roč. 1865, č. 2 (2018), s. 354-363 ISSN 0167-4889 R&D Projects: GA MŠk(CZ) LO1204; GA MŠk(CZ) LO1304 Institutional support: RVO:61389030 Keywords : Androgen receptor * Cyclin-dependent kinase * Inhibitor * Phosphorylation * Serine 81 Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology Impact factor: 4.521, year: 2016

Influence of oxygen enrichment on compression ignition engines using biodiesel blends

Directory of Open Access Journals (Sweden)

Vaiyapuri Senthil Murugan

2017-01-01

Full Text Available The influence of oxygen enrichment on performance and emission characteristics of a single cylinder diesel engine operated with biodiesel blends have been investigated in this work. The methyl ester of jatropha biodiesel was selected as bio-diesel and four blends (B10, B20, B30, and B40 were selected for experimental investigations. The performance and emission characteristics were obtained for the these blends along with three oxygen enrichment flow rates (1, 3, and 5 L per minute using an oxygen cylinder at the air intake in the diesel engine. The performance and emission characteristics were studied and compared with the diesel and biodiesel. It was observed that, oxygen enrichment enhances the brake thermal efficiency, HC, CO, and smoke. B10 biodiesel with 5 L per minute oxygen enrichment was found to be the best fuel for biodiesel operation.

Design of inhibitors of thymidylate kinase from Variola virus as new selective drugs against smallpox.

Science.gov (United States)

Guimarães, Ana P; de Souza, Felipe R; Oliveira, Aline A; Gonçalves, Arlan S; de Alencastro, Ricardo B; Ramalho, Teodorico C; França, Tanos C C

2015-02-16

Recently we constructed a homology model of the enzyme thymidylate kinase from Variola virus (VarTMPK) and proposed it as a new target to the drug design against smallpox. In the present work, we used the antivirals cidofovir and acyclovir as reference compounds to choose eleven compounds as leads to the drug design of inhibitors for VarTMPK. Docking and molecular dynamics (MD) studies of the interactions of these compounds inside VarTMPK and human TMPK (HssTMPK) suggest that they compete for the binding region of the substrate and were used to propose the structures of ten new inhibitors for VarTMPK. Further docking and MD simulations of these compounds, inside VarTMPK and HssTMPK, suggest that nine among ten are potential selective inhibitors of VarTMPK. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Enzastaurin (LY317615), a Protein Kinase C Beta Selective Inhibitor, Enhances Antiangiogenic Effect of Radiation

International Nuclear Information System (INIS)

Willey, Christopher D.; Xiao Dakai; Tu Tianxiang; Kim, Kwang Woon; Moretti, Luigi; Niermann, Kenneth J.; Tawtawy, Mohammed N.; Quarles, Chad C. Ph.D.; Lu Bo

2010-01-01

Purpose: Angiogenesis has generated interest in oncology because of its important role in cancer growth and progression, particularly when combined with cytotoxic therapies, such as radiotherapy. Among the numerous pathways influencing vascular growth and stability, inhibition of protein kinase B(Akt) or protein kinase C(PKC) can influence tumor blood vessels within tumor microvasculature. Therefore, we wanted to determine whether PKC inhibition could sensitize lung tumors to radiation. Methods and Materials: The combination of the selective PKCβ inhibitor Enzastaurin (ENZ, LY317615) and ionizing radiation were used in cell culture and a mouse model of lung cancer. Lung cancer cell lines and human umbilical vascular endothelial cells (HUVEC) were examined using immunoblotting, cytotoxic assays including cell proliferation and clonogenic assays, and Matrigel endothelial tubule formation. In vivo, H460 lung cancer xenografts were examined for tumor vasculature and proliferation using immunohistochemistry. Results: ENZ effectively radiosensitizes HUVEC within in vitro models. Furthermore, concurrent ENZ treatment of lung cancer xenografts enhanced radiation-induced destruction of tumor vasculature and proliferation by IHC. However, tumor growth delay was not enhanced with combination treatment compared with either treatment alone. Analysis of downstream effectors revealed that HUVEC and the lung cancer cell lines differed in their response to ENZ and radiation such that only HUVEC demonstrate phosphorylated S6 suppression, which is downstream of mTOR. When ENZ was combined with the mTOR inhibitor, rapamycin, in H460 lung cancer cells, radiosensitization was observed. Conclusion: PKC appears to be crucial for angiogenesis, and its inhibition by ENZ has potential to enhance radiotherapy in vivo.

Autoregulation of kinase dephosphorylation by ATP binding in AGC protein kinases.

Science.gov (United States)

Chan, Tung O; Pascal, John M; Armen, Roger S; Rodeck, Ulrich

2012-02-01

AGC kinases, including the three Akt (protein kinase B) isoforms, protein kinase A (PKA) and all protein kinase C (PKC) isoforms, require activation loop phosphorylation (threonine 308 in Akt1) as well as phosphorylation of a C-terminal residue (serine 473 in Akt1) for catalytic activity and phosphorylation of downstream targets. Conversely, phosphatases reverse these phosphorylations. Virtually all cellular processes are affected by AGC kinases, a circumstance that has led to intense scrutiny of the molecular mechanisms that regulate phosphorylation of these kinases. Here, we review a new layer of control of phosphorylation in Akt, PKA and PKC pointing to ATP binding pocket occupancy as a means to decelerate dephosphorylation of these and, potentially, other kinases. This additional level of kinase regulation opens the door to search for new functional motifs for the rational design of non- ATP-competitive kinase inhibitors that discriminate within and between protein kinase families.

Cell-selective metabolic labeling of biomolecules with bioorthogonal functionalities.

Science.gov (United States)

Xie, Ran; Hong, Senlian; Chen, Xing

2013-10-01

Metabolic labeling of biomolecules with bioorthogonal functionalities enables visualization, enrichment, and analysis of the biomolecules of interest in their physiological environments. This versatile strategy has found utility in probing various classes of biomolecules in a broad range of biological processes. On the other hand, metabolic labeling is nonselective with respect to cell type, which imposes limitations for studies performed in complex biological systems. Herein, we review the recent methodological developments aiming to endow metabolic labeling strategies with cell-type selectivity. The cell-selective metabolic labeling strategies have emerged from protein and glycan labeling. We envision that these strategies can be readily extended to labeling of other classes of biomolecules. Copyright © 2013 Elsevier Ltd. All rights reserved.

Autoregulation of kinase dephosphorylation by ATP binding to AGC protein kinases

Science.gov (United States)

Pascal, John M; Armen, Roger S

2012-01-01

AGC kinases, including the three Akt (protein kinase B) isoforms, protein kinase A (PKA) and all protein kinase C (PKC) isoforms, require activation loop phosphorylation (threonine 308 in Akt1) as well as phosphorylation of a C-terminal residue (serine 473 in Akt1) for catalytic activity and phosphorylation of downstream targets. Conversely, phosphatases reverse these phosphorylations. Virtually all cellular processes are affected by AGC kinases, a circumstance that has led to intense scrutiny of the molecular mechanisms that regulate phosphorylation of these kinases. Here, we review a new layer of control of phosphorylation in Akt, PKA and PKC pointing to ATP binding pocket occupancy as a means to decelerate dephosphorylation of these and, potentially, other kinases. This additional level of kinase regulation opens the door to search for new functional motifs for the rational design of non-ATP-competitive kinase inhibitors that discriminate within and between protein kinase families. PMID:22262182

Selective decrease of components of the creatine kinase system and ATP synthase complex in chronic Chagas disease cardiomyopathy.

Directory of Open Access Journals (Sweden)

Priscila Camillo Teixeira

2011-06-01

Full Text Available BACKGROUND: Chronic Chagas disease cardiomyopathy (CCC is an inflammatory dilated cardiomyopathy with a worse prognosis than other cardiomyopathies. CCC occurs in 30 % of individuals infected with Trypanosoma cruzi, endemic in Latin America. Heart failure is associated with impaired energy metabolism, which may be correlated to contractile dysfunction. We thus analyzed the myocardial gene and protein expression, as well as activity, of key mitochondrial enzymes related to ATP production, in myocardial samples of end-stage CCC, idiopathic dilated (IDC and ischemic (IC cardiomyopathies. METHODOLOGY/PRINCIPAL FINDINGS: Myocardium homogenates from CCC (N=5, IC (N=5 and IDC (N=5 patients, as well as from heart donors (N=5 were analyzed for protein and mRNA expression of mitochondrial creatine kinase (CKMit and muscular creatine kinase (CKM and ATP synthase subunits aplha and beta by immunoblotting and by real-time RT-PCR. Total myocardial CK activity was also assessed. Protein levels of CKM and CK activity were reduced in all three cardiomyopathy groups. However, total CK activity, as well as ATP synthase alpha chain protein levels, were significantly lower in CCC samples than IC and IDC samples. CCC myocardium displayed selective reduction of protein levels and activity of enzymes crucial for maintaining cytoplasmic ATP levels. CONCLUSIONS/SIGNIFICANCE: The selective impairment of the CK system may be associated to the loss of inotropic reserve observed in CCC. Reduction of ATP synthase alpha levels is consistent with a decrease in myocardial ATP generation through oxidative phosphorylation. Together, these results suggest that the energetic deficit is more intense in the myocardium of CCC patients than in the other tested dilated cardiomyopathies.

Protein Kinase Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Promotes Obesity-induced Hyperinsulinemia.

Science.gov (United States)

Roth Flach, Rachel J; Danai, Laura V; DiStefano, Marina T; Kelly, Mark; Menendez, Lorena Garcia; Jurczyk, Agata; Sharma, Rohit B; Jung, Dae Young; Kim, Jong Hun; Kim, Jason K; Bortell, Rita; Alonso, Laura C; Czech, Michael P

2016-07-29

Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced β cell mass, fewer proliferating β cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from β cells in mice. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Unattended safeguards instrumentation at centrifuge enrichment plants

International Nuclear Information System (INIS)

Smith, L. Eric; Lebrun, Alain R.; Labella, Rocco

2014-01-01

As global uranium enrichment capacity under international safeguards expands, the International Atomic Energy Agency (IAEA) is challenged to develop effective safeguards approaches at gaseous centrifuge enrichment plants, particularly high‑capacity plants, while working within budgetary constraints. New safeguards approaches should meet the high‑level verification objectives for such facilities (i.e., timely detection of: diversion of declared material, excess production beyond declared amounts, and production of enrichment levels higher than declared), but should also strive for efficiency advantages in implementation, for both the IAEA and operators. Under the Agency’s State- level approach to safeguards implementation, the Agency needs a flexible toolbox of technologies, allowing tailoring of safeguards measures for each individual enrichment facility. In this paper, the potential roles and development status for three different types of unattended measurement instrumentation are discussed. On‑Line Enrichment Monitors (OLEM) could provide continuous enrichment measurement for 100% of the declared gas flowing through unit header pipes. Unattended Cylinder Verification Stations (UCVS) could provide unattended verification of the declared uranium mass and enrichment of 100% of the cylinders moving through the plant, but also apply and verify an ‘NDA Fingerprint’ to preserve verification knowledge on the contents of each cylinder throughout its life in the facility. Sharing of the operator’s load cell signals from feed and withdrawal stations could count all cylinders introduced to the process and provide periodic monitoring of the uranium mass balance for in‑process material. The integration of load cell, OLEM and UCVS data streams offers the possibility for 100% verification of declared cylinder flow, and enables the periodic verification of the declared 235 U mass balance in the plant. These new capabilities would enhance the IAEA�

Phosphoproteomic insights into processes influenced by the kinase-like protein DIA1/C3orf58

Directory of Open Access Journals (Sweden)

Agnieszka Hareza

2018-04-01

Full Text Available Many kinases are still ‘orphans,’ which means knowledge about their substrates, and often also about the processes they regulate, is lacking. Here, DIA1/C3orf58, a member of a novel predicted kinase-like family, is shown to be present in the endoplasmic reticulum and to influence trafficking via the secretory pathway. Subsequently, DIA1 is subjected to phosphoproteomics analysis to cast light on its signalling pathways. A liquid chromatography–tandem mass spectrometry proteomic approach with phosphopeptide enrichment is applied to membrane fractions of DIA1-overexpressing and control HEK293T cells, and phosphosites dependent on the presence of DIA1 are elucidated. Most of these phosphosites belonged to CK2- and proline-directed kinase types. In parallel, the proteomics of proteins immunoprecipitated with DIA1 reported its probable interactors. This pilot study provides the basis for deeper studies of DIA1 signalling.

Phosphoproteomic insights into processes influenced by the kinase-like protein DIA1/C3orf58.

Science.gov (United States)

Hareza, Agnieszka; Bakun, Magda; Świderska, Bianka; Dudkiewicz, Małgorzata; Koscielny, Alicja; Bajur, Anna; Jaworski, Jacek; Dadlez, Michał; Pawłowski, Krzysztof

2018-01-01

Many kinases are still 'orphans,' which means knowledge about their substrates, and often also about the processes they regulate, is lacking. Here, DIA1/C3orf58, a member of a novel predicted kinase-like family, is shown to be present in the endoplasmic reticulum and to influence trafficking via the secretory pathway. Subsequently, DIA1 is subjected to phosphoproteomics analysis to cast light on its signalling pathways. A liquid chromatography-tandem mass spectrometry proteomic approach with phosphopeptide enrichment is applied to membrane fractions of DIA1-overexpressing and control HEK293T cells, and phosphosites dependent on the presence of DIA1 are elucidated. Most of these phosphosites belonged to CK2- and proline-directed kinase types. In parallel, the proteomics of proteins immunoprecipitated with DIA1 reported its probable interactors. This pilot study provides the basis for deeper studies of DIA1 signalling.

Capability of the electromagnetic isotope-enrichment facility at ORNL

International Nuclear Information System (INIS)

Newman, E.

1982-01-01

The isotope separation program at Oak Ridge National Laboratory (ORNL) prepares and distributes electromagnetically enriched stable isotopes to the worldwide scientific community. Among the topics discussed in the present paper are the methods of enriching isotopes, the limitations that apply to the quantity and final assay of the separation products, and a generalized production flowsheet indicating the capability of the facility. A brief description of each of the production steps, from the selection and preparation of initial feedstock to the recovery and distribution of the isotopically enriched material, is presented. The future of the facility, the continued supply of enriched isotopes, and the response of the program to new and changing requirements are emphasized

Kinases and Cancer

OpenAIRE

Jonas Cicenas; Egle Zalyte; Amos Bairoch; Pascale Gaudet

2018-01-01

Protein kinases are a large family of enzymes catalyzing protein phosphorylation. The human genome contains 518 protein kinase genes, 478 of which belong to the classical protein kinase family and 40 are atypical protein kinases [...

Resistance of Akt kinases to dephosphorylation through ATP-dependent conformational plasticity.

Science.gov (United States)

Chan, Tung O; Zhang, Jin; Rodeck, Ulrich; Pascal, John M; Armen, Roger S; Spring, Maureen; Dumitru, Calin D; Myers, Valerie; Li, Xue; Cheung, Joseph Y; Feldman, Arthur M

2011-11-15

Phosphorylation of a threonine residue (T308 in Akt1) in the activation loop of Akt kinases is a prerequisite for deregulated Akt activity frequently observed in neoplasia. Akt phosphorylation in vivo is balanced by the opposite activities of kinases and phosphatases. Here we describe that targeting Akt kinase to the cell membrane markedly reduced sensitivity of phosphorylated Akt to dephosphorylation by protein phosphatase 2A. This effect was amplified by occupancy of the ATP binding pocket by either ATP or ATP-competitive inhibitors. Mutational analysis revealed that R273 in Akt1 and the corresponding R274 in Akt2 are essential for shielding T308 in the activation loop against dephosphorylation. Thus, occupancy of the nucleotide binding pocket of Akt kinases enables intramolecular interactions that restrict phosphatase access and sustain Akt phosphorylation. This mechanism provides an explanation for the "paradoxical" Akt hyperphosphorylation induced by ATP-competitive inhibitor, A-443654. The lack of phosphatase resistance further contributes insight into the mechanism by which the human Akt2 R274H missense mutation may cause autosomal-dominant diabetes mellitus.

Laser photochemical studies on di-isopropyl ether for oxygen-18 enrichment

International Nuclear Information System (INIS)

Mathi, P.; Kumar, Awadhesh; Ghosh, Ayan; Nayak, A.K.; Parthasarathy, V.; Nataraju, V.; Jadhav, K.A.; Babu, K.Rajendra; Sarkar, S.K.

2013-05-01

Oxygen-18 is needed for the production of Fluorine-18 in medical cyclotron for use in positron emission tomography. This report deals with our work on Oxygen-18 selective photo dissociation of natural di-isopropyl ether under various conditions leading to various oxygen bearing products having different levels of 18 O enrichment. Apart from obtaining 18 O enrichment in products 2-propanol and acetaldehyde, we have observed unusually high enrichment (about 39%) in another photoproduct, acetone, as measured by mass spectrometry. This new finding is attributed to 18 O selective secondary reaction channels which is supported by molecular orbital calculations. The investigation required characterization and quantitative estimation of various chemical species, viz., di-isopropyl ether, acetaldehyde, acetone and isopropanol by various instrumental methods of analysis. These methods include gas chromatography, Fourier transform infrared spectrometry and quadrupole mass spectrometry. Detailed Gas Chromatographic (GC) studies summarize the interference problems encountered for quantitatively identifying different photo-products and establish the right experimental conditions for optimum separation. This exercise is extremely useful for an isotope enrichment scheme as it generates a valuable database to understand the processes involved for both selectivity enhancement and degradation. (author)

Method of deuterium isotope separation and enrichment

International Nuclear Information System (INIS)

Benson, S.W.

1978-01-01

A method of separating deuterium, i.e., heavy hydrogen, from certain naturally occurring sources using tuned infrared lasers to selectively decompose specified classes of organic molecules (i.e., RX) into enriched molecular products containing deuterium atoms is described. The deuterium containing molecules are easily separated from the starting material by absorption, distillation or other simple chemical separation techniques and methods. After evaporation such deuterium containing molecules can be burned to form water with an enriched deuterium content or pyrolyzed to form hydrogen gas with an enriched deuterium content. The undecomposed molecules and the other reaction products which are depleted of their deuterium containing species can be catalytically treated, preferably using normal water, to restore the natural abundance of deuterium and such restored molecules can then be recycled

Peptide microarray analysis of substrate specificity of the transmembrane Ser/Thr kinase KPI-2 reveals reactivity with cystic fibrosis transmembrane conductance regulator and phosphorylase.

Science.gov (United States)

Wang, Hong; Brautigan, David L

2006-11-01

Human lemur (Lmr) kinases are predicted to be Tyr kinases based on sequences and are related to neurotrophin receptor Trk kinases. This study used homogeneous recombinant KPI-2 (Lmr2, LMTK2, Cprk, brain-enriched protein kinase) kinase domain and a library of 1,154 peptides on a microarray to analyze substrate specificity. We found that KPI-2 is strictly a Ser/Thr kinase that reacts with Ser either preceded by or followed by Pro residues but unlike other Pro-directed kinases does not strictly require an adjacent Pro residue. The most reactive peptide in the library corresponds to Ser-737 of cystic fibrosis transmembrane conductance regulator, and the recombinant R domain of cystic fibrosis transmembrane conductance regulator was a preferred substrate. Furthermore the KPI-2 kinase phosphorylated peptides corresponding to the single site in phosphorylase and purified phosphorylase b, making this only the second known phosphorylase b kinase. Phosphorylase was used as a specific substrate to show that KPI-2 is inhibited in living cells by addition of nerve growth factor or serum. The results demonstrate the utility of the peptide library to probe specificity and discover kinase substrates and offer a specific assay that reveals hormonal regulation of the activity of this unusual transmembrane kinase.

Selective Inhibition of Casein Kinase 1 epsilon Minimally Alters Circadian Clock Period

Czech Academy of Sciences Publication Activity Database

Walton, K. M.; Fisher, K.; Rubitski, D.; Marconi, M.; Meng, Q.-J.; Sládek, Martin; Adams, J.; Bass, M.; Chandrasekaran, R.; Butler, T.; Griffor, M.; Rajamohan, F.; Serpa, M.; Chen, Y.; Claffey, M.; Hastings, M.; Loudon, A.; Maywood, E.; Ohren, J.; Doran, A.; Wager, T. T.

2009-01-01

Roč. 330, č. 2 (2009), s. 430-439 ISSN 0022-3565 Institutional research plan: CEZ:AV0Z50110509 Keywords : circadian clock * casein kinase 1 epsilon * inhibitor Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.093, year: 2009

Preparation and characterization of fluorophenylboronic acid-functionalized affinity monolithic columns for the selective enrichment of cis-diol-containing biomolecules.

Science.gov (United States)

Li, Qianjin; Liu, Zhen

2015-01-01

Boronate affinity monolithic columns have been developed into an important means for the selective recognition and capture of cis-diol-containing biomolecules, such as glycoproteins, nucleosides and saccharides. The ligands of boronic acids are playing an important role in boronate affinity monolithic columns. Although several boronate affinity monoliths with high affinity toward cis-diol-containing biomolecules have been reported, only few publications are focused on their detailed procedures for preparation and characterization. This chapter describes in detail the preparation and characterization of a boronate affinity monolithic column applying 2,4-difluoro-3-formyl-phenylboronic acid (DFFPBA) as a ligand. The DFFPBA-functionalized monolithic column not only exhibited an ultrahigh boronate affinity toward cis-diol-containing biomolecules, but also showed great potential for the selective enrichment of cis-diol-containing biomolecules in real samples.

Identification of p38α MAP kinase inhibitors by pharmacophore based virtual screening

DEFF Research Database (Denmark)

Gangwal, Rahul P; Das, Nihar R; Thanki, Kaushik

2014-01-01

The p38α mitogen-activated protein (MAP) kinase plays a vital role in treating many inflammatory diseases. In the present study, a combined ligand and structure based pharmacophore model was developed to identify potential DFG-in selective p38 MAP kinase inhibitors. Conformations of co...

Identification and Characterization of Amlexanox as a G Protein-Coupled Receptor Kinase 5 Inhibitor

Directory of Open Access Journals (Sweden)

Kristoff T. Homan

2014-10-01

Full Text Available G protein-coupled receptor kinases (GRKs have been implicated in human diseases ranging from heart failure to diabetes. Previous studies have identified several compounds that selectively inhibit GRK2, such as paroxetine and balanol. Far fewer selective inhibitors have been reported for GRK5, a target for the treatment of cardiac hypertrophy, and the mechanism of action of reported compounds is unknown. To identify novel scaffolds that selectively inhibit GRK5, a differential scanning fluorometry screen was used to probe a library of 4480 compounds. The best hit was amlexanox, an FDA-approved anti-inflammatory, anti-allergic immunomodulator. The crystal structure of amlexanox in complex with GRK1 demonstrates that its tricyclic aromatic ring system forms ATP-like interactions with the hinge of the kinase domain, which is likely similar to how this drug binds to IκB kinase ε (IKKε, another kinase known to be inhibited by this compound. Amlexanox was also able to inhibit myocyte enhancer factor 2 transcriptional activity in neonatal rat ventricular myocytes in a manner consistent with GRK5 inhibition. The GRK1 amlexanox structure thus serves as a springboard for the rational design of inhibitors with improved potency and selectivity for GRK5 and IKKε.

Selective laser melting-enabled electrospinning: Introducing complexity within electrospun membranes.

Science.gov (United States)

Paterson, Thomas E; Beal, Selina N; Santocildes-Romero, Martin E; Sidambe, Alfred T; Hatton, Paul V; Asencio, Ilida Ortega

2017-06-01

Additive manufacturing technologies enable the creation of very precise and well-defined structures that can mimic hierarchical features of natural tissues. In this article, we describe the development of a manufacturing technology platform to produce innovative biodegradable membranes that are enhanced with controlled microenvironments produced via a combination of selective laser melting techniques and conventional electrospinning. This work underpins the manufacture of a new generation of biomaterial devices that have significant potential for use as both basic research tools and components of therapeutic implants. The membranes were successfully manufactured and a total of three microenvironment designs (niches) were chosen for thorough characterisation. Scanning electron microscopy analysis demonstrated differences in fibre diameters within different areas of the niche structures as well as differences in fibre density. We also showed the potential of using the microfabricated membranes for supporting mesenchymal stromal cell culture and proliferation. We demonstrated that mesenchymal stromal cells grow and populate the membranes penetrating within the niche-like structures. These findings demonstrate the creation of a very versatile tool that can be used in a variety of tissue regeneration applications including bone healing.

Key enzymes enabling the growth of Arthrobacter sp. strain JBH1 with nitroglycerin as the sole source of carbon and nitrogen.

Science.gov (United States)

Husserl, Johana; Hughes, Joseph B; Spain, Jim C

2012-05-01

Flavoprotein reductases that catalyze the transformation of nitroglycerin (NG) to dinitro- or mononitroglycerols enable bacteria containing such enzymes to use NG as the nitrogen source. The inability to use the resulting mononitroglycerols limits most strains to incomplete denitration of NG. Recently, Arthrobacter strain JBH1 was isolated for the ability to grow on NG as the sole source of carbon and nitrogen, but the enzymes and mechanisms involved were not established. Here, the enzymes that enable the Arthrobacter strain to incorporate NG into a productive pathway were identified. Enzyme assays indicated that the transformation of nitroglycerin to mononitroglycerol is NADPH dependent and that the subsequent transformation of mononitroglycerol is ATP dependent. Cloning and heterologous expression revealed that a flavoprotein catalyzes selective denitration of NG to 1-mononitroglycerol (1-MNG) and that 1-MNG is transformed to 1-nitro-3-phosphoglycerol by a glycerol kinase homolog. Phosphorylation of the nitroester intermediate enables the subsequent denitration of 1-MNG in a productive pathway that supports the growth of the isolate and mineralization of NG.

UF6 test loop for evaluation and implementation of international enrichment plant safeguards

International Nuclear Information System (INIS)

Cooley, J.N.; Fields, L.W.; Swindle, D.W. Jr.

1987-06-01

A functional test loop capable of simulating UF 6 flows, pressures, and pipe deposits characteristic of gas centrifuge enrichment plant piping has been designed and fabricated by the Enrichment Safeguards Program of Martin Marietta Energy Systems, Inc., for use by International Atomic Energy Agency (IAEA) at its Safeguards Analytical Laboratory in Seibersdorf, Austria. Purpose of the test loop is twofold: (1) to enable the IAEA to evaluate and to calibrate enrichment safeguards measurement instrumentation to be used in limited frequency-unannounced access (LFUA) inspection strategy measurements at gas centrifuge enrichment plants and (2) to train IAEA inspectors in the use of such instrumentation. The test loop incorporates actual sections of cascade header pipes from the centrifuge enrichment plants subject to IAEA inspections. The test loop is described, applications for its use by the IAEA are detailed, and results from an initial demonstration session using the test loop are summarized

Modular enrichment measurement system for in-situ enrichment assay

International Nuclear Information System (INIS)

Stewart, J.P.

1976-01-01

A modular enrichment measurement system has been designed and is in operation within General Electric's Nuclear Fuel Fabrication Facility for the in-situ enrichment assay of uranium-bearing materials in process containers. This enrichment assay system, which is based on the ''enrichment meter'' concept, is an integral part of the site's enrichment control program and is used in the in-situ assay of the enrichment of uranium dioxide (UO 2 ) powder in process containers (five gallon pails). The assay system utilizes a commercially available modular counting system and a collimnator designed for compatability with process container transport lines and ease of operator access. The system has been upgraded to include a microprocessor-based controller to perform system operation functions and to provide data acquisition and processing functions. Standards have been fabricated and qualified for the enrichment assay of several types of uranium-bearing materials, including UO 2 powders. The assay system has performed in excess of 20,000 enrichment verification measurements annually and has significantly contributed to the facility's enrichment control program

Phosphopeptide enrichment by immobilized metal affinity chromatography

DEFF Research Database (Denmark)

Thingholm, Tine E.; Larsen, Martin R.

2016-01-01

Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively...... charged metal ions such as Fe3+, Ga3+, Al3+, Zr4+, and Ti4+ has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from...

A novel strategy for global mapping of O-GlcNAc proteins and peptides using selective enzymatic deglycosylation, HILIC enrichment and mass spectrometry identification.

Science.gov (United States)

Shen, Bingquan; Zhang, Wanjun; Shi, Zhaomei; Tian, Fang; Deng, Yulin; Sun, Changqing; Wang, Guangshun; Qin, Weijie; Qian, Xiaohong

2017-07-01

O-GlcNAcylation is a kind of dynamic O-linked glycosylation of nucleocytoplasmic and mitochondrial proteins. It serves as a major nutrient sensor to regulate numerous biological processes including transcriptional regulation, cell metabolism, cellular signaling, and protein degradation. Dysregulation of cellular O-GlcNAcylated levels contributes to the etiologies of many diseases such as diabetes, neurodegenerative disease and cancer. However, deeper insight into the biological mechanism of O-GlcNAcylation is hampered by its extremely low stoichiometry and the lack of efficient enrichment approaches for large-scale identification by mass spectrometry. Herein, we developed a novel strategy for the global identification of O-GlcNAc proteins and peptides using selective enzymatic deglycosylation, HILIC enrichment and mass spectrometry analysis. Standard O-GlcNAc peptides can be efficiently enriched even in the presence of 500-fold more abundant non-O-GlcNAc peptides and identified by mass spectrometry with a low nanogram detection sensitivity. This strategy successfully achieved the first large-scale enrichment and characterization of O-GlcNAc proteins and peptides in human urine. A total of 474 O-GlcNAc peptides corresponding to 457 O-GlcNAc proteins were identified by mass spectrometry analysis, which is at least three times more than that obtained by commonly used enrichment methods. A large number of unreported O-GlcNAc proteins related to cell cycle, biological regulation, metabolic and developmental process were found in our data. The above results demonstrated that this novel strategy is highly efficient in the global enrichment and identification of O-GlcNAc peptides. These data provide new insights into the biological function of O-GlcNAcylation in human urine, which is correlated with the physiological states and pathological changes of human body and therefore indicate the potential of this strategy for biomarker discovery from human urine. Copyright �

Hammond Postulate Mirroring Enables Enantiomeric Enrichment of Phosphorus Compounds via Two Thermodynamically Interconnected Sequential Stereoselective Processes.

Science.gov (United States)

Rajendran, Kamalraj V; Nikitin, Kirill V; Gilheany, Declan G

2015-07-29

The dynamic resolution of tertiary phosphines and phosphine oxides was monitored by NMR spectroscopy. It was found that the stereoselectivity is set during the formation of the diastereomeric alkoxyphosphonium salts (DAPS), such that their initial diastereomeric excess (de) limits the final enantiomeric excess (ee) of any phosphorus products derived from them. However, (31)P NMR monitoring of the spontaneous thermal decomposition of the DAPS shows consistent diastereomeric self-enrichment, indicating a higher rate constant for decomposition of the minor diastereomer. This crucial observation was confirmed by reductive trapping of the unreacted enriched DAPS with lithium tri-sec-butylborohydride (commercially distributed as L-Selectride reagent) at different time intervals after the start of reaction, which gives progressively higher ee of the phosphine product with time. It is proposed that the Hammond postulate operates for both formation and decomposition of DAPS intermediate so that the lower rate of formation and faster subsequent collapse of the minor isomer are thermodynamically linked. This kinetic enhancement of kinetic resolution furnishes up to 97% ee product.

Striatal-enriched Tyrosine Protein Phosphatase (STEP) in the Mechanisms of Depressive Disorders.

Science.gov (United States)

Kulikova, Elizabeth; Kulikov, Alexander

2017-08-30

Striatal-enriched tyrosine protein phosphatase (STEP) is expressed mainly in the brain. Its dysregulation is associated with Alzheimer's and Huntington's diseases, schizophrenia, fragile X syndrome, drug abuse and stroke/ischemia. However, an association between STEP and depressive disorders is still obscure. The review discusses the theoretical foundations and experimental facts concerning possible relationship between STEP dysregulation and depression risk. STEP dephosphorylates and inactivates several key neuronal signaling proteins such as extracellular signal-regulating kinase 1 and 2 (ERK1/2), stress activated protein kinases p38, the Src family tyrosine kinases Fyn, Pyk2, NMDA and AMPA glutamate receptors. The inactivation of these proteins decreases the expression of brain derived neurotrophic factor (BDNF) necessary for neurogenesis and neuronal survival. The deficit of BDNF results in progressive degeneration of neurons in the hippocampus and cortex and increases depression risk. At the same time, a STEP inhibitor, 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride (TC-2153), increases BDNF expression in the hippocampus and attenuated the depressivelike behavior in mice. Thus, STEP is involved in the mechanism of depressive disorders and it is a promising molecular target for atypical antidepressant drugs of new generation. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Fragment-Based Drug Discovery of Potent Protein Kinase C Iota Inhibitors.

Science.gov (United States)

Kwiatkowski, Jacek; Liu, Boping; Tee, Doris Hui Ying; Chen, Guoying; Ahmad, Nur Huda Binte; Wong, Yun Xuan; Poh, Zhi Ying; Ang, Shi Hua; Tan, Eldwin Sum Wai; Ong, Esther Hq; Nurul Dinie; Poulsen, Anders; Pendharkar, Vishal; Sangthongpitag, Kanda; Lee, May Ann; Sepramaniam, Sugunavathi; Ho, Soo Yei; Cherian, Joseph; Hill, Jeffrey; Keller, Thomas H; Hung, Alvin W

2018-05-24

Protein kinase C iota (PKC-ι) is an atypical kinase implicated in the promotion of different cancer types. A biochemical screen of a fragment library has identified several hits from which an azaindole-based scaffold was chosen for optimization. Driven by a structure-activity relationship and supported by molecular modeling, a weakly bound fragment was systematically grown into a potent and selective inhibitor against PKC-ι.

Induction of viral, 7-methyl-guanosine cap-independent translation and oncolysis by mitogen-activated protein kinase-interacting kinase-mediated effects on the serine/arginine-rich protein kinase.

Science.gov (United States)

Brown, Michael C; Bryant, Jeffrey D; Dobrikova, Elena Y; Shveygert, Mayya; Bradrick, Shelton S; Chandramohan, Vidyalakshmi; Bigner, Darell D; Gromeier, Matthias

2014-11-01

Protein synthesis, the most energy-consuming process in cells, responds to changing physiologic priorities, e.g., upon mitogen- or stress-induced adaptations signaled through the mitogen-activated protein kinases (MAPKs). The prevailing status of protein synthesis machinery is a viral pathogenesis factor, particularly for plus-strand RNA viruses, where immediate translation of incoming viral RNAs shapes host-virus interactions. In this study, we unraveled signaling pathways centered on the ERK1/2 and p38α MAPK-interacting kinases MNK1/2 and their role in controlling 7-methyl-guanosine (m(7)G) "cap"-independent translation at enterovirus type 1 internal ribosomal entry sites (IRESs). Activation of Raf-MEK-ERK1/2 signals induced viral IRES-mediated translation in a manner dependent on MNK1/2. This effect was not due to MNK's known functions as eukaryotic initiation factor (eIF) 4G binding partner or eIF4E(S209) kinase. Rather, MNK catalytic activity enabled viral IRES-mediated translation/host cell cytotoxicity through negative regulation of the Ser/Arg (SR)-rich protein kinase (SRPK). Our investigations suggest that SRPK activity is a major determinant of type 1 IRES competency, host cell cytotoxicity, and viral proliferation in infected cells. We are targeting unfettered enterovirus IRES activity in cancer with PVSRIPO, the type 1 live-attenuated poliovirus (PV) (Sabin) vaccine containing a human rhinovirus type 2 (HRV2) IRES. A phase I clinical trial of PVSRIPO with intratumoral inoculation in patients with recurrent glioblastoma (GBM) is showing early promise. Viral translation proficiency in infected GBM cells is a core requirement for the antineoplastic efficacy of PVSRIPO. Therefore, it is critically important to understand the mechanisms controlling viral cap-independent translation in infected host cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Proteinase K processing of rabbit muscle creatine kinase

DEFF Research Database (Denmark)

Leydier, C; Andersen, Jens S.; Couthon, F

1997-01-01

Proteinase K cleaves selectively both cytosolic and mitochondrial isoforms of creatine kinase leading to the appearance of two fragments, a large N-terminal one (K1) and a small C-terminal peptide (K2) which remain associated together. The loss of enzymatic activity correlates with the extent...... of monomer cleavage. N-terminal sequencing of the K2 fragments from rabbit cytosolic and pig mitochondrial creatine kinase shows that these peptides begin with A328 and A324, respectively. Electrospray ionization mass spectrometry demonstrates that K2 peptide is composed of 53 residues (A328-K380). However...

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay.

Science.gov (United States)

Cui, Heying; Loftus, Kyle M; Noell, Crystal R; Solmaz, Sozanne R

2018-05-03

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer. Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin α. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Mitochondrial capture enriches mito-DNA 100 fold, enabling PCR-free mitogenomics biodiversity analysis

DEFF Research Database (Denmark)

Liu, Shanlin; Wang, Xin; Xie, Lin

2016-01-01

Biodiversity analyses based on next-generation sequencing (NGS) platforms have developed by leaps and bounds in recent years. A PCR-free strategy, which can alleviate taxonomic bias, was considered as a promising approach to delivering reliable species compositions of targeted environments...... data is highly demanding on computing resources. Here, we present a mitogenome enrichment pipeline via a gene capture chip that was designed by virtue of the mitogenome sequences of the 1000 Insect Transcriptome Evolution project (1KITE, www.1kite.org). A mock sample containing 49 species was used...... in abundance. However, the frequencies of input taxa were largely maintained after capture (R2 = 0.81). We suggest that our mitogenome capture approach coupled with PCR-free shotgun sequencing could provide ecological researchers an efficient NGS method to deliver reliable biodiversity assessment....

Arctigenin protects against steatosis in WRL68 hepatocytes through activation of phosphoinositide 3-kinase/protein kinase B and AMP-activated protein kinase pathways.

Science.gov (United States)

Chen, Kung-Yen; Lin, Jui-An; Yao, Han-Yun; Hsu, An-Chih; Tai, Yu-Ting; Chen, Jui-Tai; Hsieh, Mao-Chih; Shen, Tang-Long; Hsu, Ren-Yi; Wu, Hong-Tan; Wang, Guey Horng; Ho, Bing-Ying; Chen, Yu-Pei

2018-04-01

Arctigenin (ATG), a lignin extracted from Arctium lappa (L.), exerts antioxidant and anti-inflammatory effects. We hypothesized that ATG exerts a protective effect on hepatocytes by preventing nonalcoholic fatty liver disease (NAFLD) progression associated with lipid oxidation-associated lipotoxicity and inflammation. We established an in vitro NAFLD cell model by using normal WRL68 hepatocytes to investigate oleic acid (OA) accumulation and the potential bioactive role of ATG. The results revealed that ATG inhibited OA-induced lipid accumulation, lipid peroxidation, and inflammation in WRL68 hepatocytes, as determined using Oil Red O staining, thiobarbituric acid reactive substance assay, and inflammation antibody array assays. Quantitative RT-PCR analysis demonstrated that ATG significantly mitigated the expression of acetylcoenzyme A carboxylase 1 and sterol regulatory element-binding protein-1 and significantly increased the expression of carnitine palmitoyltransferase 1 and peroxisome proliferator-activated receptor alpha. The 40 targets of the Human Inflammation Antibody Array indicated that ATG significantly inhibited the elevation of the U937 lymphocyte chemoattractant, ICAM-1, IL-1β, IL-6, IL-6sR, IL-7, and IL-8. ATG could activate the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and AMP-activated protein kinase (AMPK) pathways and could increase the phosphorylation levels of Akt and AMPK to mediate cell survival, lipid metabolism, oxidation stress, and inflammation. Thus, we demonstrated that ATG could inhibit NAFLD progression associated with lipid oxidation-associated lipotoxicity and inflammation, and we provided insights into the underlying mechanisms and revealed potential targets to enable a thorough understanding of NAFLD progression. Copyright © 2018 Elsevier Inc. All rights reserved.

Systems biology analysis of mitogen activated protein kinase inhibitor resistance in malignant melanoma.

Science.gov (United States)

Zecena, Helma; Tveit, Daniel; Wang, Zi; Farhat, Ahmed; Panchal, Parvita; Liu, Jing; Singh, Simar J; Sanghera, Amandeep; Bainiwal, Ajay; Teo, Shuan Y; Meyskens, Frank L; Liu-Smith, Feng; Filipp, Fabian V

2018-04-04

Kinase inhibition in the mitogen activated protein kinase (MAPK) pathway is a standard therapy for cancer patients with activating BRAF mutations. However, the anti-tumorigenic effect and clinical benefit are only transient, and tumors are prone to treatment resistance and relapse. To elucidate mechanistic insights into drug resistance, we have established an in vitro cellular model of MAPK inhibitor resistance in malignant melanoma. The cellular model evolved in response to clinical dosage of the BRAF inhibitor, vemurafenib, PLX4032. We conducted transcriptomic expression profiling using RNA-Seq and RT-qPCR arrays. Pathways of melanogenesis, MAPK signaling, cell cycle, and metabolism were significantly enriched among the set of differentially expressed genes of vemurafenib-resistant cells vs control. The underlying mechanism of treatment resistance and pathway rewiring was uncovered to be based on non-genomic adaptation and validated in two distinct melanoma models, SK-MEL-28 and A375. Both cell lines have activating BRAF mutations and display metastatic potential. Downregulation of dual specific phosphatases, tumor suppressors, and negative MAPK regulators reengages mitogenic signaling. Upregulation of growth factors, cytokines, and cognate receptors triggers signaling pathways circumventing BRAF blockage. Further, changes in amino acid and one-carbon metabolism support cellular proliferation despite MAPK inhibitor treatment. In addition, treatment-resistant cells upregulate pigmentation and melanogenesis, pathways which partially overlap with MAPK signaling. Upstream regulator analysis discovered significant perturbation in oncogenic forkhead box and hypoxia inducible factor family transcription factors. The established cellular models offer mechanistic insight into cellular changes and therapeutic targets under inhibitor resistance in malignant melanoma. At a systems biology level, the MAPK pathway undergoes major rewiring while acquiring inhibitor resistance

What enables size-selective trophy hunting of wildlife?

Directory of Open Access Journals (Sweden)

Chris T Darimont

Full Text Available Although rarely considered predators, wildlife hunters can function as important ecological and evolutionary agents. In part, their influence relates to targeting of large reproductive adults within prey populations. Despite known impacts of size-selective harvests, however, we know little about what enables hunters to kill these older, rarer, and presumably more wary individuals. In other mammalian predators, predatory performance varies with knowledge and physical condition, which accumulates and declines, respectively, with age. Moreover, some species evolved camouflage as a physical trait to aid in predatory performance. In this work, we tested whether knowledge-based faculty (use of a hunting guide with accumulated experience in specific areas, physical traits (relative body mass [RBM] and camouflage clothing, and age can predict predatory performance. We measured performance as do many hunters: size of killed cervid prey, using the number of antler tines as a proxy. Examining ∼ 4300 online photographs of hunters posing with carcasses, we found that only the presence of guides increased the odds of killing larger prey. Accounting for this effect, modest evidence suggested that unguided hunters presumably handicapped with the highest RBM actually had greater odds of killing large prey. There was no association with hunter age, perhaps because of our coarse measure (presence of grey hair and the performance trade-offs between knowledge accumulation and physical deterioration with age. Despite its prevalence among sampled hunters (80%, camouflage had no influence on size of killed prey. Should these patterns be representative of other areas and prey, and our interpretations correct, evolutionarily-enlightened harvest management might benefit from regulatory scrutiny on guided hunting. More broadly, we suggest that by being nutritionally and demographically de-coupled from prey and aided by efficient killing technology and road access

Wall-associated kinase-like polypeptide mediates nutritional status perception and response

Science.gov (United States)

Yang, Zhenbiao; Karr, Stephen

2014-02-11

The disclosure relates to methods for modulating plant growth and organogenesis using dominant-negative receptor-like kinases. The disclosure further provides a method for increasing plant yield relative to corresponding wild type plants comprising modulating the expression in a plant of a nucleic acid encoding a Wall-Associated Kinase-like 14 polypeptide or a homolog thereof, and selecting for plants having increased yield or growth on a nutrient deficient substrate.

Essential amino acid enriched high-protein enteral nutrition modulates insulin-like growth factor-1 system function in a rat model of trauma-hemorrhagic shock.

Directory of Open Access Journals (Sweden)

Xianfeng Xia

Full Text Available Nutrition support for critically ill patients supplemented with additional modular protein may promote skeletal muscle protein anabolism in addition to counteracting acute nitrogen loss. The present study was designed to investigate whether the essential amino acid (EAA enriched high-protein enteral nutrition (EN modulates the insulin-like growth factor-1 (IGF-1 system and activates the mammalian target of rapamycin (mTOR anabolic signaling pathway in a trauma-hemorrhagic shock (T-HS rat model.Male Sprague-Dawley rats (n = 90, 278.18 ± 0.94 g were randomly assigned to 5 groups: (1 normal control, (2 pair-fed, (3 T-HS, (4 T-HS and standard EN, and (5 T-HS and EAA enriched high-protein EN. Six animals from each group were harvested on days 2, 4, and 6 for serum, gastrocnemius, soleus, and extensor digitorum longus sample collection. T-HS significantly reduced muscle mass. Nutrition support maintained muscle mass, especially the EAA enriched high-protein EN. Meanwhile, a pronounced derangement in IGF-1-IGFBPs axis as well as impaired mTOR transduction was observed in the T-HS group. Compared with animals receiving standard EN, those receiving EAA enriched high-protein EN presented 18% higher serum free IGF-1 levels following 3 days of nutrition support and 22% higher after 5 days. These changes were consistent with the concomitant elevation in serum insulin and reduction in corticosterone levels. In addition, phosphorylations of downstream anabolic signaling effectors - including protein kinase B, mTOR, and ribosomal protein S6 kinase1 - increased significantly in rats receiving EAA enriched high-protein EN.Our findings firstly demonstrate the beneficial effect of EAA enriched high-protein EN on the metabolic modulation of skeletal muscle protein anabolism by regulating the IGF-1 system and downstream anabolic signaling transduction.

Oncogenic Receptor Tyrosine Kinases Directly Phosphorylate Focal Adhesion Kinase (FAK) as a Resistance Mechanism to FAK-Kinase Inhibitors.

Science.gov (United States)

Marlowe, Timothy A; Lenzo, Felicia L; Figel, Sheila A; Grapes, Abigail T; Cance, William G

2016-12-01

Focal adhesion kinase (FAK) is a major drug target in cancer and current inhibitors targeted to the ATP-binding pocket of the kinase domain have entered clinical trials. However, preliminary results have shown limited single-agent efficacy in patients. Despite these unfavorable data, the molecular mechanisms that drive intrinsic and acquired resistance to FAK-kinase inhibitors are largely unknown. We have demonstrated that receptor tyrosine kinases (RTK) can directly bypass FAK-kinase inhibition in cancer cells through phosphorylation of FAK's critical tyrosine 397 (Y397). We also showed that HER2 forms a direct protein-protein interaction with the FAK-FERM-F1 lobe, promoting direct phosphorylation of Y397. In addition, FAK-kinase inhibition induced two forms of compensatory RTK reprogramming: (i) the rapid phosphorylation and activation of RTK signaling pathways in RTK High cells and (ii) the long-term acquisition of RTKs novel to the parental cell line in RTK Low cells. Finally, HER2 +: cancer cells displayed resistance to FAK-kinase inhibition in 3D growth assays using a HER2 isogenic system and HER2 + cancer cell lines. Our data indicate a novel drug resistance mechanism to FAK-kinase inhibitors whereby HER2 and other RTKs can rescue and maintain FAK activation (pY397) even in the presence of FAK-kinase inhibition. These data may have important ramifications for existing clinical trials of FAK inhibitors and suggest that individual tumor stratification by RTK expression would be important to predict patient response to FAK-kinase inhibitors. Mol Cancer Ther; 15(12); 3028-39. ©2016 AACR. ©2016 American Association for Cancer Research.

Photoactivatable Caged Prodrugs of VEGFR-2 Kinase Inhibitors

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Boris Pinchuk

2016-04-01

Full Text Available In this study, we report on the design, synthesis, photokinetic properties and in vitro evaluation of photoactivatable caged prodrugs for the receptor tyrosine kinase VEGFR-2. Highly potent VEGFR-2 inhibitors 1 and 3 were caged by introduction of a photoremovable protecting group (PPG to yield the caged prodrugs 4 and 5. As expected, enzymatic and cellular proliferation assays showed dramatically diminished efficacy of caged prodrugs in vitro. Upon ultraviolet (UV irradiation of the prodrugs original inhibitory activity was completely restored and even distinctly reinforced, as was the case for the prodrug 4. The presented results are a further evidence for caging technique being an interesting approach in the protein kinase field. It could enable spatial and temporal control for the inhibition of VEGFR-2. The described photoactivatable prodrugs might be highly useful as biological probes for studying the VEGFR-2 signal transduction.

Study on behaviors and performances of universal N-glycopeptide enrichment methods.

Science.gov (United States)

Xue, Yu; Xie, Juanjuan; Fang, Pan; Yao, Jun; Yan, Guoquan; Shen, Huali; Yang, Pengyuan

2018-04-16

Glycosylation is a crucial process in protein biosynthesis. However, the analysis of glycopeptides through MS remains challenging due to the microheterogeneity and macroheterogeneity of the glycoprotein. Selective enrichment of glycopeptides from complex samples prior to MS analysis is essential for successful glycoproteome research. In this work, we systematically investigated the behaviors and performances of boronic acid chemistry, ZIC-HILIC, and PGC of glycopeptide enrichment to promote understanding of these methods. We also optimized boronic acid chemistry and ZIC-HILIC enrichment methods and applied them to enrich glycopeptides from mouse liver. The intact N-glycopeptides were interpreted using the in-house analysis software pGlyco 2.0. We found that boronic acid chemistry in this study preferred to capture glycopeptides with high mannose glycans, ZIC-HILIC enriched most N-glycopeptides and did not show significant preference during enrichment and PGC was not suitable for separating glycopeptides with a long amino acid sequence. We performed a detailed study on the behaviors and performances of boronic acid chemistry, ZIC-HILIC, and PGC enrichment methods and provide a better understanding of enrichment methods for further glycoproteomics research.

STK33 kinase inhibitor BRD-8899 has no effect on KRAS-dependent cancer cell viability.

Science.gov (United States)

Luo, Tuoping; Masson, Kristina; Jaffe, Jacob D; Silkworth, Whitney; Ross, Nathan T; Scherer, Christina A; Scholl, Claudia; Fröhling, Stefan; Carr, Steven A; Stern, Andrew M; Schreiber, Stuart L; Golub, Todd R

2012-02-21

Approximately 30% of human cancers harbor oncogenic gain-of-function mutations in KRAS. Despite interest in KRAS as a therapeutic target, direct blockade of KRAS function with small molecules has yet to be demonstrated. Based on experiments that lower mRNA levels of protein kinases, KRAS-dependent cancer cells were proposed to have a unique requirement for the serine/threonine kinase STK33. Thus, it was suggested that small-molecule inhibitors of STK33 might have therapeutic benefit in these cancers. Here, we describe the development of selective, low nanomolar inhibitors of STK33's kinase activity. The most potent and selective of these, BRD8899, failed to kill KRAS-dependent cells. While several explanations for this result exist, our data are most consistent with the view that inhibition of STK33's kinase activity does not represent a promising anti-KRAS therapeutic strategy.

Dissection of Protein Kinase Pathways in Live Cells Using Photoluminescent Probes: Surveillance or Interrogation?

Directory of Open Access Journals (Sweden)

Darja Lavogina

2018-04-01

Full Text Available Protein kinases catalyze phosphorylation, a small yet crucial modification that affects participation of the substrate proteins in the intracellular signaling pathways. The activity of 538 protein kinases encoded in human genome relies upon spatiotemporally controlled mechanisms, ensuring correct progression of virtually all physiological processes on the cellular level—from cell division to cell death. The aberrant functioning of protein kinases is linked to a wide spectrum of major health issues including cancer, cardiovascular diseases, neurodegenerative diseases, inflammatory diseases, etc. Hence, significant effort of scientific community has been dedicated to the dissection of protein kinase pathways in their natural milieu. The combination of recent advances in the field of light microscopy, the wide variety of genetically encoded or synthetic photoluminescent scaffolds, and the techniques for intracellular delivery of cargoes has enabled design of a plethora of probes that can report activation of target protein kinases in human live cells. The question remains: how much do we bias intracellular signaling of protein kinases by monitoring it? This review seeks answers to this question by analyzing different classes of probes according to their general structure, mechanism of recognition of biological target, and optical properties necessary for the reporting of intracellular events.

Cyclin dependent kinase 5 regulates endocytosis in nerve terminals via dynamin I phosphorylation

International Nuclear Information System (INIS)

Tan, T.C.; Hansra, G.; Calova, V.; Cousin, M.; Robinson, P.J.

2002-01-01

Full text: Synaptic vesicle endocytosis (SVE) in nerve terminals is essential for normal synaptic transmission and for memory retrieval. Dynamin I is a 96kDa nerve terminal phosphoprotein necessary for synaptic vesicle endocytosis in the nerve terminal. Dynamin I is dephosphorylated and rephosphorylated in a cyclical fashion with nerve terminal depolarisation and repolarisation. A number of kinases phosphorylate dynamin I in vitro including PKC, MAP kinase and cdc2. PKC phosphorylates dynamin in the proline rich domain on Ser 795 and is also thought to be the in vivo kinase for dynamin I. Another candidate is the neuron specific kinase cdk5, crucial for CNS development. The aim of this study is to identify the kinase which phosphorylates dynamin I in intact nerve terminals. Here we show that cyclin-dependent kinase 5 (cdk5) phosphorylates dynamin I in the proline-rich tail on Ser-774 or Ser-778. The phosphorylation of these sites but not Ser-795 also occurred in intact nerve terminals suggesting that cdk5 is the physiologically relevant enzyme for dynamin I. Synaptosomes prepared from rat brains (after cervical dislocations) and labelled with 32 Pi, were incubated with 100 M roscovitine (a selective inhibitor of cdks), 10 M Ro 31-8220 (a selective PKC inhibitor) and 100 M PD 98059 (a MEK kinase inhibitor). Dynamin rephosphorylation during repolarisation was reduced in synaptosomes treated with roscovitine and Ro 38-8220 but not in synaptosomes treated with PD 98059. Fluorimetric experiments on intact synaptosomes utilising FM-210 (a fluorescent dye) indicate that endocytosis was reduced in synaptosomes treated with 100 M roscovitine. Our results suggest that dynamin phosphorylation in intact nerve terminals may not be regulated by PKC or MAP kinase and that dynamin phosphorylation by cdk5 may regulate endocytosis. Copyright (2002) Australian Neuroscience Society

Real-time pathogen monitoring during enrichment: a novel nanotechnology-based approach to food safety testing.

Science.gov (United States)

Weidemaier, Kristin; Carruthers, Erin; Curry, Adam; Kuroda, Melody; Fallows, Eric; Thomas, Joseph; Sherman, Douglas; Muldoon, Mark

2015-04-02

We describe a new approach for the real-time detection and identification of pathogens in food and environmental samples undergoing culture. Surface Enhanced Raman Scattering (SERS) nanoparticles are combined with a novel homogeneous immunoassay to allow sensitive detection of pathogens in complex samples such as stomached food without the need for wash steps or extensive sample preparation. SERS-labeled immunoassay reagents are present in the cultural enrichment vessel, and the signal is monitored real-time through the wall of the vessel while culture is ongoing. This continuous monitoring of pathogen load throughout the enrichment process enables rapid, hands-free detection of food pathogens. Furthermore, the integration of the food pathogen immunoassay directly into the enrichment vessel enables fully biocontained food safety testing, thereby significantly reducing the risk of contaminating the surrounding environment with enriched pathogens. Here, we present experimental results showing the detection of E. coli, Salmonella, or Listeria in several matrices (raw ground beef, raw ground poultry, chocolate milk, tuna salad, spinach, brie cheese, hot dogs, deli turkey, orange juice, cola, and swabs and sponges used to sample a stainless steel surface) using the SERS system and demonstrate the accuracy of the approach compared to plating results. Copyright © 2014 Elsevier B.V. All rights reserved.

Enrichment of coal pulps by selective flocculation

Energy Technology Data Exchange (ETDEWEB)

Blaschke, Z

1977-01-01

The results are presented of selective flocculation of coal pulps using different reagents. In some tests the coal particles were flocculated, and in others the coal remained in suspension and the dirt was flocculated. Selective flocculation makes it possible to obtain coal concentrates with a very low ash content from slurries with a high ash content. (In Polish)

Enrichment of coal pulps by selective flocculation

Energy Technology Data Exchange (ETDEWEB)

Blaschke, Z

1977-01-01

The results are presented of selective flocculation of coal pulps using different reagents. In some tests the coal particles were flocculated, and in others the coal remained in suspension and the dirt was flocculated. Selective flocculation makes it possible to obtain coal concentrates with a very low ash content from slurries with a high ash content.

Investigation of the Flexibility of Protein Kinases Implicated in the Pathology of Alzheimer’s Disease

Directory of Open Access Journals (Sweden)

Michael P. Mazanetz

2014-06-01

Full Text Available The pathological characteristics of Alzheimer’s Disease (AD have been linked to the activity of three particular kinases—Glycogen Synthase Kinase 3β (GSK3β, Cyclin-Dependent Kinase 5 (CDK5 and Extracellular-signal Regulated Kinase 2 (ERK2. As a consequence, the design of selective, potent and drug-like inhibitors of these kinases is of particular interest. Structure-based design methods are well-established in the development of kinase inhibitors. However, progress in this field is limited by the difficulty in obtaining X-ray crystal structures suitable for drug design and by the inability of this method to resolve highly flexible regions of the protein that are crucial for ligand binding. To address this issue, we have undertaken a study of human protein kinases CDK5/p25, CDK5, ERK2 and GSK3β using both conventional molecular dynamics (MD and the new Active Site Pressurisation (ASP methodology, to look for kinase-specific patterns of flexibility that could be leveraged for the design of selective inhibitors. ASP was used to examine the intrinsic flexibility of the ATP-binding pocket for CDK5/p25, CDK5 and GSK3β where it is shown to be capable of inducing significant conformational changes when compared with X-ray crystal structures. The results from these experiments were used to quantify the dynamics of each protein, which supported the observations made from the conventional MD simulations. Additional information was also derived from the ASP simulations, including the shape of the ATP-binding site and the rigidity of the ATP-binding pocket. These observations may be exploited in the design of selective inhibitors of GSK3β, CDK5 and ERK2.

AGC kinases, mechanisms of regulation ‎and innovative drug development.

Science.gov (United States)

Leroux, Alejandro E; Schulze, Jörg O; Biondi, Ricardo M

2018-02-01

The group of AGC kinases consists of 63 evolutionarily related serine/threonine protein kinases comprising PDK1, PKB/Akt, SGK, PKC, PRK/PKN, MSK, RSK, S6K, PKA, PKG, DMPK, MRCK, ROCK, NDR, LATS, CRIK, MAST, GRK, Sgk494, and YANK, while two other families, Aurora and PLK, are the most closely related to the group. Eight of these families are physiologically activated downstream of growth factor signalling, while other AGC kinases are downstream effectors of a wide range of signals. The different AGC kinase families share aspects of their mechanisms of inhibition and activation. In the present review, we update the knowledge of the mechanisms of regulation of different AGC kinases. The conformation of the catalytic domain of many AGC kinases is regulated allosterically through the modulation of the conformation of a regulatory site on the small lobe of the kinase domain, the PIF-pocket. The PIF-pocket acts like an ON-OFF switch in AGC kinases with different modes of regulation, i.e. PDK1, PKB/Akt, LATS and Aurora kinases. In this review, we make emphasis on how the knowledge of the molecular mechanisms of regulation can guide the discovery and development of small allosteric modulators. Molecular probes stabilizing the PIF-pocket in the active conformation are activators, while compounds stabilizing the disrupted site are allosteric inhibitors. One challenge for the rational development of allosteric modulators is the lack of complete structural information of the inhibited forms of full-length AGC kinases. On the other hand, we suggest that the available information derived from molecular biology and biochemical studies can already guide screening strategies for the identification of innovative mode of action molecular probes and the development of selective allosteric drugs for the treatment of human diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Immunomagnetic separation combined with colony immunoblotting for selective enrichment and detection of piliated Lactobacillus rhamnosus strains.

Science.gov (United States)

Yang, Z Q; Wei, Y F; Rao, S Q; Gao, L; Yin, Y Q; Xue, F; Fang, W M; Gu, R X; Jiao, X A

2016-11-01

Piliated Lactobacillus rhamnosus (pLR) strains have attracted much attention owing to their excellent mucus adhering capacity and immunomodulatory effects. Here, we aimed to develop a rapid, sensitive method for isolating pLR strains in complex ecosystems using immunomagnetic separation (IMS) with colony immunoblotting (CIB). Magnetic nanobeads (diameter: 180 nm) conjugated with anti-pLR SpaA pilin antibodies (anti-SpaA) were prepared and used to preconcentrate pLR strains in samples, followed by confirmation with anti-SpaA-based CIB analysis. Under optimized experimental conditions, IMS-CIB selectively recovered pLR strains from 10 7  CFU ml -1 of faecal microbiota samples spiked with 2·9 × 10 1 to 2·4 × 10 6  CFU ml -1 of pLR strains. No positive colonies were detected in samples without addition of pLR strains. The detection limit of IMS-CIB was 29 CFU pLR ml -1 of faecal microbiota, which is much lower than that of CIB without IMS preconcentration (2·0 × 10 4  CFU ml -1 ). IMS-CIB allowed selective preconcentration of pLR strains in highly heterogeneous bacterial suspensions and direct detection of pLR colonies, which remained readily available for subsequent isolation. Our findings established an effective method for selective enrichment and detection of pLR strains. © 2016 The Society for Applied Microbiology.

Uranium enrichment: investment options for the long term

International Nuclear Information System (INIS)

Anon.

1983-01-01

The US government supplies a major portion of the enriched uranium used to fuel most of the nuclear power plants that furnish electricity in the free world. As manager of the US uranium enrichment concern, the Department of Energy (DOE) is investigating a number of technological choices to improve enrichment service and remain a significant world supplier. The Congress will ultimately select a strategy for federal investment in the uranium enrichment enterprise. A fundamental policy choice between possible future roles - that of the free world's main supplier of enrichment services, and that of a mainly domestic supplier - will underlie any investment decision the Congress makes. The technological choices are gaseous diffusion, gas centrifuge, and atomic vapor laser isotope separation (AVLIS). A base plan and four alternatives were examined by DOE and the Congressional Budget Office. In terms of total enterprise costs, Option IV, ultimately relying on advanced gas centrifuges for enrichment services, would offer the most economic approach, with costs over the full projection period totaling $123.5 billion. Option III, ultimately relying on AVLIS without gas centrifuge enrichment or gaseous diffusion, falls next in the sequence, with costs of $128.2 billion. Options I and II, involving combinations of the gas centrifuge and AVLIS technologies, follow closely with costs of $128.7 and $129.6 billion. The base plan has costs of $136.8 billion over the projection period. 1 figure, 22 tables

Protein kinase C mediates platelet secretion and thrombus formation through protein kinase D2.

Science.gov (United States)

Konopatskaya, Olga; Matthews, Sharon A; Harper, Matthew T; Gilio, Karen; Cosemans, Judith M E M; Williams, Christopher M; Navarro, Maria N; Carter, Deborah A; Heemskerk, Johan W M; Leitges, Michael; Cantrell, Doreen; Poole, Alastair W

2011-07-14

Platelets are highly specialized blood cells critically involved in hemostasis and thrombosis. Members of the protein kinase C (PKC) family have established roles in regulating platelet function and thrombosis, but the molecular mechanisms are not clearly understood. In particular, the conventional PKC isoform, PKCα, is a major regulator of platelet granule secretion, but the molecular pathway from PKCα to secretion is not defined. Protein kinase D (PKD) is a family of 3 kinases activated by PKC, which may represent a step in the PKC signaling pathway to secretion. In the present study, we show that PKD2 is the sole PKD member regulated downstream of PKC in platelets, and that the conventional, but not novel, PKC isoforms provide the upstream signal. Platelets from a gene knock-in mouse in which 2 key phosphorylation sites in PKD2 have been mutated (Ser707Ala/Ser711Ala) show a significant reduction in agonist-induced dense granule secretion, but not in α-granule secretion. This deficiency in dense granule release was responsible for a reduced platelet aggregation and a marked reduction in thrombus formation. Our results show that in the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and thrombus formation through its selective regulation of dense granule secretion.

Uranium enrichment

International Nuclear Information System (INIS)

1990-01-01

This report looks at the following issues: How much Soviet uranium ore and enriched uranium are imported into the United States and what is the extent to which utilities flag swap to disguise these purchases? What are the U.S.S.R.'s enriched uranium trading practices? To what extent are utilities required to return used fuel to the Soviet Union as part of the enriched uranium sales agreement? Why have U.S. utilities ended their contracts to buy enrichment services from DOE?

Maintaining glycogen synthase kinase-3 activity is critical for mTOR kinase inhibitors to inhibit cancer cell growth.

Science.gov (United States)

Koo, Junghui; Yue, Ping; Gal, Anthony A; Khuri, Fadlo R; Sun, Shi-Yong

2014-05-01

mTOR kinase inhibitors that target both mTORC1 and mTORC2 are being evaluated in cancer clinical trials. Here, we report that glycogen synthase kinase-3 (GSK3) is a critical determinant for the therapeutic response to this class of experimental drugs. Pharmacologic inhibition of GSK3 antagonized their suppressive effects on the growth of cancer cells similarly to genetic attenuation of GSK3. Conversely, expression of a constitutively activated form of GSK3β sensitized cancer cells to mTOR inhibition. Consistent with these findings, higher basal levels of GSK3 activity in a panel of human lung cancer cell lines correlated with more efficacious responses. Mechanistic investigations showed that mTOR kinase inhibitors reduced cyclin D1 levels in a GSK3β-dependent manner, independent of their effects on suppressing mTORC1 signaling and cap binding. Notably, selective inhibition of mTORC2 triggered proteasome-mediated cyclin D1 degradation, suggesting that mTORC2 blockade is responsible for GSK3-dependent reduction of cyclin D1. Silencing expression of the ubiquitin E3 ligase FBX4 rescued this reduction, implicating FBX4 in mediating this effect of mTOR inhibition. Together, our findings define a novel mechanism by which mTORC2 promotes cell growth, with potential implications for understanding the clinical action of mTOR kinase inhibitors. ©2014 AACR.

Enrichment Assay Methods Development for the Integrated Cylinder Verification System

Energy Technology Data Exchange (ETDEWEB)

Smith, Leon E.; Misner, Alex C.; Hatchell, Brian K.; Curtis, Michael M.

2009-10-22

International Atomic Energy Agency (IAEA) inspectors currently perform periodic inspections at uranium enrichment plants to verify UF6 cylinder enrichment declarations. Measurements are typically performed with handheld high-resolution sensors on a sampling of cylinders taken to be representative of the facility's entire product-cylinder inventory. Pacific Northwest National Laboratory (PNNL) is developing a concept to automate the verification of enrichment plant cylinders to enable 100 percent product-cylinder verification and potentially, mass-balance calculations on the facility as a whole (by also measuring feed and tails cylinders). The Integrated Cylinder Verification System (ICVS) could be located at key measurement points to positively identify each cylinder, measure its mass and enrichment, store the collected data in a secure database, and maintain continuity of knowledge on measured cylinders until IAEA inspector arrival. The three main objectives of this FY09 project are summarized here and described in more detail in the report: (1) Develop a preliminary design for a prototype NDA system, (2) Refine PNNL's MCNP models of the NDA system, and (3) Procure and test key pulse-processing components. Progress against these tasks to date, and next steps, are discussed.

Enrichment Assay Methods Development for the Integrated Cylinder Verification System

International Nuclear Information System (INIS)

Smith, Leon E.; Misner, Alex C.; Hatchell, Brian K.; Curtis, Michael M.

2009-01-01

International Atomic Energy Agency (IAEA) inspectors currently perform periodic inspections at uranium enrichment plants to verify UF6 cylinder enrichment declarations. Measurements are typically performed with handheld high-resolution sensors on a sampling of cylinders taken to be representative of the facility's entire product-cylinder inventory. Pacific Northwest National Laboratory (PNNL) is developing a concept to automate the verification of enrichment plant cylinders to enable 100 percent product-cylinder verification and potentially, mass-balance calculations on the facility as a whole (by also measuring feed and tails cylinders). The Integrated Cylinder Verification System (ICVS) could be located at key measurement points to positively identify each cylinder, measure its mass and enrichment, store the collected data in a secure database, and maintain continuity of knowledge on measured cylinders until IAEA inspector arrival. The three main objectives of this FY09 project are summarized here and described in more detail in the report: (1) Develop a preliminary design for a prototype NDA system, (2) Refine PNNL's MCNP models of the NDA system, and (3) Procure and test key pulse-processing components. Progress against these tasks to date, and next steps, are discussed.

Engineering of kinase-based protein interacting devices: active expression of tyrosine kinase domains

KAUST Repository

Diaz Galicia, Miriam Escarlet

2018-05-01

Protein-protein interactions modulate cellular processes in health and disease. However, tracing weak or rare associations or dissociations of proteins is not a trivial task. Kinases are often regulated through interaction partners and, at the same time, themselves regulate cellular interaction networks. The use of kinase domains for creating a synthetic sensor device that reads low concentration protein-protein interactions and amplifies them to a higher concentration interaction which is then translated into a FRET (Fluorescence Resonance Energy Transfer) signal is here proposed. To this end, DNA constructs for interaction amplification (split kinases), positive controls (intact kinase domains), scaffolding proteins and phosphopeptide - SH2-domain modules for the reading of kinase activity were assembled and expression protocols for fusion proteins containing Lyn, Src, and Fak kinase domains in bacterial and in cell-free systems were optimized. Also, two non-overlapping methods for measuring the kinase activity of these proteins were stablished and, finally, a protein-fragment complementation assay with the split-kinase constructs was tested. In conclusion, it has been demonstrated that features such as codon optimization, vector design and expression conditions have an impact on the expression yield and activity of kinase-based proteins. Furthermore, it has been found that the defined PURE cell-free system is insufficient for the active expression of catalytic kinase domains. In contrast, the bacterial co-expression with phosphatases produced active kinase fusion proteins for two out of the three tested Tyrosine kinase domains.

Identification of critical chemical features for Aurora kinase-B inhibitors using Hip-Hop, virtual screening and molecular docking

Science.gov (United States)

Sakkiah, Sugunadevi; Thangapandian, Sundarapandian; John, Shalini; Lee, Keun Woo

2011-01-01

This study was performed to find the selective chemical features for Aurora kinase-B inhibitors using the potent methods like Hip-Hop, virtual screening, homology modeling, molecular dynamics and docking. The best hypothesis, Hypo1 was validated toward a wide range of test set containing the selective inhibitors of Aurora kinase-B. Homology modeling and molecular dynamics studies were carried out to perform the molecular docking studies. The best hypothesis Hypo1 was used as a 3D query to screen the chemical databases. The screened molecules from the databases were sorted based on ADME and drug like properties. The selective hit compounds were docked and the hydrogen bond interactions with the critical amino acids present in Aurora kinase-B were compared with the chemical features present in the Hypo1. Finally, we suggest that the chemical features present in the Hypo1 are vital for a molecule to inhibit the Aurora kinase-B activity.

Approved and Experimental Small-Molecule Oncology Kinase Inhibitor Drugs: A Mid-2016 Overview.

Science.gov (United States)

Fischer, Peter M

2017-03-01

Kinase inhibitor research is a comparatively recent branch of medicinal chemistry and pharmacology and the first small-molecule kinase inhibitor, imatinib, was approved for clinical use only 15 years ago. Since then, 33 more kinase inhibitor drugs have received regulatory approval for the treatment of a variety of cancers and the volume of reports on the discovery and development of kinase inhibitors has increased to an extent where it is now difficult-even for those working in the field-easily to keep an overview of the compounds that are being developed, as currently there are 231 such compounds, targeting 38 different protein and lipid kinases (not counting isoforms), in clinical use or under clinical investigation. The purpose of this review is thus to provide an overview of the biomedical rationales for the kinases being targeted on the one hand, and the design principles, as well as chemical, pharmacological, pharmaceutical, and toxicological kinase inhibitor properties, on the other hand. Two issues that are especially important in kinase inhibitor research, target selectivity and drug resistance, as well as the underlying structural concepts, are discussed in general terms and in the context of relevant kinases and their inhibitors. © 2016 Wiley Periodicals, Inc.

Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

Directory of Open Access Journals (Sweden)

Lei eShi

2014-09-01

Full Text Available Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells.

Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function.

Science.gov (United States)

Grover, Prerna; Shi, Haibin; Baumgartner, Matthew; Camacho, Carlos J; Smithgall, Thomas E

2015-01-01

The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important

Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function.

Directory of Open Access Journals (Sweden)

Prerna Grover

Full Text Available The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery

Gas-phase UF6 enrichment monitor for enrichment plant safeguards

International Nuclear Information System (INIS)

Strittmatter, R.B.; Tape, J.W.

1980-03-01

An in-line enrichment monitor is being developed to provide real-time enrichment data for the gas-phase UF 6 feed stream of an enrichment plant. The nondestructive gamma-ray assay method can be used to determine the enrichment of natural UF 6 with a relative precision of better than 1% for a wide range of pressures

Tunable signal processing in synthetic MAP kinase cascades.

Science.gov (United States)

O'Shaughnessy, Ellen C; Palani, Santhosh; Collins, James J; Sarkar, Casim A

2011-01-07

The flexibility of MAPK cascade responses enables regulation of a vast array of cell fate decisions, but elucidating the mechanisms underlying this plasticity is difficult in endogenous signaling networks. We constructed insulated mammalian MAPK cascades in yeast to explore how intrinsic and extrinsic perturbations affect the flexibility of these synthetic signaling modules. Contrary to biphasic dependence on scaffold concentration, we observe monotonic decreases in signal strength as scaffold concentration increases. We find that augmenting the concentration of sequential kinases can enhance ultrasensitivity and lower the activation threshold. Further, integrating negative regulation and concentration variation can decouple ultrasensitivity and threshold from the strength of the response. Computational analyses show that cascading can generate ultrasensitivity and that natural cascades with different kinase concentrations are innately biased toward their distinct activation profiles. This work demonstrates that tunable signal processing is inherent to minimal MAPK modules and elucidates principles for rational design of synthetic signaling systems. Copyright © 2011 Elsevier Inc. All rights reserved.

Thymidine kinase enzyme selective imaging radiopharmaceutical. {sup 99m}Tc(CO){sub 3}-Ganciclovir

Energy Technology Data Exchange (ETDEWEB)

Gedik, B.; Teksoez, S.; Ichedef, C.; Kilcar, A.Y.; Medine, E.I.; Ucar, E. [Ege Univ., Bornova, Izmir (Turkey). Dept. of Nuclear Applications

2013-03-01

The aim of this study is to radiolabel Ganciclovir, known as having selective antiviral properties against thymidine kinase, with technetium tricarbonylcore ({sup 99m}Tc(CO){sub 3}{sup +}) and to investigate the biological behavior of this complex in vitro and in vivo. Commercially provided Ganciclovir (GCV) was radiolabeled with {sup 99m}Tc(CO){sub 3}{sup +}. Initially, optimum radiolabeling conditions were determined by analyzing factors such as temperature, pH and time. Quality control of the radiolabeled compound was performed. The radiolabeling yield was found to be 97%. The {sup 99m}Tc(CO){sub 3}-GCV complex also displayed good in vitro stability during the 24 h period. In vitro cell uptake studies showed that the {sup 99m}Tc(CO){sub 3}-GCV complex is highly uptaken in A-549, PC-3, HeLa cell lines according to the control group {sup 99m}Tc(I)-tricarbonyl core. The knowledge gained from in vivo and in vitro studies of {sup 99m}Tc(CO){sub 3}-GCV could contribute to the development of a new HSV1-tk gene imaging agent. (orig.)

Implementation of preference ranking organization method for enrichment evaluation (Promethee) on selection system of student’s achievement

Science.gov (United States)

Karlitasari, L.; Suhartini, D.; Nurrosikawati, L.

2018-03-01

Selection of Student Achievement is conducted every year, starting from the level of Study Program, Faculty, to University, which then rank one will be sent to Kopertis level. The criteria made for the selection are Academic and Rich Scientific, Organizational, Personality, and English. In order for the selection of Student Achievement is Objective, then in addition to the presence of the jury is expected to use methods that support the decision to be more optimal in determining the Student Achievement. One method used is the Promethee Method. Preference Ranking Organization Method for Enrichment Evaluation (Promethee) is a method of ranking in Multi Criteria Decision Making (MCDM). PROMETHEE has the advantage that there is a preference type against the criteria that can take into account alternatives with other alternatives on the same criteria. The conjecture of alternate dominance over a criterion used in PROMETHEE is the use of values in the relationships between alternative ranking values. Based on the calculation result, from 7 applicants between Manual and Promethee Matrices, rank 1, 2, and 3, did not change, only 4 to 7 positions were changed. However, after the sensitivity test, almost all criteria experience a high level of sensitivity. Although it does not affect the students who will be sent to the next level, but can bring psychological impact on prospective student’s achievement

Creative prosthetic foot selection enables successful ambulation in stiletto high heels.

Science.gov (United States)

Russell Esposito, Elizabeth; Lipe, Delbert H; Rábago, Christopher A

2017-11-01

Walking in high heels presents biomechanical challenges, yet they remain part of many women's attire. However, women with a lower limb amputation are limited in available footwear options. Case description and methods: This case study is in response to one patient's assertion that she walked better and more symmetrically in heels than flat shoes with her below-knee prosthesis. She underwent gait analysis in athletic shoes and 10-cm stiletto high heels worn with a pediatric running foot to determine if these claims could be substantiated through biomechanical measures. Global gait asymmetry indices were calculated. Findings and outcomes: Asymmetry indices were nearly identical between athletic shoes and heels but joint-level findings differed substantially. Ankle mechanics were more symmetrical in heels but hip mechanics were less. The maintenance of symmetry in stiletto high heels does not imply maintenance of gait quality, as high heels are known to adversely affect some components walking mechanics. Clinical relevance Returning to high-heel wear is achievable for prosthesis users. Accommodations can be made using creativity in prosthetic foot selection to enable successful ambulation; however, attention to gait mechanics may be important for patient safety.

Overexpression, purification and crystallographic analysis of a unique adenosine kinase from Mycobacterium tuberculosis

Energy Technology Data Exchange (ETDEWEB)

Wang, Yimin; Long, Mary C.; Ranganathan, Senthil; Escuyer, Vincent; Parker, William B.; Li, Rongbao, E-mail: li@sri.org [Southern Research Institute, 2000 Ninth Avenue South, Birmingham, Alabama 35205 (United States)

2005-06-01

Adenosine kinase from M. tuberculosis has been overexpressed, purified and crystallized in the presence of adenosine. Structure determination using molecular replacement with diffraction data collected at 2.2 Å reveals a dimeric structure. Adenosine kinase from Mycobacterium tuberculosis is the only prokaryotic adenosine kinase that has been isolated and characterized. The enzyme catalyzes the phosphorylation of adenosine to adenosine monophosphate and is involved in the activation of 2-methyladenosine, a compound that has demonstrated selective activity against M. tuberculosis. The mechanism of action of 2-methyladenosine is likely to be different from those of current tuberculosis treatments and this compound (or other adenosine analogs) may prove to be a novel therapeutic intervention for this disease. The M. tuberculosis adenosine kinase was overexpressed in Escherichia coli and the enzyme was purified with activity comparable to that reported previously. The protein was crystallized in the presence of adenosine using the vapour-diffusion method. The crystals diffracted X-rays to high resolution and a complete data set was collected to 2.2 Å using synchrotron radiation. The crystal belonged to space group P3{sub 1}21, with unit-cell parameters a = 70.2, c = 111.6 Å, and contained a single protein molecule in the asymmetric unit. An initial structural model of the protein was obtained by the molecular-replacement method, which revealed a dimeric structure. The monomers of the dimer were related by twofold crystallographic symmetry. An understanding of how the M. tuberculosis adenosine kinase differs from the human homolog should aid in the design of more potent and selective antimycobacterial agents that are selectively activated by this enzyme.

Overexpression, purification and crystallographic analysis of a unique adenosine kinase from Mycobacterium tuberculosis

International Nuclear Information System (INIS)

Wang, Yimin; Long, Mary C.; Ranganathan, Senthil; Escuyer, Vincent; Parker, William B.; Li, Rongbao

2005-01-01

Adenosine kinase from M. tuberculosis has been overexpressed, purified and crystallized in the presence of adenosine. Structure determination using molecular replacement with diffraction data collected at 2.2 Å reveals a dimeric structure. Adenosine kinase from Mycobacterium tuberculosis is the only prokaryotic adenosine kinase that has been isolated and characterized. The enzyme catalyzes the phosphorylation of adenosine to adenosine monophosphate and is involved in the activation of 2-methyladenosine, a compound that has demonstrated selective activity against M. tuberculosis. The mechanism of action of 2-methyladenosine is likely to be different from those of current tuberculosis treatments and this compound (or other adenosine analogs) may prove to be a novel therapeutic intervention for this disease. The M. tuberculosis adenosine kinase was overexpressed in Escherichia coli and the enzyme was purified with activity comparable to that reported previously. The protein was crystallized in the presence of adenosine using the vapour-diffusion method. The crystals diffracted X-rays to high resolution and a complete data set was collected to 2.2 Å using synchrotron radiation. The crystal belonged to space group P3 1 21, with unit-cell parameters a = 70.2, c = 111.6 Å, and contained a single protein molecule in the asymmetric unit. An initial structural model of the protein was obtained by the molecular-replacement method, which revealed a dimeric structure. The monomers of the dimer were related by twofold crystallographic symmetry. An understanding of how the M. tuberculosis adenosine kinase differs from the human homolog should aid in the design of more potent and selective antimycobacterial agents that are selectively activated by this enzyme

The uranium enrichment market and long-term technological options

International Nuclear Information System (INIS)

Schneider-Maunoury, A.

1992-01-01

The world enrichment market situation is clearly delineated up to the year 2000. Including the East European countries, worldwide enriched uranium requirements should reach 40 million separative work units (SWUs) a year and production capacity should reach 44 millions SWUs. Two-thirds of this capacity will be supplied by the gaseous diffusion process and one-third by the centrifuge process. The enrichment processes currently considered are: (i) the gaseous diffusion process, (ii) the centrifuge process, (iii) the chemical treatment process and (iv) the laser processes, long-term assessment of the enrichment market up to the year 2015. Two scenarios may be envisioned for the (i) Public opinion will continue to block the development of nuclear power, and requirements will level off at 40 million SWUs. (ii) Changing attitudes will favor a reasonable approach enabling a revival of nuclear power expansion around 1995. Requirements should then increase starting in 2005 and would readily attain 60 million SWUs a year by 2015. Depending on market conditions, enrichment process options will be influenced either entirely by cost considerations, without allowance for the time factor, or by need to meet demand. Demonstrations of the industrial validity of laser processes are expected by 1992 - 1995 and, if interest in nuclear power makes a comeback, decisions should be made between 1995 and 2000 to build new large-capacity enrichment plants. The gaseous diffusion process may still be used for a long time if nuclear power is judiciously employed. The centrifuge process will be fully mature by the year 2000. The uranium vapor laser processes offer the most promise and should ultimately prevail. the chemical processes, though outsiders, deserve watching. (author)

The Azaindole Framework in the Design of Kinase Inhibitors

Directory of Open Access Journals (Sweden)

Jean-Yves Mérour

2014-11-01

Full Text Available This review article illustrates the growing use of azaindole derivatives as kinase inhibitors and their contribution to drug discovery and innovation. The different protein kinases which have served as targets and the known molecules which have emerged from medicinal chemistry and Fragment-Based Drug Discovery (FBDD programs are presented. The various synthetic routes used to access these compounds and the chemical pathways leading to their synthesis are also discussed. An analysis of their mode of binding based on X-ray crystallography data gives structural insights for the design of more potent and selective inhibitors.

Survey of tyrosine kinase signaling reveals ROS kinase fusions in human cholangiocarcinoma.

Directory of Open Access Journals (Sweden)

Ting-Lei Gu

Full Text Available Cholangiocarcinoma, also known as bile duct cancer, is the second most common primary hepatic carcinoma with a median survival of less than 2 years. The molecular mechanisms underlying the development of this disease are not clear. To survey activated tyrosine kinases signaling in cholangiocarcinoma, we employed immunoaffinity profiling coupled to mass spectrometry and identified DDR1, EPHA2, EGFR, and ROS tyrosine kinases, along with over 1,000 tyrosine phosphorylation sites from about 750 different proteins in primary cholangiocarcinoma patients. Furthermore, we confirmed the presence of ROS kinase fusions in 8.7% (2 out of 23 of cholangiocarcinoma patients. Expression of the ROS fusions in 3T3 cells confers transforming ability both in vitro and in vivo, and is responsive to its kinase inhibitor. Our data demonstrate that ROS kinase is a promising candidate for a therapeutic target and for a diagnostic molecular marker in cholangiocarcinoma. The identification of ROS tyrosine kinase fusions in cholangiocarcinoma, along with the presence of other ROS kinase fusions in lung cancer and glioblastoma, suggests that a more broadly based screen for activated ROS kinase in cancer is warranted.

Uranium enrichment activities: the SILVA program

International Nuclear Information System (INIS)

Guyot, J.; Cazalet, J.; Camarcat, N.; Figuet, J.

1994-01-01

Through its commitment to a nuclear electricity generation policy, France holds today a specific position in the uranium enrichment market thanks to the modern multinational EURODIF gaseous diffusion plant. France has, altogether, a long-term goal in developing SILVA, a laser uranium enrichment process, based on the selective photo-ionization of U-235. After reviewing the fundamentals of SILVA (the laser system with copper vapor lasers and dye lasers and the separator system), a description of the general organization of the R and D program is provided going through basic research, subsystems assessment, production demonstrations and simulations (with the LACAN code), plant design and economics. The general schedule of SILVA is outlined, leading to the possible construction of a commercial plant. 7 figs., 11 refs

How Enterprise Architecture Maturity Enables Post-merger IT Integration

DEFF Research Database (Denmark)

Törmer, Robert Lorenz; Henningsson, Stefan

2017-01-01

advances the argument that a company’s pre-existing Enterprise Architecture decisively shapes the capability to implement post-merger IT integration and subsequently realize benefits from M&A. Our multiple-case study investigates three acquisition cases and develops an explanatory theory of how Enterprise...... Architecture maturity enables the implementation of distinct integration strategies. The results do not only enrich the academic literature on M&A, but also show the strategic value of Enterprise Architecture maturity....

Methods Of Using Chemical Libraries To Search For New Kinase Inhibitors

Science.gov (United States)

Gray, Nathanael S. , Schultz, Peter , Wodicka, Lisa , Meijer, Laurent , Lockhart, David J.

2003-06-03

The generation of selective inhibitors for specific protein kinases would provide new tools for analyzing signal transduction pathways and possibly new therapeutic agents. We have invented an approach to the development of selective protein kinase inhibitors based on the unexpected binding mode of 2,6,9-trisubstituted purines to the ATP binding site of human CDK2. The most potent inhibitor, purvalanol B (IC.sub.50 =6 nM), binds with a 30-fold greater affinity than the known CDK2 inhibitor, flavopiridol. The cellular effects of this class of compounds were examined and compared to those of flavopiridol by monitoring changes in mRNA expression levels for all genes in treated cells of Saccharomyces cerevisiae using high-density oligonucleotide probe arrays.

Selective blockade of protein kinase B protects the rat and human myocardium against ischaemic injury

Science.gov (United States)

Linares-Palomino, José; Husainy, Muhammad A; Lai, Vien K; Dickenson, John M; Galiñanes, Manuel

2010-01-01

Protein kinase B (PKB/Akt) plays a critical role in cell survival but the investigation of its involvement has been limited by the lack of specific pharmacological agents. In this study, using novel PKB inhibitors (VIII and XI), we investigated the role of PKB in cardioprotection of the rat and human myocardium, the location of PKB in relation to mitoKATP channels and p38 mitogen-activated protein kinase (p38 MAPK), and whether the manipulation of PKB can overcome the unresponsiveness to protection of the diabetic myocardium. Myocardial slices from rat left ventricle and from the right atrial appendage of patients undergoing elective cardiac surgery were subjected to 90 min ischaemia/120 min reoxygenation at 37°C. Tissue injury was assessed by creatine kinase (CK) released and determination of cell necrosis and apoptosis. The results showed that blockade of PKB activity caused significant reduction of CK release and cell death, a benefit that was as potent as ischaemic preconditioning and could be reproduced by blockade of phosphatidylinositol 3-kinase (PI-3K) with wortmannin and LY 294002. The protection was time dependent with maximal benefit seen when PKB and PI-3K were inhibited before ischaemia or during both ischaemia and reoxygenation. In addition, it was revealed that PKB is located downstream of mitoKATP channels but upstream of p38 MAPK. PKB inhibition induced a similar degree of protection in the human and rat myocardium and, importantly, it reversed the unresponsiveness to protection of the diabetic myocardium. In conclusion, inhibition of PKB plays a critical role in protection of the mammalian myocardium and may represent a clinical target for the reduction of ischaemic injury. PMID:20403980

Molecular Imaging of the ATM Kinase Activity

Energy Technology Data Exchange (ETDEWEB)

Williams, Terence M. [Department of Radiation Oncology, Ohio State University, Columbus, Ohio (United States); Nyati, Shyam [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Center for Molecular Imaging, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Ross, Brian D. [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Department of Radiology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Rehemtulla, Alnawaz, E-mail: alnawaz@umich.edu [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Center for Molecular Imaging, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Department of Radiology, University of Michigan Medical Center, Ann Arbor, Michigan (United States)

2013-08-01

Purpose: Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including from DNA double-strand breaks. ATM activation results in the initiation of a complex cascade of events including DNA damage repair, cell cycle checkpoint control, and survival. We sought to create a bioluminescent reporter that dynamically and noninvasively measures ATM kinase activity in living cells and subjects. Methods and Materials: Using the split luciferase technology, we constructed a hybrid cDNA, ATM-reporter (ATMR), coding for a protein that quantitatively reports on changes in ATM kinase activity through changes in bioluminescence. Results: Treatment of ATMR-expressing cells with ATM inhibitors resulted in a dose-dependent increase in bioluminescence activity. In contrast, induction of ATM kinase activity upon irradiation resulted in a decrease in reporter activity that correlated with ATM and Chk2 activation by immunoblotting in a time-dependent fashion. Nuclear targeting improved ATMR sensitivity to both ATM inhibitors and radiation, whereas a mutant ATMR (lacking the target phosphorylation site) displayed a muted response. Treatment with ATM inhibitors and small interfering (si)RNA-targeted knockdown of ATM confirm the specificity of the reporter. Using reporter expressing xenografted tumors demonstrated the ability of ATMR to report in ATM activity in mouse models that correlated in a time-dependent fashion with changes in Chk2 activity. Conclusions: We describe the development and validation of a novel, specific, noninvasive bioluminescent reporter that enables monitoring of ATM activity in real time, in vitro and in vivo. Potential applications of this reporter include the identification and development of novel ATM inhibitors or ATM-interacting partners through high-throughput screens and in vivo pharmacokinetic/pharmacodynamic studies of ATM inhibitors in preclinical models.

Coumestrol Epigenetically Suppresses Cancer Cell Proliferation: Coumestrol Is a Natural Haspin Kinase Inhibitor

Directory of Open Access Journals (Sweden)

Jong-Eun Kim

2017-10-01

Full Text Available Targeting epigenetic changes in gene expression in cancer cells may offer new strategies for the development of selective cancer therapies. In the present study, we investigated coumestrol, a natural compound exhibiting broad anti-cancer effects against skin melanoma, lung cancer and colon cancer cell growth. Haspin kinase was identified as a direct target protein of coumestrol using kinase profiling analysis. Histone H3 is a direct substrate of haspin kinase. We observed haspin kinase overexpression as well as greater phosphorylation of histone H3 at threonine 3 (Thr-3 in the cancer cells compared to normal cells. Computer modeling using the Schrödinger Suite program identified the binding interface within the ATP binding site. These findings suggest that the anti-cancer effect of coumestrol is due to the direct targeting of haspin kinase. Coumestrol has considerable potential for further development as a novel anti-cancer agent.

Cellular and molecular mechanisms of immunomodulation in the brain through environmental enrichment

Science.gov (United States)

Singhal, Gaurav; Jaehne, Emily J.; Corrigan, Frances; Baune, Bernhard T.

2014-01-01

Recent studies on environmental enrichment (EE) have shown cytokines, cellular immune components [e.g., T lymphocytes, natural killer (NK) cells], and glial cells in causal relationship to EE in bringing out changes to neurobiology and behavior. The purpose of this review is to evaluate these neuroimmune mechanisms associated with neurobiological and behavioral changes in response to different EE methods. We systematically reviewed common research databases. After applying all inclusion and exclusion criteria, 328 articles remained for this review. Physical exercise (PE), a form of EE, elicits anti-inflammatory and neuromodulatory effects through interaction with several immune pathways including interleukin (IL)-6 secretion from muscle fibers, reduced expression of Toll-like receptors on monocytes and macrophages, reduced secretion of adipokines, modulation of hippocampal T cells, priming of microglia, and upregulation of mitogen-activated protein kinase phosphatase-1 in central nervous system. In contrast, immunomodulatory roles of other enrichment methods are not studied extensively. Nonetheless, studies showing reduction in the expression of IL-1β and tumor necrosis factor-α in response to enrichment with novel objects and accessories suggest anti-inflammatory effects of novel environment. Likewise, social enrichment, though considered a necessity for healthy behavior, results in immunosuppression in socially defeated animals. This has been attributed to reduction in T lymphocytes, NK cells and IL-10 in subordinate animals. EE through sensory stimuli has been investigated to a lesser extent and the effect on immune factors has not been evaluated yet. Discovery of this multidimensional relationship between immune system, brain functioning, and EE has paved a way toward formulating environ-immuno therapies for treating psychiatric illnesses with minimal use of pharmacotherapy. While the immunomodulatory role of PE has been evaluated extensively, more research

Friedel-Crafts reaction of benzyl fluorides: selective activation of C-F bonds as enabled by hydrogen bonding.

Science.gov (United States)

Champagne, Pier Alexandre; Benhassine, Yasmine; Desroches, Justine; Paquin, Jean-François

2014-12-08

A Friedel-Crafts benzylation of arenes with benzyl fluorides has been developed. The reaction produces 1,1-diaryl alkanes in good yield under mild conditions without the need for a transition metal or a strong Lewis acid. A mechanism involving activation of the C-F bond through hydrogen bonding is proposed. This mode of activation enables the selective reaction of benzylic C-F bonds in the presence of other benzylic leaving groups. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Uranium enrichment

International Nuclear Information System (INIS)

Rae, H.K.; Melvin, J.G.

1988-06-01

Canada is the world's largest producer and exporter of uranium, most of which is enriched elsewhere for use as fuel in LWRs. The feasibility of a Canadian uranium-enrichment enterprise is therefore a perennial question. Recent developments in uranium-enrichment technology, and their likely impacts on separative work supply and demand, suggest an opportunity window for Canadian entry into this international market. The Canadian opportunity results from three particular impacts of the new technologies: 1) the bulk of the world's uranium-enrichment capacity is in gaseous diffusion plants which, because of their large requirements for electricity (more than 2000 kW·h per SWU), are vulnerable to competition from the new processes; 2) the decline in enrichment costs increases the economic incentive for the use of slightly-enriched uranium (SEU) fuel in CANDU reactors, thus creating a potential Canadian market; and 3) the new processes allow economic operation on a much smaller scale, which drastically reduces the investment required for market entry and is comparable with the potential Canadian SEU requirement. The opportunity is not open-ended. By the end of the century the enrichment supply industry will have adapted to the new processes and long-term customer/supplier relationships will have been established. In order to seize the opportunity, Canada must become a credible supplier during this century

Atrazine and its metabolites degradation in mineral salts medium and soil using an enrichment culture.

Science.gov (United States)

Kumar, Anup; Singh, Neera

2016-03-01

An atrazine-degrading enrichment culture was used to study degradation of atrazine metabolites viz. hydroxyatrazine, deethylatrazine, and deisopropylatrazine in mineral salts medium. Results suggested that the enrichment culture was able to degrade only hydroxyatrazine, and it was used as the sole source of carbon and nitrogen. Hydroxyatrazine degradation slowed down when sucrose and/or ammonium hydrogen phosphate were supplemented as the additional sources of carbon and nitrogen, respectively. The enrichment culture could degrade high concentrations of atrazine (up to 110 μg/mL) in mineral salts medium, and neutral pH was optimum for atrazine degradation. Further, except in an acidic soil, enrichment culture was able to degrade atrazine in three soil types having different physico-chemical properties. Raising the pH of acidic soil to neutral or alkaline enabled the enrichment culture to degrade atrazine suggesting that acidic pH inhibited atrazine-degrading ability. The study suggested that the enrichment culture can be successfully utilized to achieve complete degradation of atrazine and its persistent metabolite hydroxyatrazine in the contaminated soil and water.

Phenotype selection reveals coevolution of muscle glycogen and protein and PTEN as a gate keeper for the accretion of muscle mass in adult female mice.

Directory of Open Access Journals (Sweden)

Mandy Sawitzky

Full Text Available We have investigated molecular mechanisms for muscle mass accretion in a non-inbred mouse model (DU6P mice characterized by extreme muscle mass. This extreme muscle mass was developed during 138 generations of phenotype selection for high protein content. Due to the repeated trait selection a complex setting of different mechanisms was expected to be enriched during the selection experiment. In muscle from 29-week female DU6P mice we have identified robust increases of protein kinase B activation (AKT, Ser-473, up to 2-fold if compared to 11- and 54-week DU6P mice or controls. While a number of accepted effectors of AKT activation, including IGF-I, IGF-II, insulin/IGF-receptor, myostatin or integrin-linked kinase (ILK, were not correlated with this increase, phosphatase and tensin homologue deleted on chromosome 10 (PTEN was down-regulated in 29-week female DU6P mice. In addition, higher levels of PTEN phosphorylation were found identifying a second mechanism of PTEN inhibition. Inhibition of PTEN and activation of AKT correlated with specific activation of p70S6 kinase and ribosomal protein S6, reduced phosphorylation of eukaryotic initiation factor 2α (eIF2α and higher rates of protein synthesis in 29-week female DU6P mice. On the other hand, AKT activation also translated into specific inactivation of glycogen synthase kinase 3ß (GSK3ß and an increase of muscular glycogen. In muscles from 29-week female DU6P mice a significant increase of protein/DNA was identified, which was not due to a reduction of protein breakdown or to specific increases of translation initiation. Instead our data support the conclusion that a higher rate of protein translation is contributing to the higher muscle mass in mid-aged female DU6P mice. Our results further reveal coevolution of high protein and high glycogen content during the selection experiment and identify PTEN as gate keeper for muscle mass in mid-aged female DU6P mice.

Bacterial Protein-Tyrosine Kinases

DEFF Research Database (Denmark)

Shi, Lei; Kobir, Ahasanul; Jers, Carsten

2010-01-01

in exopolysaccharide production, virulence, DNA metabolism, stress response and other key functions of the bacterial cell. BY-kinases act through autophosphorylation (mainly in exopolysaccharide production) and phosphorylation of other proteins, which have in most cases been shown to be activated by tyrosine......Bacteria and Eukarya share essentially the same family of protein-serine/threonine kinases, also known as the Hanks-type kinases. However, when it comes to protein-tyrosine phosphorylation, bacteria seem to have gone their own way. Bacterial protein-tyrosine kinases (BY-kinases) are bacterial...... and highlighted their importance in bacterial physiology. Having no orthologues in Eukarya, BY-kinases are receiving a growing attention from the biomedical field, since they represent a particularly promising target for anti-bacterial drug design....

Recent Progress on Liver Kinase B1 (LKB1: Expression, Regulation, Downstream Signaling and Cancer Suppressive Function

Directory of Open Access Journals (Sweden)

Ren-You Gan

2014-09-01

Full Text Available Liver kinase B1 (LKB1, known as a serine/threonine kinase, has been identified as a critical cancer suppressor in many cancer cells. It is a master upstream kinase of 13 AMP-activated protein kinase (AMPK-related protein kinases, and possesses versatile biological functions. LKB1 gene is mutated in many cancers, and its protein can form different protein complexes with different cellular localizations in various cell types. The expression of LKB1 can be regulated through epigenetic modification, transcriptional regulation and post-translational modification. LKB1 dowcnstream pathways mainly include AMPK, microtubule affinity regulating kinase (MARK, salt-inducible kinase (SIK, sucrose non-fermenting protein-related kinase (SNRK and brain selective kinase (BRSK signalings, etc. This review, therefore, mainly discusses recent studies about the expression, regulation, downstream signaling and cancer suppressive function of LKB1, which can be helpful for better understanding of this molecular and its significance in cancers.

Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

Energy Technology Data Exchange (ETDEWEB)

Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele; Goedken, Eric R.; Gum, Rebecca J.; Borhani, David W.; Argiriadi, Maria; Groebe, Duncan R.; Jia, Yong; Clampit, Jill E.; Haasch, Deanna L.; Smith, Harriet T.; Wang, Sanyi; Song, Danying; Coen, Michael L.; Cloutier, Timothy E.; Tang, Hua; Cheng, Xueheng; Quinn, Christopher; Liu, Bo; Xin, Zhili; Liu, Gang; Fry, Elizabeth H.; Stoll, Vincent; Ng, Teresa I.; Banach, David; Marcotte, Doug; Burns, David J.; Calderwood, David J.; Hajduk, Philip J. (Abbott)

2012-03-02

Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in

A data transmission system for the centrifuge enrichment plant safeguards project

International Nuclear Information System (INIS)

Hesse, E.W.

1987-04-01

A novel data transmission system was developed to enable the Australian Safeguards Office (ASO) in Sydney to monitor remotely the running of an experimental enrichment facility at the Lucas Heights Reserch Laboratories, using Telecom telephone lines. The system allowed ASO to monitor the enrichment, flows and activity in sensitive areas of the plant by means of a master computer at ASO and a slave computer at Lucas Heights. The slave computer sent requested data and scanned video pictures to determine intrusive changes to the plant. Detailed descriptions of the transmission system and method of operation are given, together with an outline of experience obtained during a three-month surveillance of the facility

Enrichment and identification of polycyclic aromatic compound-degrading bacteria enriched from sediment samples.

Science.gov (United States)

Long, Rachel M; Lappin-Scott, Hilary M; Stevens, Jamie R

2009-07-01

The degradation of polycyclic aromatic compounds (PACs) has been widely studied. Knowledge of the degradation of PACs by microbial populations can be utilized in the remediation of contaminated sites. To isolate and identify PAC-degrading bacteria for potential use in future bioremediation programmes, we established a series of PAC enrichments under the same experimental conditions from a single sediment sample taken from a highly polluted estuarine site. Enrichment cultures were established using the pollutants: anthracene, phenanthrene and dibenzothiophene as a sole carbon source. The shift in microbial community structure on each of these carbon sources was monitored by analysis of a time series of samples from each culture using 16S rRNA polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Significantly, our findings demonstrate that shifts in the constituent species within each degradative community are directly attributable to enrichment with different PACs. Subsequently, we characterized the microorganisms comprising the degradative communities within each enrichment using 16S rRNA sequence data. Our findings demonstrate that the ability to degrade PACs is present in five divisions of the Proteobacteria and Actinobacteria. By determining the precise identity of the PAC-degrading bacterial species isolated from a single sediment sample, and by comparing our findings with previously published research, we demonstrate how bacteria with similar PAC degrading capabilities and 16S rRNA signatures are found in similarly polluted environments in geographically very distant locations, e.g., China, Italy, Japan and Hawaii. Such a finding suggests that geographical barriers do not limit the distribution of key PAC-degrading bacteria; this finding is in accordance with the Baas-Becking hypothesis "everything is everywhere; the environment selects" and may have significant consequences for the global distribution of PAC-degrading bacteria and

Optimized Enrichment of Phosphoproteomes by Fe-IMAC Column Chromatography

NARCIS (Netherlands)

Ruprecht, Benjamin; Koch, Heiner; Domasinska, Petra; Frejno, Martin; Kuster, Bernhard; Lemeer, Simone

2017-01-01

Phosphorylation is among the most important post-translational modifications of proteins and has numerous regulatory functions across all domains of life. However, phosphorylation is often substoichiometric, requiring selective and sensitive methods to enrich phosphorylated peptides from complex

Hyb-Seq: Combining Target Enrichment and Genome Skimming for Plant Phylogenomics

Directory of Open Access Journals (Sweden)

Kevin Weitemier

2014-08-01

Full Text Available Premise of the study: Hyb-Seq, the combination of target enrichment and genome skimming, allows simultaneous data collection for low-copy nuclear genes and high-copy genomic targets for plant systematics and evolution studies. Methods and Results: Genome and transcriptome assemblies for milkweed (Asclepias syriaca were used to design enrichment probes for 3385 exons from 768 genes (>1.6 Mbp followed by Illumina sequencing of enriched libraries. Hyb-Seq of 12 individuals (10 Asclepias species and two related genera resulted in at least partial assembly of 92.6% of exons and 99.7% of genes and an average assembly length >2 Mbp. Importantly, complete plastomes and nuclear ribosomal DNA cistrons were assembled using off-target reads. Phylogenomic analyses demonstrated signal conflict between genomes. Conclusions: The Hyb-Seq approach enables targeted sequencing of thousands of low-copy nuclear exons and flanking regions, as well as genome skimming of high-copy repeats and organellar genomes, to efficiently produce genome-scale data sets for phylogenomics.

Advanced enrichment techniques

International Nuclear Information System (INIS)

Johnson, A.

1988-01-01

BNFL is in a unique position in that it has commercial experience of diffusion enrichment, and of centrifuge enrichment through its associate company Urenco. In addition BNFL is developing laser enrichment techniques as part of a UK development programme in this area. The paper describes the development programme which led to the introduction of competitive centrifuge enrichment technology by Urenco and discusses the areas where improvements have and will continue to be made in the centrifuge process. It also describes the laser development programme currently being undertaken in the UK. The paper concludes by discussing the relative merits of the various methods of uranium enrichment, with particular reference to the enrichment market likely to obtain over the rest of the century

Advanced enrichment techniques

International Nuclear Information System (INIS)

Johnson, A.

1987-01-01

BNFL is in a unique position in that it has commercial experience of diffusion enrichment, and of centrifuge enrichment through its associate company Urenco. In addition BNFL is developing laser enrichment techniques as part of a UK development programme in this area. The paper describes the development programme which led to the introduction of competitive centrifuge enrichment technology by Urenco and discusses the areas where improvements have and will continue to be made in the centrifuge process. It also describes the laser development programme currently being undertaken in the UK. The paper concludes by discussing the relative merits of the various methods of uranium enrichment, with particular reference to the enrichment market likely to obtain over the rest of the century. (author)

Juvenile psittacine environmental enrichment.

Science.gov (United States)

Simone-Freilicher, Elisabeth; Rupley, Agnes E

2015-05-01

Environmental enrichment is of great import to the emotional, intellectual, and physical development of the juvenile psittacine and their success in the human home environment. Five major types of enrichment include social, occupational, physical, sensory, and nutritional. Occupational enrichment includes exercise and psychological enrichment. Physical enrichment includes the cage and accessories and the external home environment. Sensory enrichment may be visual, auditory, tactile, olfactory, or taste oriented. Nutritional enrichment includes variations in appearance, type, and frequency of diet, and treats, novelty, and foraging. Two phases of the preadult period deserve special enrichment considerations: the development of autonomy and puberty. Copyright © 2015 Elsevier Inc. All rights reserved.

Reorganization in processing of spectral and temporal input in the rat posterior auditory field induced by environmental enrichment

Science.gov (United States)

Jakkamsetti, Vikram; Chang, Kevin Q.

2012-01-01

Environmental enrichment induces powerful changes in the adult cerebral cortex. Studies in primary sensory cortex have observed that environmental enrichment modulates neuronal response strength, selectivity, speed of response, and synchronization to rapid sensory input. Other reports suggest that nonprimary sensory fields are more plastic than primary sensory cortex. The consequences of environmental enrichment on information processing in nonprimary sensory cortex have yet to be studied. Here we examine physiological effects of enrichment in the posterior auditory field (PAF), a field distinguished from primary auditory cortex (A1) by wider receptive fields, slower response times, and a greater preference for slowly modulated sounds. Environmental enrichment induced a significant increase in spectral and temporal selectivity in PAF. PAF neurons exhibited narrower receptive fields and responded significantly faster and for a briefer period to sounds after enrichment. Enrichment increased time-locking to rapidly successive sensory input in PAF neurons. Compared with previous enrichment studies in A1, we observe a greater magnitude of reorganization in PAF after environmental enrichment. Along with other reports observing greater reorganization in nonprimary sensory cortex, our results in PAF suggest that nonprimary fields might have a greater capacity for reorganization compared with primary fields. PMID:22131375

Uranium enrichment

International Nuclear Information System (INIS)

1989-01-01

GAO was asked to address several questions concerning a number of proposed uranium enrichment bills introduced during the 100th Congress. The bill would have restructured the Department of Energy's uranium enrichment program as a government corporation to allow it to compete more effectively in the domestic and international markets. Some of GAO's findings discussed are: uranium market experts believe and existing market models show that the proposed DOE purchase of a $750 million of uranium from domestic producers may not significantly increase production because of large producer-held inventories; excess uranium enrichment production capacity exists throughout the world; therefore, foreign producers are expected to compete heavily in the United States throughout the 1990s as utilities' contracts with DOE expire; and according to a 1988 agreement between DOE's Offices of Nuclear Energy and Defense Programs, enrichment decommissioning costs, estimated to total $3.6 billion for planning purposes, will be shared by the commercial enrichment program and the government

Isotope enrichment

International Nuclear Information System (INIS)

Lydtin, H-J.; Wilden, R.J.; Severin, P.J.W.

1978-01-01

The isotope enrichment method described is based on the recognition that, owing to mass diffusion and thermal diffusion in the conversion of substances at a heated substrate while depositing an element or compound onto the substrate, enrichment of the element, or a compound of the element, with a lighter isotope will occur. The cycle is repeated for as many times as is necessary to obtain the degree of enrichment required

Structural coupling of SH2-kinase domains links Fes and Abl substrate recognition and kinase activation.

Science.gov (United States)

Filippakopoulos, Panagis; Kofler, Michael; Hantschel, Oliver; Gish, Gerald D; Grebien, Florian; Salah, Eidarus; Neudecker, Philipp; Kay, Lewis E; Turk, Benjamin E; Superti-Furga, Giulio; Pawson, Tony; Knapp, Stefan

2008-09-05

The SH2 domain of cytoplasmic tyrosine kinases can enhance catalytic activity and substrate recognition, but the molecular mechanisms by which this is achieved are poorly understood. We have solved the structure of the prototypic SH2-kinase unit of the human Fes tyrosine kinase, which appears specialized for positive signaling. In its active conformation, the SH2 domain tightly interacts with the kinase N-terminal lobe and positions the kinase alphaC helix in an active configuration through essential packing and electrostatic interactions. This interaction is stabilized by ligand binding to the SH2 domain. Our data indicate that Fes kinase activation is closely coupled to substrate recognition through cooperative SH2-kinase-substrate interactions. Similarly, we find that the SH2 domain of the active Abl kinase stimulates catalytic activity and substrate phosphorylation through a distinct SH2-kinase interface. Thus, the SH2 and catalytic domains of active Fes and Abl pro-oncogenic kinases form integrated structures essential for effective tyrosine kinase signaling.

FEMO, A FLOW AND ENRICHMENT MONITOR FOR VERIFYING COMPLIANCE WITH INTERNATIONAL SAFEGUARDS REQUIREMENTS AT A GAS CENTRIFUGE ENRICHMENT FACILITY

International Nuclear Information System (INIS)

Gunning, John E.; Laughter, Mark D.; March-Leuba, Jose A.

2008-01-01

A number of countries have received construction licenses or are contemplating the construction of large-capacity gas centrifuge enrichment plants (GCEPs). The capability to independently verify nuclear material flows is a key component of international safeguards approaches, and the IAEA does not currently have an approved method to continuously monitor the mass flow of 235U in uranium hexafluoride (UF6) gas streams. Oak Ridge National Laboratory is investigating the development of a flow and enrichment monitor, or FEMO, based on an existing blend-down monitoring system (BDMS). The BDMS was designed to continuously monitor both 235U mass flow and enrichment of UF6 streams at the low pressures similar to those which exists at GCEPs. BDMSs have been installed at three sites-the first unit has operated successfully in an unattended environment for approximately 10 years. To be acceptable to GCEP operators, it is essential that the instrument be installed and maintained without interrupting operations. A means to continuously verify flow as is proposed by FEMO will likely be needed to monitor safeguards at large-capacity plants. This will enable the safeguards effectiveness that currently exists at smaller plants to be maintained at the larger facilities and also has the potential to reduce labor costs associated with inspections at current and future plants. This paper describes the FEMO design requirements, operating capabilities, and development work required before field demonstration.

The Link between Protein Kinase CK2 and Atypical Kinase Rio1

Directory of Open Access Journals (Sweden)

Konrad Kubiński

2017-02-01

Full Text Available The atypical kinase Rio1 is widespread in many organisms, ranging from Archaebacteria to humans, and is an essential factor in ribosome biogenesis. Little is known about the protein substrates of the enzyme and small-molecule inhibitors of the kinase. Protein kinase CK2 was the first interaction partner of Rio1, identified in yeast cells. The enzyme from various sources undergoes CK2-mediated phosphorylation at several sites and this modification regulates the activity of Rio1. The aim of this review is to present studies of the relationship between the two different kinases, with respect to CK2-mediated phosphorylation of Rio1, regulation of Rio1 activity, and similar susceptibility of the kinases to benzimidazole inhibitors.

Novel links in the plant TOR kinase signaling network.

Science.gov (United States)

Xiong, Yan; Sheen, Jen

2015-12-01

Nutrient and energy sensing and signaling mechanisms constitute the most ancient and fundamental regulatory networks to control growth and development in all life forms. The target of rapamycin (TOR) protein kinase is modulated by diverse nutrient, energy, hormone and stress inputs and plays a central role in regulating cell proliferation, growth, metabolism and stress responses from yeasts to plants and animals. Recent chemical, genetic, genomic and metabolomic analyses have enabled significant progress toward molecular understanding of the TOR signaling network in multicellular plants. This review discusses the applications of new chemical tools to probe plant TOR functions and highlights recent findings and predictions on TOR-mediate biological processes. Special focus is placed on novel and evolutionarily conserved TOR kinase effectors as positive and negative signaling regulators that control transcription, translation and metabolism to support cell proliferation, growth and maintenance from embryogenesis to senescence in the plant system. Copyright © 2015 Elsevier Ltd. All rights reserved.

Regulation of Autophagy by Kinases

International Nuclear Information System (INIS)

Sridharan, Savitha; Jain, Kirti; Basu, Alakananda

2011-01-01

Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets

Regulation of Autophagy by Kinases

Energy Technology Data Exchange (ETDEWEB)

Sridharan, Savitha; Jain, Kirti; Basu, Alakananda, E-mail: alakananda.basu@unthsc.edu [Department of Molecular Biology and Immunology, Institute for Cancer Research, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

2011-06-09

Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets.

Regulation of Autophagy by Kinases

Science.gov (United States)

Sridharan, Savitha; Jain, Kirti; Basu, Alakananda

2011-01-01

Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets. PMID:24212825

Regulation of Autophagy by Kinases

Directory of Open Access Journals (Sweden)

Savitha Sridharan

2011-06-01

Full Text Available Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK and protein kinase C that are often deregulated in cancer and are important therapeutic targets.

Comparative studies of a new subfamily of human Ste20-like kinases: homodimerization, subcellular localization, and selective activation of MKK3 and p38.

Science.gov (United States)

Yustein, Jason T; Xia, Liang; Kahlenburg, J Michelle; Robinson, Dan; Templeton, Dennis; Kung, Hsing-Jien

2003-09-18

The Sterile-20 or Ste20 family of serine/threonine kinases is a group of signaling molecules whose physiological roles within mammalian cells are just starting to be elucidated. Here, in this report we present the characterization of three human Ste20-like kinases with greater than 90% similarity within their catalytic domains that define a novel subfamily of Ste20s. Members of this kinase family include rat thousand and one (TAO1) and chicken KFC (kinase from chicken). For the lack of a consensus nomenclature in the literature, in this report, we shall call this family hKFC (for their homology to chicken KFC) and the three members hKFC-A, hKFC-B, and hKFC-C, respectively. These kinases have many similarities including an aminoterminal kinase domain, a serine-rich region, and a coiled-coil configuration within the C-terminus. All three kinases are able to activate the p38 MAP kinase pathway through the specific activation of the upstream MKK3 kinase. We also offer evidence, both theoretical and biochemical, showing that these kinases can undergo self-association. Despite these similarities, these kinases differ in tissue distribution, apparent subcellular localization, and feature structural differences largely within the carboxyl-terminal sequence.

Characteristics of isotope-selective chemical reactor with gas-separating device

International Nuclear Information System (INIS)

Gorshunov, N.M.; Kalitin, S.A.; Laguntsov, N.I.; Neshchimenko, Yu.P.; Sulaberidze, G.A.

1988-01-01

A study was made on characteristics of separating stage, composed of isotope-selective chemical (or photochemical) reactor and membrane separating cascade (MSC), designated for separation of isotope-enriched products from lean reagents. MSC represents the counterflow cascade for separation of two-component mixtures. Calculations show that for the process of carton isotope separation the electric power expences for MSC operation are equal to 20 kWxh/g of CO 2 final product at 13 C isotope content in it equal to 75%. Application of the membrane gas-separating cascade at rather small electric power expenses enables to perform cascading of isotope separation in the course of nonequilibrium chemical reactions

User Information Needs for Environmental Opinion-forming and Decision-making in Link-enriched Video

NARCIS (Netherlands)

A.C. Palumbo; L. Hardman (Lynda)

2013-01-01

htmlabstractLink-enriched video can support users in informative processes of environmental opinion-forming and decision-making. To enable this, we need to specify the information that should be captured in an annotation schema for describing the video. We conducted expert interviews to elicit

Kinase activity in the olfactory bulb is required for odor memory consolidation.

Science.gov (United States)

Tong, Michelle T; Kim, Tae-Young P; Cleland, Thomas A

2018-05-01

Long-term fear memory formation in the hippocampus and neocortex depends upon brain-derived neurotrophic factor (BDNF) signaling after acquisition. Incremental, appetitive odor discrimination learning is thought to depend substantially on the differentiation of adult-born neurons within the olfactory bulb (OB)-a process that is closely associated with BDNF signaling. We sought to elucidate the role of neurotrophin signaling within the OB on odor memory consolidation. Male mice were trained on odor-reward associative discriminations after bilateral infusion of the kinase inhibitor K252a, or vehicle control, into the OB. K252a is a partially selective inhibitor of tyrosine kinase (Trk) receptors, including the TrkB receptor for BDNF, though it also inhibits other plasticity-related kinases such as PKC and CaMKII/IV. K252a infusion into the OB did not impair odor acquisition or short-term (2 h) memory for the learned discriminations, but significantly impaired long-term (48 h) odor memory (LTM). This LTM deficit also was associated with reduced selectivity for the conditioned odorant in a reward-seeking digging task. Infusions of K252a immediately prior to testing did not impair LTM recall. These results indicate that kinase activation in the OB is required for the consolidation of odor memory of incrementally acquired information. © 2018 Tong et al.; Published by Cold Spring Harbor Laboratory Press.

Investigation towards link-enriched video: user information needs for environmental opinion-forming and decision-making

NARCIS (Netherlands)

A.C. Palumbo

2012-01-01

htmlabstractLink-enriched video can support users in processes of environmental opinion-forming and decision-making. For this, audiovisual content must be represented and annotated to enable automatic link generation and computer manipulation. Given the time and budget constraints of

Real Time Control of CO2 Enrichment Experiments on the Sea Floor Enabled by the MARS Cabled Observatory

Science.gov (United States)

Brewer, P. G.; Mbari Foce Team

2010-12-01

We report on progress on FOCE (Free Ocean CO2 Enrichment) techniques designed to accomplish realistic (that is not contained within land-based aquaria) experiments on the response of deep-sea animals and biogeochemical cycles to ocean acidification. Such experiments have long been carried out on ecosystems on land, and the outcome has differed significantly from CO2 enrichment in enclosed greenhouse systems, thereby undoing much of the hope for an increase in the large-scale biosphere draw down of atmospheric CO2. It is a far bigger step if deep-sea animals and systems are removed from their cold, dark, high pressure and low oxygen native habitat. The equivalent problem in the ocean is far more difficult because of (1) the very different physical forcing; (2) the complex reaction rates between CO2 and water require delay times between addition and entry to the experimental space; (3) the lack of supporting infrastructure and of adequate sensors; and (4) the need for sophisticated and robust control techniques in both hardware and software. We have overcome almost all of these challenges, and related working systems have already been successfully deployed on the Great Barrier Reef coralline flats with Australian colleagues. We have used the MBARI MARS (Monterey Accelerated Research System) cabled observatory to carry out deep-ocean (880m depth) experiments. The basic experimental unit is a 1m x 1m x 50cm chamber with side arms of ~ 3m length to provide the required chemical delay times for the reaction between admixed CO2 enriched sea water and emergence of the flow into the main chamber. Controllable thrusters, operated by user commands, help maintain a steady flow of seawater through the experiment. The site is slightly below the depth of the O2 minimum where small changes in either O2 from ocean warming, or CO2 from ocean acidification can lead to the formation of dead zones. Shallow (near shore) experiments are now also in the late planning stages. We have

New generation enrichment monitoring technology for gas centrifuge enrichment plants

International Nuclear Information System (INIS)

Ianakiev, Kiril D.; Alexandrov, Boian S.; Boyer, Brian D.; Hill, Thomas R.; Macarthur, Duncan W.; Marks, Thomas; Moss, Calvin E.; Sheppard, Gregory A.; Swinhoe, Martyn T.

2008-01-01

The continuous enrichment monitor, developed and fielded in the 1990s by the International Atomic Energy Agency, provided a go-no-go capability to distinguish between UF 6 containing low enriched (approximately 4% 235 U) and highly enriched (above 20% 235 U) uranium. This instrument used the 22-keV line from a 109 Cd source as a transmission source to achieve a high sensitivity to the UF 6 gas absorption. The 1.27-yr half-life required that the source be periodically replaced and the instrument recalibrated. The instrument's functionality and accuracy were limited by the fact that measured gas density and gas pressure were treated as confidential facility information. The modern safeguarding of a gas centrifuge enrichment plant producing low-enriched UF 6 product aims toward a more quantitative flow and enrichment monitoring concept that sets new standards for accuracy stability, and confidence. An instrument must be accurate enough to detect the diversion of a significant quantity of material, have virtually zero false alarms, and protect the operator's proprietary process information. We discuss a new concept for advanced gas enrichment assay measurement technology. This design concept eliminates the need for the periodic replacement of a radioactive source as well as the need for maintenance by experts. Some initial experimental results will be presented.

Are Janus Kinase Inhibitors Superior over Classic Biologic Agents in RA Patients?

Directory of Open Access Journals (Sweden)

Przemyslaw J. Kotyla

2018-01-01

Full Text Available The Janus Kinases (JAKs are a family of intracellular tyrosine kinases that provide transmission signals from cytokine, interferons, and many hormones receptors to the nucleus resulting in synthesis of many biologically active compounds and changing cell metabolism and function. That was theoretical background to synthetize the JAK inhibitors (Jakinibs. In recent years a substantial battery of evidence has been collected indicating the potential role of Jakinibs to interact with the specific elements of the immune system, therefore changing the inflammatory response. JAK kinase blockade offers a unique opportunity to block most of the key cytokines enabling the deep interaction into immune system functioning. Following discovery first Jakinibs were intensively studied in various forms of autoimmune diseases, including rheumatoid arthritis, and finally two Jakinibs tofacitinib and Baricitinib have been approved for the treatment of rheumatoid arthritis. Some clinical data indicated that under special circumstances Jakinibs may be even superior to biologics in the treatment of RA; however this suggestion should be verified in large clinical and observational studies.

Target-dependent enrichment of virions determines the reduction of high-throughput sequencing in virus discovery.

Directory of Open Access Journals (Sweden)

Randi Holm Jensen

Full Text Available Viral infections cause many different diseases stemming both from well-characterized viral pathogens but also from emerging viruses, and the search for novel viruses continues to be of great importance. High-throughput sequencing is an important technology for this purpose. However, viral nucleic acids often constitute a minute proportion of the total genetic material in a sample from infected tissue. Techniques to enrich viral targets in high-throughput sequencing have been reported, but the sensitivity of such methods is not well established. This study compares different library preparation techniques targeting both DNA and RNA with and without virion enrichment. By optimizing the selection of intact virus particles, both by physical and enzymatic approaches, we assessed the effectiveness of the specific enrichment of viral sequences as compared to non-enriched sample preparations by selectively looking for and counting read sequences obtained from shotgun sequencing. Using shotgun sequencing of total DNA or RNA, viral targets were detected at concentrations corresponding to the predicted level, providing a foundation for estimating the effectiveness of virion enrichment. Virion enrichment typically produced a 1000-fold increase in the proportion of DNA virus sequences. For RNA virions the gain was less pronounced with a maximum 13-fold increase. This enrichment varied between the different sample concentrations, with no clear trend. Despite that less sequencing was required to identify target sequences, it was not evident from our data that a lower detection level was achieved by virion enrichment compared to shotgun sequencing.

Identification of novel inhibitors for Pim-1 kinase using pharmacophore modeling based on a novel method for selecting pharmacophore generation subsets

Science.gov (United States)

Shahin, Rand; Swellmeen, Lubna; Shaheen, Omar; Aboalhaija, Nour; Habash, Maha

2016-01-01

Targeting Proviral integration-site of murine Moloney leukemia virus 1 kinase, hereafter called Pim-1 kinase, is a promising strategy for treating different kinds of human cancer. Headed for this a total list of 328 formerly reported Pim-1 kinase inhibitors has been explored and divided based on the pharmacophoric features of the most active molecules into 10 subsets projected to represent potential active binding manners accessible to ligands within the binding pocket of Pim-1 kinase. Discovery Studio 4.1 (DS 4.1) was employed to detect potential pharmacophoric active binding manners anticipated by Pim-1 Kinase inhibitors. The pharmacophoric models were then allowed to compete within Quantitative Structure Activity Relationship (QSAR) framework with other 2D descriptors. Accordingly Genetic algorithm and multiple linear regression investigation were engaged to find the finest QSAR equation that has the best predictive power r 262 2 = 0.70, F = 119.14, r LOO 2 = 0.693, r PRESS 2 against 66 external test inhibitors = 0.71 q2 = 0.55. Three different pharmacophores appeared in the successful QSAR equation this represents three different binding modes for inhibitors within the Pim-1 kinase binding pocket. Pharmacophoric models were later used to screen compounds within the National Cancer Institute database. Several low micromolar Pim-1 Kinase inhibitors were captured. The most potent hits show IC50 values of 0.77 and 1.03 µM. Also, upon analyzing the successful QSAR Equation we found that some polycyclic aromatic electron-rich structures namely 6-Chloro-2-methoxy-acridine can be considered as putative hits for Pim-1 kinase inhibition.

Uranium enrichment plans

International Nuclear Information System (INIS)

Thomas, D.C.; Gagne, R.W.

1978-01-01

The following topics are covered: the status of the Government's existing uranium enrichment services contracts, natural uranium requirements based on the latest contract information, uncertainty in predicting natural uranium requirements based on uranium enrichment contracts, and domestic and foreign demand assumed in enrichment planning

UF/sub 6/ test loop for evaluation and implementation of international enrichment plant safeguards

International Nuclear Information System (INIS)

Cooley, J.N.; Fields, L.W.; Swindle, D.W. Jr.

1987-01-01

A functional test loop capable of simulating UF/sub 6/ flows, pressures, and pipe deposits characteristic of gas centrifuge enrichment plant piping has been designed and fabricated by the Enrichment Safeguards Program of Martin Marietta Energy Systems, Inc., for use by the International Atomic Energy Agency (IAEA) at its Safeguards Analytical Laboratory in Seibersdorf, Austria. The purpose of the test loop is twofold: (1) to enable the IAEA to evaluate and to calibrate enrichment safeguards measurement instrumentation to be used in limited frequency-unannounced access (LFUA) inspection strategy measurements at gas centrifuge enrichment plants and (2) to train IAEA inspectors in the use of such instrumentation. The test loop incorporates actual sections of cascade header pipes from the centrifuge enrichment plants subject to IAEA inspections. The test loop is described, applications for its use by the IAEA are detailed, and results from an initial demonstration session using the test loop are summarized. By giving the IAEA the in-house capability to evaluate LFUA inspection strategy approaches, to develop inspection procedures, to calibrate instrumentation, and to train inspectors, the UF/sub 6/ cascade header pipe test loop will contribute to the IAEA's success in implementing LFUA strategy inspections at gas centrifuge enrichment facilities subject to international safeguards inspections

5-Substituted 3-isopropyl-7-[4-(2-pyridyl)benzyl]amino-1(2)H-pyrazolo[4,3-d]pyrimidines with anti-proliferative activity as potent and selective inhibitors of cyclin-dependent kinases

Czech Academy of Sciences Publication Activity Database

Vymětalová, Ladislava; Havlíček, Libor; Šturc, Antonín; Skrášková, Zuzana; Jorda, Radek; Pospíšil, Tomáš; Strnad, Miroslav; Kryštof, Vladimír

2016-01-01

Roč. 110, MAR 3 (2016), s. 291-301 ISSN 0223-5234 R&D Projects: GA MŠk(CZ) LO1204; GA ČR(CZ) GA15-15264S Institutional support: RVO:61389030 Keywords : Cyclin-dependent kinase * Inhibitor * Selectivity Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.519, year: 2016

Examination of transcript amounts and activity of protein kinase CK2 in muscle lysates of different types of human muscle pathologies.

Science.gov (United States)

Heuss, Dieter; Klascinski, Janine; Schubert, Steffen W; Moriabadi, Tehmur; Lochmüller, Hanns; Hashemolhosseini, Said

2008-09-01

Motoneurons release the heparansulfate proteoglycan agrin and thereby activate the muscle-specific receptor tyrosine kinase (MuSK), which is the main organizer of subsynaptic specializations at the neuromuscular junction. Recently, we showed that (1) the protein kinase CK2 interacts with the intracellular region of MuSK; (2) the CK2 protein is enriched and co-localized with MuSK at postsynaptic specializations; (3) CK2-mediated phosphorylation of serine residues within a specific MuSK epitope, named the kinase insert, regulates acetylcholine receptor (AChR) clustering; (4) muscle-specific CK2beta knockout mice develop a myasthenic phenotype due to impaired muscle endplate structure and function (see Genes Dev 20(13):1800-1816, 2006). Here, we investigated for the first time if CK2 is modulated in biopsies from human patients. To this end, we measured transcript amounts of the subunits CK2alpha and CK2beta and determined holoenzyme CK2 activity in 34 muscle biopsies of human patients with different muscle pathologies.

The Interaction of Src Kinase with beta 3 Integrin Tails : A Potential Therapeutic Target in Thrombosis and Cancer

NARCIS (Netherlands)

Huveneers, Stephan; Danen, Erik H. J.

2010-01-01

Activation of Src family kinases is an important event downstream of integrin adhesion signaling in many cell types. A particularly intriguing connection between an integrin and a Src family kinase was first discovered in platelets, where the selective direct interaction of alpha IIb beta 3

Radioimmunoassay measurement of creatine kinase BB in the serum of schizophrenic patients

Energy Technology Data Exchange (ETDEWEB)

Lerner, M H; Friedhoff, A J [New York Univ., NY (USA). Medical Center

1980-10-23

Brain type creatine kinase (BB) isoenzyme was measured using a highly sensitive and specific radioimmunoassay procedure (limit of detection, 1 ..mu..g/l of sample) in two schizophrenic populations, an acute non-medicated group consisting of 35 subjects and a chronic group of 15 subjects. Since the assay can also measure the B subunit of MB isoenzyme, patients were selected so as to exclude subjects with possible heart, kidney or other ailments which might result in an increased serum creatine kinase B subunit. Both the acute schizophrenics (3.0 +- 0.23) x S.E.M. and the chronic schizophrenics (2.9 +- 0.33) had serum levels of creatine kinase BB similar to those of controls (2.8 +- 0.21) and non-cardiac patients (3.5 +- 0.58). Patients having myocardial infarction or neurovascular surgery had elevated creatine kinase B subunit. Similar but much less sensitive quantitative results were obtained using agarose multizonal electrophoresis.

Radioimmunoassay measurement of creatine kinase BB in the serum of schizophrenic patients

International Nuclear Information System (INIS)

Lerner, M.H.; Friedhoff, A.J.

1980-01-01

Brain type creatine kinase (BB) isoenzyme was measured using a highly sensitive and specific radioimmunoassay procedure (limit of detection, 1 μg/l of sample) in two schizophrenic populations, an acute non-medicated group consisting of 35 subjects and a chronic group of 15 subjects. Since the assay can also measure the B subunit of MB isoenzyme, patients were selected so as to exclude subjects with possible heart, kidney or other ailments which might result in an increased serum creatine kinase B subunit. Both the acute schizophrenics (3.0 +- 0.23) x S.E.M. and the chronic schizophrenics (2.9 +- 0.33) had serum levels of creatine kinase BB similar to those of controls (2.8 +- 0.21) and non-cardiac patients (3.5 +- 0.58). Patients having myocardial infarction or neurovascular surgery had elevated creatine kinase B subunit. Similar but much less sensitive quantitative results were obtained using agarose multizonal electrophoresis. (Auth.)

Marine-Derived 2-Aminoimidazolone Alkaloids. Leucettamine B-Related Polyandrocarpamines Inhibit Mammalian and Protozoan DYRK & CLK Kinases

Directory of Open Access Journals (Sweden)

Nadège Loaëc

2017-10-01

Full Text Available A large diversity of 2-aminoimidazolone alkaloids is produced by various marine invertebrates, especially by the marine Calcareous sponges Leucetta and Clathrina. The phylogeny of these sponges and the wide scope of 2-aminoimidazolone alkaloids they produce are reviewed in this article. The origin (invertebrate cells, associated microorganisms, or filtered plankton, physiological functions, and natural molecular targets of these alkaloids are largely unknown. Following the identification of leucettamine B as an inhibitor of selected protein kinases, we synthesized a family of analogues, collectively named leucettines, as potent inhibitors of DYRKs (dual-specificity, tyrosine phosphorylation regulated kinases and CLKs (cdc2-like kinases and potential pharmacological leads for the treatment of several diseases, including Alzheimer’s disease and Down syndrome. We assembled a small library of marine sponge- and ascidian-derived 2-aminoimidazolone alkaloids, along with several synthetic analogues, and tested them on a panel of mammalian and protozoan kinases. Polyandrocarpamines A and B were found to be potent and selective inhibitors of DYRKs and CLKs. They inhibited cyclin D1 phosphorylation on a DYRK1A phosphosite in cultured cells. 2-Aminoimidazolones thus represent a promising chemical scaffold for the design of potential therapeutic drug candidates acting as specific inhibitors of disease-relevant kinases, and possibly other disease-relevant targets.

Site-directed alkylation of multiple opioid receptors. I. Binding selectivity

International Nuclear Information System (INIS)

James, I.F.; Goldstein, A.

1984-01-01

A method for measuring and expressing the binding selectivity of ligands for mu, delta, and kappa opioid binding sites is reported. Radioligands are used that are partially selective for these sites in combination with membrane preparations enriched in each site. Enrichment was obtained by treatment of membranes with the alkylating agent beta-chlornaltrexamine in the presence of appropriate protecting ligands. After enrichment for mu receptors, [ 3 H] dihydromorphine bound to a single type of site as judged by the slope of competition binding curves. After enrichment for delta or kappa receptors, binding sites for [ 3 H] [D-Ala2, D-Leu5]enkephalin and [3H]ethylketocyclazocine, respectively, were still not homogeneous. There were residual mu sites in delta-enriched membranes but no evidence for residual mu or delta sites in kappa-enriched membranes were found. This method was used to identify ligands that are highly selective for each of the three types of sites

Synthesis and Anti-Proliferative Effects of Mono- and Bis-Purinomimetics Targeting Kinases

Directory of Open Access Journals (Sweden)

Andrea Bistrović

2017-11-01

Full Text Available A series of mono-pyrrolo[2,3-d]pyrimidines 4a–4k, unsymmetrical bis-purine isosteres 5a–5e and symmetrical bis-pyrrolo[2,3-d]pyrimidines 6a and 6b connected via di(1,2,3-triazolylphenyl linker were synthesized by click chemistry. Whereas mono- 4g and bis-pseudopurine 5e showed selective inhibitory activities on cervical carcinoma (HeLa cells, bis-pyrrolo[2,3-d]pyrimidine 6b exhibited potent and selective anti-proliferative effect in the nanomolar range on pancreatic carcinoma (CFPAC-1 cells. Among these, compound 6b induced a significant reduction in the expression level of CDK9 (cyclin-dependent kinase 9/cyclin T1 in CFPAC-1 cells concomitant with attenuation of proliferative signaling mediated by c-Raf (rapidly accelerated fibrosarcoma and p38 MAP (mitogen-activated protein kinases. Our findings encourage further development of novel structurally related analog of 6b to obtain more selective anticancer agent for treating pancreatic cancer.

Role of adiponectin/phosphatidylinositol 3-kinase/protein kinase B ...

African Journals Online (AJOL)

The adiponectin/phosphatidylinositol 3-kinase/protein kinase B (ADP/PI3k/Akt) signal transduction pathway has an important role in promoting cell survival. This study was designed to determine if the ADP/PI3K/Akt signaling pathway has a role in the mechanism of ischemia–reperfusion injury in vivo. Sprague–Dawley rats ...

Selection and use of a low enriched fuel in high performance research reactors

International Nuclear Information System (INIS)

Cerles, J.M.; Schwartz, J.P.

1978-08-01

A new nuclear fuel composition for research reactors (Osiris, Siloe) is studied using low enriched (E<20%) uranium oxide. Its utilization leads to modifications in the facilities of these experimental reactors: increase of primary coolant flow, modifications in failed element detection system, handling of materials and storage

Other enrichment related contracts

International Nuclear Information System (INIS)

Hall, J.C.

1978-01-01

In addition to long-term enrichment contracts, DOE has other types of contracts: (1) short-term, fixed-commitment enrichment contract; (2) emergency sales agreement for enriched uranium; (3) feed material lease agreement; (4) enriched uranium storage agreement; and (5) feed material usage agreement

Use of enriched uranium in Canada's power reactors

International Nuclear Information System (INIS)

Dormuth, K.W.; Jackson, D.P.

2011-01-01

Recent trends in Canadian nuclear power reactor design and proposed development of nuclear power in Canada have indicated the possibility that Canada will break with its tradition of natural uranium fuelled systems, designed for superior neutron economy and, hence, superior uranium utilization. For instance, the Darlington B new reactor project procurement process included three reactor designs, all employing enriched fuel, although a natural uranium reactor design was included at a late stage in the ensuing environmental assessment for the project as an alternative technology. An evaluation of the alternative designs should include an assessment of the environmental implications through the entire fuel cycle, which unfortunately is not required by the environmental assessment process. Examples of comparative environmental implications of the reactor designs throughout the fuel cycle indicate the importance of these considerations when making a design selection. As Canada does not have enrichment capability, a move toward the use of enriched fuel would mean that Canada would be exporting natural uranium and buying back enriched uranium with value added. From a waste management perspective, Canada would need to deal with mill, refinery, and conversion tailings, as well as with the used fuel from its own reactors, while the enrichment supplier would retain depleted uranium with some commercial value. On the basis of reasoned estimates based on publicly available information, it is expected that enrichment in Canada is likely to be more profitable than exporting natural uranium and buying back enriched uranium. Further, on the basis of environmental assessments for enrichment facilities in other countries, it is expected that an environmental assessment of a properly sited enrichment facility would result in approval. (author)

Hyb-Seq: Combining target enrichment and genome skimming for plant phylogenomics1

Science.gov (United States)

Weitemier, Kevin; Straub, Shannon C. K.; Cronn, Richard C.; Fishbein, Mark; Schmickl, Roswitha; McDonnell, Angela; Liston, Aaron

2014-01-01

• Premise of the study: Hyb-Seq, the combination of target enrichment and genome skimming, allows simultaneous data collection for low-copy nuclear genes and high-copy genomic targets for plant systematics and evolution studies. • Methods and Results: Genome and transcriptome assemblies for milkweed (Asclepias syriaca) were used to design enrichment probes for 3385 exons from 768 genes (>1.6 Mbp) followed by Illumina sequencing of enriched libraries. Hyb-Seq of 12 individuals (10 Asclepias species and two related genera) resulted in at least partial assembly of 92.6% of exons and 99.7% of genes and an average assembly length >2 Mbp. Importantly, complete plastomes and nuclear ribosomal DNA cistrons were assembled using off-target reads. Phylogenomic analyses demonstrated signal conflict between genomes. • Conclusions: The Hyb-Seq approach enables targeted sequencing of thousands of low-copy nuclear exons and flanking regions, as well as genome skimming of high-copy repeats and organellar genomes, to efficiently produce genome-scale data sets for phylogenomics. PMID:25225629

High motivation for exercise is associated with altered chromatin regulators of monoamine receptor gene expression in the striatum of selectively bred mice.

Science.gov (United States)

Saul, M C; Majdak, P; Perez, S; Reilly, M; Garland, T; Rhodes, J S

2017-03-01

Although exercise is critical for health, many lack the motivation to exercise, and it is unclear how motivation might be increased. To uncover the molecular underpinnings of increased motivation for exercise, we analyzed the transcriptome of the striatum in four mouse lines selectively bred for high voluntary wheel running and four non-selected control lines. The striatum was dissected and RNA was extracted and sequenced from four individuals of each line. We found multiple genes and gene systems with strong relationships to both selection and running history over the previous 6 days. Among these genes were Htr1b, a serotonin receptor subunit and Slc38a2, a marker for both glutamatergic and γ-aminobutyric acid (GABA)-ergic signaling. System analysis of the raw results found enrichment of transcriptional regulation and kinase genes. Further, we identified a splice variant affecting the Wnt-related Golgi signaling gene Tmed5. Using coexpression network analysis, we found a cluster of interrelated coexpression modules with relationships to running behavior. From these modules, we built a network correlated with running that predicts a mechanistic relationship between transcriptional regulation by nucleosome structure and Htr1b expression. The Library of Integrated Network-Based Cellular Signatures identified the protein kinase C δ inhibitor, rottlerin, the tyrosine kinase inhibitor, Linifanib and the delta-opioid receptor antagonist 7-benzylidenenaltrexone as potential compounds for increasing the motivation to run. Taken together, our findings support a neurobiological framework of exercise motivation where chromatin state leads to differences in dopamine signaling through modulation of both the primary neurotransmitters glutamate and GABA, and by neuromodulators such as serotonin. © 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Tyrosine kinases in rheumatoid arthritis

Directory of Open Access Journals (Sweden)

Kobayashi Akiko

2011-08-01

Full Text Available Abstract Rheumatoid arthritis (RA is an inflammatory, polyarticular joint disease. A number of cellular responses are involved in the pathogenesis of rheumatoid arthritis, including activation of inflammatory cells and cytokine expression. The cellular responses involved in each of these processes depends on the specific signaling pathways that are activated; many of which include protein tyrosine kinases. These pathways include the mitogen-activated protein kinase pathway, Janus kinases/signal transducers and activators transcription pathway, spleen tyrosine kinase signaling, and the nuclear factor κ-light-chain-enhancer of activated B cells pathway. Many drugs are in development to target tyrosine kinases for the treatment of RA. Based on the number of recently published studies, this manuscript reviews the role of tyrosine kinases in the pathogenesis of RA and the potential role of kinase inhibitors as new therapeutic strategies of RA.

Two-dimensional solid-phase extraction strategy for the selective enrichment of aminoglycosides in milk.

Science.gov (United States)

Shen, Aijin; Wei, Jie; Yan, Jingyu; Jin, Gaowa; Ding, Junjie; Yang, Bingcheng; Guo, Zhimou; Zhang, Feifang; Liang, Xinmiao

2017-03-01

An orthogonal two-dimensional solid-phase extraction strategy was established for the selective enrichment of three aminoglycosides including spectinomycin, streptomycin, and dihydrostreptomycin in milk. A reversed-phase liquid chromatography material (C 18 ) and a weak cation-exchange material (TGA) were integrated in a single solid-phase extraction cartridge. The feasibility of two-dimensional clean-up procedure that experienced two-step adsorption, two-step rinsing, and two-step elution was systematically investigated. Based on the orthogonality of reversed-phase and weak cation-exchange procedures, the two-dimensional solid-phase extraction strategy could minimize the interference from the hydrophobic matrix existing in traditional reversed-phase solid-phase extraction. In addition, high ionic strength in the extracts could be effectively removed before the second dimension of weak cation-exchange solid-phase extraction. Combined with liquid chromatography and tandem mass spectrometry, the optimized procedure was validated according to the European Union Commission directive 2002/657/EC. A good performance was achieved in terms of linearity, recovery, precision, decision limit, and detection capability in milk. Finally, the optimized two-dimensional clean-up procedure incorporated with liquid chromatography and tandem mass spectrometry was successfully applied to the rapid monitoring of aminoglycoside residues in milk. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Investigating the role of class-IA PI 3-kinase isoforms in adipocyte differentiation

International Nuclear Information System (INIS)

Kim, Ji Eun; Shepherd, Peter R.; Chaussade, Claire

2009-01-01

PI 3-kinases, in particular class-IA, are key signalling molecules controlling many cellular processes including growth, proliferation, migration and differentiation. In this study, we have used a collection of isoform selective PI 3-kinase inhibitors to determine whether attenuation of signalling through class-IA PI 3-kinase isoforms will impact adipocyte differentiation. First, we analysed the expression profiles and found that fibroblastic pre-adipocytes express detectable levels of p110α and p110δ and that after differentiation, p110δ levels fall while p110α levels rise, together with C/EBPα and PPARγ. When using specific inhibitors during the differentiation process, we observed that neither p110β nor p110δ inhibition, had any significant effect. In contrast PIK-75, a selective p110α inhibitor completely abolished adipocyte differentiation as assessed by morphology, transcript and protein levels of adipocyte markers. These results indicate that long term treatment with p110α inhibitors could potentially have a severe impact on fat cell numbers in vivo.

Summary of the Effort to Use Active-induced Time Correlation Techniques to Measure the Enrichment of HEU

Energy Technology Data Exchange (ETDEWEB)

McConchie, Seth M. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Crye, Jason Michael [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States); Pena, Kirsten [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States); Sword, Eric [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Mihalczo, John T. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)

2015-09-30

This document summarizes the effort to use active-induced time correlation techniques to measure the enrichment of bulk quantities of enriched uranium. In summary, these techniques use an external source to initiate fission chains, and the time distribution of the detected fission chain neutrons is sensitive to the fissile material enrichment. The number of neutrons emitted from a chain is driven by the multiplication of the item, and the enrichment is closely coupled to the multiplication of the item. As the enrichment increases (decreases), the multiplication increases (decreases) if the geometry is held constant. The time distribution of fission chain neutrons is a complex function of the enrichment and material configuration. The enrichment contributes to the probability of a subsequent fission in a chain via the likelihood of fissioning on an even-numbered isotope versus an odd-numbered isotope. The material configuration contributes to the same probability via solid angle effects for neutrons inducing subsequent fissions and the presence of any moderating material. To simplify the ability to accurately measure the enrichment, an associated particle imaging (API) D-T neutron generator and an array of plastic scintillators are used to simultaneously image the item and detect the fission chain neutrons. The image is used to significantly limit the space of enrichment and material configuration and enable the enrichment to be determined unambiguously.

Thymidine kinases in archaea

DEFF Research Database (Denmark)

Clausen, A.R.; Matakos, A.; Sandrini, Michael

2006-01-01

Twenty-six fully sequenced archaeal genomes were searched for genes coding for putative deoxyribonucleoside kinases (dNKs). We identified only 5 human-like thymidine kinase 1 genes (TK1s) and none for non-TK1 kinases. Four TK1s were identified in the Euryarchaea and one was found in the Crenarcha...

An Enrichment Program for Migrant Students: MENTE/UOP.

Science.gov (United States)

Gilbert, Michael B.

The report describes the objectives and accomplishments of a summer enrichment program, Migrantes Envueltos en Nuevos Temas de Educacion/Migrants Engaged in New Themes in Education (MENTE), for promising and talented migrant high schoolers. The program is a cooperative one with a university. Students selected by a review committee are tested for…

Focal adhesion kinase regulates neuronal growth, synaptic plasticity and hippocampus-dependent spatial learning and memory.

Science.gov (United States)

Monje, Francisco J; Kim, Eun-Jung; Pollak, Daniela D; Cabatic, Maureen; Li, Lin; Baston, Arthur; Lubec, Gert

2012-01-01

The focal adhesion kinase (FAK) is a non-receptor tyrosine kinase abundantly expressed in the mammalian brain and highly enriched in neuronal growth cones. Inhibitory and facilitatory activities of FAK on neuronal growth have been reported and its role in neuritic outgrowth remains controversial. Unlike other tyrosine kinases, such as the neurotrophin receptors regulating neuronal growth and plasticity, the relevance of FAK for learning and memory in vivo has not been clearly defined yet. A comprehensive study aimed at determining the role of FAK in neuronal growth, neurotransmitter release and synaptic plasticity in hippocampal neurons and in hippocampus-dependent learning and memory was therefore undertaken using the mouse model. Gain- and loss-of-function experiments indicated that FAK is a critical regulator of hippocampal cell morphology. FAK mediated neurotrophin-induced neuritic outgrowth and FAK inhibition affected both miniature excitatory postsynaptic potentials and activity-dependent hippocampal long-term potentiation prompting us to explore the possible role of FAK in spatial learning and memory in vivo. Our data indicate that FAK has a growth-promoting effect, is importantly involved in the regulation of the synaptic function and mediates in vivo hippocampus-dependent spatial learning and memory. Copyright © 2011 S. Karger AG, Basel.

Selective enrichment and biochemical characterization of seven human skin fibroblasts cell types in vitro

International Nuclear Information System (INIS)

Rodemann, H.P.; Bayreuther, K.; Francz, P.I.; Dittmann, K.; Albiez, M.

1989-01-01

The mitotic and postmitotic populations of the human skin fibroblast cell line HH-8 are heterogeneous when studied in vitro. There are reproducible changes in the frequencies of the mitotic fibroblasts (MF), MF I, MF II, MF III, and the postmitotic fibroblasts (PMF), PMF IV, PMF V, PMF VI, and PMF VII. For biochemical characterization, methods for selective enrichment of homogeneous populations of these seven fibroblast cell types have been established. Clonal populations with 95% purity for the mitotic fibroblasts MF I, MF II, and MF III can be raised in uniform clone types of fibroblasts (CTF) CTF I, CTF II, and CTF III. Pure clonal subpopulations of MF I type cells are present in mass populations in the range of 1-20 cumulative population doublings (CPD). Populations of mitotic fibroblasts represent nearly homogeneous populations of MF II (75-85% purity) in the range of 28-34 CPD and MF III (73-86% purity) in the range of 48-53 CPD. These populations can be easily expanded to up to 10(7)-10(8) cells. The spontaneous transition of MF III to PMF VI takes 140-180 days. In order to shorten this period and increase the proportion of distinct postmitotic types, mitotic fibroblast mass populations (CPD 30-32, MF II: 75-85% purity) have been induced by uv-irradiation to differentiate to nearly homogeneous populations of PMF IV, PMF V, PMF VI, and PMF VII within 4 to 36 days of culture. Using this method, 10(7) cells of one differentiation stage can be obtained. Spontaneously arising and experimentally selected or induced homogeneous clonal and mass populations of MF I, MF II, MF III, PMF IV, PMF V, PMF VI, and PMF VII express an identical differentiation-dependent and cell-type-specific [35S]methionine-labeled polypeptide pattern

Novel Membranes and Processes for Oxygen Enrichment

Energy Technology Data Exchange (ETDEWEB)

Lin, Haiqing

2011-11-15

The overall goal of this project is to develop a membrane process that produces air containing 25-35% oxygen, at a cost of $25-40/ton of equivalent pure oxygen (EPO2). Oxygen-enriched air at such a low cost will allow existing air-fueled furnaces to be converted economically to oxygen-enriched furnaces, which in turn will improve the economic and energy efficiency of combustion processes significantly, and reduce the cost of CO{sub 2} capture and sequestration from flue gases throughout the U.S. manufacturing industries. During the 12-month Concept Definition project: We identified a series of perfluoropolymers (PFPs) with promising oxygen/nitrogen separation properties, which were successfully made into thin film composite membranes. The membranes showed oxygen permeance as high as 1,200 gpu and oxygen/nitrogen selectivity of 3.0, and the permeance and selectivity were stable over the time period tested (60 days). We successfully scaled up the production of high-flux PFP-based membranes, using MTR's commercial coaters. Two bench-scale spiral-wound modules with countercurrent designs were made and parametric tests were performed to understand the effect of feed flow rate and pressure, permeate pressure and sweep flow rate on the membrane module separation properties. At various operating conditions that modeled potential industrial operating conditions, the module separation properties were similar to the pure-gas separation properties in the membrane stamps. We also identified and synthesized new polymers [including polymers of intrinsic microporosity (PIMs) and polyimides] with higher oxygen/nitrogen selectivity (3.5-5.0) than the PFPs, and made these polymers into thin film composite membranes. However, these membranes were susceptible to severe aging; pure-gas permeance decreased nearly six-fold within two weeks, making them impractical for industrial applications of oxygen enrichment. We tested the effect of oxygen-enriched air on NO{sub x} emissions

Serum-dependent selective expression of EhTMKB1-9, a member of Entamoeba histolytica B1 family of transmembrane kinases.

Directory of Open Access Journals (Sweden)

Shiteshu Shrimal

Full Text Available Entamoeba histolytica transmembrane kinases (EhTMKs can be grouped into six distinct families on the basis of motifs and sequences. Analysis of the E. histolytica genome revealed the presence of 35 EhTMKB1 members on the basis of sequence identity (>or=95%. Only six homologs were full length containing an extracellular domain, a transmembrane segment and an intracellular kinase domain. Reverse transcription followed by polymerase chain reaction (RT-PCR of the kinase domain was used to generate a library of expressed sequences. Sequencing of randomly picked clones from this library revealed that about 95% of the clones were identical with a single member, EhTMKB1-9, in proliferating cells. On serum starvation, the relative number of EhTMKB1-9 derived sequences decreased with concomitant increase in the sequences derived from another member, EhTMKB1-18. The change in their relative expression was quantified by real time PCR. Northern analysis and RNase protection assay were used to study the temporal nature of EhTMKB1-9 expression after serum replenishment of starved cells. The results showed that the expression of EhTMKB1-9 was sinusoidal. Specific transcriptional induction of EhTMKB1-9 upon serum replenishment was further confirmed by reporter gene (luciferase expression and the upstream sequence responsible for serum responsiveness was identified. EhTMKB1-9 is one of the first examples of an inducible gene in Entamoeba. The protein encoded by this member was functionally characterized. The recombinant kinase domain of EhTMKB1-9 displayed protein kinase activity. It is likely to have dual specificity as judged from its sensitivity to different kinase inhibitors. Immuno-localization showed EhTMKB1-9 to be a surface protein which decreased on serum starvation and got relocalized on serum replenishment. Cell lines expressing either EhTMKB1-9 without kinase domain, or EhTMKB1-9 antisense RNA, showed decreased cellular proliferation and target cell

Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I

International Nuclear Information System (INIS)

Grose, C.; Jackson, W.; Traugh, J.A.

1989-01-01

Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, the authors investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an in vitro assay containing [γ- 32 P]ATP. The same glycoprotein was phosphorylated when [ 32 P]GTP was substituted for [ 32 P]ATP in the protein kinase assay. They also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein

Receptor-interacting protein (RIP) kinase family

OpenAIRE

Zhang, Duanwu; Lin, Juan; Han, Jiahuai

2010-01-01

Receptor-interacting protein (RIP) kinases are a group of threonine/serine protein kinases with a relatively conserved kinase domain but distinct non-kinase regions. A number of different domain structures, such as death and caspase activation and recruitment domain (CARD) domains, were found in different RIP family members, and these domains should be keys in determining the specific function of each RIP kinase. It is known that RIP kinases participate in different biological processes, incl...

On-Line Enrichment Monitor for UF{sub 6} Gas Centrifuge Enrichment Plant

Energy Technology Data Exchange (ETDEWEB)

Ianakiev, K. D.; Boyer, B.; Favalli, A.; Goda, J. M.; Hill, T.; Keller, C.; Lombardi, M.; Paffett, M.; MacArthur, D. W.; McCluskey, C.; Moss, C. E.; Parker, R.; Smith, M. K.; Swinhoe, M. T. [Los Alamos National Laboratory, Los Alamos (United States)

2012-06-15

This paper is a continuation of the Advanced Enrichment Monitoring Technology for UF{sub 6} Gas Centrifuge Enrichment Plant (GCEP) work, presented in the 2010 IAEA Safeguards Symposium. Here we will present the system architecture for a planned side-by-side field trial test of passive (186-keV line spectroscopy and pressure-based correction for UF{sub 6} gas density) and active (186-keV line spectroscopy and transmission measurement based correction for UF{sub 6} gas density) enrichment monitoring systems in URENCO's enrichment plant in Capenhurst. Because the pressure and transmission measurements of UF{sub 6} are complementary, additional information on the importance of the presence of light gases and the UF{sub 6} gas temperature can be obtained by cross-correlation between simultaneous measurements of transmission, pressure and 186-keV intensity. We will discuss the calibration issues and performance in the context of accurate, on-line enrichment measurement. It is hoped that a simple and accurate on-line enrichment monitor can be built using the UF{sub 6} gas pressure provided by the Operator, based on online mass spectrometer calibration, assuming a negligible (a small fraction of percent) contribution of wall deposits. Unaccounted-for wall deposits present at the initial calibration will lead to unwanted sensitivity to changes in theUF{sub 6} gas pressure and thus to error in the enrichment results. Because the accumulated deposits in the cascade header pipe have been identified as an issue for Go/No Go measurements with the Cascade Header Enrichment Monitor (CHEM) and Continuous Enrichment Monitor (CEMO), it is important to explore their effect. Therefore we present the expected uncertainty on enrichment measurements obtained by propagating the errors introduced by deposits, gas density, etc. and will discuss the options for a deposit correction during initial calibration of an On-Line Enrichment Monitor (OLEM).

Prevalence of extended-spectrum cephalosporinase (ESC)-producing Escherichia coli in Danish slaughter pigs and retail meat identified by selective enrichment and association with cephalosporin usage

DEFF Research Database (Denmark)

Agersø, Yvonne; Aarestrup, Frank Møller; Pedersen, Karl

2012-01-01

OBJECTIVES: To investigate the prevalence of extended-spectrum cephalosporinase (ESC)-producing Escherichia coli in pigs at slaughter and retail meat, and possible associations with the consumption of third- and fourth-generation cephalosporins. METHODS: During 2009, faecal samples from Danish pigs...... (n = 786) were collected at slaughter, and 866 meat samples [Danish: pork (153), broiler meat (121) and beef (142); and imported: pork (173), broiler meat (193) and beef (84)] were randomly collected in retail stores and outlets. E. coli was isolated after enrichment in MacConkey broth.......7%–3.3% in other meat types. ESC E. coli from imported broiler meat (n = 69) contained blaCMY-2 (48%), blaCTX-M-1 (25%) and blaSHV-12 (16%). Without selective enrichment, no ESC E. coli from pigs and only 4.1% from imported broiler meat were found. CONCLUSIONS: The usage of cephalosporins for slaughter pigs may...

Phosphatidylinositol 4-kinases: Function, structure, and inhibition

International Nuclear Information System (INIS)

Boura, Evzen; Nencka, Radim

2015-01-01

The phosphatidylinositol 4-kinases (PI4Ks) synthesize phosphatidylinositol 4-phosphate (PI4P), a key member of the phosphoinositide family. PI4P defines the membranes of Golgi and trans-Golgi network (TGN) and regulates trafficking to and from the Golgi. Humans have two type II PI4Ks (α and β) and two type III enzymes (α and β). Recently, the crystal structures were solved for both type II and type III kinase revealing atomic details of their function. Importantly, the type III PI4Ks are hijacked by +RNA viruses to create so-called membranous web, an extensively phosphorylated and modified membrane system dedicated to their replication. Therefore, selective and potent inhibitors of PI4Ks have been developed as potential antiviral agents. Here we focus on the structure and function of PI4Ks and their potential in human medicine

Phosphatidylinositol 4-kinases: Function, structure, and inhibition

Energy Technology Data Exchange (ETDEWEB)

Boura, Evzen, E-mail: boura@uochb.cas.cz; Nencka, Radim, E-mail: nencka@uochb.cas.cz

2015-10-01

The phosphatidylinositol 4-kinases (PI4Ks) synthesize phosphatidylinositol 4-phosphate (PI4P), a key member of the phosphoinositide family. PI4P defines the membranes of Golgi and trans-Golgi network (TGN) and regulates trafficking to and from the Golgi. Humans have two type II PI4Ks (α and β) and two type III enzymes (α and β). Recently, the crystal structures were solved for both type II and type III kinase revealing atomic details of their function. Importantly, the type III PI4Ks are hijacked by +RNA viruses to create so-called membranous web, an extensively phosphorylated and modified membrane system dedicated to their replication. Therefore, selective and potent inhibitors of PI4Ks have been developed as potential antiviral agents. Here we focus on the structure and function of PI4Ks and their potential in human medicine.

Structural and evolutionary adaptation of rhoptry kinases and pseudokinases, a family of coccidian virulence factors

Science.gov (United States)

2013-01-01

conserved across both active kinases and pseudokinases, and discuss these in terms of sequence motifs, evolutionary context, structural impact and potential functional relevance. By characterizing the proteins that enable these parasites to invade the host cell and co-opt its signaling mechanisms, we provide guidance on potential therapeutic targets for the diseases caused by coccidian parasites. PMID:23742205

Structural and evolutionary adaptation of rhoptry kinases and pseudokinases, a family of coccidian virulence factors.

Science.gov (United States)

Talevich, Eric; Kannan, Natarajan

2013-06-06

kinases and pseudokinases, and discuss these in terms of sequence motifs, evolutionary context, structural impact and potential functional relevance. By characterizing the proteins that enable these parasites to invade the host cell and co-opt its signaling mechanisms, we provide guidance on potential therapeutic targets for the diseases caused by coccidian parasites.

Listeria monocytogenes Strains Underrepresented during Selective Enrichment with an ISO Method Might Dominate during Passage through Simulated Gastric Fluid and In Vitro Infection of Caco-2 Cells

Science.gov (United States)

Zilelidou, Evangelia; Karmiri, Christina-Vasiliki; Zoumpopoulou, Georgia; Mavrogonatou, Eleni; Kletsas, Dimitris; Tsakalidou, Effie; Papadimitriou, Konstantinos; Drosinos, Eleftherios

2016-01-01

ABSTRACT Various Listeria monocytogenes strains may contaminate a single food product, potentially resulting in simultaneous exposure of consumers to multiple strains. However, due to bias in strain recovery, L. monocytogenes strains isolated from foods by selective enrichment (SE) might not always represent those that can better survive the immune system of a patient. We investigated the effect of cocultivation in tryptic soy broth with 0.6% yeast extract (TSB-Y) at 10°C for 8 days on (i) the detection of L. monocytogenes strains during SE with the ISO 11290-1:1996/Amd 1:2004 protocol and (ii) the in vitro virulence of strains toward the Caco-2 human colon epithelial cancer cell line following exposure to simulated gastric fluid (SGF; pH 2.0)-HCl (37°C). We determined whether the strains which were favored by SE would be effective competitors under the conditions of challenges related to gastrointestinal passage of the pathogen. Interstrain competition of L. monocytogenes in TSB-Y determined the relative population of each strain at the beginning of SE. This in turn impacted the outcome of SE (i.e., favoring survival of competitors with better fitness) and the levels exposed subsequently to SGF. However, strong growth competitors could be outcompeted after SGF exposure and infection of Caco-2 cells by strains outgrown in TSB-Y and underdetected (or even missed) during enrichment. Our data demonstrate a preferential selection of certain L. monocytogenes strains during enrichments, often not reflecting a selective advantage of strains during infection. These findings highlight a noteworthy scenario associated with the difficulty of matching the source of infection (food) with the L. monocytogenes isolate appearing to be the causative agent during listeriosis outbreak investigations. IMPORTANCE This report is relevant to understanding the processes involved in selection and prevalence of certain L. monocytogenes strains in different environments (i.e., foods or

Platelet-rich preparations to improve healing. Part II: platelet activation and enrichment, leukocyte inclusion, and other selection criteria.

Science.gov (United States)

Davis, Vicki L; Abukabda, Alaeddin B; Radio, Nicholas M; Witt-Enderby, Paula A; Clafshenkel, William P; Cairone, J Vito; Rutkowski, James L

2014-08-01

Multiple platelet-rich preparations have been reported to improve wound and bone healing, such as platelet-rich plasma (PRP) and platelet rich fibrin (PRF). The different methods employed during their preparation are important, as they influence the quality of the product applied to a wound or surgical site. Besides the general protocol for preparing the platelet-rich product (discussed in Part 1 of this review), multiple choices need to be considered during its preparation. For example, activation of the platelets is required for the release and enmeshment of growth factors, but the method of activation may influence the resulting matrix, growth factor availability, and healing. Additionally, some methods enrich leukocytes as well as platelets, but others are designed to be leukocyte-poor. Leukocytes have many important roles in healing and their inclusion in PRP results in increased platelet concentrations. Platelet and growth factor enrichment reported for the different types of platelet-rich preparations are also compared. Generally, TGF-β1 and PDGF levels were higher in preparations that contain leukocytes compared to leukocyte-poor PRP. However, platelet concentration may be the most reliable criterion for comparing different preparations. These and other criteria are described to help guide dental and medical professionals, in large and small practices, in selecting the best procedures for their patients. The healing benefits of platelet-rich preparations along with the low risk and availability of simple preparation procedures should encourage more clinicians to incorporate platelet-rich products in their practice to accelerate healing, reduce adverse events, and improve patient outcomes.

Uranium enrichment by laser: a technology for the future

International Nuclear Information System (INIS)

Cazalet, J.

1999-01-01

The SILVA (Isotopic Separation by Laser on atomic Vapor of uranium) process, developed by CEA and COGEMA, is an innovative system of production of enriched uranium, to be used as the fuel of nuclear reactors. It is a sound research program, calling on advanced technologies that are quickly changing. The goal is to cut drastically the production cost in comparison with the operating cost of the present plants based on gaseous diffusion. its industrialization is forecast for the beginning of next century. The SILVA process consists in putting a vapor of uranium through a beam of photons emitted by finely tuned lasers capable of ionising selectively the isotopes 235. The ionised isotopes are attracted on plates by an electric field, they are condensed and collected on these plates. The very high selectivity of enrichment technologies by laser, which are quite new, pave the way for compact and modular plants, which will consume little energy. Accordingly their production cost will be very low. So a new process could take a significant part of the uranium enrichment market after 2010. Even if the multinational EURODIF gaseous diffusion plant is modern and performing, it will be necessary to strengthen the French industry of uranium enrichment to maintain or improve its competitive position on the world market. This could be achieved by smoothly replacing EURODIF by a high performance laser plant. This is the common goal of CEA and COGEMA: all the efforts are concentrated on SILVA, the qualities of which (high selectivity, separation in one single step) have been demonstrated in the facilities of Saclay and Pierrelatte. 400 researchers and technicians are involved, as well as many industrial firms. The budget is equally by CEA and COGEMA through a cooperation agreement. The program includes: a phase of scientific and technical research, which has been highlighted in 1997-1998 by a demonstration of feasibility of the process; a phase of technological development, with

Robust de novo pathway enrichment with KeyPathwayMiner 5 [version 1; referees: 2 approved

Directory of Open Access Journals (Sweden)

Nicolas Alcaraz

2016-06-01

Full Text Available Identifying functional modules or novel active pathways, recently termed de novo pathway enrichment, is a computational systems biology challenge that has gained much attention during the last decade. Given a large biological interaction network, KeyPathwayMiner extracts connected subnetworks that are enriched for differentially active entities from a series of molecular profiles encoded as binary indicator matrices. Since interaction networks constantly evolve, an important question is how robust the extracted results are when the network is modified. We enable users to study this effect through several network perturbation techniques and over a range of perturbation degrees. In addition, users may now provide a gold-standard set to determine how enriched extracted pathways are with relevant genes compared to randomized versions of the original network.

Recent Advances in the Development and Application of Radiolabeled Kinase Inhibitors for PET Imaging

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Vadim Bernard-Gauthier

2015-12-01

Full Text Available Over the last 20 years, intensive investigation and multiple clinical successes targeting protein kinases, mostly for cancer treatment, have identified small molecule kinase inhibitors as a prominent therapeutic class. In the course of those investigations, radiolabeled kinase inhibitors for positron emission tomography (PET imaging have been synthesized and evaluated as diagnostic imaging probes for cancer characterization. Given that inhibitor coverage of the kinome is continuously expanding, in vivo PET imaging will likely find increasing applications for therapy monitoring and receptor density studies both in- and outside of oncological conditions. Early investigated radiolabeled inhibitors, which are mostly based on clinically approved tyrosine kinase inhibitor (TKI isotopologues, have now entered clinical trials. Novel radioligands for cancer and PET neuroimaging originating from novel but relevant target kinases are currently being explored in preclinical studies. This article reviews the literature involving radiotracer design, radiochemistry approaches, biological tracer evaluation and nuclear imaging results of radiolabeled kinase inhibitors for PET reported between 2010 and mid-2015. Aspects regarding the usefulness of pursuing selective vs. promiscuous inhibitor scaffolds and the inherent challenges associated with intracellular enzyme imaging will be discussed.

Essential role of neuron-enriched diacylglycerol kinase (DGK, DGKbeta in neurite spine formation, contributing to cognitive function.

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Yasuhito Shirai

Full Text Available BACKGROUND: Diacylglycerol (DG kinase (DGK phosphorylates DG to produce phosphatidic acid (PA. Of the 10 subtypes of mammalian DGKs, DGKbeta is a membrane-localized subtype and abundantly expressed in the cerebral cortex, hippocampus, and caudate-putamen. However, its physiological roles in neurons and higher brain function have not been elucidated. METHODOLOGY/PRINCIPAL FINDINGS: We, therefore, developed DGKbeta KO mice using the Sleeping Beauty transposon system, and found that its long-term potentiation in the hippocampal CA1 region was reduced, causing impairment of cognitive functions including spatial and long-term memories in Y-maze and Morris water-maze tests. The primary cultured hippocampal neurons from KO mice had less branches and spines compared to the wild type. This morphological impairment was rescued by overexpression of DGKbeta. In addition, overexpression of DGKbeta in SH-SY5Y cells or primary cultured mouse hippocampal neurons resulted in branch- and spine-formation, while a splice variant form of DGKbeta, which has kinase activity but loses membrane localization, did not induce branches and spines. In the cells overexpressing DGKbeta but not the splice variant form, DGK product, PA, was increased and the substrate, DG, was decreased on the plasma membrane. Importantly, lower spine density and abnormality of PA and DG contents in the CA1 region of the KO mice were confirmed. CONCLUSIONS/SIGNIFICANCE: These results demonstrate that membrane-localized DGKbeta regulates spine formation by regulation of lipids, contributing to the maintenance of neural networks in synaptic transmission of cognitive processes including memory.

Interrill sediment enrichment of P and C from organically and conventionally farmed silty loams

Science.gov (United States)

Kuhn, N. J.

2012-04-01

Globally, between 0.57 and 1.33 Pg of soil organic carbon (SOC) may be affected by interrill processes. Also, a significant amount of phosphorus (P) is contained in the surface soil layer transformed by raindrop impact, runoff and crust formation. In the EU, the P content of a crusted (2 mm) surface layer corresponds to 4 to 40 kg ha-1 of P on arable land (1.094 mil km2). Therefore, the role of interrill processes for nutrient cycling and the global carbon cycle requires close attention. Interrill erosion is a complex phenomen on involving the detachment, transport and deposition of soil particles by raindrop impacted flow. Resistance to interrill erosion varies between soils depending on their physical, chemical and mineralogical properties. In addition, significant changes in soil resistance to interrill erosion occur during storms as a result of changes in surface roughness, cohesion and particle size. As a consequence, erosion on interrill areas is selective, moving the most easily detached small and/or light soil particles. This leads to the enrichment of clay, phosphorous (P)and carbon (C). Such enrichment in interrill sediment is well documented, however, the role of interrill erosion processes on the enrichment remains unclear. Enrichment of P and C in interrill sediment is attributed to the preferential erosion of the smaller, lighter soil particles. In this study, the P and organic C content of sediment generated from two Devon silts under conventional (CS) and organic (OS) soil management were examined. Artificial rainfall was applied to the soils using two rainfall scenarios of differing intensity and kinetic energy to determine the effects on the P and C enrichment in interrill sediment. Interrill soil erodibility was lower on the OS, irrespective of rainfall intensity. Sediment from both soils showed a significant enrichment in P and C compared to the bulk soil. However, sediment from the OS displayed a much greater degree of P enrichment. This shows

syk kinase activation by a src kinase-initiated activation loop phosphorylation chain reaction

Science.gov (United States)

El-Hillal, O.; Kurosaki, T.; Yamamura, H.; Kinet, J.-P.; Scharenberg, A. M.

1997-01-01

Activation of the syk tyrosine kinase occurs almost immediately following engagement of many types of antigen receptors, including Fc receptors, but the mechanism through which syk is activated is currently unclear. Here we demonstrate that Fc receptor-induced syk activation occurs as the result of phosphorylation of the syk activation loop by both src family kinases and other molecules of activated syk, suggesting that syk activation occurs as the result of a src kinase-initiated activation loop phosphorylation chain reaction. This type of activation mechanism predicts that syk activation would exhibit exponential kinetics, providing a potential explanation for its rapid and robust activation by even weak antigen receptor stimuli. We propose that a similar mechanism may be responsible for generating rapid activation of other cytoplasmic tyrosine kinases, such as those of the Bruton tyrosine kinase/tec family, as well. PMID:9050880

Protein phosphatases active on acetyl-CoA carboxylase phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase

International Nuclear Information System (INIS)

Witters, L.A.; Bacon, G.W.

1985-01-01

The protein phosphatases in rat liver cytosol, active on rat liver acetyl-CoA carboxylase (ACC) phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase, have been partially purified by anion-exchange and gel filtration chromatography. The major phosphatase activities against all three substrates copurify through fractionation and appear to be identical to protein phosphatases 2A1 and 2A2. No unique protein phosphatase active on 32 P-ACC phosphorylated by the casein kinases was identified

Expression of Herpes Simplex Virus Thymidine Kinase/Ganciclovir by RNA Trans-Splicing Induces Selective Killing of HIV-Producing Cells

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Carin K. Ingemarsdotter

2017-06-01

Full Text Available Antiviral strategies targeting hijacked cellular processes are less easily evaded by the virus than viral targets. If selective for viral functions, they can have a high therapeutic index. We used RNA trans-splicing to deliver the herpes simplex virus thymidine kinase-ganciclovir (HSV-tk/GCV cell suicide system into HIV-producing cells. Using an extensive in silico bioinformatics and RNA structural analysis approach, ten HIV RNA trans-splicing constructs were designed targeting eight different HIV splice donor or acceptor sites and were tested in cells expressing HIV. Trans-spliced mRNAs were identified in HIV-expressing cells using qRT-PCR with successful detection of fusion RNA transcripts between HIV RNA and the HSV-tk RNA transcripts from six of ten candidate RNA trans-splicing constructs. Conventional PCR and Sanger sequencing confirmed RNA trans-splicing junctions. Measuring cell viability in the presence or absence of GCV expression of HSV-tk by RNA trans-splicing led to selective killing of HIV-producing cells using either 3′ exon replacement or 5′ exon replacement in the presence of GCV. Five constructs targeting four HIV splice donor and acceptor sites, D4, A5, A7, and A8, involved in regulating the generation of multiple HIV RNA transcripts proved to be effective for trans-splicing mediated selective killing of HIV-infected cells, within which individual constructs targeting D4 and A8 were the most efficient.

Receptor-interacting protein (RIP) kinase family

Science.gov (United States)

Zhang, Duanwu; Lin, Juan; Han, Jiahuai

2010-01-01

Receptor-interacting protein (RIP) kinases are a group of threonine/serine protein kinases with a relatively conserved kinase domain but distinct non-kinase regions. A number of different domain structures, such as death and caspase activation and recruitment domain (CARD) domains, were found in different RIP family members, and these domains should be keys in determining the specific function of each RIP kinase. It is known that RIP kinases participate in different biological processes, including those in innate immunity, but their downstream substrates are largely unknown. This review will give an overview of the structures and functions of RIP family members, and an update of recent progress in RIP kinase research. PMID:20383176

Tyrosine kinase inhibitors and mesenchymal stromal cells: effects on self-renewal, commitment and functions

Science.gov (United States)

Borriello, Adriana; Caldarelli, Ilaria; Bencivenga, Debora; Stampone, Emanuela; Perrotta, Silverio; Oliva, Adriana; Ragione, Fulvio Della

2017-01-01

The hope of selectively targeting cancer cells by therapy and eradicating definitively malignancies is based on the identification of pathways or metabolisms that clearly distinguish “normal” from “transformed” phenotypes. Some tyrosine kinase activities, specifically unregulated and potently activated in malignant cells, might represent important targets of therapy. Consequently, tyrosine kinase inhibitors (TKIs) might be thought as the “vanguard” of molecularly targeted therapy for human neoplasias. Imatinib and the successive generations of inhibitors of Bcr-Abl1 kinase, represent the major successful examples of TKI use in cancer treatment. Other tyrosine kinases have been selected as targets of therapy, but the efficacy of their inhibition, although evident, is less definite. Two major negative effects exist in this therapeutic strategy and are linked to the specificity of the drugs and to the role of the targeted kinase in non-malignant cells. In this review, we will discuss the data available on the TKIs effects on the metabolism and functions of mesenchymal stromal cells (MSCs). MSCs are widely distributed in human tissues and play key physiological roles; nevertheless, they might be responsible for important pathologies. At present, bone marrow (BM) MSCs have been studied in greater detail, for both embryological origins and functions. The available data are evocative of an unexpected degree of complexity and heterogeneity of BM-MSCs. It is conceivable that this grade of intricacy occurs also in MSCs of other organs. Therefore, in perspective, the negative effects of TKIs on MSCs might represent a critical problem in long-term cancer therapies based on such inhibitors. PMID:27750212

Removal, recovery and enrichment of metals from aqueous solutions using carbon nanotubes

International Nuclear Information System (INIS)

Jin-Gang Yu; Central South University, Changsha, Hunan; Ministry of Education; Xiu-Hui Zhao; Lin-Yan Yu; Fei-Peng Jiao; Xiao-Qing Chen; Ministry of Education; Jian-Hui Jiang

2014-01-01

Environmental pollution caused by toxic metals (heavy metals, radioactive metals, etc.) is one of the major global issues, thus removal of toxic metals from contaminated water seems to be particularly important. On the other hand, the recovery and enrichment of metals, especially noble metals, from waste water is also crucial. To address these issues, nanotechnology plays an essential role in environmental monitoring and pollution control. To remove metals from contaminated water, or enrich metals from waste water, carbon nanotubes (CNTs) and their composites have attracted great attention due to their excellent adsorption performance. The removal efficiency for metal ions by CNTs was observed around 10-80 %, which could be improved to approach 100 % by selectively functionalizing CNTs with organic ligands. Herein, we review the applications of CNTs in treatment of toxic metal-containing wastewater for environmental monitoring and metals recovery. Due to their higher sensitivity and selectivity towards the enrichment of metals or detection of toxic metal pollution of the environment, and the latest research progress of using CNT composites for metal treatment is also discussed. (author)

Regulation of the interaction between protein kinase C-related protein kinase 2 (PRK2) and its upstream kinase, 3-phosphoinositide-dependent protein kinase 1 (PDK1)

DEFF Research Database (Denmark)

Dettori, Rosalia; Sonzogni, Silvina; Meyer, Lucas

2009-01-01

of numerous AGC kinases, including the protein kinase C-related protein kinases (PRKs). Here we studied the docking interaction between PDK1 and PRK2 and analyzed the mechanisms that regulate this interaction. In vivo labeling of recombinant PRK2 by (32)P(i) revealed phosphorylation at two sites......, the activation loop and the Z/TM in the C-terminal extension. We provide evidence that phosphorylation of the Z/TM site of PRK2 inhibits its interaction with PDK1. Our studies further provide a mechanistic model to explain different steps in the docking interaction and regulation. Interestingly, we found...... that the mechanism that negatively regulates the docking interaction of PRK2 to the upstream kinase PDK1 is directly linked to the activation mechanism of PRK2 itself. Finally, our results indicate that the mechanisms underlying the regulation of the interaction between PRK2 and PDK1 are specific for PRK2 and do...

Surface modification of nanoparticles enables selective evasion of phagocytic clearance by distinct macrophage phenotypes

Science.gov (United States)

Qie, Yaqing; Yuan, Hengfeng; von Roemeling, Christina A.; Chen, Yuanxin; Liu, Xiujie; Shih, Kevin D.; Knight, Joshua A.; Tun, Han W.; Wharen, Robert E.; Jiang, Wen; Kim, Betty Y. S.

2016-05-01

Nanomedicine is a burgeoning industry but an understanding of the interaction of nanomaterials with the immune system is critical for clinical translation. Macrophages play a fundamental role in the immune system by engulfing foreign particulates such as nanoparticles. When activated, macrophages form distinct phenotypic populations with unique immune functions, however the mechanism by which these polarized macrophages react to nanoparticles is unclear. Furthermore, strategies to selectively evade activated macrophage subpopulations are lacking. Here we demonstrate that stimulated macrophages possess higher phagocytic activities and that classically activated (M1) macrophages exhibit greater phagocytic capacity than alternatively activated (M2) macrophages. We show that modification of nanoparticles with polyethylene-glycol results in decreased clearance by all macrophage phenotypes, but importantly, coating nanoparticles with CD47 preferentially lowers phagocytic activity by the M1 phenotype. These results suggest that bio-inspired nanoparticle surface design may enable evasion of specific components of the immune system and provide a rational approach for developing immune tolerant nanomedicines.

Uranium enrichment

International Nuclear Information System (INIS)

Mohrhauer, H.

1982-01-01

The separation of uranium isotopes in order to enrich the fuel for light water reactors with the light isotope U-235 is an important part of the nuclear fuel cycle. After the basic principals of isotope separation the gaseous diffusion and the centrifuge process are explained. Both these techniques are employed on an industrial scale. In addition a short review is given on other enrichment techniques which have been demonstrated at least on a laboratory scale. After some remarks on the present situation on the enrichment market the progress in the development and the industrial exploitation of the gas centrifuge process by the trinational Urenco-Centec organisation is presented. (orig.)

United States uranium enrichment policies

International Nuclear Information System (INIS)

Roberts, R.W.

1977-01-01

ERDA's uranium enrichment program policies governing the manner in which ERDA's enrichment complex is being operated and expanded to meet customer requirements for separative work, research and development activities directed at providing technology alternatives for future enrichment capacity, and establishing the framework for additional domestic uranium enrichment capacity to meet the domestic and foreign nuclear industry's growing demand for enrichment services are considered. The ERDA enrichment complex consists of three gaseous diffusion plants located in Oak Ridge, Tennessee; Paducah, Kentucky; and Portsmouth, Ohio. Today, these plants provide uranium enrichment services for commercial nuclear power generation. These enrichment services are provided under contracts between the Government and the utility customers. ERDA's program involves a major pilot plant cascade, and pursues an advanced isotope separation technique for the late 1980's. That the United States must develop additional domestic uranium enrichment capacity is discussed

High-Throughput Screening and Hit Validation of Extracellular-Related Kinase 5 (ERK5) Inhibitors.

Science.gov (United States)

Myers, Stephanie M; Bawn, Ruth H; Bisset, Louise C; Blackburn, Timothy J; Cottyn, Betty; Molyneux, Lauren; Wong, Ai-Ching; Cano, Celine; Clegg, William; Harrington, Ross W; Leung, Hing; Rigoreau, Laurent; Vidot, Sandrine; Golding, Bernard T; Griffin, Roger J; Hammonds, Tim; Newell, David R; Hardcastle, Ian R

2016-08-08

The extracellular-related kinase 5 (ERK5) is a promising target for cancer therapy. A high-throughput screen was developed for ERK5, based on the IMAP FP progressive binding system, and used to identify hits from a library of 57 617 compounds. Four distinct chemical series were evident within the screening hits. Resynthesis and reassay of the hits demonstrated that one series did not return active compounds, whereas three series returned active hits. Structure-activity studies demonstrated that the 4-benzoylpyrrole-2-carboxamide pharmacophore had excellent potential for further development. The minimum kinase binding pharmacophore was identified, and key examples demonstrated good selectivity for ERK5 over p38α kinase.

Rapid adaptation of activated sludge bacteria into a glycogen accumulating biofilm enabling anaerobic BOD uptake.

Science.gov (United States)

Hossain, Md Iqbal; Paparini, Andrea; Cord-Ruwisch, Ralf

2017-03-01

Glycogen accumulating organisms (GAO) are known to allow anaerobic uptake of biological oxygen demand (BOD) in activated sludge wastewater treatment systems. In this study, we report a rapid transition of suspended activated sludge biomass to a GAO dominated biofilm by selective enrichment using sequences of anaerobic loading followed by aerobic exposure of the biofilm to air. The study showed that within eight weeks, a fully operational, GAO dominated biofilm had developed, enabling complete anaerobic BOD uptake at a rate of 256mg/L/h. The oxygen uptake by the biofilm directly from the atmosphere had been calculated to provide significant energy savings. This study suggests that wastewater treatment plant operators can convert activated sludge systems readily into a "passive aeration" biofilm that avoids costly oxygen transfer to bulk wastewater solution. The described energy efficient BOD removal system provides an opportunity to be coupled with novel nitrogen removal processes such as anammox. Copyright © 2016. Published by Elsevier Ltd.

Blueprint for domestic uranium enrichment

International Nuclear Information System (INIS)

1981-01-01

The AEC advisory committee on domestic production of uranium enrichment has studied for more than a year how to achieve the domestic enrichment of uranium by the construction and operation of a commercial enriching plant using centrifugal separation method, and the report was submitted to the Atomic Energy Commission on August 18, 1980. Japan has depended wholly on overseas services for her uranium enrichment needs, but the development of domestic enrichment has been carried on in parallel. The AEC decided to construct a uranium enrichment pilot plant using centrifuges, and it has been forwarded as a national project. The plant is operated by the Power Reactor and Nuclear Fuel Development Corp. since 1979. The capacity of the plant will be raised to approximately 75 ton SWU a year. The centrifuges already operated have provided the first delivery of fuel of about 1 ton for the ATR ''Fugen''. The demand-supply balance of uranium enrichment service, the significance of the domestic enrichment of uranium, the evaluation of uranium enrichment technology, the target for domestic enrichment plan, the measures to promote domestic uranium enrichment, and the promotion of the construction of a demonstration plant are reported. (Kako, I.)

Downstream of tyrosine kinase/docking protein 6, as a novel substrate of tropomyosin-related kinase C receptor, is involved in neurotrophin 3-mediated neurite outgrowth in mouse cortex neurons

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Yuan Jian

2010-06-01

Full Text Available Abstract Background The downstream of tyrosine kinase/docking protein (Dok adaptor protein family has seven members, Dok1 to Dok7, that act as substrates of multiple receptor tyrosine kinase and non-receptor tyrosine kinase. The tropomyosin-related kinase (Trk receptor family, which has three members (TrkA, TrkB and TrkC, are receptor tyrosine kinases that play pivotal roles in many stages of nervous system development, such as differentiation, migration, axon and dendrite projection and neuron patterning. Upon related neurotrophin growth factor stimulation, dimerisation and autophosphorylation of Trk receptors can occur, recruiting adaptor proteins to mediate signal transduction. Results In this report, by using yeast two-hybrid assays, glutathione S-transferase (GST precipitation assays and coimmunoprecipitation (Co-IP experiments, we demonstrate that Dok6 selectively binds to the NPQY motif of TrkC through its phosphotyrosine-binding (PTB domain in a kinase activity-dependent manner. We further confirmed their interaction by coimmunoprecipitation and colocalisation in E18.5 mouse cortex neurons, which provided more in vivo evidence. Next, we demonstrated that Dok6 is involved in neurite outgrowth in mouse cortex neurons via the RNAi method. Knockdown of Dok6 decreased neurite outgrowth in cortical neurons upon neurotrophin 3 (NT-3 stimulation. Conclusions We conclude that Dok6 interacts with the NPQY motif of the TrkC receptor through its PTB domain in a kinase activity-dependent manner, and works as a novel substrate of the TrkC receptor involved in NT-3-mediated neurite outgrowth in mouse cortex neurons.

Stable-isotope-enrichment program at the Oak Ridge National Laboratory

International Nuclear Information System (INIS)

Newman, E.

1982-01-01

This paper has attempted to present a brief description of the production steps, from the selection and preparation of the initial feedstock to the recovery and distribution of the isotopically enriched materials. The facility suffers from the disadvantage of coping with utility and support systems that are rapidly becoming obsolescent and that the current operational level is insufficient to maintain sales inventory equilibrium. The electromagnetic isotope enrichment facility does, however, have the operational equipment and capability to almost triple the current production. This increased production can be achieved as rapidly as an expanded operational crew can be trained

Reduced expression of brain-enriched microRNAs in glioblastomas permits targeted regulation of a cell death gene.

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Rebecca L Skalsky

Full Text Available Glioblastoma is a highly aggressive malignant tumor involving glial cells in the human brain. We used high-throughput sequencing to comprehensively profile the small RNAs expressed in glioblastoma and non-tumor brain tissues. MicroRNAs (miRNAs made up the large majority of small RNAs, and we identified over 400 different cellular pre-miRNAs. No known viral miRNAs were detected in any of the samples analyzed. Cluster analysis revealed several miRNAs that were significantly down-regulated in glioblastomas, including miR-128, miR-124, miR-7, miR-139, miR-95, and miR-873. Post-transcriptional editing was observed for several miRNAs, including the miR-376 family, miR-411, miR-381, and miR-379. Using the deep sequencing information, we designed a lentiviral vector expressing a cell suicide gene, the herpes simplex virus thymidine kinase (HSV-TK gene, under the regulation of a miRNA, miR-128, that was found to be enriched in non-tumor brain tissue yet down-regulated in glioblastomas, Glioblastoma cells transduced with this vector were selectively killed when cultured in the presence of ganciclovir. Using an in vitro model to recapitulate expression of brain-enriched miRNAs, we demonstrated that neuronally differentiated SH-SY5Y cells transduced with the miRNA-regulated HSV-TK vector are protected from killing by expression of endogenous miR-128. Together, these results provide an in-depth analysis of miRNA dysregulation in glioblastoma and demonstrate the potential utility of these data in the design of miRNA-regulated therapies for the treatment of brain cancers.

The role of Src kinase in the biology and pathogenesis of Acanthamoeba castellanii

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Siddiqui Ruqaiyyah

2012-06-01

Full Text Available Abstract Background Acanthamoeba species are the causative agents of fatal granulomatous encephalitis in humans. Haematogenous spread is thought to be a primary step, followed by blood–brain barrier penetration, in the transmission of Acanthmaoeba into the central nervous system, but the associated molecular mechanisms remain unclear. Here, we evaluated the role of Src, a non-receptor protein tyrosine kinase in the biology and pathogenesis of Acanthamoeba. Methods Amoebistatic and amoebicidal assays were performed by incubating amoeba in the presence of Src kinase-selective inhibitor, PP2 (4-amino-5-(4-chlorophenyl-7-(t-butylpyrazolo[3,4-d]pyrimidine and its inactive analog, PP3 (4-amino-7-phenylpyrazolo[3,4-d]pyrimidine. Using this inhibitor, the role of Src kinase in A. castellanii interactions with Escherichia coli was determined. Zymographic assays were performed to study effects of Src kinase on extracellular proteolytic activities of A. castellanii. The human brain microvascular endothelial cells were used to determine the effects of Src kinase on A. castellanii adhesion to and cytotoxicity of host cells. Results Inhibition of Src kinase using a specific inhibitor, PP2 (4-amino-5-(4 chlorophenyl-7-(t-butylpyrazolo [3,4-d] pyrimidine but not its inactive analog, PP3 (4-amino-7-phenylpyrazolo[3,4-d] pyrimidine, had detrimental effects on the growth of A. castellanii (keratitis isolate, belonging to the T4 genotype. Interestingly, inhibition of Src kinase hampered the phagocytic ability of A. castellanii, as measured by the uptake of non-invasive bacteria, but, on the contrary, invasion by pathogenic bacteria was enhanced. Zymographic assays revealed that inhibition of Src kinases reduced extracellular protease activities of A. castellanii. Src kinase inhibition had no significant effect on A. castellanii binding to and cytotoxicity of primary human brain microvascular endothelial cells, which constitute the blood–brain barrier

Uranium enrichment. Enrichment processes

International Nuclear Information System (INIS)

Alexandre, M.; Quaegebeur, J.P.

2009-01-01

Despite the remarkable progresses made in the diversity and the efficiency of the different uranium enrichment processes, only two industrial processes remain today which satisfy all of enriched uranium needs: the gaseous diffusion and the centrifugation. This article describes both processes and some others still at the demonstration or at the laboratory stage of development: 1 - general considerations; 2 - gaseous diffusion: physical principles, implementation, utilisation in the world; 3 - centrifugation: principles, elementary separation factor, flows inside a centrifuge, modeling of separation efficiencies, mechanical design, types of industrial centrifuges, realisation of cascades, main characteristics of the centrifugation process; 4 - aerodynamic processes: vortex process, nozzle process; 5 - chemical exchange separation processes: Japanese ASAHI process, French CHEMEX process; 6 - laser-based processes: SILVA process, SILMO process; 7 - electromagnetic and ionic processes: mass spectrometer and calutron, ion cyclotron resonance, rotating plasmas; 8 - thermal diffusion; 9 - conclusion. (J.S.)

Pharmacologic modulation of protein kinase C isozymes: the role of RACKs and subcellular localisation.

Science.gov (United States)

Csukai, M; Mochly-Rosen, D

1999-04-01

Protein kinase C (PKC) isozymes are highly homologous kinases and several different isozymes can be present in a cell. Each isozyme is likely to mediate unique functions, but pharmacological tools to explore their isozyme-specific roles have not been available until recently. In this review, we describe the development and application of isozyme-selective inhibitors of PKC. The identification of these inhibitors stems from the observation that PKC isozymes are each localised to unique subcellular locations following activation. Inhibitors of this isozyme-unique localisation have been shown to act as selective inhibitors of the functions of individual isozymes. The identification of isozyme-specific inhibitors should allow the exploration of individual PKC isozyme function in a wide range of cell systems. Copyright 1999 The Italian Pharmacological Society.

Purification and characterization of a casein kinase 2-type protein kinase from pea nuclei

Science.gov (United States)

Li, H.; Roux, S. J.

1992-01-01

Almost all the polyamine-stimulated protein kinase activity associated with the chromatin fraction of nuclei purified from etiolated pea (Pisum sativum L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.35 molar NaCl. This protein kinase can be further purified over 2000-fold by salt fractionation and anion-exchange and casein-agarose column chromatography, after which it is more than 90% pure. The purified kinase has a specific activity of about 650 nanomoles per minute per milligram protein in the absence of polyamines, with either ATP or GTP as phosphoryl donor. Spermidine can stimulate its activity fourfold, with half-maximal activation at about 2 millimolar. Spermine and putrescine also stimulate activity, although somewhat less effectively. This kinase has a tetrameric alpha 2 beta 2 structure with a native molecular weight of 130,000, and subunit molecular weights of 36,000 for the catalytic subunit (alpha) and 29,000 for the regulatory subunit (beta). In western blot analyses, only the alpha subunit reacts strongly with polyclonal antibodies to a Drosophila casein kinase II. The pea kinase can use casein and phosvitin as artificial substrates, phosphorylating both the serine and threonine residues of casein. It has a pH optimum near 8.0, a Vmax of 1.5 micromoles per minute per milligram protein, and a Km for ATP of approximately 75 micromolar. Its activity can be almost completely inhibited by heparin at 5 micrograms per milliliter, but is relatively insensitive to concentrations of staurosporine, K252a, and chlorpromazine that strongly antagonize Ca(2+) -regulated protein kinases. These results are discussed in relation to recent findings that casein kinase 2-type kinases may phosphorylate trans-acting factors that bind to light-regulated promoters in plants.

Bioconversion of rape straw into a nutritionally enriched substrate by ...

African Journals Online (AJOL)

This work aims to select biological treatments and conditions for the bioconversion of rape straw by the mixed-strain fermentation of Ganoderma lucidum and yeasts (Saccharomyces cerevisiae, Candida tropicalis and Candida utilis), into an enriched substrate with increased crude protein and digestibility. Orthogonal ...

Choline Kinase Alpha as an Androgen Receptor Chaperone and Prostate Cancer Therapeutic Target

Science.gov (United States)

Asim, Mohammad; Massie, Charles E.; Orafidiya, Folake; Pértega-Gomes, Nelma; Warren, Anne Y.; Esmaeili, Mohsen; Selth, Luke A.; Zecchini, Heather I.; Luko, Katarina; Qureshi, Arham; Baridi, Ajoeb; Menon, Suraj; Madhu, Basetti; Escriu, Carlos; Lyons, Scott; Vowler, Sarah L.; Zecchini, Vincent R.; Shaw, Greg; Hessenkemper, Wiebke; Russell, Roslin; Mohammed, Hisham; Stefanos, Niki; Lynch, Andy G.; Grigorenko, Elena; D’Santos, Clive; Taylor, Chris; Lamb, Alastair; Sriranjan, Rouchelle; Yang, Jiali; Stark, Rory; Dehm, Scott M.; Rennie, Paul S.; Carroll, Jason S.; Griffiths, John R.; Tavaré, Simon; Mills, Ian G.; McEwan, Iain J.; Baniahmad, Aria; Tilley, Wayne D.; Neal, David E.

2016-01-01

Background: The androgen receptor (AR) is a major drug target in prostate cancer (PCa). We profiled the AR-regulated kinome to identify clinically relevant and druggable effectors of AR signaling. Methods: Using genome-wide approaches, we interrogated all AR regulated kinases. Among these, choline kinase alpha (CHKA) expression was evaluated in benign (n = 195), prostatic intraepithelial neoplasia (PIN) (n = 153) and prostate cancer (PCa) lesions (n = 359). We interrogated how CHKA regulates AR signaling using biochemical assays and investigated androgen regulation of CHKA expression in men with PCa, both untreated (n = 20) and treated with an androgen biosynthesis inhibitor degarelix (n = 27). We studied the effect of CHKA inhibition on the PCa transcriptome using RNA sequencing and tested the effect of CHKA inhibition on cell growth, clonogenic survival and invasion. Tumor xenografts (n = 6 per group) were generated in mice using genetically engineered prostate cancer cells with inducible CHKA knockdown. Data were analyzed with χ2 tests, Cox regression analysis, and Kaplan-Meier methods. All statistical tests were two-sided. Results: CHKA expression was shown to be androgen regulated in cell lines, xenografts, and human tissue (log fold change from 6.75 to 6.59, P = .002) and was positively associated with tumor stage. CHKA binds directly to the ligand-binding domain (LBD) of AR, enhancing its stability. As such, CHKA is the first kinase identified as an AR chaperone. Inhibition of CHKA repressed the AR transcriptional program including pathways enriched for regulation of protein folding, decreased AR protein levels, and inhibited the growth of PCa cell lines, human PCa explants, and tumor xenografts. Conclusions: CHKA can act as an AR chaperone, providing, to our knowledge, the first evidence for kinases as molecular chaperones, making CHKA both a marker of tumor progression and a potential therapeutic target for PCa. PMID:26657335

Promotion of uranium enrichment business

International Nuclear Information System (INIS)

Kurushima, Morihiro

1981-01-01

The Committee on Nuclear Power has studied on the basic nuclear power policy, establishing its five subcommittees, entrusted by the Ministry of Nternational Trade and Industry. The results of examination by the subcommittee on uranium enrichment business are given along with a report in this connection by the Committee. In order to establish the nuclear fuel cycle, the aspect of uranium enrichment is essential. The uranium enrichment by centrifugal process has proceeded steadily in Power Reactor and Nuclear Fuel Development Corporation. The following matters are described: the need for domestic uranium enrichment, the outlook for overseas enrichment services and the schedule for establishing domestic enrichment business, the current state of technology development, the position of the prototype enrichment plant, the course to be taken to establish enrichment business the main organization operating the prototype and commercial plants, the system of supplying centrifuges, the domestic conversion of natural uranium the subsidies for uranium enrichment business. (J.P.N.)

A homogeneous, high-throughput assay for phosphatidylinositol 5-phosphate 4-kinase with a novel, rapid substrate preparation.

Directory of Open Access Journals (Sweden)

Mindy I Davis

Full Text Available Phosphoinositide kinases regulate diverse cellular functions and are important targets for therapeutic development for diseases, such as diabetes and cancer. Preparation of the lipid substrate is crucial for the development of a robust and miniaturizable lipid kinase assay. Enzymatic assays for phosphoinositide kinases often use lipid substrates prepared from lyophilized lipid preparations by sonication, which result in variability in the liposome size from preparation to preparation. Herein, we report a homogeneous 1536-well luciferase-coupled bioluminescence assay for PI5P4Kα. The substrate preparation is novel and allows the rapid production of a DMSO-containing substrate solution without the need for lengthy liposome preparation protocols, thus enabling the scale-up of this traditionally difficult type of assay. The Z'-factor value was greater than 0.7 for the PI5P4Kα assay, indicating its suitability for high-throughput screening applications. Tyrphostin AG-82 had been identified as an inhibitor of PI5P4Kα by assessing the degree of phospho transfer of γ-(32P-ATP to PI5P; its inhibitory activity against PI5P4Kα was confirmed in the present miniaturized assay. From a pilot screen of a library of bioactive compounds, another tyrphostin, I-OMe tyrphostin AG-538 (I-OMe-AG-538, was identified as an ATP-competitive inhibitor of PI5P4Kα with an IC(50 of 1 µM, affirming the suitability of the assay for inhibitor discovery campaigns. This homogeneous assay may apply to other lipid kinases and should help in the identification of leads for this class of enzymes by enabling high-throughput screening efforts.

Foraging enrichment modulates open field response to monosodium glutamate in mice.

Science.gov (United States)

Onaolapo, Olakunle J; Onaolapo, Adejoke Y; Akanmu, Moses A; Olayiwola, Gbola

2015-07-01

Environmental enrichment can enhance expression of species-specific behaviour. While foraging enrichment is encouraged in laboratory animals, its impact on novelty induced behaviour remain largely unknown. Here, we studied behavioural response of mice to acute and subchronic oral monosodium glutamate (MSG) in an open field with /without foraging enrichment. Adult male mice, assigned to five groups were administered vehicle (distilled water), or one of four selected doses of MSG (10, 20, 40 and 80 mg/kg) for 21 days. Open field novelty induced behaviours i.e. horizontal locomotion, rearing and grooming were assessed after the first and last doses of MSG. Results were analysed using MANOVA followed by Tukey HSD multiple comparison test and expressed as mean ± S.E.M. Following acute MSG administration without enrichment, locomotor activity reduced, grooming increased, while rearing activity reduced at lower doses and increased at higher doses. Subchronic administration without enrichment was associated with increased locomotor activity and reduction in grooming, rearing activity however still showed a biphasic response. Addition of enrichment with acute administration resulted in sustained reduction in locomotor and rearing activities with a biphasic grooming response. Subchronically, there was reduction in horizontal locomotion, biphasic rearing response and sustained increase in grooming activity. Behavioural response to varying doses of MSG as observed in the open field is affected by modifications such as foraging enrichment, which can reverse or dampen the central effects seen irrespective of duration of administration.

Enrichment of gadolinium-157 and gadolinium-155 by laser method

International Nuclear Information System (INIS)

Xinjun, Su; Xiaowei, Zhang; Zhiquan, Li

2008-01-01

Laser isotope separation experiments of gadolinium by atomic vapor method have been performed. Gadolinium-157 and gadolinium-155 were selectively photoionized by means of three linearly polarized dye lasers, the excitation process of which is based on the polarization selection rules. Gramme-magnitude of enriched gadolinium was obtained and the total abundance of gadolinium-157 and gadolinium-155 was in excess of 80%, and the product rating exceeded 200 mg/h. (author)

Rapid Column-Free Enrichment of Mononuclear Cells from Solid Tissues

Science.gov (United States)

Scoville, Steven D.; Keller, Karen A.; Cheng, Stephanie; Zhang, Michael; Zhang, Xiaoli; Caligiuri, Michael A.; Freud, Aharon G.

2015-01-01

We have developed a rapid negative selection method to enrich rare mononuclear cells from human tissues. Unwanted and antibody-tethered cells are selectively depleted during a Ficoll separation step, and there is no need for magnetic-based reagents and equipment. The new method is fast, customizable, inexpensive, remarkably efficient, and easy to perform, and per sample the overall cost is less than one-tenth the cost associated with a magnetic column-based method. PMID:26223896

Selective inhibition of Sarcocystis neurona calcium-dependent protein kinase 1 for equine protozoal myeloencephalitis therapy

Science.gov (United States)

Sarcocystis neurona is the most frequent cause of Equine Protozoal Myeloencephalitis (EPM), a debilitating neurologic disease of horses that can be difficult to treat. We identified SnCDPK1, the S. neurona homologue of calcium dependent protein kinase 1 (CDPK1), a validated drug target in Toxoplasma...

Cocoa Procyanidins Suppress Transformation by Inhibiting Mitogen-activated Protein Kinase Kinase*S⃞

Science.gov (United States)

Kang, Nam Joo; Lee, Ki Won; Lee, Dong Eun; Rogozin, Evgeny A.; Bode, Ann M.; Lee, Hyong Joo; Dong, Zigang

2008-01-01

Cocoa was shown to inhibit chemically induced carcinogenesis in animals and exert antioxidant activity in humans. However, the molecular mechanisms of the chemopreventive potential of cocoa and its active ingredient(s) remain unknown. Here we report that cocoa procyanidins inhibit neoplastic cell transformation by suppressing the kinase activity of mitogen-activated protein kinase kinase (MEK). A cocoa procyanidin fraction (CPF) and procyanidin B2 at 5 μg/ml and 40 μm, respectively, inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation of JB6 P+ mouse epidermal (JB6 P+) cells by 47 and 93%, respectively. The TPA-induced promoter activity and expression of cyclooxygenase-2, which is involved in tumor promotion and inflammation, were dose-dependently inhibited by CPF or procyanidin B2. The activation of activator protein-1 and nuclear factor-κB induced by TPA was also attenuated by CPF or procyanidin B2. The TPA-induced phosphorylation of MEK, extracellular signal-regulated kinase, and p90 ribosomal s6 kinase was suppressed by CPF or procyanidin B2. In vitro and ex vivo kinase assay data demonstrated that CPF or procyanidin B2 inhibited the kinase activity of MEK1 and directly bound with MEK1. CPF or procyanidin B2 suppressed JB6 P+ cell transformation induced by epidermal growth factor or H-Ras, both of which are known to be involved in MEK/ERK signal activation. In contrast, theobromine (up to 80 μm) had no effect on TPA-induced transformation, cyclooxygenase-2 expression, the transactivation of activator protein-1 or nuclear factor-κB, or MEK. Notably, procyanidin B2 exerted stronger inhibitory effects compared with PD098059 (a well known pharmacological inhibitor of MEK) on MEK1 activity and neoplastic cell transformation. PMID:18519570

Identifying kinase dependency in cancer cells by integrating high-throughput drug screening and kinase inhibition data.

Science.gov (United States)

Ryall, Karen A; Shin, Jimin; Yoo, Minjae; Hinz, Trista K; Kim, Jihye; Kang, Jaewoo; Heasley, Lynn E; Tan, Aik Choon

2015-12-01

Targeted kinase inhibitors have dramatically improved cancer treatment, but kinase dependency for an individual patient or cancer cell can be challenging to predict. Kinase dependency does not always correspond with gene expression and mutation status. High-throughput drug screens are powerful tools for determining kinase dependency, but drug polypharmacology can make results difficult to interpret. We developed Kinase Addiction Ranker (KAR), an algorithm that integrates high-throughput drug screening data, comprehensive kinase inhibition data and gene expression profiles to identify kinase dependency in cancer cells. We applied KAR to predict kinase dependency of 21 lung cancer cell lines and 151 leukemia patient samples using published datasets. We experimentally validated KAR predictions of FGFR and MTOR dependence in lung cancer cell line H1581, showing synergistic reduction in proliferation after combining ponatinib and AZD8055. KAR can be downloaded as a Python function or a MATLAB script along with example inputs and outputs at: http://tanlab.ucdenver.edu/KAR/. aikchoon.tan@ucdenver.edu. Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Silver surface enrichment controlled by simultaneous RBS for reliable PIXE analysis of ancient coins

International Nuclear Information System (INIS)

Beck, L.; Alloin, E.; Berthier, C.; Reveillon, S.; Costa, V.

2008-01-01

Evidence of silver surface enrichment of ancient silver-copper coins has been pointed out in the past years. Surface enrichment can be fortuitous or intentional. In this paper, we have investigated the cleaning procedures usually performed after excavation or in museums. We have shown that chemicals or commercial products routinely used dissolve preferentially the copper phase and consequently contribute to the silver surface enrichment. As a result, surface analyses such as PIXE or XRF can be strongly affected by this effect. By using simultaneously RBS and PIXE, it is possible to check through the silver surface enrichment and then select the reliable measurements, characteristic of the bulk composition. Results on coins recently discovered and mechanically or chemically cleaned are presented

From high enriched to low enriched uranium fuel in research reactors

Energy Technology Data Exchange (ETDEWEB)

Van Den Berghe, S.; Leenaers, A.; Koonen, E.; Moons, F.; Sannen, L. [Nuclear Materials Science Institute, SCK.CEN, Boeretang 200, B-2400 Mol (Belgium)

2010-07-01

Since the 1970's, global efforts have been going on to replace the high-enriched (>90% {sup 235}U), low-density UAlx research reactor fuel with high-density, low enriched (<20% {sup 235}U) replacements. This search is driven by the attempt to reduce the civil use of high-enriched material because of proliferation risks and terrorist threats. American initiatives, such as the Global Threat Reduction Initiative (GTRI) and the Reduced Enrichment for Research and Test Reactors (RERTR) program have triggered the development of reliable low-enriched fuel types for these reactors, which can replace the high enriched ones without loss of performance. Most success has presently been obtained with U{sub 3}Si{sub 2} dispersion fuel, which is currently used in many research reactors in the world. However, efforts to search for a replacement with even higher density, which will also allow the conversion of some high flux research reactors that currently cannot change to U{sub 3}Si{sub 2} (eg. BR2 in Belgium), have continued and are for the moment mainly directed towards the U(Mo) alloy fuel (7-10 w% Mo). This paper provides an overview of the past efforts and presents the current status of the U(Mo) development. (authors)

From high enriched to low enriched uranium fuel in research reactors

International Nuclear Information System (INIS)

Van Den Berghe, S.; Leenaers, A.; Koonen, E.; Moons, F.; Sannen, L.

2010-01-01

Since the 1970's, global efforts have been going on to replace the high-enriched (>90% 235 U), low-density UAlx research reactor fuel with high-density, low enriched ( 235 U) replacements. This search is driven by the attempt to reduce the civil use of high-enriched material because of proliferation risks and terrorist threats. American initiatives, such as the Global Threat Reduction Initiative (GTRI) and the Reduced Enrichment for Research and Test Reactors (RERTR) program have triggered the development of reliable low-enriched fuel types for these reactors, which can replace the high enriched ones without loss of performance. Most success has presently been obtained with U 3 Si 2 dispersion fuel, which is currently used in many research reactors in the world. However, efforts to search for a replacement with even higher density, which will also allow the conversion of some high flux research reactors that currently cannot change to U 3 Si 2 (eg. BR2 in Belgium), have continued and are for the moment mainly directed towards the U(Mo) alloy fuel (7-10 w% Mo). This paper provides an overview of the past efforts and presents the current status of the U(Mo) development. (authors)

Uranium enrichment by gas centrifuge

International Nuclear Information System (INIS)

Heriot, I.D.

1988-01-01

After recalling the physical principles and the techniques of centrifuge enrichment the report describes the centrifuge enrichment programmes of the various countries concerned and compares this technology with other enrichment technologies like gaseous diffusion, laser, aerodynamic devices and chemical processes. The centrifuge enrichment process is said to be able to replace with advantage the existing enrichment facilities in the short and medium term. Future prospects of the process are also described, like recycled uranium enrichment and economic improvements; research and development needs to achieve the economic prospects are also indicated. Finally the report takes note of the positive aspect of centrifuge enrichment as far as safeguards and nuclear safety are concerned. 27 figs, 113 refs

Derived enriched uranium market

International Nuclear Information System (INIS)

Rutkowski, E.

1996-01-01

The potential impact on the uranium market of highly enriched uranium from nuclear weapons dismantling in the Russian Federation and the USA is analyzed. Uranium supply, conversion, and enrichment factors are outlined for each country; inventories are also listed. The enrichment component and conversion components are expected to cause little disruption to uranium markets. The uranium component of Russian derived enriched uranium hexafluoride is unresolved; US legislation places constraints on its introduction into the US market

Immune-related genetic enrichment in frontotemporal dementia: An analysis of genome-wide association studies.

Directory of Open Access Journals (Sweden)

Iris Broce

2018-01-01

Full Text Available Converging evidence suggests that immune-mediated dysfunction plays an important role in the pathogenesis of frontotemporal dementia (FTD. Although genetic studies have shown that immune-associated loci are associated with increased FTD risk, a systematic investigation of genetic overlap between immune-mediated diseases and the spectrum of FTD-related disorders has not been performed.Using large genome-wide association studies (GWASs (total n = 192,886 cases and controls and recently developed tools to quantify genetic overlap/pleiotropy, we systematically identified single nucleotide polymorphisms (SNPs jointly associated with FTD-related disorders-namely, FTD, corticobasal degeneration (CBD, progressive supranuclear palsy (PSP, and amyotrophic lateral sclerosis (ALS-and 1 or more immune-mediated diseases including Crohn disease, ulcerative colitis (UC, rheumatoid arthritis (RA, type 1 diabetes (T1D, celiac disease (CeD, and psoriasis. We found up to 270-fold genetic enrichment between FTD and RA, up to 160-fold genetic enrichment between FTD and UC, up to 180-fold genetic enrichment between FTD and T1D, and up to 175-fold genetic enrichment between FTD and CeD. In contrast, for CBD and PSP, only 1 of the 6 immune-mediated diseases produced genetic enrichment comparable to that seen for FTD, with up to 150-fold genetic enrichment between CBD and CeD and up to 180-fold enrichment between PSP and RA. Further, we found minimal enrichment between ALS and the immune-mediated diseases tested, with the highest levels of enrichment between ALS and RA (up to 20-fold. For FTD, at a conjunction false discovery rate < 0.05 and after excluding SNPs in linkage disequilibrium, we found that 8 of the 15 identified loci mapped to the human leukocyte antigen (HLA region on Chromosome (Chr 6. We also found novel candidate FTD susceptibility loci within LRRK2 (leucine rich repeat kinase 2, TBKBP1 (TBK1 binding protein 1, and PGBD5 (piggyBac transposable element

Glycogen synthase kinase 3: more than a namesake.

Science.gov (United States)

Rayasam, Geetha Vani; Tulasi, Vamshi Krishna; Sodhi, Reena; Davis, Joseph Alex; Ray, Abhijit

2009-03-01

Glycogen synthase kinase 3 (GSK3), a constitutively acting multi-functional serine threonine kinase is involved in diverse physiological pathways ranging from metabolism, cell cycle, gene expression, development and oncogenesis to neuroprotection. These diverse multiple functions attributed to GSK3 can be explained by variety of substrates like glycogen synthase, tau protein and beta catenin that are phosphorylated leading to their inactivation. GSK3 has been implicated in various diseases such as diabetes, inflammation, cancer, Alzheimer's and bipolar disorder. GSK3 negatively regulates insulin-mediated glycogen synthesis and glucose homeostasis, and increased expression and activity of GSK3 has been reported in type II diabetics and obese animal models. Consequently, inhibitors of GSK3 have been demonstrated to have anti-diabetic effects in vitro and in animal models. However, inhibition of GSK3 poses a challenge as achieving selectivity of an over achieving kinase involved in various pathways with multiple substrates may lead to side effects and toxicity. The primary concern is developing inhibitors of GSK3 that are anti-diabetic but do not lead to up-regulation of oncogenes. The focus of this review is the recent advances and the challenges surrounding GSK3 as an anti-diabetic therapeutic target.

Gene and miRNA expression signature of Lewis lung carcinoma LLC1 cells in extracellular matrix enriched microenvironment

International Nuclear Information System (INIS)

Stankevicius, Vaidotas; Vasauskas, Gintautas; Bulotiene, Danute; Butkyte, Stase; Jarmalaite, Sonata; Rotomskis, Ricardas; Suziedelis, Kestutis

2016-01-01

The extracellular matrix (ECM), one of the key components of tumor microenvironment, has a tremendous impact on cancer development and highly influences tumor cell features. ECM affects vital cellular functions such as cell differentiation, migration, survival and proliferation. Gene and protein expression levels are regulated in cell-ECM interaction dependent manner as well. The rate of unsuccessful clinical trials, based on cell culture research models lacking the ECM microenvironment, indicates the need for alternative models and determines the shift to three-dimensional (3D) laminin rich ECM models, better simulating tissue organization. Recognized advantages of 3D models suggest the development of new anticancer treatment strategies. This is among the most promising directions of 3D cell cultures application. However, detailed analysis at the molecular level of 2D/3D cell cultures and tumors in vivo is still needed to elucidate cellular pathways most promising for the development of targeted therapies. In order to elucidate which biological pathways are altered during microenvironmental shift we have analyzed whole genome mRNA and miRNA expression differences in LLC1 cells cultured in 2D or 3D culture conditions. In our study we used DNA microarrays for whole genome analysis of mRNA and miRNA expression differences in LLC1 cells cultivated in 2D or 3D culture conditions. Next, we indicated the most common enriched functional categories using KEGG pathway enrichment analysis. Finally, we validated the microarray data by quantitative PCR in LLC1 cells cultured under 2D or 3D conditions or LLC1 tumors implanted in experimental animals. Microarray gene expression analysis revealed that 1884 genes and 77 miRNAs were significantly altered in LLC1 cells after 48 h cell growth under 2D and ECM based 3D cell growth conditions. Pathway enrichment results indicated metabolic pathway, MAP kinase, cell adhesion and immune response as the most significantly altered

Src kinase regulation by phosphorylation and dephosphorylation

International Nuclear Information System (INIS)

Roskoski, Robert

2005-01-01

Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTPα, PTPε, and PTPλ. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined

Selective enhancement of radiation response of herpes simplex virus thymidine kinase transduced 9L gliosarcoma cells in vitro and in vivo by antiviral agents

International Nuclear Information System (INIS)

Kim, Jae Ho; Kim, Sang Hie; Kolozsvary, A.

1995-01-01

The purpose of this investigation was to demonstrate in a well-characterized tumor model that the radiosensitivity of tumor cells transduced with a herpes simplex virus thymidine kinase gene (HS-tk) would be selectively enhanced by antiviral agents. Rat 9L gliosarcoma cells transduced with a retroviral vector containing an HS-tk gene, 9L-tk cells were exposed to various doses or irradiation under either in vitro or in vivo conditions. The radiation sensitizing potential of two antiviral drugs, bromovinyl deoxyuridine (BVdU) and dihydroxymethyl ethyl methyl guanine (acyclovir), was evaluated in vitro. The radiosensitizing ability of BVdU was also evaluated with a 9L-tk tumor growing in the rat brain. Tumors growing in the right hemisphere of rat brains were irradiated stereotactically with single-dose irradiation. The radiation response of 9L-tk cells was selectively enhanced by antiviral agents relative to nontransduced cells. In the cell culture, when a 24-h drug exposure (20 μg/ml) preceded radiation, the sensitizer enhancement ratio (SER) for BVdU and acyclovir was 1.4 ± 0.1 and 1.3 ± 0.1, respectively. Exposure of cells to 10 μg/ml acyclovir for two 24-h periods both pre- and postirradiation resulted in a SER of 1.6 ± 0.1. In vivo, a significant increase in median survival time of rats with 9L-tk tumors was found when BVdU was administered prior to single-dose irradiation relative to the survival time of similar rats receiving radiation alone. An antiviral agent can enhance cell killing by radiation with selective action in cells transduced with the herpes simplex virus thymidine kinase gene. The results suggest that the three-pronged therapy of HS-tk gene transduction, systemically administered antiviral drug, and stereotactically targeted radiation therapy will improve the effectiveness of radiation therapy for the treatment of radioresistant tumors. 25 refs., 6 figs

Selective enhancement of radiation response of herpes simplex virus thymidine kinase transduced 9L gliosarcoma cells in vitro and in vivo by antiviral agents

Energy Technology Data Exchange (ETDEWEB)

Kim, Jae Ho; Kim, Sang Hie; Kolozsvary, A. [Henry Ford Hospital, Detroit, MI (United States)] [and others

1995-11-01

The purpose of this investigation was to demonstrate in a well-characterized tumor model that the radiosensitivity of tumor cells transduced with a herpes simplex virus thymidine kinase gene (HS-tk) would be selectively enhanced by antiviral agents. Rat 9L gliosarcoma cells transduced with a retroviral vector containing an HS-tk gene, 9L-tk cells were exposed to various doses or irradiation under either in vitro or in vivo conditions. The radiation sensitizing potential of two antiviral drugs, bromovinyl deoxyuridine (BVdU) and dihydroxymethyl ethyl methyl guanine (acyclovir), was evaluated in vitro. The radiosensitizing ability of BVdU was also evaluated with a 9L-tk tumor growing in the rat brain. Tumors growing in the right hemisphere of rat brains were irradiated stereotactically with single-dose irradiation. The radiation response of 9L-tk cells was selectively enhanced by antiviral agents relative to nontransduced cells. In the cell culture, when a 24-h drug exposure (20 {mu}g/ml) preceded radiation, the sensitizer enhancement ratio (SER) for BVdU and acyclovir was 1.4 {plus_minus} 0.1 and 1.3 {plus_minus} 0.1, respectively. Exposure of cells to 10 {mu}g/ml acyclovir for two 24-h periods both pre- and postirradiation resulted in a SER of 1.6 {plus_minus} 0.1. In vivo, a significant increase in median survival time of rats with 9L-tk tumors was found when BVdU was administered prior to single-dose irradiation relative to the survival time of similar rats receiving radiation alone. An antiviral agent can enhance cell killing by radiation with selective action in cells transduced with the herpes simplex virus thymidine kinase gene. The results suggest that the three-pronged therapy of HS-tk gene transduction, systemically administered antiviral drug, and stereotactically targeted radiation therapy will improve the effectiveness of radiation therapy for the treatment of radioresistant tumors. 25 refs., 6 figs.

Selective enhancement of radiation response of herpes simplex virus thymidine kinase transduced 9L gliosarcoma cells in vitro and in vivo by antiviral agents

International Nuclear Information System (INIS)

Jae, Ho Kim; Sang, Hie Kim; Kolozsvary, Andrew; Brown, Stephen L.; Ok, Bae Kim; Freytag, Svend O.

1995-01-01

Purpose: To demonstrate in a well-characterized tumor model that the radiosensitivity of tumor cells transduced with a herpes simplex virus thymidine kinase gene (HS-tk) would be selectively enhanced by antiviral agents. Methods and Materials: Rat 9L gliosarcoma cells transduced with a retroviral vector containing an HS-tk gene, 9L-tk cells were exposed to various doses of irradiation under either in vitro or in vivo conditions. The radiation sensitizing potential of two antiviral drugs, bromovinyl deoxyuridine (BVdU) and dihydroxymethyl ethyl methyl guanine (acyclovir), was evaluated in vitro. The radiosensitizing ability of BVdU was also evaluated with a 9L-tk tumor growing in the rat brain. Tumors growing in the right hemisphere of rat brains were irradiated stereotactically with single-dose irradiation. Results: The radiation response of 9L-tk cells was selectively enhanced by antiviral agents relative to nontransduced cells. In the cell culture, when a 24-h drug exposure (20 μg/ml) preceded radiation, the sensitizer enhancement ratio (SER) for BVdU and acyclovir was 1.4 ± 0.1 and 1.3 ± 0.1, respectively. Exposure of cells to 10 μg/ml acyclovir for two 24-h periods both pre- and postirradiation resulted in a SER of 1.6 ± 0.1. In vivo, a significant increase in median survival time of rats with 9L-tk tumors was found when BVdU was administered prior to single-dose irradiation relative to the survival time of similar rats receiving radiation alone. Conclusion: An antiviral agent can enhance cell killing by radiation with selective action in cells transduced with the herpes simplex virus thymidine kinase gene. The results suggest that the three-pronged therapy of HS-tk gene transduction, systemically administered antiviral drug, and stereotactically targeted radiation therapy will improve the effectiveness of radiation therapy for the treatment of radioresistant tumors

Radiometric enrichment of nonradioactive ores

International Nuclear Information System (INIS)

Mokrousov, V.A.; Lileev, V.A.

1979-01-01

Considered are the methods of mineral enrichment based on the use of the radioation of various types. The physical essence of enrichment processes is presented, their classification is given. Described are the ore properties influencing the efficiency of radiometric enrichment, methods of the properties study and estimation of ore enrichment. New possibilities opened by radiometric enrichment in the technology of primary processing of mineral raw materials are elucidated. A considerable attention is paid to the main and auxiliary equipment for radiometric enrichment. The foundations of the safety engineering are presented in a brief form. Presented are also results of investigations and practical works in the field of enrichment of ores of non-ferrous, ferrous and non-metallic minerals with the help of radiometric methods

Specific RSK kinase inhibition by dibenzyl trisulfide and implication for therapeutic treatment of cancer.

Science.gov (United States)

Lowe, Henry I C; Facey, Caroline O B; Toyang, Ngeh J; Bryant, Joseph L

2014-04-01

The Jamaican "Guinea Hen Weed" (Petiveria alliacea L.) plant has been traditionally used in folklore medicine to treat a variety of diseases including cancer. In the present study we investigated on the therapeutic feasibility of dibenzyl trisulfide (DTS) (isolated from the Jamaican Guinea Hen Weed) as a potent small-molecule kinase inhibitor to treat cancer. We investigated the inhibitory effects of DTS against a large panel of kinases using a well-established competitive binding assay. Cell proliferation data were obtained using the WST-1 colorimetric assay. DTS inhibited the activity of the C-terminal kinase domain of RSK1 (80% compared to control) with a Kd of 1.3 μM. Anti-proliferative effects of DTS were observed in small lung, pancreatic, breast, and prostate cancer cells with IC50 values ranging from 0.34-0.84 μM. We have identified DTS as a highly selective and isoform-specific RSK1 kinase inhibitor with broad cancer therapeutic potential.

PWR fuel of high enrichment with erbia and enriched gadolinia

International Nuclear Information System (INIS)

Bejmer, Klaes-Håkan; Malm, Christian

2011-01-01

Today standard PWR fuel is licensed for operation up to 65-70 MWd/kgU, which in most cases corresponds to an enrichment of more than 5 w/o "2"3"5U. Due to criticality safety reason of storage and transportation, only fuel up to 5 w/o "2"3"5U enrichment is so far used. New fuel storage installations and transportation casks are necessary investments before the reactivity level of the fresh fuel can be significantly increased. These investments and corresponding licensing work takes time, and in the meantime a solution that requires burnable poisons in all pellets of the fresh high-enriched fuel might be used. By using very small amounts of burnable absorber in every pellet the initial reactivity can be reduced to today's levels. This study presents core calculations with fuel assemblies enriched to almost 6 w/o "2"3"5U mixed with a small amount of erbia. Some of the assemblies also contain gadolinia. The results are compared to a reference case containing assemblies with 4.95 w/o "2"3"5U without erbia, utilizing only gadolinia as burnable poison. The comparison shows that the number of fresh fuel assemblies can be reduced by 21% (which increases the batch burnup by 24%) by utilizing the erbia fuel concept. However, increased cost of uranium due to higher enrichment is not fully compensated for by the cost gain due to the reduction of the number assemblies. Hence, the fuel cycle cost becomes slightly higher for the high enrichment erbia case than for the reference case. (author)

Preparation of hydrophilic monolithic capillary column by in situ photo-polymerization of N-vinyl-2-pyrrolidinone and acrylamide for highly selective and sensitive enrichment of N-linked glycopeptides.

Science.gov (United States)

Jiang, Hao; Yuan, Huiming; Qu, Yanyan; Liang, Yu; Jiang, Bo; Wu, Qi; Deng, Nan; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

2016-01-01

In this study, a novel kind of amide functionalized hydrophilic monolith was synthesized by the in situ photo-polymerization of N-vinyl-2-pyrrolidinone (NVP), acrylamide (AM), and N, N'-methylenebisacrylamide (MBA) in a UV transparent capillary, and successfully applied for hydrophilic interaction chromatography (HILIC) based enrichment of N-linked glycopeptides. With 2 μg of the tryptic digests of IgG as the sample, after enrichment, 18 glycopeptides could be identified by MALDI-TOF/TOF MS analysis. Furthermore, with the mixture of BSA and IgG digests (10,000:1, m/m) as the sample, 6 N-linked glycopeptides were unambiguously identified after enrichment, indicating the high selectivity and good specificity of such material. Moreover, such a monolithic capillary column was also applied for the N-glycosylation sites profiling of 6 μg protein digests from HeLa cells and 1 μL human serum. In total, 530 and 262 unique N-glycosylated peptides were identified, respectively, corresponding to 282 and 124N-glycoproteins, demonstrating its great potential for the large scale glycoproteomics analysis. Copyright © 2015 Elsevier B.V. All rights reserved.

Profiling plasma extracellular vesicle by pluronic block-copolymer based enrichment method unveils features associated with breast cancer aggression, metastasis and invasion.

Science.gov (United States)

Zhong, Zhenyu; Rosenow, Matthew; Xiao, Nick; Spetzler, David

2018-01-01

Extracellular vesicle (EV)-based liquid biopsies have been proposed to be a readily obtainable biological substrate recently for both profiling and diagnostics purposes. Development of a fast and reliable preparation protocol to enrich such small particles could accelerate the discovery of informative, disease-related biomarkers. Though multiple EV enrichment protocols are available, in terms of efficiency, reproducibility and simplicity, precipitation-based methods are most amenable to studies with large numbers of subjects. However, the selectivity of the precipitation becomes critical. Here, we present a simple plasma EV enrichment protocol based on pluronic block copolymer. The enriched plasma EV was able to be verified by multiple platforms. Our results showed that the particles enriched from plasma by the copolymer were EV size vesicles with membrane structure; proteomic profiling showed that EV-related proteins were significantly enriched, while high-abundant plasma proteins were significantly reduced in comparison to other precipitation-based enrichment methods. Next-generation sequencing confirmed the existence of various RNA species that have been observed in EVs from previous studies. Small RNA sequencing showed enriched species compared to the corresponding plasma. Moreover, plasma EVs enriched from 20 advanced breast cancer patients and 20 age-matched non-cancer controls were profiled by semi-quantitative mass spectrometry. Protein features were further screened by EV proteomic profiles generated from four breast cancer cell lines, and then selected in cross-validation models. A total of 60 protein features that highly contributed in model prediction were identified. Interestingly, a large portion of these features were associated with breast cancer aggression, metastasis as well as invasion, consistent with the advanced clinical stage of the patients. In summary, we have developed a plasma EV enrichment method with improved precipitation selectivity

A strictly monofunctional bacterial hydroxymethylpyrimidine phosphate kinase precludes damaging errors in thiamin biosynthesis.

Science.gov (United States)

Thamm, Antje M; Li, Gengnan; Taja-Moreno, Marlene; Gerdes, Svetlana Y; de Crécy-Lagard, Valérie; Bruner, Steven D; Hanson, Andrew D

2017-07-20

The canonical kinase (ThiD) that converts the thiamin biosynthesis intermediate hydroxymethylpyrimidine (HMP) monophosphate to the diphosphate can also very efficiently convert free HMP to the monophosphate in prokaryotes, plants, and fungi. This HMP kinase activity enables salvage of HMP, but it is not substrate-specific and so allows toxic HMP analogs and damage products to infiltrate the thiamin biosynthesis pathway. Comparative analysis of bacterial genomes uncovered a gene, thiD2 , that is often fused to the thiamin synthesis gene thiE and could potentially encode a replacement for ThiD. Standalone ThiD2 proteins and ThiD2 fusion domains are small (~130-residues) and do not belong to any previously known protein family. Genetic and biochemical analyses showed that representative standalone and fused ThiD2 proteins catalyze phosphorylation of HMP monophosphate, but not of HMP or its toxic analogs and damage products such as bacimethrin and 5-(hydroxymethyl)-2-methylpyrimidin-4-ol. As strictly monofunctional HMP monophosphate kinases, ThiD2 proteins eliminate a potentially fatal vulnerability of canonical ThiD, at the cost of the ability to reclaim HMP formed by thiamin turnover. ©2017 The Author(s).

Use of mathematics to guide target selection in systems pharmacology; application to receptor tyrosine kinase (RTK) pathways.

Science.gov (United States)

Benson, Neil; van der Graaf, Piet H; Peletier, Lambertus A

2017-11-15

A key element of the drug discovery process is target selection. Although the topic is subject to much discussion and experimental effort, there are no defined quantitative rules around optimal selection. Often 'rules of thumb', that have not been subject to rigorous exploration, are used. In this paper we explore the 'rule of thumb' notion that the molecule that initiates a pathway signal is the optimal target. Given the multi-factorial and complex nature of this question, we have simplified an example pathway to its logical minimum of two steps and used a mathematical model of this to explore the different options in the context of typical small and large molecule drugs. In this paper, we report the conclusions of our analysis and describe the analysis tool and methods used. These provide a platform to enable a more extensive enquiry into this important topic. Copyright © 2017 Elsevier B.V. All rights reserved.

Structure-function similarities between a plant receptor-like kinase and the human interleukin-1 receptor-associated kinase-4.

Science.gov (United States)

Klaus-Heisen, Dörte; Nurisso, Alessandra; Pietraszewska-Bogiel, Anna; Mbengue, Malick; Camut, Sylvie; Timmers, Ton; Pichereaux, Carole; Rossignol, Michel; Gadella, Theodorus W J; Imberty, Anne; Lefebvre, Benoit; Cullimore, Julie V

2011-04-01

Phylogenetic analysis has previously shown that plant receptor-like kinases (RLKs) are monophyletic with respect to the kinase domain and share an evolutionary origin with the animal interleukin-1 receptor-associated kinase/Pelle-soluble kinases. The lysin motif domain-containing receptor-like kinase-3 (LYK3) of the legume Medicago truncatula shows 33% amino acid sequence identity with human IRAK-4 over the kinase domain. Using the structure of this animal kinase as a template, homology modeling revealed that the plant RLK contains structural features particular to this group of kinases, including the tyrosine gatekeeper and the N-terminal extension α-helix B. Functional analysis revealed the importance of these conserved features for kinase activity and suggests that kinase activity is essential for the biological role of LYK3 in the establishment of the root nodule nitrogen-fixing symbiosis with rhizobia bacteria. The kinase domain of LYK3 has dual serine/threonine and tyrosine specificity, and mass spectrometry analysis identified seven serine, eight threonine, and one tyrosine residue as autophosphorylation sites in vitro. Three activation loop serine/threonine residues are required for biological activity, and molecular dynamics simulations suggest that Thr-475 is the prototypical phosphorylated residue that interacts with the conserved arginine in the catalytic loop, whereas Ser-471 and Thr-472 may be secondary sites. A threonine in the juxtamembrane region and two threonines in the C-terminal lobe of the kinase domain are important for biological but not kinase activity. We present evidence that the structure-function similarities that we have identified between LYK3 and IRAK-4 may be more widely applicable to plant RLKs in general.

77 FR 14838 - General Electric-Hitachi Global Laser Enrichment LLC, Commercial Laser-Based Uranium Enrichment...

Science.gov (United States)

2012-03-13

... Laser Enrichment LLC, Commercial Laser-Based Uranium Enrichment Facility, Wilmington, North Carolina... a license to General Electric-Hitachi Global Laser Enrichment LLC (GLE or the applicant) to authorize construction of a laser-based uranium enrichment facility and possession and use of byproduct...

Role of contractile prostaglandins and Rho-kinase in growth factor-induced airway smooth muscle contraction

Directory of Open Access Journals (Sweden)

Zaagsma Johan

2005-07-01

Full Text Available Abstract Background In addition to their proliferative and differentiating effects, several growth factors are capable of inducing a sustained airway smooth muscle (ASM contraction. These contractile effects were previously found to be dependent on Rho-kinase and have also been associated with the production of eicosanoids. However, the precise mechanisms underlying growth factor-induced contraction are still unknown. In this study we investigated the role of contractile prostaglandins and Rho-kinase in growth factor-induced ASM contraction. Methods Growth factor-induced contractions of guinea pig open-ring tracheal preparations were studied by isometric tension measurements. The contribution of Rho-kinase, mitogen-activated protein kinase (MAPK and cyclooxygenase (COX to these reponses was established, using the inhibitors Y-27632 (1 μM, U-0126 (3 μM and indomethacin (3 μM, respectively. The Rho-kinase dependency of contractions induced by exogenously applied prostaglandin F2α (PGF2α and prostaglandin E2 (PGE2 was also studied. In addition, the effects of the selective FP-receptor antagonist AL-8810 (10 μM and the selective EP1-antagonist AH-6809 (10 μM on growth factor-induced contractions were investigated, both in intact and epithelium-denuded preparations. Growth factor-induced PGF2α-and PGE2-release in the absence and presence of Y-27632, U-0126 and indomethacin, was assessed by an ELISA-assay. Results Epidermal growth factor (EGF-and platelet-derived growth factor (PDGF-induced contractions of guinea pig tracheal smooth muscle preparations were dependent on Rho-kinase, MAPK and COX. Interestingly, growth factor-induced PGF2α-and PGE2-release from tracheal rings was significantly reduced by U-0126 and indomethacin, but not by Y-27632. Also, PGF2α-and PGE2-induced ASM contractions were largely dependent on Rho-kinase, in contrast to other contractile agonists like histamine. The FP-receptor antagonist AL-8810 (10 μM significantly

Stable acetate production in extreme-thermophilic (70°C) mixed culture fermentation by selective enrichment of hydrogenotrophic methanogens

Science.gov (United States)

Zhang, Fang; Zhang, Yan; Ding, Jing; Dai, Kun; van Loosdrecht, Mark C. M.; Zeng, Raymond J.

2014-06-01

The control of metabolite production is difficult in mixed culture fermentation. This is particularly related to hydrogen inhibition. In this work, hydrogenotrophic methanogens were selectively enriched to reduce the hydrogen partial pressure and to realize efficient acetate production in extreme-thermophilic (70°C) mixed culture fermentation. The continuous stirred tank reactor (CSTR) was stable operated during 100 days, in which acetate accounted for more than 90% of metabolites in liquid solutions. The yields of acetate, methane and biomass in CSTR were 1.5 +/- 0.06, 1.0 +/- 0.13 and 0.4 +/- 0.05 mol/mol glucose, respectively, close to the theoretical expected values. The CSTR effluent was stable and no further conversion occurred when incubated for 14 days in a batch reactor. In fed-batch experiments, acetate could be produced up to 34.4 g/L, significantly higher than observed in common hydrogen producing fermentations. Acetate also accounted for more than 90% of soluble products formed in these fed-batch fermentations. The microbial community analysis revealed hydrogenotrophic methanogens (mainly Methanothermobacter thermautotrophicus and Methanobacterium thermoaggregans) as 98% of Archaea, confirming that high temperature will select hydrogenotrophic methanogens over aceticlastic methanogens effectively. This work demonstrated a potential application to effectively produce acetate as a value chemical and methane as an energy gas together via mixed culture fermentation.

Design and synthesis of selective CDK8/19 dual inhibitors: Discovery of 4,5-dihydrothieno[3',4':3,4]benzo[1,2-d]isothiazole derivatives.

Science.gov (United States)

Ono, Koji; Banno, Hiroshi; Okaniwa, Masanori; Hirayama, Takaharu; Iwamura, Naoki; Hikichi, Yukiko; Murai, Saomi; Hasegawa, Maki; Hasegawa, Yuka; Yonemori, Kazuko; Hata, Akito; Aoyama, Kazunobu; Cary, Douglas R

2017-04-15

To develop a novel series of CDK8/19 dual inhibitors, we employed structure-based drug design using docking models based on a library compound, 4,5-dihydroimidazolo[3',4':3,4]benzo[1,2-d]isothiazole 16 bound to CDK8. We designed various [5,6,5]-fused tricyclic scaffolds bearing a carboxamide group to maintain predicted interactions with the backbone CO and NH of Ala100 in the CDK8 kinase hinge region. We found that 4,5-dihydrothieno[3',4':3,4]benzo[1,2-d]isothiazole derivative 29a showed particularly potent enzymatic inhibitory activity in both CDK8/19 (CDK8 IC 50 : 0.76nM, CDK19 IC 50 : 1.7nM). To improve the physicochemical properties and kinase selectivity of this compound, we introduced a substituted 3-pyridyloxy group into the scaffold 8-position. The resulting optimized compound 52h showed excellent in vitro potency (CDK8 IC 50 : 0.46nM, CDK19 IC 50 : 0.99nM), physicochemical properties, and kinase selectivity (only 5 kinases showed DMG activation loop. In vitro pharmacological evaluation of 52h revealed potent suppression of phosphorylated STAT1 in various cancer cells. The high oral bioavailability found for this compound enabled in vivo studies, in which we demonstrated a mechanism-based in vivo PD effect as well as tumor growth suppression in an RPMI8226 human hematopoietic and lymphoid xenograft model in mouse [T/C: -1% (2.5mg/kg, qd)]. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mung bean nuclease treatment increases capture specificity of microdroplet-PCR based targeted DNA enrichment.

Directory of Open Access Journals (Sweden)

Zhenming Yu

Full Text Available Targeted DNA enrichment coupled with next generation sequencing has been increasingly used for interrogation of select sub-genomic regions at high depth of coverage in a cost effective manner. Specificity measured by on-target efficiency is a key performance metric for target enrichment. Non-specific capture leads to off-target reads, resulting in waste of sequencing throughput on irrelevant regions. Microdroplet-PCR allows simultaneous amplification of up to thousands of regions in the genome and is among the most commonly used strategies for target enrichment. Here we show that carryover of single-stranded template genomic DNA from microdroplet-PCR constitutes a major contributing factor for off-target reads in the resultant libraries. Moreover, treatment of microdroplet-PCR enrichment products with a nuclease specific to single-stranded DNA alleviates off-target load and improves enrichment specificity. We propose that nuclease treatment of enrichment products should be incorporated in the workflow of targeted sequencing using microdroplet-PCR for target capture. These findings may have a broad impact on other PCR based applications for which removal of template DNA is beneficial.

A rice kinase-protein interaction map.

Science.gov (United States)

Ding, Xiaodong; Richter, Todd; Chen, Mei; Fujii, Hiroaki; Seo, Young Su; Xie, Mingtang; Zheng, Xianwu; Kanrar, Siddhartha; Stevenson, Rebecca A; Dardick, Christopher; Li, Ying; Jiang, Hao; Zhang, Yan; Yu, Fahong; Bartley, Laura E; Chern, Mawsheng; Bart, Rebecca; Chen, Xiuhua; Zhu, Lihuang; Farmerie, William G; Gribskov, Michael; Zhu, Jian-Kang; Fromm, Michael E; Ronald, Pamela C; Song, Wen-Yuan

2009-03-01

Plants uniquely contain large numbers of protein kinases, and for the vast majority of the 1,429 kinases predicted in the rice (Oryza sativa) genome, little is known of their functions. Genetic approaches often fail to produce observable phenotypes; thus, new strategies are needed to delineate kinase function. We previously developed a cost-effective high-throughput yeast two-hybrid system. Using this system, we have generated a protein interaction map of 116 representative rice kinases and 254 of their interacting proteins. Overall, the resulting interaction map supports a large number of known or predicted kinase-protein interactions from both plants and animals and reveals many new functional insights. Notably, we found a potential widespread role for E3 ubiquitin ligases in pathogen defense signaling mediated by receptor-like kinases, particularly by the kinases that may have evolved from recently expanded kinase subfamilies in rice. We anticipate that the data provided here will serve as a foundation for targeted functional studies in rice and other plants. The application of yeast two-hybrid and TAPtag analyses for large-scale plant protein interaction studies is also discussed.

Structural basis for basal activity and autoactivation of abscisic acid (ABA) signaling SnRK2 kinases

Energy Technology Data Exchange (ETDEWEB)

Ng, Ley-Moy; Soon, Fen-Fen; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Suino-Powell, Kelly M.; Chalmers, Michael J.; Li, Jun; Yong, Eu-Leong; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric (Van Andel); (Scripps); (Purdue); (NU Singapore)

2014-10-02

Abscisic acid (ABA) is an essential hormone that controls plant growth, development, and responses to abiotic stresses. Central for ABA signaling is the ABA-mediated autoactivation of three monomeric Snf1-related kinases (SnRK2.2, -2.3, and -2.6). In the absence of ABA, SnRK2s are kept in an inactive state by forming physical complexes with type 2C protein phosphatases (PP2Cs). Upon relief of this inhibition, SnRK2 kinases can autoactivate through unknown mechanisms. Here, we report the crystal structures of full-length Arabidopsis thaliana SnRK2.3 and SnRK2.6 at 1.9- and 2.3-{angstrom} resolution, respectively. The structures, in combination with biochemical studies, reveal a two-step mechanism of intramolecular kinase activation that resembles the intermolecular activation of cyclin-dependent kinases. First, release of inhibition by PP2C allows the SnRK2s to become partially active because of an intramolecular stabilization of the catalytic domain by a conserved helix in the kinase regulatory domain. This stabilization enables SnRK2s to gain full activity by activation loop autophosphorylation. Autophosphorylation is more efficient in SnRK2.6, which has higher stability than SnRK2.3 and has well-structured activation loop phosphate acceptor sites that are positioned next to the catalytic site. Together, these data provide a structural framework that links ABA-mediated release of PP2C inhibition to activation of SnRK2 kinases.

A selective inhibitor of protein kinase A induces behavioural and neurological antidepressant-like effects in rats

DEFF Research Database (Denmark)

Liebenberg, Nico; Müller, Heidi Kaastrup; Elfving, Betina

2011-01-01

Background: It is well established that cyclic adenosine monophosphate (AMP) signalling via cAMP-dependent protein kinase (PKA) within neurons plays an important role in depression and antidepressant treatment. However, the importance of several newly discovered targets that function independentl...

Epigenetic Mechanisms Regulating Adaptive Responses to Targeted Kinase Inhibitors in Cancer.

Science.gov (United States)

Angus, Steven P; Zawistowski, Jon S; Johnson, Gary L

2018-01-06

Although targeted inhibition of oncogenic kinase drivers has achieved remarkable patient responses in many cancers, the development of resistance has remained a significant challenge. Numerous mechanisms have been identified, including the acquisition of gatekeeper mutations, activating pathway mutations, and copy number loss or gain of the driver or alternate nodes. These changes have prompted the development of kinase inhibitors with increased selectivity, use of second-line therapeutics to overcome primary resistance, and combination treatment to forestall resistance. In addition to genomic resistance mechanisms, adaptive transcriptional and signaling responses seen in tumors are gaining appreciation as alterations that lead to a phenotypic state change-often observed as an epithelial-to-mesenchymal shift or reversion to a cancer stem cell-like phenotype underpinned by remodeling of the epigenetic landscape. This epigenomic modulation driving cell state change is multifaceted and includes modulation of repressive and activating histone modifications, DNA methylation, enhancer remodeling, and noncoding RNA species. Consequently, the combination of kinase inhibitors with drugs targeting components of the transcriptional machinery and histone-modifying enzymes has shown promise in preclinical and clinical studies. Here, we review mechanisms of resistance to kinase inhibition in cancer, with special emphasis on the rewired kinome and transcriptional signaling networks and the potential vulnerabilities that may be exploited to overcome these adaptive signaling changes.

Pyruvate dehydrogenase kinase inhibition: Reversing the Warburg effect in cancer therapy

Directory of Open Access Journals (Sweden)

Hayden Bell

2016-06-01

Full Text Available The poor efficacy of many cancer chemotherapeutics, which are often non-selective and highly toxic, is attributable to the remarkable heterogeneity and adaptability of cancer cells. The Warburg effect describes the up regulation of glycolysis as the main source of adenosine 5’-triphosphate in cancer cells, even under normoxic conditions, and is a unique metabolic phenotype of cancer cells. Mitochondrial suppression is also observed which may be implicated in apoptotic suppression and increased funneling of respiratory substrates to anabolic processes, conferring a survival advantage. The mitochondrial pyruvate dehydrogenase complex is subject to meticulous regulation, chiefly by pyruvate dehydrogenase kinase. At the interface between glycolysis and the tricarboxylic acid cycle, the pyruvate dehydrogenase complex functions as a metabolic gatekeeper in determining the fate of glucose, making pyruvate dehydrogenase kinase an attractive candidate in a bid to reverse the Warburg effect in cancer cells. The small pyruvate dehydrogenase kinase inhibitor dichloroacetate has, historically, been used in conditions associated with lactic acidosis but has since gained substantial interest as a potential cancer chemotherapeutic. This review considers the Warburg effect as a unique phenotype of cancer cells in-line with the history of and current approaches to cancer therapies based on pyruvate dehydrogenase kinase inhibition with particular reference to dichloroacetate and its derivatives.

Chitin and stress induced protein kinase activation

DEFF Research Database (Denmark)

Kenchappa, Chandra Shekar; Azevedo da Silva, Raquel; Bressendorff, Simon

2017-01-01

The assays described here are pertinent to protein kinase studies in any plant. They include an immunoblot phosphorylation/activation assay and an in-gel activity assay for MAP kinases (MPKs) using the general protein kinase substrate myelin basic protein. They also include a novel in-gel peptide...... substrate assay for Snf1-related kinase family 2 members (SnRK2s). This kinase family-specific assay overcomes some limitations of in-gel assays and permits the identification of different types of kinase activities in total protein extracts....

Efficiency of tritium measurement in the environmental water by electrolysis enrichment

Energy Technology Data Exchange (ETDEWEB)

Koganezawa, T.; Iida, T. [Nagoya Univ., Graduate School of Engineering, Nagoya, Aichi (Japan); Sakuma, Y.; Yamanishi, H. [National Inst. for Fusion Science, Toki, Gifu (Japan); Ogata, Y. [Nagoya Univ., School of Health Sciences, Nagoya, Aichi (Japan); Tsuji, N. [Japan Air-conditioning Service Co. and Ltd., Nagoya, Aichi (Japan); Kakiuchi, M. [Gakushuin Univ., Faculty of Science, Tokyo (Japan); Satake, H. [Toyama Univ., Faculty of Science, Toyama (Japan)

2002-06-01

Now tritium concentration in the environmental water is 0.5-2 Bq{center_dot}L{sup -1} in Japan. Tritium concentration cannot be measured accurately by liquid scintillation method, because the minimum detectable limits of liquid scintillation method is 0.5 Bq{center_dot}L{sup -1}. Therefore, one needs to enrich tritium concentration in the environmental water. Although the most popular method for tritium enrichment is electrolysis, the electrolysis takes much time and labor for distilling sample water at before and after the electrolysis. The purpose of this study is to investigate the possibility of more convenient method for tritium measurement. The method substitutes filtration for distillation at before electrolysis and omits distillation at after electrolysis. The method enables by using the electrolysis with solid polymer electrode. We performed two kinds of experiment to confirm the possibility of the method. First, impurities eluted from electrolysis installation with ultra pure water as sample was measured. Some impurities were eluted into the sample, but they brought noneffective quenching. Secondly, we applied new method to the environmental waters. Substituting for distillation, two filtration, 0.1 {mu}m filtration and reverse osmosis method, were investigated. Impurities in the samples by the filtrations were somewhat higher than that by the distillation, they brought noneffective quenching. We, however, observed distemper of the electrolysis happened by electrolysing filtered sample. Distillation is substituted filtration at before enrichment and omitted at after enrichment, leaving the influence of quenching out of consideration. (author)

Efficiency of tritium measurement in the environmental water by electrolysis enrichment

International Nuclear Information System (INIS)

Koganezawa, T.; Iida, T.; Sakuma, Y.; Yamanishi, H.; Ogata, Y.; Tsuji, N.; Kakiuchi, M.; Satake, H.

2002-01-01

Now tritium concentration in the environmental water is 0.5-2 Bq·L -1 in Japan. Tritium concentration cannot be measured accurately by liquid scintillation method, because the minimum detectable limits of liquid scintillation method is 0.5 Bq·L -1 . Therefore, one needs to enrich tritium concentration in the environmental water. Although the most popular method for tritium enrichment is electrolysis, the electrolysis takes much time and labor for distilling sample water at before and after the electrolysis. The purpose of this study is to investigate the possibility of more convenient method for tritium measurement. The method substitutes filtration for distillation at before electrolysis and omits distillation at after electrolysis. The method enables by using the electrolysis with solid polymer electrode. We performed two kinds of experiment to confirm the possibility of the method. First, impurities eluted from electrolysis installation with ultra pure water as sample was measured. Some impurities were eluted into the sample, but they brought noneffective quenching. Secondly, we applied new method to the environmental waters. Substituting for distillation, two filtration, 0.1 μm filtration and reverse osmosis method, were investigated. Impurities in the samples by the filtrations were somewhat higher than that by the distillation, they brought noneffective quenching. We, however, observed distemper of the electrolysis happened by electrolysing filtered sample. Distillation is substituted filtration at before enrichment and omitted at after enrichment, leaving the influence of quenching out of consideration. (author)

TRIGA low enrichment fuel

International Nuclear Information System (INIS)

Gietzen, A.

1993-01-01

Sixty TRIGA reactors have been sold and the earliest of these are now passing twenty years of operation. All of these reactors use the uranium zirconium hydride fuel (UZrH) which provides certain unique advantages arising out of its large prompt negative temperature coefficient, very low fission product release, and high temperature capability. Eleven of these Sixty reactors are conversions from plate fuel to TRIGA fuel which were made as a result of these advantages. With only a few exceptions, TRIGA reactors have always used low-enriched uranium (LEU) fuel with an enrichment of 19.9%. The exceptions have either been converted from the standard low-enriched fuel to the 70% enriched FLIP fuel in order to achieve extended lifetime, or are higher powered reactors which were designed for long life using 93%-enriched uranium during the time when the use and export of highly enriched uranium (HEU) was not restricted. The advent of international policies focusing attention on nonproliferation and safeguards made the HEU fuels obsolete. General Atomic immediately undertook a development effort (nearly two years ago) in order to be in a position to comply with these policies for all future export sales and also to provide a low-enriched alternative to fully enriched plate-type fuels. This important work was subsequently partially supported by the U.S. Department of Energy. The laboratory and production tests have shown that higher uranium densities can be achieved to compensate for reducing the enrichment to 20%, and that the fuels maintain the characteristics of the very thoroughly proven standard TRIGA fuels. In May of 1978, General Atomic announced that these fuels were available for TRIGA reactors and for plate-type reactors with power levels up to 15 MW with General Atomic's standard commercial warranty

TRIGA low enrichment fuel

International Nuclear Information System (INIS)

Gietzen, A.

1993-01-01

Sixty TRIGA reactors have been sold and the earliest of these are now passing twenty years of operation. All of these reactors use the uranium-zirconium hydride fuel (UZrH) which provides certain unique advantages arising out of its large prompt negative temperature coefficient, very low fission product release, and high temperature capability. Eleven of these Sixty reactors are conversions from plate fuel to TRIGA fuel which were made as a result of these advantages. With only a few exceptions, TRIGA reactors have always used low-enriched-uranium (LEU) fuel with an enrichment of 19.9%. The exceptions have either been converted from the standard low-enriched fuel to the 70% enriched FLIP fuel in order to achieve extended lifetime, or are higher powered reactors which were designed for long life using 93%-enriched uranium during the time when the use and export of highly enriched uranium (HEU) was not restricted. The advent of international policies focusing attention on nonproliferation and safeguards made the HEU fuels obsolete. General Atomic immediately undertook a development effort (nearly two years ago) in order to be in a position to comply with these policies for all future export sales and also to provide a low-enriched alternative to fully enriched plate-type fuels. This important work was subsequently partially supported by the U.S. Department of Energy. The laboratory and production tests have shown that higher uranium densities can be achieved to compensate for reducing the enrichment to 20%, and that the fuels maintain the characteristics of the very thoroughly proven standard TRIGA fuels. In May of 1978, General Atomic announced that these fuels were available for TRIGA reactors and for plate-type reactors with power levels up to 15 MW with GA's standard commercial warranty

Engineering of kinase-based protein interacting devices: active expression of tyrosine kinase domains

KAUST Repository

Diaz Galicia, Miriam Escarlet

2018-01-01

is then translated into a FRET (Fluorescence Resonance Energy Transfer) signal is here proposed. To this end, DNA constructs for interaction amplification (split kinases), positive controls (intact kinase domains), scaffolding proteins and phosphopeptide - SH2-domain

Receptor Tyrosine Kinases in Drosophila Development

Science.gov (United States)

Sopko, Richelle; Perrimon, Norbert

2013-01-01

Tyrosine phosphorylation plays a significant role in a wide range of cellular processes. The Drosophila genome encodes more than 20 receptor tyrosine kinases and extensive studies in the past 20 years have illustrated their diverse roles and complex signaling mechanisms. Although some receptor tyrosine kinases have highly specific functions, others strikingly are used in rather ubiquitous manners. Receptor tyrosine kinases regulate a broad expanse of processes, ranging from cell survival and proliferation to differentiation and patterning. Remarkably, different receptor tyrosine kinases share many of the same effectors and their hierarchical organization is retained in disparate biological contexts. In this comprehensive review, we summarize what is known regarding each receptor tyrosine kinase during Drosophila development. Astonishingly, very little is known for approximately half of all Drosophila receptor tyrosine kinases. PMID:23732470

Turkey's regulatory plans for high enriched to low enriched conversion of TR-2 reactor core

International Nuclear Information System (INIS)

Guelol Oezdere, Oya

2003-01-01

Turkey is a developing country and has three nuclear facilities two of which are research reactors and one pilot fuel production plant. One of the two research reactors is TR-2 which is located in Cekmece site in Istanbul. TR-2 Reactor's core is composed of both high enriched and low enriched fuel and from high enriched to low enriched core conversion project will take place in year 2005. This paper presents the plans for drafting regulations on the safety analysis report updates for high enriched to low enriched core conversion of TR-2 reactor, the present regulatory structure of Turkey and licensing activities of nuclear facilities. (author)

Virtual screening filters for the design of type II p38 MAP kinase inhibitors: a fragment based library generation approach.

Science.gov (United States)

Badrinarayan, Preethi; Sastry, G Narahari

2012-04-01

In this work, we introduce the development and application of a three-step scoring and filtering procedure for the design of type II p38 MAP kinase leads using allosteric fragments extracted from virtual screening hits. The design of the virtual screening filters is based on a thorough evaluation of docking methods, DFG-loop conformation, binding interactions and chemotype specificity of the 138 p38 MAP kinase inhibitors from Protein Data Bank bound to DFG-in and DFG-out conformations using Glide, GOLD and CDOCKER. A 40 ns molecular dynamics simulation with the apo, type I with DFG-in and type II with DFG-out forms was carried out to delineate the effects of structural variations on inhibitor binding. The designed docking-score and sub-structure filters were first tested on a dataset of 249 potent p38 MAP kinase inhibitors from seven diverse series and 18,842 kinase inhibitors from PDB, to gauge their capacity to discriminate between kinase and non-kinase inhibitors and likewise to selectively filter-in target-specific inhibitors. The designed filters were then applied in the virtual screening of a database of ten million (10⁷) compounds resulting in the identification of 100 hits. Based on their binding modes, 98 allosteric fragments were extracted from the hits and a fragment library was generated. New type II p38 MAP kinase leads were designed by tailoring the existing type I ATP site binders with allosteric fragments using a common urea linker. Target specific virtual screening filters can thus be easily developed for other kinases based on this strategy to retrieve target selective compounds. Copyright © 2012 Elsevier Inc. All rights reserved.

Isotopic enrichments via altered first and second solution electron affinities

International Nuclear Information System (INIS)

Stevenson, G.R.; Espe, M.P.; Reiter, R.C.

1986-01-01

Electron spin resonance experiments have been utilized to show that the solution electron affinity of benzene- 13 C 6 is less than that of benzene by 0.24 kcal/mol and that the solution EA of benzene-d 6 is less than that of benzene by 0.44 kcal/mol. Perdeuteration of naphthalene, anthracene, or perylene results in a very similar lowering of the solution EA of the hydrocarbon as evidenced by the fact that the equilibrium constant for the electron transfer between the hydrocarbon anion radical, X/sup .-/, and the perdeuterated hydrocarbon, Xd (X/sup .-/ + Xd = Xd/sup .-/ + X), is less than unity. Likewise the second EAs of perdeuterated perylene and anthracene are lower than those of the unsubstituted hydrocarbons (K/sub eq/ for X 2- + Xd/sup .-/ = X/sup .-/ + Xd 2- is less than unity). The free energy and enthalpy of electron transfer from the anthracene anion radical to perdeuterated anthracene is 0.41 kcal/mol and that from the anthracene dianion to the perdeuterated anion radical is 0.10 kcal/mol. The fact that these equilibrium constants are not equal to 1 enables one to use the difference in the chemical reactivity of the ions and neutral molecules to selectively isotopically enrich the hydrocarbons involved

High enrichment to low enrichment core's conversion. Technical securities

International Nuclear Information System (INIS)

Abbate, P.; Madariaga, M.R.

1990-01-01

This work presents the fulfillment of the technical securities subscribed by INVAP S.E. for the conversion of a high enriched uranium core. The reactor (of 5 thermal Mw), built in the 50's and 60's, is of the 'swimming pool' type, with light water and fuel elements of the curve plates MTR type, enriched at 93.15 %. These are neutronic and thermohydraulic securities. (Author) [es

Beta activity of enriched uranium

International Nuclear Information System (INIS)

Nambiar, P.P.V.J.; Ramachandran, V.

1975-01-01

Use of enriched uranium as reactor fuel necessitates its handling in various forms. For purposes of planning and organising radiation protection measures in enriched uranium handling facilities, it is necessary to have a basic knowledge of the radiation status of enriched uranium systems. The theoretical variations in beta activity and energy with U 235 enrichment are presented. Depletion is considered separately. Beta activity build up is also studied for two specific enrichments, in respect of which experimental values for specific alpha activity are available. (author)

A cGMP kinase mutant with increased sensitivity to the protein kinase inhibitor peptide PKI(5-24).

Science.gov (United States)

Ruth, P; Kamm, S; Nau, U; Pfeifer, A; Hofmann, F

1996-01-01

Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein PKI are very potent inhibitors of cAMP-dependent protein kinase, but are extremely weak inhibitors of cGMP-dependent protein kinase. In this study, we tried to confer PKI sensitivity to cGMP kinase by site-directed mutagenesis. The molecular requirements for high affinity inhibition by PKI were deduced from the crystal structure of the cAMP kinase/PKI complex. A prominent site of interaction are residues Tyr235 and Phe239 in the catalytic subunit, which from a sandwich-like structure with Phe10 of the PKI(5-24) peptide. To increase the sensitivity for PKI, the cGMP kinase codons at the corresponding sites, Ser555 and Ser559, were changed to Tyr and Phe. The mutant cGMP kinase was stimulated half maximally by cGMP at 3-fold higher concentrations (240 nM) than the wild type (77 nM). Wild type and mutant cGMP kinase did not differ significantly in their Km and Vmax for three different substrate peptides. The PKI(5-24) peptide inhibited phosphotransferase activity of the mutant cGMP kinase with higher potency than that of wild type, with Ki values of 42 +/- .3 microM and 160 +/- .7 microM, respectively. The increased affinity of the mutant cGMP kinase was specific for the PKI(5-24) peptide. Mutation of the essential Phe10 in the PKI(5-24) sequence to an Ala yielded a peptide that inhibited mutant and wild type cGMP kinase with similar potency, with Ki values of 160 +/- 11 and 169 +/- 27 microM, respectively. These results suggest that the mutations Ser555Tyr and Ser559Phe are required, but not sufficient, for high affinity inhibition of cGMP kinase by PKI.

ROS and CDPK-like kinase-mediated activation of MAP kinase in rice roots exposed to lead.

Science.gov (United States)

Huang, Tsai-Lien; Huang, Hao-Jen

2008-04-01

Lead (Pb2+) is a cytotoxic metal ion in plants, the mechanism of which is not yet established. The aim of this study is to investigate the signalling pathways that are activated by elevated concentrations of Pb2+ in rice roots. Root growth was stunted and cell death was accelerated when exposed to different dosages of Pb2+ during extended time periods. Using ROS-sensitive dye and Ca2+ indicator, we demonstrated that Pb2+ induced ROS production and Ca2+ accumulation, respectively. In addition, Pb2+ elicited a remarkable increase in myelin basic protein (MBP) kinase activities. By immunoblot and immunoprecipitation analysis, 40- and 42-kDa MBP kinases that were activated by Pb2+ were identified to be mitogen-activated protein (MAP) kinases. Pre-treatment of rice roots with an antioxidant and a NADPH oxidase inhibitor, glutathione (GSH) and diphenylene iodonium (DPI), effectively reduced Pb2+-induced cell death and MAP kinase activation. Moreover, calcium-dependent protein kinase (CDPK) antagonist, W7, attenuated Pb2+-induced cell death and MAP kinase activation. These results suggested that the ROS and CDPK may function in the Pb2+-triggered cell death and MAP kinase signalling pathway in rice roots.

Laser and gas centrifuge enrichment

Energy Technology Data Exchange (ETDEWEB)

Heinonen, Olli [Senior Fellow, Belfer Center for Science and International Affairs, Harvard Kennedy School, Cambridge, Massachusetts (United States)

2014-05-09

Principles of uranium isotope enrichment using various laser and gas centrifuge techniques are briefly discussed. Examples on production of high enriched uranium are given. Concerns regarding the possibility of using low end technologies to produce weapons grade uranium are explained. Based on current assessments commercial enrichment services are able to cover the global needs of enriched uranium in the foreseeable future.

Enrichment technology. Dependable vendor of gas centrifuges; Enrichment Technology. Zuverlaessiger Lieferant von Gaszentrifugen

Energy Technology Data Exchange (ETDEWEB)

Anon.

2011-10-15

Enrichment Technology is an innovative, high-tech company that develops, manufactures and installs gas centrifuges for enriching uranium. In addition, Enrichment Technology designs enrichment plants that use gas centrifuge technology. This technology offers the most efficient and cost-effective method for enriching uranium yet: high-performance, safe technology that dominates the market with a global share of 45 percent. A determining factor in Enrichment Technology's success is its mission: supplying its customers with safe, reliable technology. Production of the centrifuges requires versatile know-how and collaboration between different departments as well as interdisciplinary teams at the various sites. More than 2000 operators at 8 sites in 5 countries contribute their individual knowledge and personal skills in order to produce this exceptional technology. The head office is in Beaconsfield near London and the operational headquarters are in Almelo in the Netherlands. There are other sites in Germany (Juelich und Gronau), Great Britain (Capenhurst) as well as project sites in the USA and France. Capenhurst is where experienced engineers design new enrichment plants and organise their construction. Centrifuge components are manufactured in Almelo and Juelich, while the pipework needed to connect up the centrifuges is produced at the site in Gronau. In Juelich, highly qualified scientists in interdisciplinary teams are continuously researching ways of improving the current centrifuges. Communication between specialists in the fields of chemistry, physics and engineering forms the basis for the company's success and the key to extending this leading position in the global enrichment market. (orig.)