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Sample records for encoding ump kinase

  1. Regulatory mechanisms differ in UMP kinases from gram-negative and gram-positive bacteria.

    PubMed

    Evrin, Cécile; Straut, Monica; Slavova-Azmanova, Neli; Bucurenci, Nadia; Onu, Adrian; Assairi, Liliane; Ionescu, Mihaela; Palibroda, Nicolae; Bârzu, Octavian; Gilles, Anne-Marie

    2007-03-01

    In this work, we examined the regulation by GTP and UTP of the UMP kinases from eight bacterial species. The enzyme from Gram-positive organisms exhibited cooperative kinetics with ATP as substrate. GTP decreased this cooperativity and increased the affinity for ATP. UTP had the opposite effect, as it decreased the enzyme affinity for ATP. The nucleotide analogs 5-bromo-UTP and 5-iodo-UTP were 5-10 times stronger inhibitors than the parent compound. On the other hand, UMP kinases from the Gram-negative organisms did not show cooperativity in substrate binding and catalysis. Activation by GTP resulted mainly from the reversal of inhibition caused by excess UMP, and inhibition by UTP was accompanied by a strong increase in the apparent K(m) for UMP. Altogether, these results indicate that, depending on the bacteria considered, GTP and UTP interact with different enzyme recognition sites. In Gram-positive bacteria, GTP and UTP bind to a single site or largely overlapping sites, shifting the T R equilibrium to either the R or T form, a scenario corresponding to almost all regulatory proteins, commonly called K systems. In Gram-negative organisms, the GTP-binding site corresponds to the unique allosteric site of the Gram-positive bacteria. In contrast, UTP interacts cooperatively with a site that overlaps the catalytic center, i.e. the UMP-binding site and part of the ATP-binding site. These characteristics make UTP an original regulator of UMP kinases from Gram-negative organisms, beyond the common scheme of allosteric control.

  2. Site-specific mutations of conserved residues in the phosphate-binding loop of the Arabidopsis UMP/CMP kinase alter ATP and UMP binding.

    PubMed

    Zhou, L; Thornburg, R

    1998-10-15

    All eukaryotic UMP/CMP kinases contain a glycine-rich sequence GGPG(S/A)GK at the N-terminus. This sequence is homologous to the conserved sequence GXXGXGK found in other ATP-binding proteins. To study the role of this conserved sequence in Arabidopsis UMP/CMP kinase, five conserved residues were mutated by site-directed mutagenesis to generate seven mutant enzymes: G21A, G22A, G24A, G26A, K27R, K27M, and K27E. The G21A and G26A mutants were degraded during the purification phase and were thus unable to be purified. Kinetic studies on the other mutants, when compared to studies on the wild-type enzyme, revealed that this sequence is important for ATP binding and enzyme catalysis. All mutants had a decreased kcat/KATPm value. The G22A and G24A mutants had about half of the kcat value of wildtype and 3.9-fold and 3.3-fold increases in KATPm values, respectively. The kcat/KATPm values in the K27M and K27E mutants were changed significantly and decreased by 1000-fold and 2600-fold, respectively. The removal of the terminal positive charge of Lys27 in the K27M and K27E mutants resulted in 20% of the kcat value of wildtype. However, both mutants had a remarkable increase in KATPm value by 241-fold and 552-fold, respectively. Therefore, the positive charge of Lys27 plays an important role on both ATP binding and enzyme catalysis. Interestingly, the results also showed that the mutations that affected ATP binding also had an effect on UMP binding. PMID:9784243

  3. Prognostic significance of nuclear expression of UMP-CMP kinase in triple negative breast cancer patients.

    PubMed

    Liu, Ning Qing; De Marchi, Tommaso; Timmermans, Annemieke; Trapman-Jansen, Anita M A C; Foekens, Renée; Look, Maxime P; Smid, Marcel; van Deurzen, Carolien H M; Span, Paul N; Sweep, Fred C G J; Brask, Julie Benedicte; Timmermans-Wielenga, Vera; Foekens, John A; Martens, John W M; Umar, Arzu

    2016-01-01

    We have previously identified UMP-CMP kinase (CMPK1) as a prognostic marker for triple negative breast cancer (TNBC) by mass spectrometry (MS). In this study we evaluated CMPK1 association to prognosis in an independent set of samples by immunohistochemistry (IHC) and assessed biological pathways associated to its expression through gene set enrichment analysis (GSEA). A total of 461 TNBC paraffin-embedded tissues were collected from different academic hospitals in Europe, incorporated into tissue micro-arrays (TMA), and stained for CMPK1 expression. We also collected gene expression data of 60 samples, which were also present in the TMA, for GSEA correlation analysis. CMPK1 IHC staining showed both cytoplasmic and nuclear components. While cytoplasmic CMPK1 did not show any association to metastasis free survival (MFS), nuclear CMPK1 was associated to poor prognosis independently from other prognostic factors in stratified Cox regression analyses. GSEA correlation analysis of the nuclear CMPK1-stratified gene expression dataset showed a significant enrichment of extracellular matrix (ECM; positive correlation) and cell cycle (negative correlation) associated genes. We have shown here that nuclear CMPK1 is indicative of poor prognosis in TNBCs and that its expression may be related to dysregulation of ECM and cell cycle molecules. PMID:27558661

  4. Prognostic significance of nuclear expression of UMP-CMP kinase in triple negative breast cancer patients

    PubMed Central

    Liu, Ning Qing; De Marchi, Tommaso; Timmermans, Annemieke; Trapman-Jansen, Anita M. A. C.; Foekens, Renée; Look, Maxime P.; Smid, Marcel; van Deurzen, Carolien H. M.; Span, Paul N.; Sweep, Fred C. G. J.; Brask, Julie Benedicte; Timmermans-Wielenga, Vera; Foekens, John A.; Martens, John W. M.; Umar, Arzu

    2016-01-01

    We have previously identified UMP-CMP kinase (CMPK1) as a prognostic marker for triple negative breast cancer (TNBC) by mass spectrometry (MS). In this study we evaluated CMPK1 association to prognosis in an independent set of samples by immunohistochemistry (IHC) and assessed biological pathways associated to its expression through gene set enrichment analysis (GSEA). A total of 461 TNBC paraffin-embedded tissues were collected from different academic hospitals in Europe, incorporated into tissue micro-arrays (TMA), and stained for CMPK1 expression. We also collected gene expression data of 60 samples, which were also present in the TMA, for GSEA correlation analysis. CMPK1 IHC staining showed both cytoplasmic and nuclear components. While cytoplasmic CMPK1 did not show any association to metastasis free survival (MFS), nuclear CMPK1 was associated to poor prognosis independently from other prognostic factors in stratified Cox regression analyses. GSEA correlation analysis of the nuclear CMPK1-stratified gene expression dataset showed a significant enrichment of extracellular matrix (ECM; positive correlation) and cell cycle (negative correlation) associated genes. We have shown here that nuclear CMPK1 is indicative of poor prognosis in TNBCs and that its expression may be related to dysregulation of ECM and cell cycle molecules. PMID:27558661

  5. The crystallization of apo-form UMP kinase from Xanthomonas campestris is significantly improved in a strong magnetic field

    SciTech Connect

    Tu, Jhe-Le; Chin, Ko-Hsin; Wang, Andrew H.-J.; Chou, Shan-Ho

    2007-05-01

    A bacterial UMP kinase from the plant pathogen X. campestris pathovar campestris has been overexpressed in E. coli, purified and crystallized in a strong magnetic field. The crystals diffracted to 2.35 Å. Bacterial UMP kinases (UMPKs) are crucial enzymes that are responsible for microbial UTP biosynthesis. Interestingly, eukaryotic and prokaryotic cells use different enzymes for UMP-phosphorylation reactions. Prokaryotic UMPKs are thus believed to be potential targets for antimicrobial drug development. Here, the cloning, expression and crystallization of SeMet-substituted XC1936, a bacterial UMPK from Xanthomonas campestris pathovar campestris, are reported. The crystallization of the apo-form UMPK was found to be significantly improved in a strong magnetic field; the crystals diffracted to a resolution of 2.35 Å, a dramatic improvement over the original value of 3.6 Å. Preliminary structural analyses of apo-form XC1936 using crystals grown in a strong magnetic field clearly reveal well defined loop regions involved in substrate-analogue binding that were previously not visible. Crystallization in a strong magnetic field thus was found to be indispensable in determining the flexible region of the XC1936 UMPK structure.

  6. Screening-based discovery of the first novel ATP competitive inhibitors of the Staphylococcus aureus essential enzyme UMP kinase.

    PubMed

    Doig, Peter; Gorseth, Elise; Nash, Tory; Patten, Arthur; Gao, Ning; Blackett, Carolyn

    2013-07-19

    UMP kinase (PyrH) is an essential enzyme found only in bacteria, making it ideal as a target for the discovery of antibacterials. To identify inhibitors of PyrH, an assay employing Staphylococcus aureus PyrH coupled to pyruvate kinase/lactate dehydrogenase was developed and was used to perform a high throughput screen. A validated aminopyrimidine series was identified from screening. Kinetic characterization of this aminopyrimidine indicated it was a competitive inhibitor of ATP. We have shown that HTS can be used to identify potential leads for this novel target, the first ATP competitive inhibitor of PyrH reported.

  7. The novel fluorescent CDP-analogue (Pbeta)MABA-CDP is a specific probe for the NMP binding site of UMP/CMP kinase.

    PubMed Central

    Rudolph, M. G.; Veit, T. J.; Reinstein, J.

    1999-01-01

    Direct thermodynamic and kinetic investigations of the binding of nucleotides to the nucleoside monophosphate (NMP) site of NMP kinases have not been possible so far because a spectroscopic probe was not available. By coupling a fluorescent N-methylanthraniloyl- (mant) group to the beta-phosphate of CDP via a butyl linker, a CDP analogue [(Pbeta)MABA-CDP] was obtained that still binds specifically to the NMP site of UmpKdicty, because the base and the ribose moieties, which are involved in specific interactions, are not modified. This allows the direct determination of binding constants for its substrates in competition experiments. PMID:10631985

  8. Bacillus halodurans Strain C125 Encodes and Synthesizes Enzymes from Both Known Pathways To Form dUMP Directly from Cytosine Deoxyribonucleotides.

    PubMed

    Oehlenschlæger, Christian Berg; Løvgreen, Monika Nøhr; Reinauer, Eva; Lehtinen, Emilia; Pind, Marie-Louise Lindberg; Harris, Pernille; Martinussen, Jan; Willemoës, Martin

    2015-05-15

    Analysis of the genome of Bacillus halodurans strain C125 indicated that two pathways leading from a cytosine deoxyribonucleotide to dUMP, used for dTMP synthesis, were encoded by the genome of the bacterium. The genes that were responsible, the comEB gene and the dcdB gene, encoding dCMP deaminase and the bifunctional dCTP deaminase:dUTPase (DCD:DUT), respectively, were both shown to be expressed in B. halodurans, and both genes were subject to repression by the nucleosides thymidine and deoxycytidine. The latter nucleoside presumably exerts its repression after deamination by cytidine deaminase. Both comEB and dcdB were cloned, overexpressed in Escherichia coli, and purified to homogeneity. Both enzymes were active and displayed the expected regulatory properties: activation by dCTP for dCMP deaminase and dTTP inhibition for both enzymes. Structurally, the B. halodurans enzyme resembled the Mycobacterium tuberculosis enzyme the most. An investigation of sequenced genomes from other species of the genus Bacillus revealed that not only the genome of B. halodurans but also the genomes of Bacillus pseudofirmus, Bacillus thuringiensis, Bacillus hemicellulosilyticus, Bacillus marmarensis, Bacillus cereus, and Bacillus megaterium encode both the dCMP deaminase and the DCD:DUT enzymes. In addition, eight dcdB homologs from Bacillus species within the genus for which the whole genome has not yet been sequenced were registered in the NCBI Entrez database.

  9. Bacillus halodurans Strain C125 Encodes and Synthesizes Enzymes from Both Known Pathways To Form dUMP Directly from Cytosine Deoxyribonucleotides

    PubMed Central

    Oehlenschlæger, Christian Berg; Løvgreen, Monika Nøhr; Reinauer, Eva; Lehtinen, Emilia; Pind, Marie-Louise Lindberg; Martinussen, Jan

    2015-01-01

    Analysis of the genome of Bacillus halodurans strain C125 indicated that two pathways leading from a cytosine deoxyribonucleotide to dUMP, used for dTMP synthesis, were encoded by the genome of the bacterium. The genes that were responsible, the comEB gene and the dcdB gene, encoding dCMP deaminase and the bifunctional dCTP deaminase:dUTPase (DCD:DUT), respectively, were both shown to be expressed in B. halodurans, and both genes were subject to repression by the nucleosides thymidine and deoxycytidine. The latter nucleoside presumably exerts its repression after deamination by cytidine deaminase. Both comEB and dcdB were cloned, overexpressed in Escherichia coli, and purified to homogeneity. Both enzymes were active and displayed the expected regulatory properties: activation by dCTP for dCMP deaminase and dTTP inhibition for both enzymes. Structurally, the B. halodurans enzyme resembled the Mycobacterium tuberculosis enzyme the most. An investigation of sequenced genomes from other species of the genus Bacillus revealed that not only the genome of B. halodurans but also the genomes of Bacillus pseudofirmus, Bacillus thuringiensis, Bacillus hemicellulosilyticus, Bacillus marmarensis, Bacillus cereus, and Bacillus megaterium encode both the dCMP deaminase and the DCD:DUT enzymes. In addition, eight dcdB homologs from Bacillus species within the genus for which the whole genome has not yet been sequenced were registered in the NCBI Entrez database. PMID:25746996

  10. The Leishmania donovani UMP Synthase Is Essential for Promastigote Viability and Has an Unusual Tetrameric Structure That Exhibits Substrate-controlled Oligomerization

    SciTech Connect

    French, Jarrod B.; Yates, Phillip A.; Soysa, D.Radika; Boitz, Jan M.; Carter, Nicola S.; Chang, Bailey; Ullman, Buddy; Ealick, Steven E.

    2011-08-09

    The final two steps of de novo uridine 5'-monophosphate (UMP) biosynthesis are catalyzed by orotate phosphoribosyltransferase (OPRT) and orotidine 5'-monophosphate decarboxylase (OMPDC). In most prokaryotes and simple eukaryotes these two enzymes are encoded by separate genes, whereas in mammals they are expressed as a bifunctional gene product called UMP synthase (UMPS), with OPRT at the N terminus and OMPDC at the C terminus. Leishmania and some closely related organisms also express a bifunctional enzyme for these two steps, but the domain order is reversed relative to mammalian UMPS. In this work we demonstrate that L. donovani UMPS (LdUMPS) is an essential enzyme in promastigotes and that it is sequestered in the parasite glycosome. We also present the crystal structure of the LdUMPS in complex with its product, UMP. This structure reveals an unusual tetramer with two head to head and two tail to tail interactions, resulting in two dimeric OMPDC and two dimeric OPRT functional domains. In addition, we provide structural and biochemical evidence that oligomerization of LdUMPS is controlled by product binding at the OPRT active site. We propose a model for the assembly of the catalytically relevant LdUMPS tetramer and discuss the implications for the structure of mammalian UMPS.

  11. Message from Vice Chancellor, UMP

    NASA Astrophysics Data System (ADS)

    Nasir Ibrahim, Daing

    2012-09-01

    Assalamualaikumwarahmatullahiwabarakatuh and Salam i Malaysia First and foremost, I want to thank the International Conference Mechanical Engineering Research (ICMER) organisers for inviting me to address and officiate at this conference. It is a privilege and an honour for me on this momentous occasion to grace the ceremony. The ICMER provides a platform to bring together not only researchers but also postgraduate students in Mechanical Engineering, Automotive Engineering, Manufacturing Engineering, Biomechanical Engineering, Material Engineering and Industrial Engineering. With this platform, ICMER will embark on a whole process of making new discoveries and then translating them into products and services for the marketplace; this is only made possible by people like all of you. It might be only a starting point but with hard work and perseverance I am sure you will succeed with flying colours. As one of Malaysia's Public Universities, UMP's main challenge is to remain competitive and relevant by offering high quality technical academic programmes and research activities, focusing on its niche areas. New knowledge and findings cannot be generated without research and development (R&D) therefore, Malaysia has had substantial investment in research and development facilities. These efforts will undoubtedly generate lots of interesting results and new knowledge as either further investigation or commercial activities. Therefore, researchers like you must see this as the generator of new knowledge to extend your research outcomes from laboratory experiments to the marketplace and towards commercialisation. Naybe this doesn't appear significant in the short term but it may make a tremendous impact in the future. The Malaysian government has invested a huge sum of Ringgits in R&D over the years. Therefore, public universities such as UMP must produce more quality researchers and graduates to ensure Malaysia reaps the returns from these investments and consequently

  12. Message from Vice Chancellor, UMP

    NASA Astrophysics Data System (ADS)

    Nasir Ibrahim, Daing

    2012-09-01

    Assalamualaikumwarahmatullahiwabarakatuh and Salam i Malaysia First and foremost, I want to thank the International Conference Mechanical Engineering Research (ICMER) organisers for inviting me to address and officiate at this conference. It is a privilege and an honour for me on this momentous occasion to grace the ceremony. The ICMER provides a platform to bring together not only researchers but also postgraduate students in Mechanical Engineering, Automotive Engineering, Manufacturing Engineering, Biomechanical Engineering, Material Engineering and Industrial Engineering. With this platform, ICMER will embark on a whole process of making new discoveries and then translating them into products and services for the marketplace; this is only made possible by people like all of you. It might be only a starting point but with hard work and perseverance I am sure you will succeed with flying colours. As one of Malaysia's Public Universities, UMP's main challenge is to remain competitive and relevant by offering high quality technical academic programmes and research activities, focusing on its niche areas. New knowledge and findings cannot be generated without research and development (R&D) therefore, Malaysia has had substantial investment in research and development facilities. These efforts will undoubtedly generate lots of interesting results and new knowledge as either further investigation or commercial activities. Therefore, researchers like you must see this as the generator of new knowledge to extend your research outcomes from laboratory experiments to the marketplace and towards commercialisation. Naybe this doesn't appear significant in the short term but it may make a tremendous impact in the future. The Malaysian government has invested a huge sum of Ringgits in R&D over the years. Therefore, public universities such as UMP must produce more quality researchers and graduates to ensure Malaysia reaps the returns from these investments and consequently

  13. Nucleic acid molecules encoding isopentenyl monophosphate kinase, and methods of use

    DOEpatents

    Croteau, Rodney B.; Lange, Bernd M.

    2001-01-01

    A cDNA encoding isopentenyl monophosphate kinase (IPK) from peppermint (Mentha x piperita) has been isolated and sequenced, and the corresponding amino acid sequence has been determined. Accordingly, an isolated DNA sequence (SEQ ID NO:1) is provided which codes for the expression of isopentenyl monophosphate kinase (SEQ ID NO:2), from peppermint (Mentha x piperita). In other aspects, replicable recombinant cloning vehicles are provided which code for isopentenyl monophosphate kinase, or for a base sequence sufficiently complementary to at least a portion of isopentenyl monophosphate kinase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding isopentenyl monophosphate kinase. Thus, systems and methods are provided for the recombinant expression of the aforementioned recombinant isopentenyl monophosphate kinase that may be used to facilitate its production, isolation and purification in significant amounts. Recombinant isopentenyl monophosphate kinase may be used to obtain expression or enhanced expression of isopentenyl monophosphate kinase in plants in order to enhance the production of isopentenyl monophosphate kinase, or isoprenoids derived therefrom, or may be otherwise employed for the regulation or expression of isopentenyl monophosphate kinase, or the production of its products.

  14. Conversion of UMP, an allosteric inhibitor of carbamyl phosphate synthetase, to an activator by modification of the UMP ribose moiety.

    PubMed

    Boettcher, B; Meister, A

    1981-06-25

    UMP is known to be an allosteric inhibitor of carbamyl phosphate synthetase, whereas IMP activates the enzyme. Surprisingly, dialdehyde UMP (prepared by periodate oxidation of UMP) was found to be a potent activator of the enzyme. Dialdehyde IMP, like IMP, produced activation. The corresponding dialcohol analogs of UMP and IMP (prepared by borohydride reduction of the dialdehyde analogs) had no effect on activity. These nucleotide interactions were further characterized by sedimentation velocity studies and by examination of the effects of inorganic phosphate on enzymatic activity. Although UMP promotes formation of an enzyme dimer, and IMP promotes formation of a tetramer (Powers, S. G., Meister, A., and Haschemeyer, R. H. (1980) J. Biol. Chem. 255, 1554-1558), the dialdehyde analogs of UMP and IMP both promote formation of mixed species. Low levels (less than 10 mM) of inorganic phosphate decrease the extent of activation by IMP, dialdehyde IMP, and dialdehyde UMP, but increase the extent of inhibition by UMP. The marked activation observed with dialdehyde UMP, and other considerations, suggest that the binding sites on the enzyme for IMP and UMP may overlap substantially. The findings also suggest that physiological levels of inorganic phosphate function in the modulation of the allosteric regulation of this enzyme by nucleotides. PMID:7240186

  15. Casein kinase I-like protein kinases encoded by YCK1 and YCK2 are required for yeast morphogenesis.

    PubMed Central

    Robinson, L C; Menold, M M; Garrett, S; Culbertson, M R

    1993-01-01

    Casein kinase I is an acidotropic protein kinase class that is widely distributed among eukaryotic cell types. In the yeast Saccharomyces cerevisiae, the casein kinase I isoform encoded by the gene pair YCK1 and YCK2 is a 60- to 62-kDa membrane-associated form. The Yck proteins perform functions essential for growth and division; either alone supports growth, but loss of function of both is lethal. We report here that casein kinase I-like activity is associated with a soluble Yck2-beta-galactosidase fusion protein in vitro and that thermolabile protein kinase activity is exhibited by a protein encoded by fusion of a temperature-sensitive yck2 allele with lacZ. Cells carrying the yck2-2ts allele arrest at restrictive temperature with multiple, elongated buds containing multiple nuclei. This phenotype suggests that the essential functions of the Yck proteins include roles in bud morphogenesis, possibly in control of cell growth polarity, and in cytokinesis or cell separation. Further, a genetic relationship between the yck2ts allele and deletion of CDC55 indicates that the function of Yck phosphorylation may be related to that of protein phosphatase 2A activity. Images PMID:8474447

  16. Yeast spindle pole body duplication gene MPS1 encodes an essential dual specificity protein kinase.

    PubMed Central

    Lauzé, E; Stoelcker, B; Luca, F C; Weiss, E; Schutz, A R; Winey, M

    1995-01-01

    The MPS1 gene has been previously identified by a mutant allele that shows defects in spindle pole body (SPB) duplication and cell cycle control. The SPB is the centrosome-equivalent organelle in the yeast Saccharomyces cerevisiae, and it nucleates all the microtubules in the cell. We report the isolation of the MPS1 gene, which encodes an essential protein kinase homolog. The MPS1 open reading frame has been fused to those that encode the LexA protein or the GST protein and both of these constructs function in yeast. The fusion proteins have been affinity-purified from yeast extracts and the GST chimeric protein has been found to be a phosphoprotein. Both proteins have been used to demonstrate intrinsic in vitro protein kinase activity of Mps1p against exogenous substrates and itself (autophosphorylation). A mutation predicted to abolish kinase function not only eliminates in vitro protein kinase activity, but also behaves like a null mutation in vivo, suggesting that kinase activity contributes to the essential function of the protein. Phosphoamino acid analysis of substrates phosphorylated by Mps1p indicates that this kinase can phosphorylate serine, threonine and tyrosine residues, identifying Mps1p as a dual specificity protein kinase. Images PMID:7737118

  17. African swine fever virus encodes a serine protein kinase which is packaged into virions.

    PubMed Central

    Baylis, S A; Banham, A H; Vydelingum, S; Dixon, L K; Smith, G L

    1993-01-01

    Nucleotide sequencing of the SalI j region of the virulent Malawi (LIL20/1) strain of African swine fever virus (ASFV) identified an open reading frame (ORF), designated j9L, with extensive similarity to the family of protein kinases. This ORF encodes a 35.1-kDa protein of 299 amino acids which shares 24.6% amino acid identity with the human pim-1 proto-oncogene and 21.0% identity with the vaccinia virus B1R-encoded protein kinase. The ASFV ORF contains the motifs characteristic of serine-threonine protein kinases, with the exception of the presumed ATP-binding site, which is poorly conserved. The ORF was expressed to high levels in Escherichia coli, and the recombinant enzyme phosphorylated a calf thymus histone protein on serine residues in vitro. An antibody raised to an amino-terminal peptide of the ASFV protein kinase was reactive with the recombinant protein in Western immunoblot analyses and was used to demonstrate the presence of the protein kinase in ASF virions. Images PMID:8331722

  18. Detail of ump that is attached to rectangular rearing tanks ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    Detail of ump that is attached to rectangular rearing tanks (pair). Pump located on the north end of rearing tank. View to the northwest. - Prairie Creek Fish Hatchery, Hwy. 101, Orick, Humboldt County, CA

  19. Homology probing: identification of cDNA clones encoding members of the protein-serine kinase family

    SciTech Connect

    Hanks, S.K.

    1987-01-01

    Mixed /sup 32/P-labeled oligonucleotide probes were used to screen a HeLa cDNA library for clones encoding amino acid contiguities whose conservation is characteristic of the protein-serine kinase family. Eighty thousand clones were screened, from which 19 were identified as showing strong hybridization to two distinct probes. Four clones were chosen for characterization by partial DNA sequence analysis and 3 of these were found to encode amino acid sequences typical of protein-serine kinases. One deduced amino acid sequence shares 72% identify with rabbit skeletal muscle phosphorylase kinase ..gamma..-subunit, while another is closely related to the yeast protein-serine kinases CDC2 in Schizosaccharomyces pombe and CDC28 in Saccharomyces cerevisiae. This screening approach should have applications in the identification of clones encoding previously unknown or poorly characterized members of other protein families.

  20. Lipopolysaccharide phosphorylating enzymes encoded in the genomes of Gram-negative bacteria are related to the eukaryotic protein kinases

    PubMed Central

    Krupa, A.; Srinivasan, N.

    2002-01-01

    By means of profile-matching procedures, conservation of functionally important residues, and fold-recognition techniques, we show that two distinct families of lipopolysaccharide kinases encoded in the genomes of Gram-negative bacteria are related to each other and to two distinct classes of proteins, namely eukaryotic protein kinases and right open reading frame (RIO1). Members of one of the lipopolysaccharide kinase families are identified only in pathogenic bacteria. Phosphorylation by these enzymes is relevant in the construction of outer membrane, immune response, and pathogenic virulence. The class of proteins called RIO1, also related to eukaryotic protein kinases and previously known to occur only in archaea and eukaryotes, are now identified in eubacteria as well. It has been suggested here that RIO1 proteins are intermediately related to lipopolysaccharide kinases and eukaryotic protein kinases implying an evolutionary relationship between the three classes of proteins. PMID:12021457

  1. The HER-2/neu receptor tyrosine kinase gene encodes a secreted autoinhibitor

    PubMed Central

    Doherty, Joni K.; Bond, Chris; Jardim, Armando; Adelman, John P.; Clinton, Gail M.

    1999-01-01

    HER-2/neu (erbB-2) encodes an 185-kDa orphan receptor tyrosine kinase that is constitutively active as a dimer and displays potent oncogenic activity when overexpressed. Here we describe a secreted protein of ≈68 kDa, designated herstatin, as the product of an alternative HER-2 transcript that retains intron 8. This alternative transcript specifies 340 residues identical to subdomains I and II from the extracellular domain of p185HER-2 followed by a unique C-terminal sequence of 79 aa encoded by intron 8. The recombinant product of the alternative transcript specifically binds to HER-2-transfected cells with a KD of ≈14 nM and was chemically crosslinked to p185HER-2, whereas the intron encoded sequence alone also binds with high affinity to transfected cells and associates with p185 solubilized from cell extracts. The herstatin mRNA is expressed in normal human fetal kidney and liver, but is at reduced levels relative to p185HER-2 mRNA in carcinoma cells that contain an amplified HER-2 gene. Herstatin appears to be an inhibitor of p185HER-2, because it disrupts dimers, reduces tyrosine phosphorylation of p185, and inhibits the anchorage-independent growth of transformed cells that overexpress HER-2. PMID:10485918

  2. MDS1, a dosage suppressor of an mck1 mutant, encodes a putative yeast homolog of glycogen synthase kinase 3.

    PubMed Central

    Puziss, J W; Hardy, T A; Johnson, R B; Roach, P J; Hieter, P

    1994-01-01

    The yeast gene MCK1 encodes a serine/threonine protein kinase that is thought to function in regulating kinetochore activity and entry into meiosis. Disruption of MCK1 confers a cold-sensitive phenotype, a temperature-sensitive phenotype, and sensitivity to the microtubule-destabilizing drug benomyl and leads to loss of chromosomes during growth on benomyl. A dosage suppression selection was used to identify genes that, when present at high copy number, could suppress the cold-sensitive phenotype of mck1::HIS3 mutant cells. Several unique classes of clones were identified, and one of these, designated MDS1, has been characterized in some detail. Nucleotide sequence data reveal that MDS1 encodes a serine/threonine protein kinase that is highly homologous to the shaggy/zw3 kinase in Drosophila melanogaster and its functional homolog, glycogen synthase kinase 3, in rats. The presence of MDS1 in high copy number rescues both the cold-sensitive and the temperature-sensitive phenotypes, but not the benomyl-sensitive phenotype, associated with the disruption of MCK1. Analysis of strains harboring an mds1 null mutation demonstrates that MDS1 is not essential during normal vegetative growth but appears to be required for meiosis. Finally, in vitro experiments indicate that the proteins encoded by both MCK1 and MDS1 possess protein kinase activity with substrate specificity similar to that of mammalian glycogen synthase kinase 3. Images PMID:8264650

  3. Arabidopsis DOK1 encodes a functional dolichol kinase involved in reproduction.

    PubMed

    Kanehara, Kazue; Cho, Yueh; Lin, Ying-Chen; Chen, Chia-En; Yu, Chao-Yuan; Nakamura, Yuki

    2015-01-01

    Dolichol phosphate (Dol-P) serves as a carrier of complex polysaccharides during protein glycosylation. Dol-P is synthesized by the phosphorylation of dolichol or the monodephosphorylation of dolichol pyrophosphate (Dol-PP); however, the enzymes that catalyze these reactions remain unidentified in Arabidopsis thaliana. We performed a genome-wide search for cytidylyltransferase motif-containing proteins in Arabidopsis, and found that At3g45040 encodes a protein homologous with Sec59p, a dolichol kinase (DOK) in Saccharomyces cerevisiae. At3g45040, designated AtDOK1, complemented defects in the growth and N-linked glycosylation of the S. cerevisiae sec59 mutant, suggesting that AtDOK1 encodes a functional DOK. To characterize the physiological roles of AtDOK1 in planta, we isolated two independent lines of T-DNA-tagged AtDOK1 mutants, dok1-1 and dok1-2. The heterozygous plants showed developmental defects in male and female gametophytes, including an aberrant pollen structure, low pollen viability, and short siliques. Additionally, the mutations had incomplete penetrance. These results suggest that AtDOK1 is a functional DOK required for reproductive processes in Arabidopsis.

  4. Hybrid and Rogue Kinases Encoded in the Genomes of Model Eukaryotes

    PubMed Central

    Rakshambikai, Ramaswamy; Gnanavel, Mutharasu; Srinivasan, Narayanaswamy

    2014-01-01

    The highly modular nature of protein kinases generates diverse functional roles mediated by evolutionary events such as domain recombination, insertion and deletion of domains. Usually domain architecture of a kinase is related to the subfamily to which the kinase catalytic domain belongs. However outlier kinases with unusual domain architectures serve in the expansion of the functional space of the protein kinase family. For example, Src kinases are made-up of SH2 and SH3 domains in addition to the kinase catalytic domain. A kinase which lacks these two domains but retains sequence characteristics within the kinase catalytic domain is an outlier that is likely to have modes of regulation different from classical src kinases. This study defines two types of outlier kinases: hybrids and rogues depending on the nature of domain recombination. Hybrid kinases are those where the catalytic kinase domain belongs to a kinase subfamily but the domain architecture is typical of another kinase subfamily. Rogue kinases are those with kinase catalytic domain characteristic of a kinase subfamily but the domain architecture is typical of neither that subfamily nor any other kinase subfamily. This report provides a consolidated set of such hybrid and rogue kinases gleaned from six eukaryotic genomes–S.cerevisiae, D. melanogaster, C.elegans, M.musculus, T.rubripes and H.sapiens–and discusses their functions. The presence of such kinases necessitates a revisiting of the classification scheme of the protein kinase family using full length sequences apart from classical classification using solely the sequences of kinase catalytic domains. The study of these kinases provides a good insight in engineering signalling pathways for a desired output. Lastly, identification of hybrids and rogues in pathogenic protozoa such as P.falciparum sheds light on possible strategies in host-pathogen interactions. PMID:25255313

  5. Structure and expression of chicken protein kinase PITSLRE-encoding genes.

    PubMed

    Li, H; Grenet, J; Valentine, M; Lahti, J M; Kidd, V J

    1995-02-14

    The human PITSLRE protein kinases (PK), members of the p34cdc2 kinase family named according to the single amino acid (aa) code of an important (PSTAIRE) regulatory region [Meyerson et al., EMBO J. 11 (1992) 2909-2917], are candidate tumor suppressor gene(s) localized to human chromosome 1p36.2 and a syntenic region of mouse chromosome 4 [Lahti et al., Nature Genet. 7 (1994) 370-375; Mock et al., Mammal. Genome 5 (1994) 191-192]. At least ten isoforms of this PK family are expressed from three duplicated and tandemly linked genes in humans [Xiang et al., J. Biol. Chem. 269 (1994) 15786-15794]. We have now isolated two different species of PITSLRE PK cDNAs from chicken that encode identical polypeptides, but are clearly expressed from different genes, based on nucleotide (nt) differences. Isolation of one of the corresponding chicken PITSLRE PK genes confirms that only one of the two species of PITSLRE mRNA is expressed from this gene. Comparison of the predicted avian PITSLRE PK aa sequence to human and mouse sequences shows a high degree of sequence identity (> 91%). Like humans, the PITSLRE PK genes in chickens must be closely linked, based on fluorescent in situ hybridization (FISH) localization of these genes to a single chicken microchromosome. PITSLRE PK mRNAs are expressed in two avian B- and T-cell lines. These results suggest that the PITSLRE PK gene family has been well conserved evolutionarily, that the gene duplication observed in humans is not a recent event, and that expression of redundant PITSLRE mRNAs is observed in different vertebrate species.

  6. rlk/TXK Encodes Two Forms of a Novel Cysteine String Tyrosine Kinase Activated by Src Family Kinases

    PubMed Central

    Debnath, Jayantha; Chamorro, Mario; Czar, Michael J.; Schaeffer, Edward M.; Lenardo, Michael J.; Varmus, Harold E.; Schwartzberg, Pamela L.

    1999-01-01

    Rlk/Txk is a member of the BTK/Tec family of tyrosine kinases and is primarily expressed in T lymphocytes. Unlike other members of this kinase family, Rlk lacks a pleckstrin homology (PH) domain near the amino terminus and instead contains a distinctive cysteine string motif. We demonstrate here that Rlk protein consists of two isoforms that arise by alternative initiation of translation from the same cDNA. The shorter, internally initiated protein species lacks the cysteine string motif and is located in the nucleus when expressed in the absence of the larger form. In contrast, the larger form is cytoplasmic. We show that the larger form is palmitoylated and that mutation of its cysteine string motif both abolishes palmitoylation and allows the protein to migrate to the nucleus. The cysteine string, therefore, is a critical determinant of both fatty acid modification and protein localization for the larger isoform of Rlk, suggesting that Rlk regulation is distinct from the other Btk family kinases. We further show that Rlk is phosphorylated and changes localization in response to T-cell-receptor (TCR) activation and, like the other Btk family kinases, can be phosphorylated and activated by Src family kinases. However, unlike the other Btk family members, Rlk is activated independently of the activity of phosphatidylinositol 3-kinase, consistent with its lack of a PH domain. Thus, Rlk has two distinct isoforms, each of which may have unique properties in signaling downstream from the TCR. PMID:9891083

  7. Activation of protein kinase C or cAMP-dependent protein kinase increases phosphorylation of the c-erbA-encoded thyroid hormone receptor and of the v-erbA-encoded protein.

    PubMed Central

    Goldberg, Y; Glineur, C; Gesquière, J C; Ricouart, A; Sap, J; Vennström, B; Ghysdael, J

    1988-01-01

    The c-erbA proto-oncogene encodes a nuclear receptor for thyroid hormone (T3), which is believed to stimulate transcription from specific target promoters upon binding to cis-acting DNA sequence elements. The v-erbA oncogene of avian erythroblastosis virus (AEV) encodes a ligand-independent version of this nuclear receptor. The v-erbA product inhibits terminal differentiation of avian erythroblasts, presumably by affecting the transcription of specific genes. We show here that the c-erbA-encoded nuclear receptor (p46c-erbA) is phosphorylated on serine residues on two distinct sites. One of these sites, defined by the limit tryptic phosphopeptide 28SSQCLVK, is retained on the v-erbA-encoded P75gag-v-erbA protein. This site is located in the amino-terminal domain of these molecules, 21 amino acids upstream of the DNA-binding region. Phosphorylation of this site in both p46c-erbA and P75gag-v-erbA is enhanced 10-fold following treatment of cells with activators of either protein kinase C or cAMP-dependent protein kinase. Since cAMP-dependent protein kinase phosphorylates both p46c-erbA and P75gag-v-erbA in vitro at the same site as that observed in vivo, at least part of the cAMP-dependent phosphorylation of erbA molecules in cells could result from direct phosphorylation by this enzyme. The possible role phosphorylation may play in the function of the erbA-encoded transcriptional factors is discussed. Images PMID:2903825

  8. The pim-1 oncogene encodes two related protein-serine/threonine kinases by alternative initiation at AUG and CUG.

    PubMed Central

    Saris, C J; Domen, J; Berns, A

    1991-01-01

    The pim-1 gene is frequently found activated by proviral insertion in murine T cell lymphomas. Overexpression of pim-1 in lymphoid cells by transgenesis formally proved its oncogenic potential. The pim-1 cDNA sequence predicts that both murine and human pim-1 encode a 34 kd protein with homology to protein kinases. In this study, we show that the murine pim-1 gene encodes a 44 kd protein in addition to the predicted 34 kd protein. The 44 kd protein is an amino-terminal extension of the 34 kd protein and is synthesized by alternative translation initiation at an upstream CUG codon. Contrary to previous findings by others, we provide evidence that both murine and human pim-1 gene products are protein-serine/threonine kinases. Murine 44 kd and 34 kd pim-1 proteins exhibit comparable in vitro kinase activity and are both mainly cytoplasmic, but they differ in in vivo association state and half-life. Images PMID:1825810

  9. SMALL GRAIN 1, which encodes a mitogen-activated protein kinase kinase 4, influences grain size in rice.

    PubMed

    Duan, Penggen; Rao, Yuchun; Zeng, Dali; Yang, Yaolong; Xu, Ran; Zhang, Baolan; Dong, Guojun; Qian, Qian; Li, Yunhai

    2014-02-01

    Although grain size is one of the most important components of grain yield, little information is known about the mechanisms that determine final grain size in crops. Here we characterize rice small grain1 (smg1) mutants, which exhibit small and light grains, dense and erect panicles and comparatively slightly shorter plants. The short grain and panicle phenotypes of smg1 mutants are caused by a defect in cell proliferation. The smg1 mutations were identified, using a map-based cloning approach, in mitogen-activated protein kinase kinase 4 (OsMKK4). Relatively higher expression of OsMKK4/SMG1 was detected in younger organs than in older ones, consistent with its role in cell proliferation. Green fluorescent protein (GFP)-OsMKK4/SMG1 fusion proteins appear to be distributed ubiquitously in plant cells. Further results revealed that OsMKK4 influenced brassinosteroid (BR) responses and the expression of BR-related genes. Thus, our findings have identified OsMKK4 as a factor for grain size, and suggest a possible link between the MAPK pathways and BRs in grain growth.

  10. Receptor protein kinase gene encoded at the self-incompatibility locus

    DOEpatents

    Nasrallah, June B.; Nasrallah, Mikhail E.; Stein, Joshua

    1996-01-01

    Described herein is a S receptor kinase gene (SRK), derived from the S locus in Brassica oleracea, having a extracellular domain highly similar to the secreted product of the S-locus glycoprotein gene.

  11. Molecular cloning of a putative receptor protein kinase gene encoded at the self-incompatibility locus of Brassica oleracea

    SciTech Connect

    Stein, J.C.; Howlett, B.; Boyes, D.C.; Nasrallah, M.E.; Nasrallah, J.B. )

    1991-10-01

    Self-recognition between pollen and stigma during pollination in Brassica oleracea is genetically controlled by the multiallelic self-incompatibility locus (S). The authors describe the S receptor kinase (SRK) gene, a previously uncharacterized gene that residues at the S locus. The nucleotide sequences of genomic DNA and of cDNAs corresponding to SRK predict a putative transmembrane receptor having serine/threonine-specific protein kinase activity. Its extracellular domain exhibits striking homology to the secreted product of the S-locus genotypes are highly polymorphic and have apparently evolved in unison with genetically linked alleles of SLG. SRK directs the synthesis of several alternative transcripts, which potentially encode different protein products, and these transcripts were detected exclusively in reproductive organs. The identification of SRK may provide new perspectives into the signal transduction mechanism underlying pollen recognition.

  12. Myxococcus xanthus mokA Encodes a Histidine Kinase-Response Regulator Hybrid Sensor Required for Development and Osmotic Tolerance

    PubMed Central

    Kimura, Yoshio; Nakano, Hiromi; Terasaka, Hideaki; Takegawa, Kaoru

    2001-01-01

    A gene, mokA, encoding a protein with similarities to histidine kinase-response regulator hybrid sensor, was cloned from a Myxococcus xanthus genomic library. The predicted mokA gene product was found to contain three domains: an amino-terminal input domain, a central transmitter domain, and a carboxy-terminal receiver domain. mokA mutants placed under starvation conditions exhibited reduced sporulation. Mutation of mokA also caused marked growth retardation at high osmolarity. These results indicated that M. xanthus MokA is likely a transmembrane sensor that is required for development and osmotic tolerance. The putative function of MokA is similar to that of the hybrid histidine kinase, DokA, of the eukaryotic slime mold Dictyostelium discoideum. PMID:11157925

  13. Transcription factor Reb1p regulates DGK1-encoded diacylglycerol kinase and lipid metabolism in Saccharomyces cerevisiae.

    PubMed

    Qiu, Yixuan; Fakas, Stylianos; Han, Gil-Soo; Barbosa, Antonio Daniel; Siniossoglou, Symeon; Carman, George M

    2013-10-01

    In the yeast Saccharomyces cerevisiae, the DGK1-encoded diacylglycerol kinase catalyzes the CTP-dependent phosphorylation of diacylglycerol to form phosphatidate. This enzyme, in conjunction with PAH1-encoded phosphatidate phosphatase, controls the levels of phosphatidate and diacylglycerol for phospholipid synthesis, membrane growth, and lipid droplet formation. In this work, we showed that a functional level of diacylglycerol kinase is regulated by the Reb1p transcription factor. In the electrophoretic mobility shift assay, purified recombinant Reb1p was shown to specifically bind its consensus recognition sequence (CGGGTAA, -166 to -160) in the DGK1 promoter. Analysis of cells expressing the PDGK1-lacZ reporter gene showed that mutations (GT→TG) in the Reb1p-binding sequence caused an 8.6-fold reduction in β-galactosidase activity. The expression of DGK1(reb1), a DGK1 allele containing the Reb1p-binding site mutation, was greatly lower than that of the wild type allele, as indicated by analyses of DGK1 mRNA, Dgk1p, and diacylglycerol kinase activity. In the presence of cerulenin, an inhibitor of de novo fatty acid synthesis, the dgk1Δ mutant expressing DGK1(reb1) exhibited a significant defect in growth as well as in the synthesis of phospholipids from triacylglycerol mobilization. Unlike DGK1, the DGK1(reb1) expressed in the dgk1Δ pah1Δ mutant did not result in the nuclear/endoplasmic reticulum membrane expansion, which occurs in cells lacking phosphatidate phosphatase activity. Taken together, these results indicate that the Reb1p-mediated regulation of diacylglycerol kinase plays a major role in its in vivo functions in lipid metabolism.

  14. Identification of two eukaryote-like serine/threonine kinases encoded by Chlamydia trachomatis serovar L2 and characterization of interacting partners of Pkn1.

    PubMed

    Verma, Anita; Maurelli, Anthony T

    2003-10-01

    Genome sequencing of C. trachomatis serovar D revealed the presence of three putative open reading frames (ORFs), CT145 (Pkn1), CT673 (Pkn5), and CT301 (PknD), encoding eukaryote-like serine/threonine kinases (Ser/Thr kinases). Two of these putative kinase genes, CT145 and CT301, were PCR amplified from serovar L2, cloned, and sequenced. Predicted translation products of the ORFs showed the presence of conserved kinase motifs at the N terminus of the proteins. CT145 and CT301 (encoding Pkn1 and PknD, respectively) were expressed in Escherichia coli as GST fusion proteins. In vitro kinase assays with Escherichia coli-derived glutathione S-transferase fusion proteins showed autophosphorylation of Pkn1 and PknD, indicating that they are functional kinases. Gene expression analysis of these kinase genes in Chlamydia by reverse transcriptase PCR indicated expression of these kinases at the early mid phase of the developmental cycle. Immunoprecipitated native chlamydial Pkn1 and PknD proteins also showed autophosphorylation in an in vitro kinase assay. Phosphoamino acid analysis by thin-layer chromatography confirmed that Pkn1 and PknD are phosphorylated on both serine and threonine residues. Interaction of Pkn1 and PknD with each other as well as interaction of Pkn1 with inclusion membrane protein G (IncG) was demonstrated by using a bacterial two-hybrid system. These interactions were further suggested by phosphorylation of the proteins in in vitro kinase assays. This report is the first description of the existence of functional Ser/Thr kinases in Chlamydia. The results of these findings should lead to a better understanding of how Chlamydia interact and interfere with host signaling pathways, since kinases represent potential mediators of the intimate host-pathogen interactions that are essential to the intracellular life cycle of Chlamydia.

  15. Structure and transforming potential of the human cot oncogene encoding a putative protein kinase.

    PubMed Central

    Miyoshi, J; Higashi, T; Mukai, H; Ohuchi, T; Kakunaga, T

    1991-01-01

    A new transforming gene has been molecularly cloned from hamster SHOK cells transformed with DNA extracted from a human thyroid carcinoma cell line and named the cot (cancer Osaka thyroid) oncogene. cDNA sequencing disclosed that this oncogene codes for a protein with 415 amino acid residues, and computer matching showed 42 to 48% similarity matches with serine protein kinases. Its gene product was identified as a 52-kDa protein by transcription and translation in vitro. Expression of cot cDNA under transcriptional control by a retroviral long terminal repeat induced morphological transformation of NIH 3T3 cells as well as SHOK cells. Protein kinase activity associated with constructed p60gag-cot was detected by immune complex kinase assay with anti-gag antiserum. The cot oncogene was overexpressed in transformed SHOK cells and found to have a rearranged 3' end in the last coding exon, which probably resulted in a deletion and an altered C' terminus in the transforming protein. This DNA rearrangement appeared to have occurred during transfection of the tumor DNA into hamster SHOK cells and not in the original thyroid tumor. Images PMID:2072910

  16. Identification and chromosome assignment of a human gene encoding a novel phosphatidylinositol-3 kinase.

    PubMed

    Seki, N; Nimura, Y; Ohira, M; Saito, T; Ichimiya, S; Nomura, N; Nakagawara, A

    1997-10-31

    We identified a novel phosphatidylinositol (PI) 3-kinase by screening human brain cDNA libraries with probes designed from the conserved kinase-domain sequence. Analysis of cDNAs indicated that two different forms of transcripts are present: one is the full-length form composed of 1,044 amino acid residues and the other is the short form that the N-terminal 216 amino acid residues including a putative p85 binding domain has been truncated (828 amino acid residues). Database search revealed the sequence of the full-length form to be identical to that recently registered by D. Chantry et al. (Accession No. U86453 in GenBank release, August 1997). Northern blot analysis showed this mRNA to be ubiquitously expressed in various tissues, with relatively higher expression was observed in spleen, thymus and leukocytes. Based on fluorescence in situ hybridization and PCR-based analyses with both human/rodent mono-chromosomal hybrid cell panels and radiation hybrid mapping panels, this gene was localized to chromosome region 1p36.2. This region is frequently lost in a variety of human malignancies, including neuroblastoma. The novel PI3K could be a candidate target of the 1p36 alteration that occurs in neuroendocrine tumors.

  17. Heterozygous Mutations in MAP3K7, Encoding TGF-β-Activated Kinase 1, Cause Cardiospondylocarpofacial Syndrome.

    PubMed

    Le Goff, Carine; Rogers, Curtis; Le Goff, Wilfried; Pinto, Graziella; Bonnet, Damien; Chrabieh, Maya; Alibeu, Olivier; Nistchke, Patrick; Munnich, Arnold; Picard, Capucine; Cormier-Daire, Valérie

    2016-08-01

    Cardiospondylocarpofacial (CSCF) syndrome is characterized by growth retardation, dysmorphic facial features, brachydactyly with carpal-tarsal fusion and extensive posterior cervical vertebral synostosis, cardiac septal defects with valve dysplasia, and deafness with inner ear malformations. Whole-exome sequencing identified heterozygous MAP3K7 mutations in six distinct CSCF-affected individuals from four families and ranging in age from 5 to 37 years. MAP3K7 encodes transforming growth factor β (TGF-β)-activated kinase 1 (TAK1), which is involved in the mitogen-activated protein kinase (MAPK)-p38 signaling pathway. MAPK-p38 signaling was markedly altered when expression of non-canonical TGF-β-driven target genes was impaired. These findings support the loss of transcriptional control of the TGF-β-MAPK-p38 pathway in fibroblasts obtained from affected individuals. Surprisingly, although TAK1 is located at the crossroad of inflammation, immunity, and cancer, this study reports MAP3K7 mutations in a developmental disorder affecting mainly cartilage, bone, and heart. PMID:27426734

  18. Mutations in CIT, encoding citron rho-interacting serine/threonine kinase, cause severe primary microcephaly in humans.

    PubMed

    Shaheen, Ranad; Hashem, Amal; Abdel-Salam, Ghada M H; Al-Fadhli, Fatima; Ewida, Nour; Alkuraya, Fowzan S

    2016-10-01

    Primary microcephaly is a clinical phenotype in which the head circumference is significantly reduced at birth due to abnormal brain development, primarily at the cortical level. Despite the marked genetic heterogeneity, most primary microcephaly-linked genes converge on mitosis regulation. Two consanguineous families segregating the phenotype of severe primary microcephaly, spasticity and failure to thrive had overlapping autozygomes in which exome sequencing identified homozygous splicing variants in CIT that segregate with the phenotype within each family. CIT encodes citron, an effector of the Rho signaling that is required for cytokinesis specifically in proliferating neuroprogenitors, as well as for postnatal brain development. In agreement with the critical role assigned to the kinase domain in effecting these biological roles, we show that both splicing variants predict variable disruption of this domain. The striking phenotypic overlap between CIT-mutated individuals and the knockout mice and rats that are specifically deficient in the kinase domain supports the proposed causal link between CIT mutation and primary microcephaly in humans. PMID:27503289

  19. Osmoregulation and fungicide resistance: the Neurospora crassa os-2 gene encodes a HOG1 mitogen-activated protein kinase homologue.

    PubMed

    Zhang, Yan; Lamm, Randy; Pillonel, Christian; Lam, Stephen; Xu, Jin-Rong

    2002-02-01

    Neurospora crassa osmosensitive (os) mutants are sensitive to high osmolarity and therefore are unable to grow on medium containing 4% NaCl. We found that os-2 and os-5 mutants were resistant to the phenylpyrrole fungicides fludioxonil and fenpiclonil. To understand the relationship between osmoregulation and fungicide resistance, we cloned the os-2 gene by using sib selection. os-2 encodes a putative mitogen-activated protein (MAP) kinase homologous to HOG1 and can complement the osmosensitive phenotype of a Saccharomyces cerevisiae hog1 mutant. We sequenced three os-2 alleles and found that all of them were null with either frameshift or nonsense point mutations. An os-2 gene replacement mutant also was generated and was sensitive to high osmolarity and resistant to phenylpyrrole fungicides. Conversely, os-2 mutants transformed with the wild-type os-2 gene could grow on media containing 4% NaCl and were sensitive to phenylpyrrole fungicides. Fludioxonil stimulated intracellular glycerol accumulation in wild-type strains but not in os-2 mutants. Fludioxonil also caused wild-type conidia and hyphal cells to swell and burst. These results suggest that the hyperosmotic stress response pathway of N. crassa is the target of phenylpyrrole fungicides and that fungicidal effects may result from a hyperactive os-2 MAP kinase pathway.

  20. Welcoming speech from Dean Faculty of Mechanical Engineering, UMP

    NASA Astrophysics Data System (ADS)

    Taha, Zahari

    2012-09-01

    In the Name of Allah, the Most Beneficent, the Most Merciful. It is with great pleasure that I welcome the participants of the International Conference of Mechanical Engineering Research 2011. The Prophet Muhammad (peace be upon him) said 'Acquire knowledge and impart it to the people.' (Al Tirmidhi). The quest for knowledge has been from the beginning of time but knowledge only becomes valuable when it is disseminated and applied to benefit humankind. It is hoped that ICMER 2011 will be a platform to gather and disseminate the latest knowledge in mechanical engineering. Academicians, Scientist, Researchers and practitioners of mechanical engineering will be able to share and discuss new findings and applications of mechanical engineering. It is envisaged that the intellectual discourse will result in future collaborations between universities, research institutions and industry both locally and internationally. In particular it is expected that focus will be given to issues on environmental and energy sustainability. Researchers in the mechanical engineering faculty at UMP have a keen interest in technology to harness energy from the ocean. Lowering vehicle emissions has been a primary goal of researchers in the mechanical engineering faculty and the automotive engineering centre as well including developing vehicles using alternative fuels such as biodiesel and renewable sources such as solar driven electric vehicles. Finally I would like to congratulate the organizing committee for their tremendous efforts in organizing the conference. As I wrote this in the Holy Land of Makkah, I pray to Allah swt that the conference will be a success. Prof. Dr. Zahari Taha CEng, MIED, FASc Dean, Faculty of Mechanical Engineering Universiti Malaysia Pahang

  1. Determination of the promoter region of an early vaccinia virus gene encoding thymidine kinase.

    PubMed

    Weir, J P; Moss, B

    1987-05-01

    Nine recombinant vaccinia viruses that contain overlapping segments of the putative promoter region of the vaccinia virus thymidine kinase (TK) gene linked to DNA coding for the prokaryotic enzyme chloramphenicol acetyltransferase (CAT) were constructed. In each case, the RNA start site and 5 bp of DNA downstream were retained. No significant difference in CAT expression occurred as the deletion was extended from 352 to 32 bp before the RNA start site. Deletion of a further 10 bp, however, led to complete cessation of early promoter activity. Primer extension analysis of the 5' ends of the transcripts verified that the natural TK RNA start site was still used when only 32 bp of upstream DNA remained. Loss of early promoter activity was previously found when deletions were extended from 31 to 24 bp before the RNA start site of another vaccinia gene that is expressed constitutively throughout infection (M.A. Cochran, C. Puckett, and B. Moss, 1985, Proc. Natl. Acad. Sci. USA 82, 19-23). Sequence similarities in the promoter regions of these two genes were noted.

  2. Characterization and distribution of a maize cDNA encoding a peptide similar to the catalytic region of second messenger dependent protein kinases

    NASA Technical Reports Server (NTRS)

    Biermann, B.; Johnson, E. M.; Feldman, L. J.

    1990-01-01

    Maize (Zea mays) roots respond to a variety of environmental stimuli which are perceived by a specialized group of cells, the root cap. We are studying the transduction of extracellular signals by roots, particularly the role of protein kinases. Protein phosphorylation by kinases is an important step in many eukaryotic signal transduction pathways. As a first phase of this research we have isolated a cDNA encoding a maize protein similar to fungal and animal protein kinases known to be involved in the transduction of extracellular signals. The deduced sequence of this cDNA encodes a polypeptide containing amino acids corresponding to 33 out of 34 invariant or nearly invariant sequence features characteristic of protein kinase catalytic domains. The maize cDNA gene product is more closely related to the branch of serine/threonine protein kinase catalytic domains composed of the cyclic-nucleotide- and calcium-phospholipid-dependent subfamilies than to other protein kinases. Sequence identity is 35% or more between the deduced maize polypeptide and all members of this branch. The high structural similarity strongly suggests that catalytic activity of the encoded maize protein kinase may be regulated by second messengers, like that of all members of this branch whose regulation has been characterized. Northern hybridization with the maize cDNA clone shows a single 2400 base transcript at roughly similar levels in maize coleoptiles, root meristems, and the zone of root elongation, but the transcript is less abundant in mature leaves. In situ hybridization confirms the presence of the transcript in all regions of primary maize root tissue.

  3. Isolation and characterization of human cDNA clones encoding the. alpha. and the. alpha. prime subunits of casein kinase II

    SciTech Connect

    Lozeman, F.J.; Litchfield, D.W.; Piening, C.; Takio, Koji; Walsh, K.A.; Krebs E.G. )

    1990-09-11

    Casein kinase II is a widely distributed protein serine/threonine kinase. The holoenzyme appears to be a tetramer, containing two {alpha} or {alpha}{prime} subunits (or one of each) and two {beta} subunits. Complementary DNA clones encoding the subunits of casein kinase II were isolated from a human T-cell {lambda}gt 10 library using cDNA clones isolated from Drosophila melanogasten. One of the human cDNA clones (hT4.1) was 2.2 kb long, including a coding region of 1176 bp preceded by 156 bp (5{prime} untranslated region) and followed by 871 bp (3{prime} untranslated region). The hT4.1 close was nearly identical in size and sequence with a cDNA clone from HepG2 human hepatoma cultured cells. Another of the human T-cell cDNA clones (hT9.1) was 1.8 kb long, containing a coding region of 1053 bp preceded by 171 by (5{prime} untranslated region) and followed by 550 bp (3{prime} untranslated region). Amino acid sequences deduced from these two cDNA clones were about 85% identical. Most of the difference between the two encoded polypeptides was in the carboxy-terminal region, but heterogeneity was distributed throughout the molecules. Partial amino acid sequence was determined in a mixture of {alpha} and {alpha}{prime} subunits from bovine lung casein kinase II. The bovine sequences aligned with the 2 human cDNA-encoded polypeptides with only 2 discrepancies out of 535 amino acid positions. This confirmed that the two human T-cell cDNA clones encoded the {alpha} and {alpha}{prime} subunits of casein kinase II. These studies show that there are two distinct catalytic subunits for casein II ({alpha} and {alpha}{prime}) and that the sequence of these subunits is largely conserved between the bovine and the human.

  4. A plant receptor-like gene, the S-locus receptor kinase of Brassica oleracea L. , encodes a functional serine/threonine kinase

    SciTech Connect

    Stein, J.C.; Nasrallah, J.B. )

    1993-03-01

    To investigate the catalytic properties of the Brassica oleracea S-locus receptor kinase (SRK), the authors have expressed the domain that is homologous to protein kinases as a fusion protein in Escherichia coli. Following in vivo labeling of cultures with [sup 32]P-labeled inorganic phosphate, they observed phosphorylation of the fusion protein on serine and threonine, but not on tyrosine. In contrast, labeling was not observed when lysine-524, a residue conserved among all protein kinases, was mutated to arginine, thus confirmed that SRK phosphorylation was the result of intrinsic serine/threonine kinase activity. 26 refs., 3 figs.

  5. Cloning of a human cDNA encoding a CDC2-related kinase by complementation of a budding yeast cdc28 mutation

    SciTech Connect

    Ninomiya-Tsuji, Jun ); Nomoto, Satoshi; Matsumoto, Kunihiro ); Yasuda, Hideyo ); Reed, S.I. )

    1991-10-15

    The authors have cloned two different human cDNAs that can complement cdc28 mutations of budding yeast Saccharomyces cerevisiae. One corresponds to a gene encoding human p34{sup CDC2} kinase, and the other to a gene (CDK2; cell division kinase) that has not been characterized previously. The CDK2 protein is highly homologous to p34{sup CDC2} kinase and more significantly is homologous to Xenopus Eg1 kinase, suggesting that CDK2 is the human homolog of Eg1. The human CDC2 and CDK2 genes were both able to complement the inviability of a null allele of S. cerevisiae CDC28. This result indicates that the CDK2 protein has a biological activity closely related to the CDC28 and p34{sup CDC2} kinases. However, CDK2 was unable to complement cdc2 mutants in fission yeast Schizosaccharomyces pombe under the condition where the human CDC2 gene could complement them. CDK2 mRNA appeared late in G{sub 1} or in early S phase, slightly before CDC2 mRNA, after growth stimulation in normal human fibroblast cells. These results suggest that in human cells, two different CDC2-like kinases may regulate the cell cycle at distinct stages.

  6. An unusual UMP C-5 methylase in nucleoside antibiotic polyoxin biosynthesis.

    PubMed

    Chen, Wenqing; Li, Yan; Li, Jie; Wu, Lian; Li, Yan; Wang, Renxiao; Deng, Zixin; Zhou, Jiahai

    2016-09-01

    Polyoxin is a group of structurally-related peptidyl nucleoside antibiotics bearing C-5 modifications on the nucleoside skeleton. Although the structural diversity and bioactivity preference of polyoxin are, to some extent, affected by such modifications, the biosynthetic logic for their occurence remains obscure. Here we report the identification of PolB in polyoxin pathway as an unusual UMP C-5 methylase with thymidylate synthase activity which is responsible for the C-5 methylation of the nucleoside skeleton. To probe its molecular mechanism, we determined the crystal structures of PolB alone and in complexes with 5-Br UMP and 5-Br dUMP at 2.15 Å, 1.76 Å and 2.28 Å resolutions, respectively. Loop 1 (residues 117-131), Loop 2 (residues 192-201) and the substrate recognition peptide (residues 94-102) of PolB exhibit considerable conformational flexibility and adopt distinct structures upon binding to different substrate analogs. Consistent with the structural findings, a PolB homolog that harbors an identical function from Streptomyces viridochromogenes DSM 40736 was identified. The discovery of UMP C5-methylase opens the way to rational pathway engineering for polyoxin component optimization, and will also enrich the toolbox for natural nucleotide chemistry. PMID:27412636

  7. Expression and functional analysis of genes encoding cytokinin receptor-like histidine kinase in maize (Zea mays L.).

    PubMed

    Wang, Bo; Chen, Yanhong; Guo, Baojian; Kabir, Muhammad Rezaul; Yao, Yingyin; Peng, Huiru; Xie, Chaojie; Zhang, Yirong; Sun, Qixin; Ni, Zhongfu

    2014-08-01

    Cytokinin signaling is vital for plant growth and development which function via the two-component system (TCS). As one of the key component of TCS, transmembrane histidine kinases (HK) are encoded by a small gene family in plants. In this study, we focused on expression and functional analysis of cytokinin receptor-like HK genes (ZmHK) in maize. Firstly, bioinformatics analysis revealed that seven cloned ZmHK genes have different expression patterns during maize development. Secondly, ectopic expression by CaMV35S promoter in Arabidopsis further revealed that functional differentiation exists among these seven members. Among them, the ZmHK1a2-OX transgenic line has the lowest germination rate in the dark, ZmHK1-OX and ZmHK2a2-OX can delay leaf senescence, and seed size of ZmHK1-OX, ZmHK1a2-OX, ZmHK2-OX, ZmHK3b-OX and ZmHK2a2-OX was obviously reduced as compared to wild type. Additionally, ZmHK genes play opposite roles in shoot and root development; all ZmHK-OX transgenic lines display obvious shorter root length and reduced number of lateral roots, but enhanced shoot development compared with the wild type. Most notably, Arabidopsis response regulator ARR5 gene was up-regulated in ZmHK1-OX, ZmHK1a2-OX, ZmHK2-OX, ZmHK3b-OX and ZmHK2a2-OX as compared to wild type. Although the causal link between ZmHK genes and cytokinin signaling pathway is still an area to be further elucidated, these findings reflected that the diversification of ZmHK genes expression patterns and functions occurred in the course of maize evolution, indicating that some ZmHK genes might play different roles during maize development.

  8. Expression and functional analysis of genes encoding cytokinin receptor-like histidine kinase in maize (Zea mays L.).

    PubMed

    Wang, Bo; Chen, Yanhong; Guo, Baojian; Kabir, Muhammad Rezaul; Yao, Yingyin; Peng, Huiru; Xie, Chaojie; Zhang, Yirong; Sun, Qixin; Ni, Zhongfu

    2014-08-01

    Cytokinin signaling is vital for plant growth and development which function via the two-component system (TCS). As one of the key component of TCS, transmembrane histidine kinases (HK) are encoded by a small gene family in plants. In this study, we focused on expression and functional analysis of cytokinin receptor-like HK genes (ZmHK) in maize. Firstly, bioinformatics analysis revealed that seven cloned ZmHK genes have different expression patterns during maize development. Secondly, ectopic expression by CaMV35S promoter in Arabidopsis further revealed that functional differentiation exists among these seven members. Among them, the ZmHK1a2-OX transgenic line has the lowest germination rate in the dark, ZmHK1-OX and ZmHK2a2-OX can delay leaf senescence, and seed size of ZmHK1-OX, ZmHK1a2-OX, ZmHK2-OX, ZmHK3b-OX and ZmHK2a2-OX was obviously reduced as compared to wild type. Additionally, ZmHK genes play opposite roles in shoot and root development; all ZmHK-OX transgenic lines display obvious shorter root length and reduced number of lateral roots, but enhanced shoot development compared with the wild type. Most notably, Arabidopsis response regulator ARR5 gene was up-regulated in ZmHK1-OX, ZmHK1a2-OX, ZmHK2-OX, ZmHK3b-OX and ZmHK2a2-OX as compared to wild type. Although the causal link between ZmHK genes and cytokinin signaling pathway is still an area to be further elucidated, these findings reflected that the diversification of ZmHK genes expression patterns and functions occurred in the course of maize evolution, indicating that some ZmHK genes might play different roles during maize development. PMID:24585212

  9. Us3 kinase encoded by herpes simplex virus 1 mediates downregulation of cell surface major histocompatibility complex class I and evasion of CD8+ T cells.

    PubMed

    Imai, Takahiko; Koyanagi, Naoto; Ogawa, Ryo; Shindo, Keiko; Suenaga, Tadahiro; Sato, Ayuko; Arii, Jun; Kato, Akihisa; Kiyono, Hiroshi; Arase, Hisashi; Kawaguchi, Yasushi

    2013-01-01

    Detection and elimination of virus-infected cells by CD8(+) cytotoxic T lymphocytes (CTLs) depends on recognition of virus-derived peptides presented by major histocompatibility complex class I (MHC-I) molecules on the surface of infected cells. In the present study, we showed that inactivation of the activity of viral kinase Us3 encoded by herpes simplex virus 1 (HSV-1), the etiologic agent of several human diseases and a member of the alphaherpesvirinae, significantly increased cell surface expression of MHC-I, thereby augmenting CTL recognition of infected cells in vitro. Overexpression of Us3 by itself had no effect on cell surface expression of MHC-I and Us3 was not able to phosphorylate MHC-I in vitro, suggesting that Us3 indirectly downregulated cell surface expression of MHC-I in infected cells. We also showed that inactivation of Us3 kinase activity induced significantly more HSV-1-specific CD8(+) T cells in mice. Interestingly, depletion of CD8(+) T cells in mice significantly increased replication of a recombinant virus encoding a kinase-dead mutant of Us3, but had no effect on replication of a recombinant virus in which the kinase-dead mutation was repaired. These results indicated that Us3 kinase activity is required for efficient downregulation of cell surface expression of MHC-I and mediates evasion of HSV-1-specific CD8(+) T cells. Our results also raised the possibility that evasion of HSV-1-specific CD8(+) T cells by HSV-1 Us3-mediated inhibition of MHC-I antigen presentation might in part contribute to viral replication in vivo.

  10. Regulation of the double-stranded RNA-dependent protein kinase PKR by RNAs encoded by a repeated sequence in the Epstein-Barr virus genome.

    PubMed Central

    Elia, A; Laing, K G; Schofield, A; Tilleray, V J; Clemens, M J

    1996-01-01

    During the initial infection of B lymphocytes by Epstein-Barr virus (EBV) only a few viral genes are expressed, six of which encode the EBV nuclear antigens, EBNAs 1-6. The majority of EBNA mRNAs share common 5'-ends containing a variable number of two alternating and repeated exons transcribed from the BamHI W major internal repeats of the viral DNA. These sequences can also exist as independent small RNA species in some EBV-infected cell types. We present evidence that transcripts from these W repeat regions can exert a trans-acting effect on protein synthesis, through their ability to activate the dsRNA-dependent protein kinase PKR. UV cross-linking and filter binding assays have demonstrated that the W transcripts bind specifically to PKR and can compete with another EBV-encoded small RNA, EBER-1, which was shown previously to bind this kinase. In the reticulocyte lysate system the W RNAs shut off protein synthesis through an ability to activate PKR. In contrast to EBER-1, the W RNAs are unable to block the dsRNA-dependent activation of PKR. Using a purified preparation of the protein kinase we have shown that the W transcripts directly activate PKR in vitro. The results suggest that EBV has the ability both to activate and to inhibit PKR through the actions of different products of viral transcription. PMID:8948637

  11. [Characterization of a putative S locus encoded receptor protein kinase and its role in self-incompatibility

    SciTech Connect

    Not Available

    1993-01-01

    The serine/threonine protein kinase (SRK) protein was predicted to be similar to the growth factor receptor tyrosine kinases in animals but its amino acid sequence of the catalytic domain is more similar to that of the catalytic domains of protein serine/threonine kinases than to protein tyrosine kinases. We have shown that the SRK protein has intrinsic scrine/threonine kinase activity. We subcloned the protein kinase-homologous domain of the SRK[sub 6] cDNA into the bacterial expression vector pGEX-3X and we have constructed a second plasmid identical to the first except that it carried a conservative mutation that substituted Arg for the Lys[sup 524] codon of SRK6 This lysine corresponds to the ATP-binding site, is essential in protein kinases, and is a common target for site-directed mutagenesis as a means to obtain kinase-defective proteins. Cultures bearing the wild-type and mutant SRK catalytic domains each produced an approximately 64 kD protein that reacted with anti-SRK6 antibodies. Following pulse-labeling with [sup 32]P we found that the wild-type SRK6 protein but not the mutant form was detectably phosphorylated. Phosphoamino acid analysis of the affinity purified [sup 32]p-labeled GST-SRK6 fusion protein demonstrated that SRK was phosphorylated predominantly on semine and to a lesser extent on threonine, but not on tyrosine. Thus, SRK6 is a functional serine/threonine protein kinase.

  12. Vaccinia virus gene H5R encodes a protein that is phosphorylated by the multisubstrate vaccinia virus B1R protein kinase.

    PubMed Central

    Beaud, G; Beaud, R; Leader, D P

    1995-01-01

    Vaccinia virus gene B1R encodes a protein kinase, the previously identified substrates of which include the proteins S2 and Sa of 40S ribosomal subunits. This work characterizes another substrate of the B1R kinase: a 36-kDa protein induced at the early stage of infection. Partially purified 36-kDa protein, eluted from a single-stranded DNA-cellulose column by 0.5 M NaCl, was separated by two-dimensional gel electrophoresis. Phosphorylation in vitro yielded multiple forms of the 36-kDa protein with approximate isoelectric points (pI) of 5.5, 5.7, 5.9, and 6.3, in addition to the apparently unphosphorylated form with a pI of approximately 6.8. The tryptic peptides derived from 36-kDa proteins with pI values of 5.7, 5.9, and 6.3 yielded almost identical high-pressure liquid chromatography profiles, strongly suggesting that the 36-kDa protein was modified by the phosphorylation of at least four sites, which were characterized as threonine residues. The amino acid sequence of several tryptic peptides derived from the 36-kDa protein showed that the 36-kDa protein was encoded by gene H5R of vaccinia virus. Consistent with this, the B1R kinase--either expressed in Escherichia coli or highly purified from HeLa cells--phosphorylated a recombinant trpE-H5R fusion protein in vitro. Fingerprints of the trpE-H5R and 36-kDa proteins phosphorylated by recombinant B1R kinase revealed common sites of phosphorylation, although some tryptic peptides were specific to either protein. Comparison was made of fingerprints of tryptic phosphopeptides derived from 36-kDa single-stranded DNA-binding protein labelled in vivo or in vitro. A common subset of peptides was observed, suggesting that some sites on H5R protein are phosphorylated by the B1R kinase in infected cells. These results suggest that some of the multiple threonine sites in the H5R protein are phosphorylated in vivo by the B1R protein kinase. PMID:7853522

  13. Cloning and characterization of a cDNA encoding an A-kinase anchoring protein located in the centrosome, AKAP450.

    PubMed Central

    Witczak, O; Skålhegg, B S; Keryer, G; Bornens, M; Taskén, K; Jahnsen, T; Orstavik, S

    1999-01-01

    A combination of protein kinase A type II (RII) overlay screening, database searches and PCR was used to identify a centrosomal A-kinase anchoring protein. A cDNA with an 11.7 kb open reading frame was characterized and found to correspond to 50 exons of genomic sequence on human chromosome 7q21-22. This cDNA clone encoded a 3908 amino acid protein of 453 kDa, that was designated AKAP450 (DDBJ/EMBL/GenBank accession No. AJ131693). Sequence comparison demonstrated that the open reading frame contained a previously characterized cDNA encoding Yotiao, as well as the human homologue of AKAP120. Numerous coiled-coil structures were predicted from AKAP450, and weak homology to pericentrin, giantin and other structural proteins was observed. A putative RII-binding site was identified involving amino acid 2556 of AKAP450 by mutation analysis combined with RII overlay and an amphipatic helix was predicted in this region. Immunoprecipitation of RII from RIPA-buffer extracts of HeLa cells demonstrated co-precipitation of AKAP450. By immunofluorecent labeling with specific antibodies it was demonstrated that AKAP450 localized to centrosomes. Furthermore, AKAP450 was shown to co-purify in centrosomal preparations. The observation of two mRNAs and several splice products suggests additional functions for the AKAP450 gene. PMID:10202149

  14. Identification of a novel operon in Lactococcus lactis encoding three enzymes for lactic acid synthesis: phosphofructokinase, pyruvate kinase, and lactate dehydrogenase.

    PubMed Central

    Llanos, R M; Harris, C J; Hillier, A J; Davidson, B E

    1993-01-01

    The discovery of a novel multicistronic operon that encodes phosphofructokinase, pyruvate kinase, and lactate dehydrogenase in the lactic acid bacterium Lactococcus lactis is reported. The three genes in the operon, designated pfk, pyk, and ldh, contain 340, 502, and 325 codons, respectively. The intergenic distances are 87 bp between pfk and pyk and 117 bp between pyk and ldh. Plasmids containing pfk and pyk conferred phosphofructokinase and pyruvate kinase activity, respectively, on their host. The identity of ldh was established previously by the same approach (R. M. Llanos, A. J. Hillier, and B. E. Davidson, J. Bacteriol. 174:6956-6964, 1992). Each of the genes is preceded by a potential ribosome binding site. The operon is expressed in a 4.1-kb transcript. The 5' end of the transcript was determined to be a G nucleotide positioned 81 bp upstream from the pfk start codon. The pattern of codon usage within the operon is highly biased, with 11 unused amino acid codons. This degree of bias suggests that the operon is highly expressed. The three proteins encoded on the operon are key enzymes in the Embden-Meyerhoff pathway, the central pathway of energy production and lactic acid synthesis in L. lactis. For this reason, we have called the operon the las (lactic acid synthesis) operon. Images PMID:8478320

  15. ENV7 and YCK3, which encode vacuolar membrane protein kinases, genetically interact to impact cell fitness and vacuole morphology.

    PubMed

    Manandhar, Surya P; Gharakhanian, Editte

    2014-05-01

    Saccharomyces cerevisiae vacuoles serve as a model for membrane fusion and fission. Yck3, a vacuolar membrane kinase, has been implicated in regulation of vacuole fusion. Recently, we established Env7 as another vacuolar membrane protein kinase with similar but nonredundant function to Yck3. Here, we report that native Env7 localizes to the vacuole independent of Yck3, where as its phosphorylation is YCK3 dependent. We also show that env7Δyck3Δ double mutant exhibits severely compromised fitness, altered cell size and bud vacuoles, and F-class vacuolar morphology. Our results establish negative genetic interactions between ENV7 and YCK3 and suggest cooperative roles for the two conserved genes in regulation of membrane dynamics. Such genetic buffering supports a critical role for membrane flux in global cell fitness.

  16. The Epstein-Barr virus (EBV)-encoded protein kinase, EBV-PK, but not the thymidine kinase (EBV-TK), is required for ganciclovir and acyclovir inhibition of lytic viral production.

    PubMed

    Meng, Qiao; Hagemeier, Stacy R; Fingeroth, Joyce D; Gershburg, Edward; Pagano, Joseph S; Kenney, Shannon C

    2010-05-01

    Ganciclovir (GCV) and acyclovir (ACV) are guanine nucleoside analogues that inhibit lytic herpesvirus replication. GCV and ACV must be monophosphorylated by virally encoded enzymes to be converted into nucleotides and incorporated into viral DNA. However, whether GCV and/or ACV phosphorylation in Epstein-Barr virus (EBV)-infected cells is mediated primarily by the EBV-encoded protein kinase (EBV-PK), the EBV-encoded thymidine kinase (EBV-TK), or both is controversial. To examine this question, we constructed EBV mutants containing stop codons in either the EBV-PK or EBV-TK open reading frame and selected for stable 293T clones latently infected with wild-type EBV or each of the mutant viruses. Cells were induced to the lytic form of viral replication with a BZLF1 expression vector in the presence and absence of various doses of GCV and ACV, and infectious viral titers were determined by a green Raji cell assay. As expected, virus production in wild-type EBV-infected 293T cells was inhibited by both GCV (50% inhibitory concentration [IC(50)] = 1.5 microM) and ACV (IC(50) = 4.1 microM). However, the EBV-PK mutant (which replicates as well as the wild-type (WT) virus in 293T cells) was resistant to both GCV (IC(50) = 19.6 microM) and ACV (IC(50) = 36.4 microM). Expression of the EBV-PK protein in trans restored GCV and ACV sensitivity in cells infected with the PK mutant virus. In contrast, in 293T cells infected with the TK mutant virus, viral replication remained sensitive to both GCV (IC(50) = 1.2 microM) and ACV (IC(50) = 2.8 microM), although susceptibility to the thymine nucleoside analogue, bromodeoxyuridine, was reduced. Thus, EBV-PK but not EBV-TK mediates ACV and GCV susceptibilities.

  17. Characterization of a putative S-locus encoded receptor protein kinase and its role in self-incompatibility. Progress report

    SciTech Connect

    Nasrallah, J.B.

    1994-05-01

    The major results of our research effort include the determination of the S-Receptor Kinase (SRK) gene structure, the demonstration of S-haplotype-associated SRK polymorphisms and possible co-evolution of SRK and SLG, the characterization of the temporal and spatial expression patterns of SRK, and the demonstration that SRK has intrinsic serine/threonine kinase activity. Our results have indicated that SLG originated from an SRK-like gene by a gene duplication event and suggested a possible molecular basis for leaky S haplotypes. The data have allowed us to develop a model of self-incompatibility based on the interaction of SRK and SLG and the activation of SRK in response to self-pollination. More generally, the information that we have obtained is potentially relevant to understanding mechanisms of signalling inplants. Thus, the interaction of membrane-based receptor protein kinases with secreted forms of their extracellular domains may represent a generalized mechanism by which receptors signal across the plant cell wall.

  18. Sequence analysis of a Molluscum contagiosum virus DNA region which includes the gene encoding protein kinase 2 and other genes with unique organization.

    PubMed

    Martin-Gallardo, A; Moratilla, M; Funes, J M; Agromayor, M; Nuñez, A; Varas, A J; Collado, M; Valencia, A; Lopez-Estebaranz, J L; Esteban, M

    1996-01-01

    The nucleotide sequence of a near left-terminal region from the genome of Molluscum contagiosum virus subtype I (MCVI) was determined. This region was contained within three adjacent BamHI fragments, designated L (2.4 kilobases (kb)), M (1.8 kb), and N (1.6 kb). BamHI cleavage of MCVI DNA produced another 1.6-kb fragment (N'), which had been mapped 30-50 kb from the L,M region. The MCVI restriction fragments were cloned and end-sequenced. The N fragment that maps at the L,M region was identified by the polymerase chain reaction, using primers devised from the sequence of each fragment. The results from this analysis led to establish the relative position of these fragments within the MCVI genome. The analysis of 3.6 kb of DNA sequence revealed the presence of ten open reading frames (ORFs). Comparison of the amino acid sequence of these ORFs to the amino acid sequence of vaccinia virus (VAC) proteins revealed that two complete MCVI ORFs, termed N1L and L1L, showed high degree of homology with VAC F9 and F10 genes, respectively. The F10 gene encodes a 52-kDa serine/threonine protein kinase (protein kinase 2), an essential protein involved in virus morphogenesis. The MCVI homologue (L1L) encoded a putative polypeptide of 443 aa, with a calculated molecular mass of 53 kDa, and 60.5/30.2% sequence identity/similarity to VAC F10. The MCV N1L (213 aa, 24 kDa) showed 42.6/40.6% amino acid sequence identity/similarity to VAC F9, a gene of unknown function encoding a 24-kDa protein with a hydrophobic C-terminal domain, which was conserved in MCVI. The genomic arrangement of MCVI N1L and L1L was equivalent to that of the vaccinia and variola virus homologues. However, the ORFs contained within MCVI fragment M (leftward) showed no homology, neither similarity in genetic organization, to the genes encoded by the corresponding regions of vaccinia and variola viruses.

  19. Splicing of Receptor-Like Kinase-Encoding SNC4 and CERK1 is Regulated by Two Conserved Splicing Factors that Are Required for Plant Immunity

    PubMed Central

    Zhang, Zhibin; Liu, Yanan; Ding, Pingtao; Li, Yan; Kong, Qing; Zhang, Yuelin

    2014-01-01

    Plant immune receptors belonging to the receptor-like kinase (RLK) family play important roles in the recognition of microbial pathogens and activation of downstream defense responses. The Arabidopsis mutant snc4-1D contains a gain-of-function mutation in the RLK SNC4 (SUPPRESSOR OF NPR1-1, CONSTITUTIVE4), which leads to constitutive activation of defense responses. Analysis of suppressor mutants of snc4-1D identified two conserved splicing factors, SUA (SUPPRESSOR OF ABI3-5) and RSN2 (REQUIRED FOR SNC4-1D 2), that are required for the constitutive defense responses in snc4-1D. In sua and rsn2 mutants, SNC4 splicing is altered and the amount of SNC4 transcripts is reduced. Further analysis showed that SUA and RSN2 are also required for the proper splicing of CERK1 (CHITIN ELICITOR RECEPTOR KINASE1), which encodes another RLK that functions as a receptor for chitin. In sua and rsn2 mutants, induction of reactive oxygen species by chitin is reduced and the non-pathogenic bacteria Pseudomonas syringae pv. tomato DC3000hrcC grows to higher titers than in wild-type plants. Our study suggests that pre-mRNA splicing plays important roles in the regulation of plant immunity mediated by the RLKs SNC4 and CERK1. PMID:25267732

  20. Splicing of receptor-like kinase-encoding SNC4 and CERK1 is regulated by two conserved splicing factors that are required for plant immunity.

    PubMed

    Zhang, Zhibin; Liu, Yanan; Ding, Pingtao; Li, Yan; Kong, Qing; Zhang, Yuelin

    2014-12-01

    Plant immune receptors belonging to the receptor-like kinase (RLK) family play important roles in the recognition of microbial pathogens and activation of downstream defense responses. The Arabidopsis mutant snc4-1D contains a gain-of-function mutation in the RLK SNC4 (SUPPRESSOR OF NPR1-1, CONSTITUTIVE4), which leads to constitutive activation of defense responses. Analysis of suppressor mutants of snc4-1D identified two conserved splicing factors, SUA (SUPPRESSOR OF ABI3-5) and RSN2 (REQUIRED FOR SNC4-1D 2), that are required for the constitutive defense responses in snc4-1D. In sua and rsn2 mutants, SNC4 splicing is altered and the amount of SNC4 transcripts is reduced. Further analysis showed that SUA and RSN2 are also required for the proper splicing of CERK1 (CHITIN ELICITOR RECEPTOR KINASE1), which encodes another RLK that functions as a receptor for chitin. In sua and rsn2 mutants, induction of reactive oxygen species by chitin is reduced and the non-pathogenic bacteria Pseudomonas syringae pv. tomato DC3000hrcC grows to higher titers than in wild-type plants. Our study suggests that pre-mRNA splicing plays important roles in the regulation of plant immunity mediated by the RLKs SNC4 and CERK1. PMID:25267732

  1. The tricornered gene, which is required for the integrity of epidermal cell extensions, encodes the Drosophila nuclear DBF2-related kinase.

    PubMed

    Geng, W; He, B; Wang, M; Adler, P N

    2000-12-01

    During their differentiation epidermal cells of Drosophila form a rich variety of polarized structures. These include the epidermal hairs that decorate much of the adult cuticular surface, the shafts of the bristle sense organs, the lateral extensions of the arista, and the larval denticles. These cuticular structures are produced by cytoskeletal-mediated outgrowths of epidermal cells. Mutations in the tricornered gene result in the splitting or branching of all of these structures. Thus, tricornered function appears to be important for maintaining the integrity of the outgrowths. tricornered mutations however do not have major effects on the growth or shape of these cellular extensions. Inhibiting actin polymerization in differentiating cells by cytochalasin D or latrunculin A treatment also induces the splitting of hairs and bristles, suggesting that the actin cytoskeleton might be a target of tricornered. However, the drugs also result in short, fat, and occasionally malformed hairs and bristles. The data suggest that the function of the actin cytoskeleton is important for maintaining the integrity of cellular extensions as well as their growth and shape. Thus, if tricornered causes the splitting of cellular extensions by interacting with the actin cytoskeleton it likely does so in a subtle way. Consistent with this possibility we found that a weak tricornered mutant is hypersensitive to cytochalasin D. We have cloned the tricornered gene and found that it encodes the Drosophila NDR kinase. This is a conserved ser/thr protein kinase found in Caenorhabditis elegans and humans that is related to a number of kinases that have been found to be important in controlling cell structure and proliferation.

  2. Cloning and characterisation of the pknD gene encoding an eukaryotic-type protein kinase in the cyanobacterium Anabaena sp. PCC7120.

    PubMed

    Zhang, C C; Libs, L

    1998-04-01

    Protein phosphorylation catalysed by protein kinases is an important mechanism for signal transduction in both prokaryotes and eukaryotes. A novel gene, pknD, encoding a protein similar to eukaryotic-type protein kinases, was cloned from Anabaena sp. PCC7120. The N-terminal region of PknD is 60% identical to that of PknA, another putative Ser/Thr kinase from the same strain. Both PknA and PknD have C-terminal regions that are rich in Pro and Thr residues. Expression of pknD was undetectable by RNA/DNA hybridisation and was thus examined by RT-PCR. The pknD transcript was detected in filaments cultured in the presence of either nitrate or ammonium as a source of combined nitrogen, and also in filaments transferred from nitrate-sufficient to N2-fixing conditions. pknD mutants were created, and their growth characteristics under different nitrogen regimes and their capacity for heterocyst development were investigated. The growth rates of the mutants were similar to those of the wild-type strain in the presence of either nitrate or ammonium, but were only 20% that of the wild type under N2-fixing conditions. The rate of nitrogenase activity is normal in pknD mutant under aerobic conditions. Under nitrogen-fixing conditions, the inactivation of pknD led to enhanced modification of the PII protein compared to the weak phosphorylation of PII observed in the wild-type strain. This high level of PII phosphorylation in the pknD mutant is reminiscent of the situation in nitrogen-starved Synechococcus PCC7942 cells. PknD might be involved in regulating nitrogen metabolism or nitrogen trafficking from heterocysts to vegetative cells.

  3. The tricornered gene, which is required for the integrity of epidermal cell extensions, encodes the Drosophila nuclear DBF2-related kinase.

    PubMed Central

    Geng, W; He, B; Wang, M; Adler, P N

    2000-01-01

    During their differentiation epidermal cells of Drosophila form a rich variety of polarized structures. These include the epidermal hairs that decorate much of the adult cuticular surface, the shafts of the bristle sense organs, the lateral extensions of the arista, and the larval denticles. These cuticular structures are produced by cytoskeletal-mediated outgrowths of epidermal cells. Mutations in the tricornered gene result in the splitting or branching of all of these structures. Thus, tricornered function appears to be important for maintaining the integrity of the outgrowths. tricornered mutations however do not have major effects on the growth or shape of these cellular extensions. Inhibiting actin polymerization in differentiating cells by cytochalasin D or latrunculin A treatment also induces the splitting of hairs and bristles, suggesting that the actin cytoskeleton might be a target of tricornered. However, the drugs also result in short, fat, and occasionally malformed hairs and bristles. The data suggest that the function of the actin cytoskeleton is important for maintaining the integrity of cellular extensions as well as their growth and shape. Thus, if tricornered causes the splitting of cellular extensions by interacting with the actin cytoskeleton it likely does so in a subtle way. Consistent with this possibility we found that a weak tricornered mutant is hypersensitive to cytochalasin D. We have cloned the tricornered gene and found that it encodes the Drosophila NDR kinase. This is a conserved ser/thr protein kinase found in Caenorhabditis elegans and humans that is related to a number of kinases that have been found to be important in controlling cell structure and proliferation. PMID:11102376

  4. Imprinting of the gene encoding a human cyclin-dependent kinase inhibitor, p57KIP2, on chromosome 11p15.

    PubMed Central

    Matsuoka, S; Thompson, J S; Edwards, M C; Bartletta, J M; Grundy, P; Kalikin, L M; Harper, J W; Elledge, S J; Feinberg, A P

    1996-01-01

    Parental origin-specific alterations of chromosome 11p15 in human cancer suggest the involvement of one or more maternally expressed imprinted genes involved in embryonal tumor suppression and the cancer-predisposing Beckwith-Wiedemann syndrome (BWS). The gene encoding cyclin-dependent kinase inhibitor p57KIP2, whose overexpression causes G1 phase arrest, was recently cloned and mapped to this band. We find that the p57KIP2 gene is imprinted, with preferential expression of the maternal allele. However, the imprint is not absolute, as the paternal allele is also expressed at low levels in most tissues, and at levels comparable to the maternal allele in fetal brain and some embryonal tumors. The biochemical function, chromosomal location, and imprinting of the p57KIP2 gene match the properties predicted for a tumor suppressor gene at 11p15.5. However, as the p57KIP2 gene is 500 kb centromeric to the gene encoding insulin-like growth factor 2, it is likely to be part of a large domain containing other imprinted genes. Thus, loss of heterozygosity or loss of imprinting might simultaneously affect several genes at this locus that together contribute to tumor and/or growth- suppressing functions that are disrupted in BWS and embryonal tumors. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8610162

  5. Localization of eight additional genes in the human major histocompatibility complex, including the gene encoding the casein kinase II {beta} subunit (CSNK2B)

    SciTech Connect

    Albertella, M.R.; Jones, H.; Thomson, W.

    1996-09-01

    A wide range of autoimmune and other diseases are known to be associated with the major histocompatibility complex. Many of these diseases are linked to the genes encoding the polymorphic histocompatibility complex. Many of these diseases are linked to the genes encoding the polymorphic histocompatibility antigens in the class I and class II regions, but some appear to be more strongly associated with genes in the central 1100-kb class III region, making it important to characterize this region fully for the presence of novel genes. An {approximately}220-kb segment of DNA in the class III region separating the Hsp70 (HSPA1L) and BAT1 (D6S8IE) genes, which was previously known to contain 14 genes. Genomic DNA fragments spanning the gaps between the known genes were used as probes to isolate cDNAs corresponding to five new genes within this region. Evidence from Northern blot analysis and exon trapping experiments that suggested the presence of at least two more new genes was also obtained. Partial cDNA and complete exonic genomic sequencing of one of the new genes has identified it as the casein kinase II{beta} subunit (CSNK2B). Two of the other novel genes lie within a region syntenic to that implicated in susceptibility to experimental allergic orchitis in the mouse, an autoimmune disease of the testis, and represent additional candidates for the Orch-1 locus associated with this disease. In addition, characterization of the 13-kb intergenic gap separating the RD (D6545) and G11 (D6S60E) genes has revealed the presence of a gene encoding a 1246-amino-acid polypeptide that shows significant sequence similarity to the yeast anti-viral Ski2p gene product. 49 refs., 8 figs.

  6. Mutations in TBCK, Encoding TBC1-Domain-Containing Kinase, Lead to a Recognizable Syndrome of Intellectual Disability and Hypotonia

    PubMed Central

    Bhoj, Elizabeth J.; Li, Dong; Harr, Margaret; Edvardson, Shimon; Elpeleg, Orly; Chisholm, Elizabeth; Juusola, Jane; Douglas, Ganka; Guillen Sacoto, Maria J.; Siquier-Pernet, Karine; Saadi, Abdelkrim; Bole-Feysot, Christine; Nitschke, Patrick; Narravula, Alekhya; Walke, Maria; Horner, Michele B.; Day-Salvatore, Debra-Lynn; Jayakar, Parul; Vergano, Samantha A. Schrier; Tarnopolsky, Mark A.; Hegde, Madhuri; Colleaux, Laurence; Crino, Peter; Hakonarson, Hakon

    2016-01-01

    Through an international multi-center collaboration, 13 individuals from nine unrelated families and affected by likely pathogenic biallelic variants in TBC1-domain-containing kinase (TBCK) were identified through whole-exome sequencing. All affected individuals were found to share a core phenotype of intellectual disability and hypotonia, and many had seizures and showed brain atrophy and white-matter changes on neuroimaging. Minor non-specific facial dysmorphism was also noted in some individuals, including multiple older children who developed coarse features similar to those of storage disorders. TBCK has been shown to regulate the mammalian target of rapamycin (mTOR) signaling pathway, which is also stimulated by exogenous leucine supplementation. TBCK was absent in cells from affected individuals, and decreased phosphorylation of phospho-ribosomal protein S6 was also observed, a finding suggestive of downregulation of mTOR signaling. Lastly, we demonstrated that activation of the mTOR pathway in response to L-leucine supplementation was retained, suggesting a possible avenue for directed therapies for this condition. PMID:27040691

  7. Rickettsia prowazekii transports UMP and GMP, but not CMP, as building blocks for RNA synthesis.

    PubMed

    Winkler, H H; Daugherty, R; Hu, F

    1999-05-01

    Rickettsia prowazekii, the etiological agent of epidemic typhus, is an obligate intracellular bacterium and is apparently unable to synthesize ribonucleotides de novo. Here, we show that as an alternative, isolated, purified R. prowazekii organisms transported exogenous uridyl- and guanylribonucleotides and incorporated these labeled precursors into their RNA in a rifampin-sensitive manner. Transport systems for nucleotides, which we have shown previously and show here are present in rickettsiae, have never been reported in free-living bacteria, and the usual nucleobase and nucleoside transport systems are absent in rickettsiae. There was a clear preference for the monophosphate form of ribonucleotides as the transported substrate. In contrast, rickettsiae did not transport cytidylribonucleotides. The source of rickettsial CTP appears to be the transport of UMP followed by its phosphorylation and the amination of intrarickettsial UTP to CTP by CTP synthetase. A complete schema of nucleotide metabolism in rickettsiae is presented that is based on a combination of biochemical, physiological, and genetic information. PMID:10322027

  8. Modulated expression of genes encoding estrogen metabolizing enzymes by G1-phase cyclin-dependent kinases 6 and 4 in human breast cancer cells.

    PubMed

    Jia, Yi; Domenico, Joanne; Swasey, Christina; Wang, Meiqin; Gelfand, Erwin W; Lucas, Joseph J

    2014-01-01

    G1-phase cell cycle defects, such as alterations in cyclin D1 or cyclin-dependent kinase (cdk) levels, are seen in most tumors. For example, increased cyclin D1 and decreased cdk6 levels are seen in many human breast tumors. Overexpression of cdk6 in breast tumor cells in culture has been shown to suppress proliferation, unlike the growth stimulating effects of its close homolog, cdk4. In addition to directly affecting proliferation, alterations in cdk6 or cdk4 levels in breast tumor cells also differentially influence levels of numerous steroid metabolic enzymes (SMEs), including those involved in estrogen metabolism. Overexpression of cdk6 in tumor cell lines having low cdk6 resulted in decreased levels of mRNAs encoding aldo-keto reductase (AKR)1C1, AKR1C2 and AKR1C3, which are hydroxysteroid dehydrogenases (HSDs) involved in steroid hormone metabolism. In contrast, increasing cdk4 dramatically increased these transcript levels, especially those encoding AKR1C3, an enzyme that converts estrone to 17β-estradiol, a change that could result in a pro-estrogenic state favoring tumor growth. Effects on other estrogen metabolizing enzymes, including cytochrome P450 (CYP) 19 aromatase, 17β-HSD2, and CYP1B1 transcripts, were also observed. Interactions of cdk6 and cdk4, but not cyclin D1, with the promoter region of a cdk-regulated gene, 17β-HSD2, were detected. The results uncover a previously unsuspected link between the cell cycle and hormone metabolism and differential roles for cdk6 and cdk4 in a novel mechanism for pre-receptor control of steroid hormone action, with important implications for the origin and treatment of steroid hormone-dependent cancers. PMID:24848372

  9. Mutations and polymorphisms in the gene encoding regulatory subunit type 1-alpha of protein kinase A (PRKAR1A): an update.

    PubMed

    Horvath, Anélia; Bertherat, Jérôme; Groussin, Lionel; Guillaud-Bataille, Marine; Tsang, Kitman; Cazabat, Laure; Libé, Rosella; Remmers, Elaine; René-Corail, Fernande; Faucz, Fabio Rueda; Clauser, Eric; Calender, Alain; Bertagna, Xavier; Carney, J Aidan; Stratakis, Constantine A

    2010-04-01

    PRKAR1A encodes the regulatory subunit type 1-alpha (RIalpha) of the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA). Inactivating PRKAR1A mutations are known to be responsible for the multiple neoplasia and lentiginosis syndrome Carney complex (CNC). To date, at least 117 pathogenic variants in PRKAR1A have been identified (online database: http://prkar1a.nichd.nih.gov). The majority are subject to nonsense mediated mRNA decay (NMD), leading to RIalpha haploinsufficiency and, as a result, activated cAMP signaling. Recently, it became apparent that CNC may be caused not only by RIalpha haploinsufficiency, but also by the expression of altered RIalpha protein, as proven by analysis of expressed mutations in the gene, consisting of amino acid substitutions and in-frame genetic alterations. In addition, a new subgroup of mutations that potentially escape NMD and result in CNC through altered (rather than missing) protein has been analyzed-these are frame-shifts in the 3' end of the coding sequence that shift the stop codon downstream of the normal one. The mutation detection rate in CNC patients is recently estimated at above 60%; PRKAR1A mutation-negative CNC patients are characterized by significant phenotypic heterogeneity. In this report, we present a comprehensive analysis of all presently known PRKAR1A sequence variations and discuss their molecular context and clinical phenotype. PMID:20358582

  10. Molecular and biochemical identification of inositol 1,3,4,5,6-pentakisphosphate 2-kinase encoding mRNA variants in castor bean (Ricinus communis L.) seeds.

    PubMed

    Yu, Jaeju; Saiardi, Adolfo; Greenwood, John S; Bewley, J Derek

    2014-05-01

    During seed development, phytic acid (PA) associated with mineral cations is stored as phytin and mobilized following germination in support of seedling growth. Two parallel biosynthetic pathways for PA have been proposed; yet the pathway is still poorly understood in terms of its regulation and the enzymes involved. Here, the castor bean (Ricinus communis L.) gene for inositol 1,3,4,5,6-pentakisphosphate 2-kinase (RcIPK1) has been identified. This encodes the enzyme implicated in catalyzing the final reaction in PA biosynthesis, and its expression is enhanced in isolated germinated embryos by application of phosphate and myo-inositol (Ins). Even though only one copy of the RcIPK1 gene is present in the genome, numerous RNA variants are present, most likely due to alternative splicing. These are translated into six closely related protein isoforms according to in silico analysis. Functional analyses using yeast ipk1Δ revealed that only three of the mRNA variants can rescue a temperature-sensitive growth phenotype of this strain. High-performance liquid chromatography (HPLC) analysis of the synthesized inositol phosphates demonstrated that the ability to complement the missing yeast IPK1 enzyme is associated with the production of enzyme activity. The three active isoforms possess unique conserved motifs important for IPK1 catalytic activity.

  11. The Role of the Equine Herpesvirus Type 1 (EHV-1) US3-Encoded Protein Kinase in Actin Reorganization and Nuclear Egress

    PubMed Central

    Proft, Alexandra; Spiesschaert, Bart; Izume, Satoko; Taferner, Selina; Lehmann, Maik J.; Azab, Walid

    2016-01-01

    The serine-threonine protein kinase encoded by US3 gene (pUS3) of alphaherpesviruses was shown to modulate actin reorganization, cell-to-cell spread, and virus egress in a number of virus species. However, the role of the US3 orthologues of equine herpesvirus type 1 and 4 (EHV-1 and EHV-4) has not yet been studied. Here, we show that US3 is not essential for virus replication in vitro. However, growth rates and plaque diameters of a US3-deleted EHV-1 and a mutant in which the catalytic active site was destroyed were significantly reduced when compared with parental and revertant viruses or a virus in which EHV-1 US3 was replaced with the corresponding EHV-4 gene. The reduced plaque sizes were consistent with accumulation of primarily enveloped virions in the perinuclear space of the US3-negative EHV-1, a phenotype that was also rescued by the EHV-4 orthologue. Furthermore, actin stress fiber disassembly was significantly more pronounced in cells infected with parental EHV-1, revertant, or the recombinant EHV-1 expressing EHV-4 US3. Finally, we observed that deletion of US3 in EHV-1 did not affect the expression of adhesion molecules on the surface of infected cells. PMID:27754319

  12. Molecular and biochemical identification of inositol 1,3,4,5,6-pentakisphosphate 2-kinase encoding mRNA variants in castor bean (Ricinus communis L.) seeds.

    PubMed

    Yu, Jaeju; Saiardi, Adolfo; Greenwood, John S; Bewley, J Derek

    2014-05-01

    During seed development, phytic acid (PA) associated with mineral cations is stored as phytin and mobilized following germination in support of seedling growth. Two parallel biosynthetic pathways for PA have been proposed; yet the pathway is still poorly understood in terms of its regulation and the enzymes involved. Here, the castor bean (Ricinus communis L.) gene for inositol 1,3,4,5,6-pentakisphosphate 2-kinase (RcIPK1) has been identified. This encodes the enzyme implicated in catalyzing the final reaction in PA biosynthesis, and its expression is enhanced in isolated germinated embryos by application of phosphate and myo-inositol (Ins). Even though only one copy of the RcIPK1 gene is present in the genome, numerous RNA variants are present, most likely due to alternative splicing. These are translated into six closely related protein isoforms according to in silico analysis. Functional analyses using yeast ipk1Δ revealed that only three of the mRNA variants can rescue a temperature-sensitive growth phenotype of this strain. High-performance liquid chromatography (HPLC) analysis of the synthesized inositol phosphates demonstrated that the ability to complement the missing yeast IPK1 enzyme is associated with the production of enzyme activity. The three active isoforms possess unique conserved motifs important for IPK1 catalytic activity. PMID:24463774

  13. A protein kinase C-encoding gene, pkcA, is essential to the viability of the filamentous fungus Aspergillus nidulans.

    PubMed

    Ichinomiya, Masayuki; Uchida, Hirotaka; Koshi, Yukako; Ohta, Akinori; Horiuchi, Hiroyuki

    2007-11-01

    A protein kinase C (PKC)-encoding gene (pkcA) was isolated from the filamentous fungus Aspergillus nidulans. Although we attempted to isolate pkcA deletion mutants, we obtained only heterokaryons that had both DeltapkcA and pkcA(+) nuclei. Conidia produced by the heterokaryon germinated. The germ tubes, however, lysed frequently and no colony formation was observed, indicating that the pkcA gene is essential to the viability of A. nidulans. We constructed conditional mutants (alcA(p)-pkcA mutants) that expressed pkcA under the control of the alcA promoter (alcA(p)). Under alcA(p)-repressing conditions, their colonies were smaller than those of the wild-type strains and their hyphae lysed frequently. These phenotypes were not remedied under moderate- or high-osmolarity conditions; the growth defect deteriorated further under the latter. Under alcA(p)-inducing conditions, the alcA(p)-pkcA mutants also showed growth-sensitivity to cell wall destabilizing agents. These results indicate that pkcA plays an important role in the maintenance of cell integrity.

  14. Domain structure of the large subunit of Escherichia coli carbamoyl phosphate synthetase. Location of the binding site for the allosteric inhibitor UMP in the COOH-terminal domain.

    PubMed

    Rubio, V; Cervera, J; Lusty, C J; Bendala, E; Britton, H G

    1991-01-29

    The large subunit of Escherichia coli carbamoyl phosphate synthetase (a polypeptide of 117.7 kDa that consists of two homologous halves) is responsible for carbamoyl phosphate synthesis from NH3 and for the binding of the allosteric activators ornithine and IMP and of the inhibitor UMP. Elastase, trypsin, and chymotrypsin inactivate the enzyme and cleave the large subunit at a site approximately 15 kDa from the COOH terminus (demonstrated by NH2-terminal sequencing). UMP, IMP, and ornithine prevent this cleavage and the inactivation. Upon irradiation with ultraviolet light in the presence of [14C]UMP, the large subunit is labeled selectively and specifically. The labeling is inhibited by ornithine and IMP. Cleavage of the 15-kDa COOH-terminal region by prior treatment of the enzyme with trypsin prevents the labeling on subsequent irradiation with [14C]UMP. The [14C]UMP-labeled large subunit is resistant to proteolytic cleavage, but if it is treated with SDS the resistance is lost, indicating that UMP is cross-linked to its binding site and that the protection is due to conformational factors. In the presence of SDS, the labeled large subunit is cleaved by trypsin or by V8 staphylococcal protease at a site located 15 or 25 kDa, respectively, from the COOH terminus (shown by NH2-terminal sequencing), and only the 15- or 25-kDa fragments are labeled. Similarly, upon cleavage of the aspartyl-prolyl bonds of the [14C]UMP-labeled enzyme with 70% formic acid, labeling was found only in the 18.5-kDa fragment that contains the COOH terminus of the subunit. Thus, UMP binds to the COOH-terminal domain.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1989678

  15. Trypanosoma brucei (UMP synthase null mutants) are avirulent in mice, but recover virulence upon prolonged culture in vitro while retaining pyrimidine auxotrophy

    PubMed Central

    Ong, Han B; Sienkiewicz, Natasha; Wyllie, Susan; Patterson, Stephen; Fairlamb, Alan H

    2013-01-01

    African trypanosomes are capable of both de novo synthesis and salvage of pyrimidines. The last two steps in de novo synthesis are catalysed by UMP synthase (UMPS) – a bifunctional enzyme comprising orotate phosphoribosyl transferase (OPRT) and orotidine monophosphate decarboxylase (OMPDC). To investigate the essentiality of pyrimidine biosynthesis in Trypanosoma brucei, we generated a umps double knockout (DKO) line by gene replacement. The DKO was unable to grow in pyrimidine-depleted medium in vitro, unless supplemented with uracil, uridine, deoxyuridine or UMP. DKO parasites were completely resistant to 5-fluoroorotate and hypersensitive to 5-fluorouracil, consistent with loss of UMPS, but remained sensitive to pyrazofurin indicating that, unlike mammalian cells, the primary target of pyrazofurin is not OMPDC. The null mutant was unable to infect mice indicating that salvage of host pyrimidines is insufficient to support growth. However, following prolonged culture in vitro, parasites regained virulence in mice despite retaining pyrimidine auxotrophy. Unlike the wild-type, both pyrimidine auxotrophs secreted substantial quantities of orotate, significantly higher in the virulent DKO line. We propose that this may be responsible for the recovery of virulence in mice, due to host metabolism converting orotate to uridine, thereby bypassing the loss of UMPS in the parasite. PMID:23980694

  16. The Aspergillus niger D-xylulose kinase gene is co-expressed with genes encoding arabinan degrading enzymes, and is essential for growth on D-xylose and L-arabinose.

    PubMed

    vanKuyk, P A; de Groot, M J; Ruijter, G J; de Vries, R P; Visser, J

    2001-10-01

    The Aspergillus niger D-xylulose kinase encoding gene has been cloned by complementation of a strain deficient in D-xylulose kinase activity. Expression of xkiA was observed in the presence of L-arabinose, L-arabitol and D-xylose. Expression of xkiA is not mediated by XLNR, the xylose-dependent positively-acting xylanolytic regulator. Although the expression of xkiA is subject to carbon catabolite repression, the wide domain regulator CREA is not directly involved. The A. niger D-xylulose kinase was purified to homogeneity, and the molecular mass determined using electrospray ionization mass spectrometry agreed with the calculated molecular mass of 62816.6 Da. The activity of XKIA is highly specific for D-xylulose. Kinetic parameters were determined as Km(D-xylulose) = 0.76 mM and Km(ATP) = 0.061 mM. Increased transcript levels of the genes encoding arabinan and xylan degrading enzymes, observed in the xylulose kinase deficient strain, correlate with increased accumulation of L-arabitol and xylitol, respectively. This result supports the suggestion that L-arabitol may be the specific low molecular mass inducer of the genes involved in arabinan degradation. It also suggests a possible role for xylitol in the induction of xylanolytic genes. Conversely, overproduction of XKIA did not reduce the size of the intracellular arabitol and xylitol pools, and therefore had no effect on expression of genes encoding xylan and arabinan degrading enzymes nor on the activity of the enzymes of the catabolic pathway.

  17. Domain structure of the large subunit of Escherichia coli carbamoyl phosphate synthetase. Location of the binding site for the allosteric inhibitor UMP in the COOH-terminal domain

    SciTech Connect

    Rubio, V.; Cervera, J.; Bendala, E. ); Lusty, C.J. ); Britton, H.G. )

    1991-01-29

    The large subunit of Escherichia coli carbamoyl phosphate synthetase is responsible for carbamoyl phosphate synthesis from NH{sub 3} and for the binding of the allosteric activators ornithine and IMP and of the inhibitor UMP. Elastase, trypsin, and chymotrypsin inactivate the enzyme and cleave the large subunit at a site approximately 15 kDa from the COOH terminus UMP, IMP, and ornithine prevent this cleavage and the inactivation. Upon irradiation with ultraviolet light in the presence of ({sup 14}C)UMP, the large subunit is labeled selectively and specifically. The labeling is inhibited by ornithine and IMP. Cleavage of the 15-kDa COOH-terminal region by prior treatment of the enzyme with trypsin prevents the labeling on subsequent irradation with ({sup 14}C)UMP. The ({sup 14}C)UMP-labeled large subunit is resistant to proteolytic cleavage, but if it is treated with SDS the resistance is lost, indicating that UMP is cross-linked to its binding site and that the protection is due to conformational factors. Since the binding sites for IMP and UMP overlap, most probably IMP also binds in this domain. The protection from proteolysis by ornithine suggests that ornithine binds in the same domain. To account for the effects of the allosteric effectors on the binding of ATP, the authors propose a scheme where the two halves of the large subunit form a pseudohomodimer by complementary isologous association, thus placing the NH{sub 2} half, which is involved in the binding of the molecule of ATP that yields P{sub i}, close to the regulatory domain.

  18. [Characterization of a putative S locus encoded receptor protein kinase and its role in self-incompatibility]. Progress report, January 1993

    SciTech Connect

    Not Available

    1993-06-01

    The serine/threonine protein kinase (SRK) protein was predicted to be similar to the growth factor receptor tyrosine kinases in animals but its amino acid sequence of the catalytic domain is more similar to that of the catalytic domains of protein serine/threonine kinases than to protein tyrosine kinases. We have shown that the SRK protein has intrinsic scrine/threonine kinase activity. We subcloned the protein kinase-homologous domain of the SRK{sub 6} cDNA into the bacterial expression vector pGEX-3X and we have constructed a second plasmid identical to the first except that it carried a conservative mutation that substituted Arg for the Lys{sup 524} codon of SRK6 This lysine corresponds to the ATP-binding site, is essential in protein kinases, and is a common target for site-directed mutagenesis as a means to obtain kinase-defective proteins. Cultures bearing the wild-type and mutant SRK catalytic domains each produced an approximately 64 kD protein that reacted with anti-SRK6 antibodies. Following pulse-labeling with {sup 32}P we found that the wild-type SRK6 protein but not the mutant form was detectably phosphorylated. Phosphoamino acid analysis of the affinity purified {sup 32}p-labeled GST-SRK6 fusion protein demonstrated that SRK was phosphorylated predominantly on semine and to a lesser extent on threonine, but not on tyrosine. Thus, SRK6 is a functional serine/threonine protein kinase.

  19. Ethanol yield and volatile compound content in fermentation of agave must by Kluyveromyces marxianus UMPe-1 comparing with Saccharomyces cerevisiae baker's yeast used in tequila production.

    PubMed

    López-Alvarez, Arnoldo; Díaz-Pérez, Alma Laura; Sosa-Aguirre, Carlos; Macías-Rodríguez, Lourdes; Campos-García, Jesús

    2012-05-01

    In tequila production, fermentation is an important step. Fermentation determines the ethanol productivity and organoleptic properties of the beverage. In this study, a yeast isolated from native residual agave must was identified as Kluyveromyces marxianus UMPe-1 by 26S rRNA sequencing. This yeast was compared with the baker's yeast Saccharomyces cerevisiae Pan1. Our findings demonstrate that the UMPe-1 yeast was able to support the sugar content of agave must and glucose up to 22% (w/v) and tolerated 10% (v/v) ethanol concentration in the medium with 50% cells survival. Pilot and industrial fermentation of agave must tests showed that the K. marxianus UMPe-1 yeast produced ethanol with yields of 94% and 96% with respect to fermentable sugar content (glucose and fructose, constituting 98%). The S. cerevisiae Pan1 baker's yeast, however, which is commonly used in some tequila factories, showed 76% and 70% yield. At the industrial level, UMPe-1 yeast shows a maximum velocity of fermentable sugar consumption of 2.27g·L(-1)·h(-1) and ethanol production of 1.38g·L(-1)·h(-1), providing 58.78g ethanol·L(-1) at 72h fermentation, which corresponds to 96% yield. In addition, the major and minor volatile compounds in the tequila beverage obtained from UMPe-1 yeast were increased. Importantly, 29 volatile compounds were identified, while the beverage obtained from Pan1-yeast contained fewer compounds and in lower concentrations. The results suggest that the K. marxianus UMPe-1 is a suitable yeast for agave must fermentation, showing high ethanol productivity and increased volatile compound content comparing with a S. cerevisiae baker's yeast used in tequila production.

  20. Ethanol yield and volatile compound content in fermentation of agave must by Kluyveromyces marxianus UMPe-1 comparing with Saccharomyces cerevisiae baker's yeast used in tequila production.

    PubMed

    López-Alvarez, Arnoldo; Díaz-Pérez, Alma Laura; Sosa-Aguirre, Carlos; Macías-Rodríguez, Lourdes; Campos-García, Jesús

    2012-05-01

    In tequila production, fermentation is an important step. Fermentation determines the ethanol productivity and organoleptic properties of the beverage. In this study, a yeast isolated from native residual agave must was identified as Kluyveromyces marxianus UMPe-1 by 26S rRNA sequencing. This yeast was compared with the baker's yeast Saccharomyces cerevisiae Pan1. Our findings demonstrate that the UMPe-1 yeast was able to support the sugar content of agave must and glucose up to 22% (w/v) and tolerated 10% (v/v) ethanol concentration in the medium with 50% cells survival. Pilot and industrial fermentation of agave must tests showed that the K. marxianus UMPe-1 yeast produced ethanol with yields of 94% and 96% with respect to fermentable sugar content (glucose and fructose, constituting 98%). The S. cerevisiae Pan1 baker's yeast, however, which is commonly used in some tequila factories, showed 76% and 70% yield. At the industrial level, UMPe-1 yeast shows a maximum velocity of fermentable sugar consumption of 2.27g·L(-1)·h(-1) and ethanol production of 1.38g·L(-1)·h(-1), providing 58.78g ethanol·L(-1) at 72h fermentation, which corresponds to 96% yield. In addition, the major and minor volatile compounds in the tequila beverage obtained from UMPe-1 yeast were increased. Importantly, 29 volatile compounds were identified, while the beverage obtained from Pan1-yeast contained fewer compounds and in lower concentrations. The results suggest that the K. marxianus UMPe-1 is a suitable yeast for agave must fermentation, showing high ethanol productivity and increased volatile compound content comparing with a S. cerevisiae baker's yeast used in tequila production. PMID:22280963

  1. The Crystal Structure of the Leishmania major Deoxyuridine Triphosphate Nucleotidohydrolase in Complex with Nucleotide Analogues, dUMP, and Deoxyuridine*

    PubMed Central

    Hemsworth, Glyn R.; Moroz, Olga V.; Fogg, Mark J.; Scott, Benjamin; Bosch-Navarrete, Cristina; González-Pacanowska, Dolores; Wilson, Keith S.

    2011-01-01

    Members of the Leishmania genus are the causative agents of the life-threatening disease leishmaniasis. New drugs are being sought due to increasing resistance and adverse side effects with current treatments. The knowledge that dUTPase is an essential enzyme and that the all α-helical dimeric kinetoplastid dUTPases have completely different structures compared with the trimeric β-sheet type dUTPase possessed by most organisms, including humans, make the dimeric enzymes attractive drug targets. Here, we present crystal structures of the Leishmania major dUTPase in complex with substrate analogues, the product dUMP and a substrate fragment, and of the homologous Campylobacter jejuni dUTPase in complex with a triphosphate substrate analogue. The metal-binding properties of both enzymes are shown to be dependent upon the ligand identity, a previously unseen characteristic of this family. Furthermore, structures of the Leishmania enzyme in the presence of dUMP and deoxyuridine coupled with tryptophan fluorescence quenching indicate that occupation of the phosphate binding region is essential for induction of the closed conformation and hence for substrate binding. These findings will aid in the development of dUTPase inhibitors as potential new lead anti-trypanosomal compounds. PMID:21454646

  2. The genes encoding the Eph-related receptor tyrosine kinase ligands LERK-1 (EPLG1, Epl1), LERK-3 (EPLG3, Epl3), and LERK-4 (EPLG4, Epl4) are clustered on human chromosome 1 and mouse chromosome 3

    SciTech Connect

    Cerretti, D.P.; Lyman, S.D.; Kozlosky, C.J.

    1996-04-15

    Hek and elk are members of the eph-related family of receptor tyrosine kinases. Recently, we isolated five cDNAs encoding membrane-bound ligands to hek and elk. Because of the promiscuous nature of their binding, we have termed these proteins ligands of the eph-related kinases or LERKs. The LERKs can be divided into two subgroups by virtue of their sequence identity, binding properties, and mode of cell membrane attachment. For example, LERK-2 (EPLG2, Epl2) and LERK-5 (EPLG5, Epl5) are type 1 transmembrane proteins, while LERK-1 (EPLG4, Epl4) are anchored to the membrane by glycosyl-phosphatidylinositol (GPI) linkage. Using Southern hybridization analysis of human x rodent somatic cell hybrid DNAs, we have assigned the genes that encode the GPI-anchored LERKs (EPLG1, EPLG3, and EPLG4) to human chromosome 1. Fluorescence in situ hybridization to metaphase chromosome preparations using genomic clones from each locus refined this localization to chromosome 1, bands q21-q22. In addition, Southern blot analysis of DNA from interspecific backcross mice indicated that the mouse homologues Epl1, Epl3, and Epl4 map to a homologous region on mouse chromosome 3. 36 refs., 2 figs.

  3. Functional characterization of the MKC1 gene of Candida albicans, which encodes a mitogen-activated protein kinase homolog related to cell integrity.

    PubMed Central

    Navarro-García, F; Sánchez, M; Pla, J; Nombela, C

    1995-01-01

    Mitogen-activated protein (MAP) kinases represent a group of serine/threonine protein kinases playing a central role in signal transduction processes in eukaryotic cells. Using a strategy based on the complementation of the thermosensitive autolytic phenotype of slt2 null mutants, we have isolated a Candida albicans homolog of Saccharomyces cerevisiae MAP kinase gene SLT2 (MPK1), which is involved in the recently outlined PKC1-controlled signalling pathway. The isolated gene, named MKC1 (MAP kinase from C. albicans), coded for a putative protein, Mkc1p, of 58,320 Da that displayed all the characteristic domains of MAP kinases and was 55% identical to S. cerevisiae Slt2p (Mpk1p). The MKC1 gene was deleted in a diploid Candida strain, and heterozygous and homozygous strains, in both Ura+ and Ura- backgrounds, were obtained to facilitate the analysis of the function of the gene. Deletion of the two alleles of the MKC1 gene gave rise to viable cells that grew at 28 and 37 degrees C but, nevertheless, displayed a variety of phenotypic traits under more stringent conditions. These included a low growth yield and a loss of viability in cultures grown at 42 degrees C, a high sensitivity to thermal shocks at 55 degrees C, an enhanced susceptibility to caffeine that was osmotically remediable, and the formation of a weak cell wall with a very low resistance to complex lytic enzyme preparations. The analysis of the functions downstream of the MKC1 gene should contribute to understanding of the connection of growth and morphogenesis in pathogenic fungi. PMID:7891715

  4. A novel oncogene, v-ryk, encoding a truncated receptor tyrosine kinase is transduced into the RPL30 virus without loss of viral sequences.

    PubMed Central

    Jia, R; Mayer, B J; Hanafusa, T; Hanafusa, H

    1992-01-01

    The RPL viruses are acute oncogenic avian retroviruses isolated from chicken tumors. We carried out a genetic analysis of three of the viruses, RPL25, RPL28, and RPL30. While RPL25 and RPL28 were shown to contain the erbB oncogene, RPL30 appeared to contain a novel protein tyrosine kinase oncogene. This gene, v-ryk, was cloned and sequenced. The v-ryk oncogene contains a 1.39-kb nonretroviral sequence that includes a tyrosine kinase domain which was inserted into the viral envelope protein gp37-coding region and fused in frame with upstream gp37 to generate a P69gp37-ryk fusion oncoprotein. Unlike that of other acutely transforming retroviruses, transduction of the v-ryk gene into RPL30 did not result in deletion of viral sequences. Sequence analysis suggested that v-Ryk is more homologous to receptor-type tyrosine kinases than to nonreceptor-type kinases. By reconstitution of a virus from its cDNA, the v-ryk oncogene has been shown to be fully responsible for the transforming activity of the RPL30 virus. Antibodies specific to v-Ryk immunoprecipitated the v-Ryk oncoprotein from cells transformed by the RPL30 virus. The v-Ryk protein was shown to be first synthesized as a 150-kDa precursor and then cleaved into the mature 69-kDa gp37-Ryk fusion protein, both parts of which were found to be localized to the membrane fraction. As expected from the sequence of v-Ryk, immunoprecipitates of v-Ryk from RPL30-transformed cells were found to display a protein tyrosine kinase activity in vitro, and the levels of tyrosine-phosphorylated proteins are elevated in v-ryk-transformed cells. Images PMID:1527848

  5. Extraction, purification, identification and metabolism of 3',5'-cyclic UMP, 3',5'-cyclic IMP and 3',5'-cyclic dTMP from rat tissues.

    PubMed Central

    Newton, R P; Kingston, E E; Hakeem, N A; Salih, S G; Beynon, J H; Moyse, C D

    1986-01-01

    The large-scale extraction and partial purification of endogenous 3',5'-cyclic UMP, 3',5'-cyclic IMP and 3',5'-cyclic dTMP are described. Rat liver, kidney, heart, spleen and lung tissues were subjected to a sequential purification procedure involving freeze-clamping, perchlorate extraction, alumina and Sephadex ion-exchange chromatography and preparative electrophoresis. The samples thus obtained co-chromatographed with authentic cyclic UMP, cyclic IMP and cyclic dTMP on t.l.c. and h.p.l.c. and the u.v. spectra of the extracted samples were identical with those of the standards. Fast atom bombardment of the three cyclic nucleotide standards yielded mass spectra containing a molecular protonated ion in each case; mass-analysed ion kinetic-energy spectrometry ('m.i.k.e.s') of these ions produced a spectrum unique to the parent cyclic nucleotide. The extracted putative cyclic UMP, cyclic IMP and cyclic dTMP each produced a m.i.k.e.s. identical with that obtained with the corresponding cyclic nucleotide standard. Rat liver, heart, kidney, brain, intestine, spleen, testis and lung protein preparations were each found capable of the synthesis of cyclic UMP, cyclic IMP and cyclic dTMP from the corresponding nucleoside triphosphate, of the hydrolysis of these cyclic nucleotides and of their binding, with the exception that cyclic dTMP was not synthesized by the kidney preparation. PMID:3019316

  6. ATP-binding site of adenylate kinase: mechanistic implications of its homology with ras-encoded p21, F1-ATPase, and other nucleotide-binding proteins.

    PubMed

    Fry, D C; Kuby, S A; Mildvan, A S

    1986-02-01

    The MgATP binding site of adenylate kinase, located by a combination of NMR and x-ray diffraction, is near three protein segments, five to seven amino acids in length, that are homologous in sequence to segments found in other nucleotide-binding phosphotransferases, such as myosin and F1-ATPase, ras p21 and transducin GTPases, and cAMP-dependent and src protein kinases, suggesting equivalent mechanistic roles of these segments in all of these proteins. Segment 1 is a glycine-rich flexible loop that, on adenylate kinase, may control access to the ATP-binding site by changing its conformation. Segment 2 is an alpha-helix containing two hydrophobic residues that interact with the adenine-ribose moiety of ATP, and a lysine that may bind to the beta- and gamma-phosphates of ATP. Segment 3 is a hydrophobic strand of parallel beta-pleated sheet, terminated by a carboxylate, that flanks the triphosphate binding site. The various reported mutations of ras p21 that convert it to a transforming agent all appear to involve segment 1, and such substitutions may alter the properties of p21 by hindering a conformational change at this segment. In F1-ATPase, the flexible loop may, by its position, control both the accessibility and the ATP/ADP equilibrium constant on the enzyme.

  7. Repression of protein kinase C and stimulation of cyclic AMP response elements by fumonisin, a fungal encoded toxin which is a carcinogen.

    PubMed

    Huang, C; Dickman, M; Henderson, G; Jones, C

    1995-04-15

    Fusarium moniliforme (FM) is a major fungal pathogen of corn and is involved with stalk rot disease. FM is widely spread throughout the world, including the United States. Most strains of FM produce several mycotoxins, the most prominent of which is called fumonisin. Recent epidemiological studies indicated that ingestion of fumonisin correlates with a higher incidence of esophageal cancer in Southern and Northern Africa and China. Furthermore, fumonisin causes a neurodegenerative disease in horses, induces hepatic cancer in rats, and induces pulmonary edema in swine. Considering that high levels of fumonisin have been detected in healthy and diseased corn grown in the United States, fumonisin may pose a health threat to humans and livestock animals. Structurally, fumonisin resembles sphingolipids which are present in the membranes of animal and plant cells. At the present time, very little is known concerning the mechanism by which fumonisin elicits its carcinogenic effect. Our studies indicate that fumonisin represses expression of protein kinase C and AP-1-dependent transcription. In contrast, fumonisin stimulated a simple promoter containing a single cyclic AMP response element. Since fumonisin did not alter protein kinase A activity, it appears that cyclic AMP response element activation was independent of protein kinase A. It is hypothesized that the ability of fumonisin to alter signal transduction pathways plays a role in carcinogenesis. PMID:7712470

  8. The Fus3/Kss1 MAP kinase homolog Amk1 regulates the expression of genes encoding hydrolytic enzymes in Alternaria brassicicola.

    PubMed

    Cho, Yangrae; Cramer, Robert A; Kim, Kwang-Hyung; Davis, Josh; Mitchell, Thomas K; Figuli, Patricia; Pryor, Barry M; Lemasters, Emily; Lawrence, Christopher B

    2007-06-01

    Mitogen-activated protein (MAP) kinases have been shown to be required for virulence in diverse phytopathogenic fungi. To study its role in pathogenicity, we disrupted the Amk1 MAP kinase gene, a homolog of the Fus3/Kss1 MAP kinases in Saccharomyces cerevisiae, in the necrotrophic Brassica pathogen, Alternaria brassicicola. The amk1 disruption mutants showed null pathogenicity on intact host plants. However, amk1 mutants were able to colonize host plants when they were inoculated on a physically damaged host surface, or when they were inoculated along with nutrient supplements. On intact plants, mutants expressed extremely low amounts of several hydrolytic enzyme genes that were induced over 10-fold in the wild-type during infection. These genes were also dramatically induced in the mutants on wounded plants. These results imply a correlation between virulence and the expression level of specific hydrolytic enzyme genes plus the presence of an unidentified pathway controlling these genes in addition to or in conjunction with the Amk1 pathway. PMID:17280842

  9. Repression of protein kinase C and stimulation of cyclic AMP response elements by fumonisin, a fungal encoded toxin which is a carcinogen.

    PubMed

    Huang, C; Dickman, M; Henderson, G; Jones, C

    1995-04-15

    Fusarium moniliforme (FM) is a major fungal pathogen of corn and is involved with stalk rot disease. FM is widely spread throughout the world, including the United States. Most strains of FM produce several mycotoxins, the most prominent of which is called fumonisin. Recent epidemiological studies indicated that ingestion of fumonisin correlates with a higher incidence of esophageal cancer in Southern and Northern Africa and China. Furthermore, fumonisin causes a neurodegenerative disease in horses, induces hepatic cancer in rats, and induces pulmonary edema in swine. Considering that high levels of fumonisin have been detected in healthy and diseased corn grown in the United States, fumonisin may pose a health threat to humans and livestock animals. Structurally, fumonisin resembles sphingolipids which are present in the membranes of animal and plant cells. At the present time, very little is known concerning the mechanism by which fumonisin elicits its carcinogenic effect. Our studies indicate that fumonisin represses expression of protein kinase C and AP-1-dependent transcription. In contrast, fumonisin stimulated a simple promoter containing a single cyclic AMP response element. Since fumonisin did not alter protein kinase A activity, it appears that cyclic AMP response element activation was independent of protein kinase A. It is hypothesized that the ability of fumonisin to alter signal transduction pathways plays a role in carcinogenesis.

  10. Microtubule Affinity Regulating Kinase Activity in Living Neurons Was Examined by a Genetically Encoded Fluorescence Resonance Energy Transfer/Fluorescence Lifetime Imaging-based Biosensor

    PubMed Central

    Timm, Thomas; von Kries, Jens Peter; Li, Xiaoyu; Zempel, Hans; Mandelkow, Eckhard; Mandelkow, Eva-Maria

    2011-01-01

    Protein kinases of the microtubule affinity regulating kinase (MARK)/Par-1 family play important roles in the establishment of cellular polarity, cell cycle control, and intracellular signal transduction. Disturbance of their function is linked to cancer and brain diseases, e.g. lissencephaly and Alzheimer disease. To understand the biological role of MARK family kinases, we searched for specific inhibitors and a biosensor for MARK activity. A screen of the ChemBioNet library containing ∼18,000 substances yielded several compounds with inhibitory activity in the low micromolar range and capable of inhibiting MARK activity in cultured cells and primary neurons, as judged by MARK-dependent phosphorylation of microtubule-associated proteins and its consequences for microtubule integrity. Four of the compounds share a 9-oxo-9H-acridin-10-yl structure as a basis that will serve as a lead for optimization of inhibition efficiency. To test these inhibitors, we developed a cellular biosensor for MARK activity based on a MARK target sequence attached to the 14-3-3 scaffold protein and linked to enhanced cyan or teal and yellow fluorescent protein as FRET donor and acceptor pairs. Transfection of the teal/yellow fluorescent protein sensor into neurons and imaging by fluorescence lifetime imaging revealed that MARK was particularly active in the axons and growth cones of differentiating neurons. PMID:21984823

  11. Disruption of Glycerol Metabolism by RNAi Targeting of Genes Encoding Glycerol Kinase Results in a Range of Phenotype Severity in Drosophila

    PubMed Central

    Wightman, Patrick J.; Jackson, George R.; Dipple, Katrina M.

    2013-01-01

    In Drosophila, RNAi targeting of either dGyk or dGK can result in two alternative phenotypes: adult glycerol hypersensitivity or larval lethality. Here we compare these two phenotypes at the level of glycerol kinase (GK) phosphorylation activity, dGyk and dGK-RNA expression, and glycerol levels. We found both phenotypes exhibit reduced but similar levels of GK phosphorylation activity. Reduced RNA expression levels of dGyk and dGK corresponded with RNAi progeny that developed into glycerol hypersensitive adult flies. However, quantification of dGyk/dGK expression levels for the larval lethality phenotype revealed unexpected levels possibly due to a compensatory mechanism between dGyk and dGK or RNAi inhibition. The enzymatic role of glycerol kinase converts glycerol to glycerol 3-phosphate. As expected, elevated glycerol levels were observed in larvae that went on to develop into glycerol hypersensitive adults. Interestingly, larvae that died before eclosion revealed extremely low glycerol levels. Further characterization identified a wing phenotype that is enhanced by a dGpdh null mutation, indicating disrupted glycerol metabolism underlies the wing phenotype. In humans, glycerol kinase deficiency (GKD) exhibits a wide range of phenotypic variation with no obvious genotype-phenotype correlations. Additionally, disease severity often does not correlate with GK phosphorylation activity. It is intriguing that both human GKD patients and our GKD Drosophila model show a range of phenotype severity. Additionally, the lack of correlation between GK phosphorylation and dGyk/dGK-RNA expression with phenotypic severity suggests further study including understanding the alternative functions of the GK protein, could provide insights into the complex pathogenic mechanism observed in human GKD patients. PMID:24039719

  12. Physical structure and chromosomal localization of a gene encoding human p58[sup clk-1], a cell division control related protein kinase

    SciTech Connect

    Eipers, P.G.

    1992-01-01

    The gene for the human p58[sup clk[minus]1] protein kinase, a cell division control-related gene, has been mapped by somatic cell hybrid analyses, in situ localization with the chromosomal gene, and nested polymerase chain reaction amplification of microdissected chromosomes. These studies indicate that the expressed p58[sup clk[minus]1] chromosomal gene maps to 1p36, while a highly related p58[sup clk[minus]1] sequence of unknown nature maps to chromosome 15. Assignment of a p34[sup cdc2]-related gene to 1p36 region, including neuroblastoma, ductal carcinoma of the breast, malignant melanoma, Merkel cell carcinoma and endocrine neoplasia among others. Aberrant expression of this protein kinase negatively regulates normal cellular growth. The p58[sup clk[minus]1] protein contains a central domain of 299 amino acids that is 46% identical to human p34[sup cdc2], the master mitotic protein kinase. This dissertation details the complete structure of the p58[sup clk[minus]1] chromosomal gene, including its putative promoter region, transcriptional start sites, exonic sequences, and intron/exon boundary sequences. The gene is 10 kb in size and contains 12 exons and 11 introns. Interestingly, the rather large 2.0 kb 3[prime] untranslated region is interrupted by an intron that separates a region containing numerous AUUUA destabilization motifs from the coding region. Furthermore, the expression of this gene in normal human tissues, as well as several human tumor cell samples and lines, is examined. The origin of multiple human transcripts from the same chromosomal gene, and the possible differential stability of these various transcripts, is discussed with regard to the transcriptional and post-transcriptional regulation of this gene. This is the first report of the chromosomal gene structure of a member of the p34[sup cdc2] supergene family.

  13. A Potential Regulatory Role for Intronic microRNA-338-3p for Its Host Gene Encoding Apoptosis-Associated Tyrosine Kinase

    PubMed Central

    Kos, Aron; Olde Loohuis, Nikkie F. M.; Wieczorek, Martha L.; Glennon, Jeffrey C.; Martens, Gerard J. M.; Kolk, Sharon M.; Aschrafi, Armaz

    2012-01-01

    MicroRNAs (miRNAs) are important gene regulators that are abundantly expressed in both the developing and adult mammalian brain. These non-coding gene transcripts are involved in post-transcriptional regulatory processes by binding to specific target mRNAs. Approximately one third of known miRNA genes are located within intronic regions of protein coding and non-coding regions, and previous studies have suggested a role for intronic miRNAs as negative feedback regulators of their host genes. In the present study, we monitored the dynamic gene expression changes of the intronic miR-338-3p and miR-338-5p and their host gene Apoptosis-associated Tyrosine Kinase (AATK) during the maturation of rat hippocampal neurons. This revealed an uncorrelated expression pattern of mature miR-338 strands with their host gene. Sequence analysis of the 3′ untranslated region (UTR) of rat AATK mRNA revealed the presence of two putative binding sites for miR-338-3p. Thus, miR-338-3p may have the capacity to modulate AATK mRNA levels in neurons. Transfection of miR-338-3p mimics into rat B35 neuroblastoma cells resulted in a significant decrease of AATK mRNA levels, while the transfection of synthetic miR-338-5p mimics did not alter AATK levels. Our results point to a possible molecular mechanism by which miR-338-3p participates in the regulation of its host gene by modulating the levels of AATK mRNA, a kinase which plays a role during differentiation, apoptosis and possibly in neuronal degeneration. PMID:22363537

  14. Mutation analysis of the gene encoding Bruton`s tyrosine kinase in a family with a sporadic case of X-linked agammaglobulinemia reveals three female carriers

    SciTech Connect

    Hagemann, T.L.; Kwan, Sau-Ping; Assa`ad, A.H.

    1995-11-06

    Bruton`s tyrosine kinase (Btk) has been identified as the protein responsible for the primary immunodeficiency X-linked agammaglobulinemia (XLA). We and others have cloned the gene for Btk and recently reported the genomic organization. Nineteen exons were positioned within the 37 kb gene. With the sequence data derived from our genomic map, we have designed a PCR based assay to directly identify mutations of the Btk gene in germline DNA of patients with XLA. In this report, the assay was used to analyze a family with a sporadic case of XLA to determine if other female relatives carry the disease. A four base-pair deletion was found in the DNA of the affected boy and was further traced through three generations. With the direct identification of the mutations responsible for XLA, we can now diagnose conclusively the disease and identify the immunologically normal female carriers. This same technique can easily be applied to prenatal diagnosis in families where the mutation can be identified. 34 refs., 3 figs.

  15. Oxidative stress induction as a cause of Ba2+-dependent fungicidal action of UMP-derivative on the yeast Shizosaccharomyces pombe.

    PubMed

    Tanaka, Toshio; Usuki, Yoshinosuke

    2003-01-01

    A UMP-derivative, uridine 5'-hexadecylphosphate (UMPC16), exhibited a fungicidal action against various yeast strains including the fission yeast Schizosaccharomyces pombe in combination with Ba2+ ion. UMPC16 accelerated reactive oxygen species (ROS) generation in medium with Ba2+ ion in a dose- and time-dependent manner. Additional supplementation of Ca2+ ion into medium could suppress such a combined fungicidal action due to oxidative stress induction. PMID:16233563

  16. Expression analysis of genes encoding mitogen-activated protein kinases in maize provides a key link between abiotic stress signaling and plant reproduction.

    PubMed

    Sun, Wei; Chen, Hao; Wang, Juan; Sun, Hong Wei; Yang, Shu Ke; Sang, Ya Lin; Lu, Xing Bo; Xu, Xiao Hui

    2015-01-01

    Mitogen-activated protein kinases (MAPKs) play important roles in stress responses and development in plants. Maize (Zea mays), an important cereal crop, is a model plant species for molecular studies. In the last decade, several MAPKs have been identified in maize; however, their functions have not been studied extensively. Genome-wide identification and expression analysis of maize MAPK genes could provide valuable information for understanding their functions. In this study, 20 non-redundant maize MAPK genes (ZmMPKs) were identified via a genome-wide survey. Phylogenetic analysis of MAPKs from maize, rice (Oryza sativa), Arabidopsis (Arabidopsis thaliana), poplar (Populus trichocarpa), and tomato (Solanum lycopersicum) classified them into four major classes. ZmMPKs in the same class had similar domains, motifs, and genomic structures. Gene duplication investigations suggested that segmental duplications made a large contribution to the expansion of ZmMPKs. A number of cis-acting elements related to plant development and response to stress and hormones were identified in the promoter regions of ZmMPKs. Furthermore, transcript profile analysis in eight tissues and organs at various developmental stages demonstrated that most ZmMPKs were preferentially expressed in reproductive tissues and organs. The transcript abundance of most ZmMPKs changed significantly under salt, drought, cold, or abscisic acid (ABA) treatments, implying that they might participate in abiotic stress and ABA signaling. These expression analyses indicated that ZmMPKs might serve as linkers between abiotic stress signaling and plant reproduction. Our data will deepen our understanding of the complexity of the maize MAPK gene family and provide new clues to investigate their functions.

  17. Terminal uridylyl transferase of Vigna unguiculata: purification and characterization of an enzyme catalyzing the addition of a single UMP residue to the 3'-end of an RNA primer.

    PubMed Central

    Zabel, P; Dorssers, L; Wernars, K; Van Kammen, A

    1981-01-01

    An enzyme which catalyzes the addition of a single UMP residue from UTP to the 3'-end of an RNA primer and which is referred to as terminal uridylyl transferase (TUT) has been extensively purified from the membrane fraction of vigna unguiculata leaves. The purification procedure involved (i) solubilization by cation depletion (ii) DEAE-Sepharose CL-6B column chromatography (iii) affinity chromatography of poly(U)-Sepharose 4B and (iv) glycerol gradient centrifugation. The molecular weight of the native enzyme was approximately 50,000 as determined by velocity sedimentation. Under conditions that were optimal for UMP-incorporation (5 mM Mg2+, low salt, 30 degrees C) TUT displayed a marked specificity for UTP as substrate, was unable to incorporate deoxyribonucleoside triphosphates and required a single-stranded oligo- or polyribonucleotide as primer. When oligoA20, tRNAasp of E. coli or alfalfa mosaic virus RNA 4 were used as primers at various substrate to primer ratio's, the vast majority of the product appeared to consist of primer molecules elongated with a single UMP residue as shown by polyacrylamide gelelectrophoresis and nearest neighbour analysis. We believe TUT to be a novel enzyme which has not been reported before and which may be a feasible tool in RNA sequencing as it enables the specific 3'-terminal labeling of RNA molecules. Images PMID:6269049

  18. Assignment of the gene (EPLG2) encoding a high-affinity binding protein for the receptor tyrosine kinase elk to a 200-kilobasepair region in human chromosome Xq12

    SciTech Connect

    Fletcher, F.A.; Beckmann, M.P.; Lyman, S.D.

    1995-01-01

    Elk is a member of the eph family of receptor tyrosine kinases. Elk is expressed only in the brain and testes of the developing and adult rat, and the interaction of elk with its ligand(s) has been suggested to play a role in the development or maintenance of the nervous system. The mouse gene Eplg2 encodes a potential elk ligand that is highly conserved among rat, mouse, and human. Eplg2 has been mapped to the central portion of the mouse X chromosome, tightly linked to the androgen receptor (Ar) locus. Linkage conservation between the mouse and the human X chromosomes suggested that the human homologue (EPLG2) would map near human AR, in the interval Xq11-q12. In the present study, we have confirmed this prediction and have localized EPLG2 to a 200-kb interval in Xq12 by somatic cell hybrid analysis, two-color fluorescence in situ hybridization (FISH), and yeast artificial chromosome (YAC) hybridization. 12 refs., 1 fig.

  19. Encoding Dictionaries.

    ERIC Educational Resources Information Center

    Ide, Nancy

    1995-01-01

    Describes problems in devising a Text Encoding Initiative (TEI) encoding format for dictionaries. Asserts that the high degree of structuring and compression of information are among the most complex text types treated in the TEI. Concludes that the source of some TEI problems lies in the design of Standard Generalized Markup Language (SGML). (CFR)

  20. Folding Properties of Cytosine Monophosphate Kinase from E. coli Indicate Stabilization through an Additional Insert in the NMP Binding Domain

    PubMed Central

    Beitlich, Thorsten; Lorenz, Thorsten; Reinstein, Jochen

    2013-01-01

    The globular 25 kDa protein cytosine monophosphate kinase (CMPK, EC ID: 2.7.4.14) from E. coli belongs to the family of nucleoside monophosphate (NMP) kinases (NMPK). Many proteins of this family share medium to high sequence and high structure similarity including the frequently found α/β topology. A unique feature of CMPK in the family of NMPKs is the positioning of a single cis-proline residue in the CORE-domain (cis-Pro124) in conjunction with a large insert in the NMP binding domain. This insert is not found in other well studied NMPKs such as AMPK or UMP/CMPK. We have analyzed the folding pathway of CMPK using time resolved tryptophan and FRET fluorescence as well as CD. Our results indicate that unfolding at high urea concentrations is governed by a single process, whereas refolding in low urea concentrations follows at least a three step process which we interpret as follows: Pro124 in the CORE-domain is in cis in the native state (Nc) and equilibrates with its trans-isomer in the unfolded state (Uc - Ut). Under refolding conditions, at least the Ut species and possibly also the Uc species undergo a fast initial collapse to form intermediates with significant amount of secondary structure, from which the trans-Pro124 fraction folds to the native state with a 100-fold lower rate constant than the cis-Pro124 species. CMPK thus differs from homologous NMP kinases like UMP/CMP kinase or AMP kinase, where folding intermediates show much lower content of secondary structure. Importantly also unfolding is up to 100-fold faster compared to CMPK. We therefore propose that the stabilizing effect of the long NMP-domain insert in conjunction with a subtle twist in the positioning of a single cis-Pro residue allows for substantial stabilization compared to other NMP kinases with α/β topology. PMID:24205218

  1. Chromosomal assignment of the gene encoding the human 58-kDa inhibitor (PRKRI) of the interferon-induced dsRNA-activated protein kinase to chromosome 13q32

    SciTech Connect

    Korth, M.J.; Katze, M.G.; Edelhoff, S.; Disteche, C.M.

    1996-01-15

    The 58-kDa inhibitor (p58) of the interferon-induced dsRNA-activated protein kinase (PKR) is a cellular protein recruited by the influenza virus to down-regulate the activity of PKR during virus infection. The inhibitor also appears to play a role in the regulation of cellular gene expression in the absence of viral infection and has oncogenic properties when overexpressed. Using fluorescence in situ hybridization, we have mapped the p58 gene (PRKRI) to human chromosome 13 band q32. Aberrations in the structure or number of chromosome 13 have been identified in a variety of human cancers, particularly in acute leukemia. 15 refs., 1 fig.

  2. Receptor Tyrosine Kinases in Drosophila Development

    PubMed Central

    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

  3. Phosphorylation of thymidylate synthase affects slow-binding inhibition by 5-fluoro-dUMP and N(4)-hydroxy-dCMP.

    PubMed

    Ludwiczak, Jan; Maj, Piotr; Wilk, Piotr; Frączyk, Tomasz; Ruman, Tomasz; Kierdaszuk, Borys; Jarmuła, Adam; Rode, Wojciech

    2016-04-01

    Endogenous thymidylate synthases, isolated from tissues or cultured cells of the same specific origin, have been reported to show differing slow-binding inhibition patterns. These were reflected by biphasic or linear dependence of the inactivation rate on time and accompanied by differing inhibition parameters. Considering its importance for chemotherapeutic drug resistance, the possible effect of thymidylate synthase inhibition by post-translational modification was tested, e.g. phosphorylation, by comparing sensitivities to inhibition by two slow-binding inhibitors, 5-fluoro-dUMP and N(4)-hydroxy-dCMP, of two fractions of purified recombinant mouse enzyme preparations, phosphorylated and non-phosphorylated, separated by metal oxide/hydroxide affinity chromatography on Al(OH)3 beads. The modification, found to concern histidine residues and influence kinetic properties by lowering Vmax, altered both the pattern of dependence of the inactivation rate on time from linear to biphasic, as well as slow-binding inhibition parameters, with each inhibitor studied. Being present on only one subunit of at least a great majority of phosphorylated enzyme molecules, it probably introduced dimer asymmetry, causing the altered time dependence of the inactivation rate pattern (biphasic with the phosphorylated enzyme) and resulting in asymmetric binding of each inhibitor studied. The latter is reflected by the ternary complexes, stable under denaturing conditions, formed by only the non-phosphorylated subunit of the phosphorylated enzyme with each of the two inhibitors and N(5,10)-methylenetetrahydrofolate. Inhibition of the phosphorylated enzyme by N(4)-hydroxy-dCMP was found to be strongly dependent on [Mg(2+)], cations demonstrated previously to also influence the activity of endogenous mouse TS isolated from tumour cells.

  4. Oncoprotein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2001-02-27

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  5. Rac-1 and Raf-1 kinases, components of distinct signaling pathways, activate myotonic dystrophy protein kinase

    NASA Technical Reports Server (NTRS)

    Shimizu, M.; Wang, W.; Walch, E. T.; Dunne, P. W.; Epstein, H. F.

    2000-01-01

    Myotonic dystrophy protein kinase (DMPK) is a serine-threonine protein kinase encoded by the myotonic dystrophy (DM) locus on human chromosome 19q13.3. It is a close relative of other kinases that interact with members of the Rho family of small GTPases. We show here that the actin cytoskeleton-linked GTPase Rac-1 binds to DMPK, and coexpression of Rac-1 and DMPK activates its transphosphorylation activity in a GTP-sensitive manner. DMPK can also bind Raf-1 kinase, the Ras-activated molecule of the MAP kinase pathway. Purified Raf-1 kinase phosphorylates and activates DMPK. The interaction of DMPK with these distinct signals suggests that it may play a role as a nexus for cross-talk between their respective pathways and may partially explain the remarkable pleiotropy of DM.

  6. Inactivation of the Carney complex gene 1 (PRKAR1A) alters spatiotemporal regulation of cAMP and cAMP-dependent protein kinase: a study using genetically encoded FRET-based reporters.

    PubMed

    Cazabat, Laure; Ragazzon, Bruno; Varin, Audrey; Potier-Cartereau, Marie; Vandier, Christophe; Vezzosi, Delphine; Risk-Rabin, Marthe; Guellich, Aziz; Schittl, Julia; Lechêne, Patrick; Richter, Wito; Nikolaev, Viacheslav O; Zhang, Jin; Bertherat, Jérôme; Vandecasteele, Grégoire

    2014-03-01

    Carney complex (CNC) is a hereditary disease associating cardiac myxoma, spotty skin pigmentation and endocrine overactivity. CNC is caused by inactivating mutations in the PRKAR1A gene encoding PKA type I alpha regulatory subunit (RIα). Although PKA activity is enhanced in CNC, the mechanisms linking PKA dysregulation to endocrine tumorigenesis are poorly understood. In this study, we used Förster resonance energy transfer (FRET)-based sensors for cAMP and PKA activity to define the role of RIα in the spatiotemporal organization of the cAMP/PKA pathway. RIα knockdown in HEK293 cells increased basal as well as forskolin or prostaglandin E1 (PGE1)-stimulated total cellular PKA activity as reported by western blots of endogenous PKA targets and the FRET-based global PKA activity reporter, AKAR3. Using variants of AKAR3 targeted to subcellular compartments, we identified similar increases in the response to PGE1 in the cytoplasm and at the outer mitochondrial membrane. In contrast, at the plasma membrane, the response to PGE1 was decreased along with an increase in basal FRET ratio. These results were confirmed by western blot analysis of basal and PGE1-induced phosphorylation of membrane-associated vasodilator-stimulated phosphoprotein. Similar differences were observed between the cytoplasm and the plasma membrane in human adrenal cells carrying a RIα inactivating mutation. RIα inactivation also increased cAMP in the cytoplasm, at the outer mitochondrial membrane and at the plasma membrane, as reported by targeted versions of the cAMP indicator Epac1-camps. These results show that RIα inactivation leads to multiple, compartment-specific alterations of the cAMP/PKA pathway revealing new aspects of signaling dysregulation in tumorigenesis. PMID:24122441

  7. Discovering the first tyrosine kinase

    PubMed Central

    Hunter, Tony

    2015-01-01

    In the middle of the 20th century, animal tumor viruses were heralded as possible models for understanding human cancer. By the mid-1970s, the molecular basis by which tumor viruses transform cells into a malignant state was beginning to emerge as the first viral genomic sequences were reported and the proteins encoded by their transforming genes were identified and characterized. This was a time of great excitement and rapid progress. In 1978, prompted by the discovery from Ray Erikson’s group that the Rous sarcoma virus (RSV) v-Src–transforming protein had an associated protein kinase activity specific for threonine, my group at the Salk Institute set out to determine whether the polyomavirus middle T-transforming protein had a similar kinase activity. Here, I describe the experiments that led to the identification of a kinase activity associated with middle T antigen and our serendipitous discovery that this activity was specific for tyrosine in vitro, and how this in turn led to the fortuitous observation that the v-Src–associated kinase activity was also specific for tyrosine. Our finding that v-Src increased the level of phosphotyrosine in cellular proteins in RSV-transformed cells confirmed that v-Src is a tyrosine kinase and transforms cells by phosphorylating proteins on tyrosine. My colleague Bart Sefton and I reported these findings in the March issue of PNAS in 1980. Remarkably, all of the experiments in this paper were accomplished in less than one month. PMID:26130799

  8. Soybean nodule autoregulation receptor kinase phosphorylates two kinase-associated protein phosphatases in vitro.

    PubMed

    Miyahara, Akira; Hirani, Tripty A; Oakes, Marie; Kereszt, Attila; Kobe, Bostjan; Djordjevic, Michael A; Gresshoff, Peter M

    2008-09-12

    The NARK (nodule autoregulation receptor kinase) gene, a negative regulator of cell proliferation in nodule primordia in several legumes, encodes a receptor kinase that consists of an extracellular leucine-rich repeat and an intracellular serine/threonine protein kinase domain. The putative catalytic domain of NARK was expressed and purified as a maltose-binding or a glutathione S-transferase fusion protein in Escherichia coli. The recombinant NARK proteins showed autophosphorylation activity in vitro. Several regions of the NARK kinase domain were shown by mass spectrometry to possess phosphoresidues. The kinase-inactive protein K724E failed to autophosphorylate, as did three other proteins corresponding to phenotypically detected mutants defective in whole plant autoregulation of nodulation. A wild-type NARK fusion protein transphosphorylated a kinase-inactive mutant NARK fusion protein, suggesting that it is capable of intermolecular autophosphorylation in vitro. In addition, Ser-861 and Thr-963 in the NARK kinase catalytic domain were identified as phosphorylation sites through site-directed mutagenesis. The genes coding for the kinase-associated protein phosphatases KAPP1 and KAPP2, two putative interacting components of NARK, were isolated. NARK kinase domain phosphorylated recombinant KAPP proteins in vitro. Autophosphorylated NARK kinase domain was, in turn, dephosphorylated by both KAPP1 and KAPP2. Our results suggest a model for signal transduction involving NARK in the control of nodule development.

  9. Updated rice kinase database RKD 2.0: enabling transcriptome and functional analysis of rice kinase genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein kinases catalyze the transfer of a phosphate moiety from a phosphate donor to the substrate molecule, thus, playing critical roles in cell signaling and metabolism. Although plant genomes contain more than 1,000 genes that encode kinases, knowledge is limited about the precise roles for the...

  10. Characterization of mouse UDP-glucose pyrophosphatase, a Nudix hydrolase encoded by the Nudt14 gene

    SciTech Connect

    Heyen, Candy A.; Tagliabracci, Vincent S.; Zhai, Lanmin; Roach, Peter J.

    2009-12-25

    Recombinant mouse UDP-glucose pyrophosphatase (UGPPase), encoded by the Nudt14 gene, was produced in Escherichia coli and purified close to homogeneity. The enzyme catalyzed the conversion of [{beta}-{sup 32}P]UDP-glucose to [{sup 32}P]glucose-1-P and UMP, confirming that it hydrolyzed the pyrophosphate of the nucleoside diphosphate sugar to generate glucose-1-P and UMP. The enzyme was also active toward ADP-ribose. Activity is dependent on the presence of Mg{sup 2+} and was greatest at alkaline pH above 8. Kinetic analysis indicated a K{sub m} of {approx}4 mM for UDP-glucose and {approx}0.3 mM for ADP-ribose. Based on V{sub max}/K{sub m} values, the enzyme was {approx}20-fold more active toward ADP-ribose. UGPPase behaves as a dimer in solution and can be cross-linked to generate a species of M{sub r} 54,000 from a monomer of 30,000 as judged by SDS-PAGE. The dimerization was not affected by the presence of glucose-1-P or UDP-glucose. Using antibodies raised against the recombinant protein, Western analysis indicated that UGPPase was widely expressed in mouse tissues, including skeletal muscle, liver, kidney, heart, lung, fat, heart and pancreas with a lower level in brain. It was generally present as a doublet when analyzed by SDS-PAGE, suggesting the occurrence of some form of post-translational modification. Efforts to interconvert the species by adding or inhibiting phosphatase activity were unsuccessful, leaving the nature of the modification unknown. Sequence alignments and database searches revealed related proteins in species as distant as Drosophila melanogaster and Caenorhabditis elegans.

  11. ENCODE data at the ENCODE portal.

    PubMed

    Sloan, Cricket A; Chan, Esther T; Davidson, Jean M; Malladi, Venkat S; Strattan, J Seth; Hitz, Benjamin C; Gabdank, Idan; Narayanan, Aditi K; Ho, Marcus; Lee, Brian T; Rowe, Laurence D; Dreszer, Timothy R; Roe, Greg; Podduturi, Nikhil R; Tanaka, Forrest; Hong, Eurie L; Cherry, J Michael

    2016-01-01

    The Encyclopedia of DNA Elements (ENCODE) Project is in its third phase of creating a comprehensive catalog of functional elements in the human genome. This phase of the project includes an expansion of assays that measure diverse RNA populations, identify proteins that interact with RNA and DNA, probe regions of DNA hypersensitivity, and measure levels of DNA methylation in a wide range of cell and tissue types to identify putative regulatory elements. To date, results for almost 5000 experiments have been released for use by the scientific community. These data are available for searching, visualization and download at the new ENCODE Portal (www.encodeproject.org). The revamped ENCODE Portal provides new ways to browse and search the ENCODE data based on the metadata that describe the assays as well as summaries of the assays that focus on data provenance. In addition, it is a flexible platform that allows integration of genomic data from multiple projects. The portal experience was designed to improve access to ENCODE data by relying on metadata that allow reusability and reproducibility of the experiments.

  12. Targeting cancer with small-molecular-weight kinase inhibitors.

    PubMed

    Fabbro, Doriano; Cowan-Jacob, Sandra W; Möbitz, Henrik; Martiny-Baron, Georg

    2012-01-01

    Protein and lipid kinases fulfill essential roles in many signaling pathways that regulate normal cell functions. Deregulation of these kinase activities lead to a variety of pathologies ranging from cancer to inflammatory diseases, diabetes, infectious diseases, cardiovascular disorders, cell growth and survival. 518 protein kinases and about 20 lipid-modifying kinases are encoded by the human genome, and a much larger proportion of additional kinases are present in parasite, bacterial, fungal, and viral genomes that are susceptible to exploitation as drug targets. Since many human diseases result from overactivation of protein and lipid kinases due to mutations and/or overexpression, this enzyme class represents an important target for the pharmaceutical industry. Approximately one third of all protein targets under investigation in the pharmaceutical industry are protein or lipid kinases.The kinase inhibitors that have been launched, thus far, are mainly in oncology indications and are directed against a handful of protein and lipid kinases. With one exception, all of these registered kinase inhibitors are directed toward the ATP-site and display different selectivities, potencies, and pharmacokinetic properties. At present, about 150 kinase-targeted drugs are in clinical development and many more in various stages of preclinical development. Kinase inhibitor drugs that are in clinical trials target all stages of signal transduction from the receptor protein tyrosine kinases that initiate intracellular signaling, through second-messenger-dependent lipid and protein kinases, and protein kinases that regulate the cell cycle.This review provides an insight into protein and lipid kinase drug discovery with respect to achievements, binding modes of inhibitors, and novel avenues for the generation of second-generation kinase inhibitors to treat cancers.

  13. Varicella-Zoster Virus Open Reading Frame 66 Protein Kinase and Its Relationship to Alphaherpesvirus US3 Kinases

    PubMed Central

    Erazo, Angela

    2014-01-01

    The varicella-zoster virus (VZV) open reading frame (ORF) 66 encodes a basophilic kinase orthologous to the US3 protein kinases found in all alphaherpesviruses. This review summarizes current information on the ORF66 kinase, and outlines apparent differences from other US3 kinases, as well as some of the conserved functions. One critical difference is the VZV ORF66 kinase targeting of the major regulatory VZV IE62 protein to control its nuclear import and assembly into the VZV virion, which is so far unprecedented in the alphaherpesviruses. However, ORF66 targets some cellular targets which are also targeted by US3 kinases of other herpesviruses, including the histone deacetylase-1 and 2 proteins, pathways that lead to changes in actin dynamics, and the targeting of substrates of protein kinase A, including the nuclear matrix protein matrin 3. PMID:20186610

  14. Feedback Regulation of Kinase Signaling Pathways by AREs and GREs

    PubMed Central

    Vlasova-St. Louis, Irina; Bohjanen, Paul R.

    2016-01-01

    In response to environmental signals, kinases phosphorylate numerous proteins, including RNA-binding proteins such as the AU-rich element (ARE) binding proteins, and the GU-rich element (GRE) binding proteins. Posttranslational modifications of these proteins lead to a significant changes in the abundance of target mRNAs, and affect gene expression during cellular activation, proliferation, and stress responses. In this review, we summarize the effect of phosphorylation on the function of ARE-binding proteins ZFP36 and ELAVL1 and the GRE-binding protein CELF1. The networks of target mRNAs that these proteins bind and regulate include transcripts encoding kinases and kinase signaling pathways (KSP) components. Thus, kinase signaling pathways are involved in feedback regulation, whereby kinases regulate RNA-binding proteins that subsequently regulate mRNA stability of ARE- or GRE-containing transcripts that encode components of KSP. PMID:26821046

  15. Changes in dynamics upon oligomerization regulate substrate binding and allostery in amino acid kinase family members.

    PubMed

    Marcos, Enrique; Crehuet, Ramon; Bahar, Ivet

    2011-09-01

    Oligomerization is a functional requirement for many proteins. The interfacial interactions and the overall packing geometry of the individual monomers are viewed as important determinants of the thermodynamic stability and allosteric regulation of oligomers. The present study focuses on the role of the interfacial interactions and overall contact topology in the dynamic features acquired in the oligomeric state. To this aim, the collective dynamics of enzymes belonging to the amino acid kinase family both in dimeric and hexameric forms are examined by means of an elastic network model, and the softest collective motions (i.e., lowest frequency or global modes of motions) favored by the overall architecture are analyzed. Notably, the lowest-frequency modes accessible to the individual subunits in the absence of multimerization are conserved to a large extent in the oligomer, suggesting that the oligomer takes advantage of the intrinsic dynamics of the individual monomers. At the same time, oligomerization stiffens the interfacial regions of the monomers and confers new cooperative modes that exploit the rigid-body translational and rotational degrees of freedom of the intact monomers. The present study sheds light on the mechanism of cooperative inhibition of hexameric N-acetyl-L-glutamate kinase by arginine and on the allosteric regulation of UMP kinases. It also highlights the significance of the particular quaternary design in selectively determining the oligomer dynamics congruent with required ligand-binding and allosteric activities.

  16. Conservation and early expression of zebrafish tyrosine kinases support the utility of zebrafish as a model for tyrosine kinase biology.

    PubMed

    Challa, Anil Kumar; Chatti, Kiranam

    2013-09-01

    Tyrosine kinases have significant roles in cell growth, apoptosis, development, and disease. To explore the use of zebrafish as a vertebrate model for tyrosine kinase signaling and to better understand their roles, we have identified all of the tyrosine kinases encoded in the zebrafish genome and quantified RNA expression of selected tyrosine kinases during early development. Using profile hidden Markov model analysis, we identified 122 zebrafish tyrosine kinase genes and proposed unambiguous gene names where needed. We found them to be organized into 39 nonreceptor and 83 receptor type, and 30 families consistent with human tyrosine kinase family assignments. We found five human tyrosine kinase genes (epha1, bmx, fgr, srm, and insrr) with no identifiable zebrafish ortholog, and one zebrafish gene (yrk) with no identifiable human ortholog. We also found that receptor tyrosine kinase genes were duplicated more often than nonreceptor tyrosine kinase genes in zebrafish. We profiled expression levels of 30 tyrosine kinases representing all families using direct digital detection at different stages during the first 24 hours of development. The profiling experiments clearly indicate regulated expression of tyrosine kinases in the zebrafish, suggesting their role during early embryonic development. In summary, our study has resulted in the first comprehensive description of the zebrafish tyrosine kinome. PMID:23234507

  17. Conservation and early expression of zebrafish tyrosine kinases support the utility of zebrafish as a model for tyrosine kinase biology.

    PubMed

    Challa, Anil Kumar; Chatti, Kiranam

    2013-09-01

    Tyrosine kinases have significant roles in cell growth, apoptosis, development, and disease. To explore the use of zebrafish as a vertebrate model for tyrosine kinase signaling and to better understand their roles, we have identified all of the tyrosine kinases encoded in the zebrafish genome and quantified RNA expression of selected tyrosine kinases during early development. Using profile hidden Markov model analysis, we identified 122 zebrafish tyrosine kinase genes and proposed unambiguous gene names where needed. We found them to be organized into 39 nonreceptor and 83 receptor type, and 30 families consistent with human tyrosine kinase family assignments. We found five human tyrosine kinase genes (epha1, bmx, fgr, srm, and insrr) with no identifiable zebrafish ortholog, and one zebrafish gene (yrk) with no identifiable human ortholog. We also found that receptor tyrosine kinase genes were duplicated more often than nonreceptor tyrosine kinase genes in zebrafish. We profiled expression levels of 30 tyrosine kinases representing all families using direct digital detection at different stages during the first 24 hours of development. The profiling experiments clearly indicate regulated expression of tyrosine kinases in the zebrafish, suggesting their role during early embryonic development. In summary, our study has resulted in the first comprehensive description of the zebrafish tyrosine kinome.

  18. Kinase active Misshapen regulates Notch signaling in Drosophila melanogaster.

    PubMed

    Mishra, Abhinava K; Sachan, Nalani; Mutsuddi, Mousumi; Mukherjee, Ashim

    2015-11-15

    Notch signaling pathway represents a principal cellular communication system that plays a pivotal role during development of metazoans. Drosophila misshapen (msn) encodes a protein kinase, which is related to the budding yeast Ste20p (sterile 20 protein) kinase. In a genetic screen, using candidate gene approach to identify novel kinases involved in Notch signaling, we identified msn as a novel regulator of Notch signaling. Data presented here suggest that overexpression of kinase active form of Msn exhibits phenotypes similar to Notch loss-of-function condition and msn genetically interacts with components of Notch signaling pathway. Kinase active form of Msn associates with Notch receptor and regulate its signaling activity. We further show that kinase active Misshapen leads to accumulation of membrane-tethered form of Notch. Moreover, activated Msn also depletes Armadillo and DE-Cadherin from adherens junctions. Thus, this study provides a yet unknown mode of regulation of Notch signaling by Misshapen. PMID:26431585

  19. Quantitative Targeted Proteomics of Pancreatic Cancer: Deoxycytidine Kinase Protein Level Correlates to Progression-Free Survival of Patients Receiving Gemcitabine Treatment.

    PubMed

    Ohmine, Ken; Kawaguchi, Kei; Ohtsuki, Sumio; Motoi, Fuyuhiko; Ohtsuka, Hideo; Kamiie, Junichi; Abe, Takaaki; Unno, Michiaki; Terasaki, Tetsuya

    2015-09-01

    The purpose of the present study is to identify the determinant(s) of gemcitabine (dFdC)-sensitivity in pancreatic cancer tissues of patients treated with dFdC alone and in pancreatic cancer cell lines exposed to dFdC in vitro. Protein expression levels of 12 enzymes and 13 transporters potentially involved in transport and metabolism of dFdC in pancreatic cancer cell lines and tissues were quantified by means of our LC-MS/MS-based quantitative targeted proteomics technology. Protein expression levels of deoxycytidine kinase (dCK), uridine monophosphate-cytidine monophosphate (UMP-CMP) kinase, cytosolic nucleotidase III (cN-III), and equilibrative nucleoside transporter 1 (ENT1) were significantly correlated with IC50 or 1/IC50 in five cell lines with different sensitivities to dFdC (p < 0.05). Expression levels of the selected proteins in pancreatic cancer tissues of 10 patients with different progression-free survival (PFS) (49-955 days) were quantified, and their relationship with PFS was examined. Only the protein expression level of dCK was significantly correlated with PFS (p < 0.05). Multiple regression analysis was also performed, and combinations of ENT1, UMP-CMP kinase, CTPS1, and dCK were highly correlated with PFS. Our results indicate that the protein expression level of dCK in pancreatic cancer tissue is a good predictor of PFS, and thus dCK may be the best biomarker of dFdC sensitivity in pancreatic cancer patients treated with dFdC, although other proteins would also contribute to dFdC-sensitivity at the cellular level in vivo and in vitro.

  20. Crystal structure of pyridoxal kinase from the Escherichia coli pdxK gene: implications for the classification of pyridoxal kinases.

    PubMed

    Safo, Martin K; Musayev, Faik N; di Salvo, Martino L; Hunt, Sharyn; Claude, Jean-Baptiste; Schirch, Verne

    2006-06-01

    The pdxK and pdxY genes have been found to code for pyridoxal kinases, enzymes involved in the pyridoxal phosphate salvage pathway. Two pyridoxal kinase structures have recently been published, including Escherichia coli pyridoxal kinase 2 (ePL kinase 2) and sheep pyridoxal kinase, products of the pdxY and pdxK genes, respectively. We now report the crystal structure of E. coli pyridoxal kinase 1 (ePL kinase 1), encoded by a pdxK gene, and an isoform of ePL kinase 2. The structures were determined in the unliganded and binary complexes with either MgATP or pyridoxal to 2.1-, 2.6-, and 3.2-A resolutions, respectively. The active site of ePL kinase 1 does not show significant conformational change upon binding of either pyridoxal or MgATP. Like sheep PL kinase, ePL kinase 1 exhibits a sequential random mechanism. Unlike sheep pyridoxal kinase, ePL kinase 1 may not tolerate wide variation in the size and chemical nature of the 4' substituent on the substrate. This is the result of differences in a key residue at position 59 on a loop (loop II) that partially forms the active site. Residue 59, which is His in ePL kinase 1, interacts with the formyl group at C-4' of pyridoxal and may also determine if residues from another loop (loop I) can fill the active site in the absence of the substrate. Both loop I and loop II are suggested to play significant roles in the functions of PL kinases.

  1. Mevalonate kinase deficiency: current perspectives

    PubMed Central

    Favier, Leslie A; Schulert, Grant S

    2016-01-01

    Mevalonate kinase deficiency (MKD) is a recessively inherited autoinflammatory disorder with a spectrum of manifestations, including the well-defined clinical phenotypes of hyperimmunoglobulinemia D and periodic fever syndrome and mevalonic aciduria. Patients with MKD have recurrent attacks of hyperinflammation associated with fever, abdominal pain, arthralgias, and mucocutaneous lesions, and more severely affected patients also have dysmorphisms and central nervous system anomalies. MKD is caused by mutations in the gene encoding mevalonate kinase, with the degree of residual enzyme activity largely determining disease severity. Mevalonate kinase is essential for the biosynthesis of nonsterol isoprenoids, which mediate protein prenylation. Although the precise pathogenesis of MKD remains unclear, increasing evidence suggests that deficiency in protein prenylation leads to innate immune activation and systemic hyperinflammation. Given the emerging understanding of MKD as an autoinflammatory disorder, recent treatment approaches have largely focused on cytokine-directed biologic therapy. Herein, we review the current genetic and pathologic understanding of MKD, its various clinical phenotypes, and the evolving treatment approach for this multifaceted disorder. PMID:27499643

  2. A novel human STE20-related protein kinase, HGK, that specifically activates the c-Jun N-terminal kinase signaling pathway.

    PubMed

    Yao, Z; Zhou, G; Wang, X S; Brown, A; Diener, K; Gan, H; Tan, T H

    1999-01-22

    The yeast serine/threonine kinase STE20 activates a signaling cascade that includes STE11 (mitogen-activated protein kinase kinase kinase), STE7 (mitogen-activated protein kinase kinase), and FUS3/KSS1 (mitogen-activated protein kinase) in response to signals from both Cdc42 and the heterotrimeric G proteins associated with transmembrane pheromone receptors. Using degenerate polymerase chain reaction, we have isolated a human cDNA encoding a protein kinase homologous to STE20. This protein kinase, designated HPK/GCK-like kinase (HGK), has nucleotide sequences that encode an open reading frame of 1165 amino acids with 11 kinase subdomains. HGK was a serine/threonine protein kinase that specifically activated the c-Jun N-terminal kinase (JNK) signaling pathway when transfected into 293T cells, but it did not stimulate either the extracellular signal-regulated kinase or p38 kinase pathway. HGK also increased AP-1-mediated transcriptional activity in vivo. HGK-induced JNK activation was inhibited by the dominant-negative MKK4 and MKK7 mutants. The dominant-negative mutant of TAK1, but not MEKK1 or MAPK upstream kinase (MUK), strongly inhibited HGK-induced JNK activation. TNF-alpha activated HGK in 293T cells, as well as the dominant-negative HGK mutants, inhibited TNF-alpha-induced JNK activation. These results indicate that HGK, a novel activator of the JNK pathway, may function through TAK1, and that the HGK --> TAK1 --> MKK4, MKK7 --> JNK kinase cascade may mediate the TNF-alpha signaling pathway. PMID:9890973

  3. Selection for Genes Encoding Secreted Proteins and Receptors

    NASA Astrophysics Data System (ADS)

    Klein, Robert D.; Gu, Qimin; Goddard, Audrey; Rosenthal, Arnon

    1996-07-01

    Extracellular proteins play an essential role in the formation, differentiation, and maintenance of multicellular organisms. Despite that, the systematic identification of genes encoding these proteins has not been possible. We describe here a highly efficient method to isolate genes encoding secreted and membrane-bound proteins by using a single-step selection in yeast. Application of this method, termed signal peptide selection, to various tissues yielded 559 clones that appear to encode known or novel extracellular proteins. These include members of the transforming growth factor and epidermal growth factor protein families, endocrine hormones, tyrosine kinase receptors, serine/threonine kinase receptors, seven transmembrane receptors, cell adhesion molecules, extracellular matrix proteins, plasma proteins, and ion channels. The eventual identification of most, or all, extracellular signaling molecules will advance our understanding of fundamental biological processes and our ability to intervene in disease states.

  4. Miniaturised optical encoder

    NASA Astrophysics Data System (ADS)

    Carr, John; Desmulliez, Marc P. Y.; Weston, Nick; McKendrick, David; Cunningham, Graeme; McFarland, Geoff; Meredith, Wyn; McKee, Andrew; Langton, Conrad; Eddie, Iain

    2008-08-01

    Optical encoders are pervasive in many sectors of industry including metrology, motion systems, electronics, medical, scanning/ printing, scientific instruments, space research and specialist machine tools. The precision of automated manufacture and assembly has been revolutionised by the adoption of optical diffractive measurement methods. Today's optical encoders comprise discrete components: light source(s), reference and analyser gratings, and a photodiode array that utilise diffractive optic methods to achieve high resolution. However the critical alignment requirements between the optical gratings and to the photodiode array, the bulky nature of the encoder devices and subsequent packaging mean that optical encoders can be prohibitively expensive for many applications and unsuitable for others. We report here on the design, manufacture and test of a miniaturised optical encoder to be used in precision measurement systems. Microsystems manufacturing techniques facilitate the monolithic integration of the traditional encoder components onto a single compound semiconductor chip, radically reducing the size, cost and set-up time. Fabrication of the gratings at the wafer level, by standard photo-lithography, allows for the simultaneous alignment of many devices in a single process step. This development coupled with a unique photodiode configuration not only provides increased performance but also significantly improves the alignment tolerances in both manufacture and set-up. A National Research and Development Corporation type optical encoder chip has been successfully demonstrated under test conditions on both amplitude and phase scales with pitches of 20 micron, 8 micron and 4 micron, showing significantly relaxed alignment tolerances with signal-to-noise ratios greater than 60:1. Various reference mark schemes have also been investigated. Results are presented here.

  5. Two Kinase Family Dramas

    PubMed Central

    Leonard, Thomas A.; Hurley, James H.

    2007-01-01

    In this issue, Lietha and colleagues (2007) report the structure of focal adhesion kinase (FAK) and reveal how FAK maintains an autoinhibited state. Together with the structure of another tyrosine kinase, ZAP-70 (Deindl et al., 2007), this work highlights the diversity of mechanisms that nature has evolved within the kinase superfamily to regulate their activity through autoinhibition. PMID:17574014

  6. Polarization encoded color camera.

    PubMed

    Schonbrun, Ethan; Möller, Guðfríður; Di Caprio, Giuseppe

    2014-03-15

    Digital cameras would be colorblind if they did not have pixelated color filters integrated into their image sensors. Integration of conventional fixed filters, however, comes at the expense of an inability to modify the camera's spectral properties. Instead, we demonstrate a micropolarizer-based camera that can reconfigure its spectral response. Color is encoded into a linear polarization state by a chiral dispersive element and then read out in a single exposure. The polarization encoded color camera is capable of capturing three-color images at wavelengths spanning the visible to the near infrared. PMID:24690806

  7. Prokaryotic Diacylglycerol Kinase and Undecaprenol Kinase

    PubMed Central

    Van Horn, Wade D.; Sanders, Charles R.

    2013-01-01

    Prokaryotic diacylglycerol kinase (DAGK) and undecaprenol kinase (UDPK) are the lone members of a family of multispan membrane enzymes that are very small, lack relationships to any other family of proteins—including water soluble kinases, and that exhibit an unusual structure and active site architecture. Escherichia coli DAGK plays an important role in recycling diacylglycerol produced as a byproduct of biosynthesis of molecules located in the periplasmic space. UDPK seems to play an analogous role in Gram-positive bacteria, where its importance is evident by the fact that UDPK is essential for biofilm formation by the oral pathogen Streptococcus mutans. DAGK has also long served as a model system for studies of membrane protein biocatalysis, folding, stability, and structure. This review explores our current understanding of the microbial physiology, enzymology, structural biology, and folding of the prokaryotic diacylglycerol kinase family, which is based on over 40 years of studies. PMID:22224599

  8. Plasmids encoding therapeutic agents

    DOEpatents

    Keener, William K.

    2007-08-07

    Plasmids encoding anti-HIV and anti-anthrax therapeutic agents are disclosed. Plasmid pWKK-500 encodes a fusion protein containing DP178 as a targeting moiety, the ricin A chain, an HIV protease cleavable linker, and a truncated ricin B chain. N-terminal extensions of the fusion protein include the maltose binding protein and a Factor Xa protease site. C-terminal extensions include a hydrophobic linker, an L domain motif peptide, a KDEL ER retention signal, another Factor Xa protease site, an out-of-frame buforin II coding sequence, the lacZ.alpha. peptide, and a polyhistidine tag. More than twenty derivatives of plasmid pWKK-500 are described. Plasmids pWKK-700 and pWKK-800 are similar to pWKK-500 wherein the DP178-encoding sequence is substituted by RANTES- and SDF-1-encoding sequences, respectively. Plasmid pWKK-900 is similar to pWKK-500 wherein the HIV protease cleavable linker is substituted by a lethal factor (LF) peptide-cleavable linker.

  9. Video Time Encoding Machines

    PubMed Central

    Lazar, Aurel A.; Pnevmatikakis, Eftychios A.

    2013-01-01

    We investigate architectures for time encoding and time decoding of visual stimuli such as natural and synthetic video streams (movies, animation). The architecture for time encoding is akin to models of the early visual system. It consists of a bank of filters in cascade with single-input multi-output neural circuits. Neuron firing is based on either a threshold-and-fire or an integrate-and-fire spiking mechanism with feedback. We show that analog information is represented by the neural circuits as projections on a set of band-limited functions determined by the spike sequence. Under Nyquist-type and frame conditions, the encoded signal can be recovered from these projections with arbitrary precision. For the video time encoding machine architecture, we demonstrate that band-limited video streams of finite energy can be faithfully recovered from the spike trains and provide a stable algorithm for perfect recovery. The key condition for recovery calls for the number of neurons in the population to be above a threshold value. PMID:21296708

  10. Video time encoding machines.

    PubMed

    Lazar, Aurel A; Pnevmatikakis, Eftychios A

    2011-03-01

    We investigate architectures for time encoding and time decoding of visual stimuli such as natural and synthetic video streams (movies, animation). The architecture for time encoding is akin to models of the early visual system. It consists of a bank of filters in cascade with single-input multi-output neural circuits. Neuron firing is based on either a threshold-and-fire or an integrate-and-fire spiking mechanism with feedback. We show that analog information is represented by the neural circuits as projections on a set of band-limited functions determined by the spike sequence. Under Nyquist-type and frame conditions, the encoded signal can be recovered from these projections with arbitrary precision. For the video time encoding machine architecture, we demonstrate that band-limited video streams of finite energy can be faithfully recovered from the spike trains and provide a stable algorithm for perfect recovery. The key condition for recovery calls for the number of neurons in the population to be above a threshold value.

  11. Time-Encoded Imagers.

    SciTech Connect

    Marleau, Peter; Brubaker, Erik

    2014-11-01

    This report provides a short overview of the DNN R&D funded project, Time-Encoded Imagers. The project began in FY11 and concluded in FY14. The Project Description below provides the overall motivation and objectives for the project as well as a summary of programmatic direction. It is followed by a short description of each task and the resulting deliverables.

  12. A (U)MP2(full) and (U)CCSD(T) theoretical investigation into the substituent effects on the intermolecular T-shaped F-H...π interactions between HF and LBBL (L = -H, : CO, :NN, -Cl, -CN and -NC).

    PubMed

    Yang, Zhao-ming; Zhang, Lin; Chen, Li-zhen; Ren, Fu-de; Du, Shan; Yang, Lei

    2012-07-01

    The substituent effects on the intermolecular T-shaped F-H...π interactions are investigated between HF and LBBL (L = -H, : CO, :NN, -Cl, -CN and -NC) using the (U)MP2(full) and (U)CCSD(T) methods with the 6-311++G(2 d,p) basis set. The B ≡ B triple-bond contraction is found in the complexes with lone-pair-electron donors while the B = B double-bond is lengthened in the systems with the single-electron substituents upon complexation. The T-shaped F-H...π interaction energies follow the order of ClB = BCl...HF>HB = BH...HF>NNB ≡ BNN...HF>OCB ≡ BCO...HF>CNB = BNC...HF>NCB = BCN...HF. The electron-donating substituents : CO and :NN increases electron density of the B ≡ B triple bond by the delocalization interaction E ((2)) π ((CO/NN) → Lp(B)) while the electron-withdrawing substituents -CN and -NC decrease electron density of the B = B double bond by means of the π-π conjugative effect. The analyses of the APT atomic charge, "truncated" model, natural bond orbital (NBO), atoms in molecules (AIM) and electron density shifts reveal the nature of the substituent effect and explain the origin of the B ≡ B bond contraction.

  13. A FRET biosensor reveals spatiotemporal activation and functions of aurora kinase A in living cells

    PubMed Central

    Bertolin, Giulia; Sizaire, Florian; Herbomel, Gaëtan; Reboutier, David; Prigent, Claude; Tramier, Marc

    2016-01-01

    Overexpression of AURKA is a major hallmark of epithelial cancers. It encodes the multifunctional serine/threonine kinase aurora A, which is activated at metaphase and is required for cell cycle progression; assessing its activation in living cells is mandatory for next-generation drug design. We describe here a Förster's resonance energy transfer (FRET) biosensor detecting the conformational changes of aurora kinase A induced by its autophosphorylation on Thr288. The biosensor functionally replaces the endogenous kinase in cells and allows the activation of the kinase to be followed throughout the cell cycle. Inhibiting the catalytic activity of the kinase prevents the conformational changes of the biosensor. Using this approach, we discover that aurora kinase A activates during G1 to regulate the stability of microtubules in cooperation with TPX2 and CEP192. These results demonstrate that the aurora kinase A biosensor is a powerful tool to identify new regulatory pathways controlling aurora kinase A activation. PMID:27624869

  14. A FRET biosensor reveals spatiotemporal activation and functions of aurora kinase A in living cells.

    PubMed

    Bertolin, Giulia; Sizaire, Florian; Herbomel, Gaëtan; Reboutier, David; Prigent, Claude; Tramier, Marc

    2016-01-01

    Overexpression of AURKA is a major hallmark of epithelial cancers. It encodes the multifunctional serine/threonine kinase aurora A, which is activated at metaphase and is required for cell cycle progression; assessing its activation in living cells is mandatory for next-generation drug design. We describe here a Förster's resonance energy transfer (FRET) biosensor detecting the conformational changes of aurora kinase A induced by its autophosphorylation on Thr288. The biosensor functionally replaces the endogenous kinase in cells and allows the activation of the kinase to be followed throughout the cell cycle. Inhibiting the catalytic activity of the kinase prevents the conformational changes of the biosensor. Using this approach, we discover that aurora kinase A activates during G1 to regulate the stability of microtubules in cooperation with TPX2 and CEP192. These results demonstrate that the aurora kinase A biosensor is a powerful tool to identify new regulatory pathways controlling aurora kinase A activation. PMID:27624869

  15. Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho.

    PubMed Central

    Matsui, T; Amano, M; Yamamoto, T; Chihara, K; Nakafuku, M; Ito, M; Nakano, T; Okawa, K; Iwamatsu, A; Kaibuchi, K

    1996-01-01

    The small GTP binding protein Rho is implicated in cytoskeletal responses to extracellular signals such as lysophosphatidic acid to form stress fibers and focal contacts. Here we have purified a Rho-interacting protein with a molecular mass of approximately 164 kDa (p164) from bovine brain. This protein bound to GTPgammaS (a non-hydrolyzable GTP analog).RhoA but not to GDP.RhoA or GTPgammaS.RhoA with a mutation in the effector domain (RhoAA37).p164 had a kinase activity which was specifically stimulated by GTPgammaS.RhoA. We obtained the cDNA encoding p164 on the basis of its partial amino acid sequences and named it Rho-associated kinase (Rho-kinase). Rho-kinase has a catalytic domain in the N-terminal portion, a coiled coil domain in the middle portion and a zinc finger-like motif in the C-terminal portion. The catalytic domain shares 72% sequence homology with that of myotonic dystrophy kinase and the coiled coil domain contains a Rho-interacting interface. When COS7 cells were cotransfected with Rho-kinase and activated RhoA, some Rho-kinase was recruited to membranes. Thus it is likely that Rho-kinase is a putative target serine/threonine kinase for Rho and serves as a mediator of the Rho-dependent signaling pathway. Images PMID:8641286

  16. Expression of a gibberellin-induced leucine-rich repeat receptor-like protein kinase in deepwater rice and its interaction with kinase-associated protein phosphatase

    SciTech Connect

    Knaap, E. van der; Sauter, M.; Kende, H. . DOE Plant Research Lab.); Song, W.Y.; Ruan, D.L.; Ronald, P.C. . Dept. of Plant Pathology)

    1999-06-01

    The authors identified in deepwater rice (Oryza sativa L.) a gene encoding a leucine-rich repeat receptor-like transmembrane protein kinase, OsTMK (O. sativa transmembrane kinase). The transcript levels of OsTMK increased in the rice internode in response to gibberellin. Expression of OsTMK was especially high in regions undergoing cell division and elongation. The kinase domain of OsTMK was enzymatically active autophosphorylating on serine and threonine residues. A cDNA encoding a rice ortholog of a kinase-associated type 2C protein phosphatase (OsKAPP) was cloned. KAPPs are putative downstream components in kinase-mediated signal transduction pathways. The kinase interaction domain of OsKAPP was phosphorylated in vitro by the kinase domain of OsTMK. RNA gel-blot analysis indicated that the expression of OsTMK and OsKAPP was similar in different tissues of the rice plant. In protein-binding assays, OsKAPP interacted with a receptor-like protein kinase, RLK5 of Arabidopsis, but not with the protein kinase domains of the rice and maize receptor-like protein kinases Xa21 and ZmPK1, respectively.

  17. The Secretory Pathway Kinases

    PubMed Central

    Sreelatha, Anju; Kinch, Lisa N.; Tagliabracci, Vincent S.

    2015-01-01

    Protein phosphorylation is a nearly universal post-translation modification involved in a plethora of cellular events. Even though phosphorylation of extracellular proteins had been observed, the identity of the kinases that phosphorylate secreted proteins remained a mystery until recently. Advances in genome sequencing and genetic studies have paved the way for the discovery of a new class of kinases that localize within the endoplasmic reticulum, Golgi apparatus and the extracellular space. These novel kinases phosphorylate proteins and proteoglycans in the secretory pathway and appear to regulate various extracellular processes. Mutations in these kinases cause human disease, thus underscoring the biological importance of phosphorylation within the secretory pathway. PMID:25862977

  18. Molecular cloning and characterization of human JNKK2, a novel Jun NH2-terminal kinase-specific kinase.

    PubMed Central

    Wu, Z; Wu, J; Jacinto, E; Karin, M

    1997-01-01

    At least three mitogen-activated protein kinase (MAPK) cascades were identified in mammals, each consisting of a well-defined three-kinase module composed of a MAPK, a MAPK kinase (MAPKK), and a MAPKK kinase (MAPKKK). These cascades play key roles in relaying various physiological, environmental, or pathological signals from the environment to the transcriptional machinery in the nucleus. One of these MAPKs, c-Jun N-terminal kinase (JNK), stimulates the transcriptional activity of c-Jun in response to growth factors, proinflammatory cytokines, and certain environmental stresses, such as short wavelength UV light or osmotic shock. The JNKs are directly activated by the MAPKK JNKK1/SEK1/MKK4. However, inactivation of the gene encoding this MAPKK by homologous recombination suggested the existence of at least one more JNK-activating kinase. Recently, the JNK cascade was found to be structurally and functionally conserved in Drosophila, where DJNK is activated by the MAPKK DJNKK (hep). By a database search, we identified an expressed sequence tag (EST) encoding a portion of human MAPKK that is highly related to DJNKK (hep). We used this EST to isolate a full-length cDNA clone encoding a human JNKK2. We show that JNKK2 is a highly specific JNK kinase. Unlike JNKK1, it does not activate the related MAPK, p38. Although the regulation of JNKK1 activities and that of JNKK2 activities could be very similar, the two kinases may play somewhat different regulatory roles in a cell-type-dependent manner. PMID:9372971

  19. The Text Encoding Initiative: Flexible and Extensible Document Encoding.

    ERIC Educational Resources Information Center

    Barnard, David T.; Ide, Nancy M.

    1997-01-01

    The Text Encoding Initiative (TEI), an international collaboration aimed at producing a common encoding scheme for complex texts, examines the requirement for generality versus the requirement to handle specialized text types. Discusses how documents and users tax the limits of fixed schemes requiring flexible extensible encoding to support…

  20. Chemoproteomic characterization of protein kinase inhibitors using immobilized ATP.

    PubMed

    Duncan, James S; Haystead, Timothy A J; Litchfield, David W

    2012-01-01

    Protein kinase inhibitors have emerged as indispensable tools for the elucidation of the biological functions of specific signal transduction pathways and as promising candidates for molecular-targeted therapy. However, because many protein kinase inhibitors are ATP-competitive inhibitors targeting the catalytic site of specific protein kinases, the large number of protein kinases that are encoded within eukaryotic genomes and the existence of many other cellular proteins that bind ATP result in the prospect of off-target effects for many of these compounds. Many of the potential off-target effects remain unrecognized because protein kinase inhibitors are often developed and tested primarily on the basis of in vitro assays using purified components. To overcome this limitation, we describe a systematic approach to characterize ATP-competitive protein kinase inhibitors employing ATP-sepharose to capture the purine-binding proteome from cell extracts. Protein kinase inhibitors can be used in competition experiments to prevent binding of specific cellular proteins to ATP-sepharose or to elute bound proteins from ATP-sepharose. Collectively, these strategies can enable validation of interactions between a specific protein kinase and an inhibitor in complex mixtures and can yield the identification of inhibitor targets.

  1. Regulation of tomato Prf by Pto-like protein kinases.

    PubMed

    Mucyn, Tatiana S; Wu, Ai-Jiuan; Balmuth, Alexi L; Arasteh, Julia Maryam; Rathjen, John P

    2009-04-01

    Tomato Prf encodes a nucleotide-binding domain shared by Apaf-1, certain R proteins, and CED-4 fused to C-terminal leucine-rich repeats (NBARC-LRR) protein that is required for bacterial immunity to Pseudomonas syringae and sensitivity to the organophosphate fenthion. The signaling pathways involve two highly related protein kinases. Pto kinase mediates direct recognition of the bacterial effector proteins AvrPto or AvrPtoB. Fen kinase is required for fenthion sensitivity and recognition of bacterial effectors related to AvrPtoB. The role of Pto and its association with Prf has been characterized but Fen is poorly described. We show that, similar to Pto, Fen requires N-myristoylation and kinase activity for signaling and interacts with the N-terminal domain of Prf. Thus, the mechanisms of activation of Prf by the respective protein kinases are similar. Prf-Fen interaction is underlined by coregulatory mechanisms in which Prf negatively regulates Fen, most likely by controlling kinase activity. We further characterized negative regulation of Prf by Pto, and show that regulation is mediated by the previously described negative regulatory patch. Remarkably, the effectors released negative regulation of Prf in a manner dependent on Pto kinase activity. The data suggest a model in which Prf associates generally with Pto-like kinases in tightly regulated complexes, which are activated by effector-mediated disruption of negative regulation. Release of negative regulation may be a general feature of activation of NBARC-LRR proteins by cognate effectors.

  2. Disorders of phonological encoding.

    PubMed

    Butterworth, B

    1992-03-01

    Studies of phonological disturbances in aphasic speech are reviewed. It is argued that failure to test for error consistency in individual patients makes it generally improper to draw inferences about specific disorders of phonological encoding. A minimalist interpretation of available data on phonological errors is therefore proposed that involves variable loss of information in transmission between processing subsystems. Proposals for systematic loss or corruption of phonological information in lexical representations or in translation subsystems is shown to be inadequately grounded. The review concludes with some simple methodological prescriptions for future research.

  3. Receptor Tyrosine Kinase and Tyrosine Kinase Inhibitors

    PubMed Central

    Mirshafiey, Abbas; Ghalamfarsa, Ghasem; Asghari, Babak

    2014-01-01

    Receptor tyrosine kinases (RTKs) are essential components of signal transduction pathways that mediate cell-to-cell communication and their function as relay points for signaling pathways. They have a key role in numerous processes that control cellular proliferation and differentiation, regulate cell growth and cellular metabolism, and promote cell survival and apoptosis. Recently, the role of RTKs including TCR, FLT-3, c-Kit, c-Fms, PDGFR, ephrin, neurotrophin receptor, and TAM receptor in autoimmune disorder, especially rheumatoid arthritis and multiple sclerosis has been suggested. In multiple sclerosis pathogenesis, RTKs and their tyrosine kinase enzymes are selective important targets for tyrosine kinase inhibitor (TKI) agents. TKIs, compete with the ATP binding site of the catalytic domain of several tyrosine kinases, and act as small molecules that have a favorable safety profile in disease treatment. Up to now, the efficacy of TKIs in numerous animal models of MS has been demonstrated, but application of these drugs in human diseases should be tested in future clinical trials. PMID:25337443

  4. Mycobacterium tuberculosis Serine/Threonine Protein Kinases

    PubMed Central

    PRISIC, SLADJANA; HUSSON, ROBERT N.

    2014-01-01

    The Mycobacterium tuberculosis genome encodes 11 serine/threonine protein kinases (STPKs). A similar number of two-component systems are also present, indicating that these two signal transduction mechanisms are both important in the adaptation of this bacterial pathogen to its environment. The M. tuberculosis phosphoproteome includes hundreds of Ser- and Thr-phosphorylated proteins that participate in all aspects of M. tuberculosis biology, supporting a critical role for the STPKs in regulating M. tuberculosis physiology. Nine of the STPKs are receptor type kinases, with an extracytoplasmic sensor domain and an intracellular kinase domain, indicating that these kinases transduce external signals. Two other STPKs are cytoplasmic and have regulatory domains that sense changes within the cell. Structural analysis of some of the STPKs has led to advances in our understanding of the mechanisms by which these STPKs are activated and regulated. Functional analysis has provided insights into the effects of phosphorylation on the activity of several proteins, but for most phosphoproteins the role of phosphorylation in regulating function is unknown. Major future challenges include characterizing the functional effects of phosphorylation for this large number of phosphoproteins, identifying the cognate STPKs for these phosphoproteins, and determining the signals that the STPKs sense. Ultimately, combining these STPK-regulated processes into larger, integrated regulatory networks will provide deeper insight into M. tuberculosis adaptive mechanisms that contribute to tuberculosis pathogenesis. Finally, the STPKs offer attractive targets for inhibitor development that may lead to new therapies for drug-susceptible and drug-resistant tuberculosis. PMID:25429354

  5. Rotary encoding device

    NASA Technical Reports Server (NTRS)

    Leviton, Douglas B. (Inventor)

    1993-01-01

    A device for position encoding of a rotating shaft in which a polygonal mirror having a number of facets is mounted to the shaft and a light beam is directed towards the facets is presented. The facets of the polygonal mirror reflect the light beam such that a light spot is created on a linear array detector. An analog-to-digital converter is connected to the linear array detector for reading the position of the spot on the linear array detector. A microprocessor with memory is connected to the analog-to-digital converter to hold and manipulate the data provided by the analog-to-digital converter on the position of the spot and to compute the position of the shaft based upon the data from the analog-to-digital converter.

  6. Time encoded radiation imaging

    DOEpatents

    Marleau, Peter; Brubaker, Erik; Kiff, Scott

    2014-10-21

    The various technologies presented herein relate to detecting nuclear material at a large stand-off distance. An imaging system is presented which can detect nuclear material by utilizing time encoded imaging relating to maximum and minimum radiation particle counts rates. The imaging system is integrated with a data acquisition system that can utilize variations in photon pulse shape to discriminate between neutron and gamma-ray interactions. Modulation in the detected neutron count rates as a function of the angular orientation of the detector due to attenuation of neighboring detectors is utilized to reconstruct the neutron source distribution over 360 degrees around the imaging system. Neutrons (e.g., fast neutrons) and/or gamma-rays are incident upon scintillation material in the imager, the photons generated by the scintillation material are converted to electrical energy from which the respective neutrons/gamma rays can be determined and, accordingly, a direction to, and the location of, a radiation source identified.

  7. Linear encoding device

    NASA Technical Reports Server (NTRS)

    Leviton, Douglas B. (Inventor)

    1993-01-01

    A Linear Motion Encoding device for measuring the linear motion of a moving object is disclosed in which a light source is mounted on the moving object and a position sensitive detector such as an array photodetector is mounted on a nearby stationary object. The light source emits a light beam directed towards the array photodetector such that a light spot is created on the array. An analog-to-digital converter, connected to the array photodetector is used for reading the position of the spot on the array photodetector. A microprocessor and memory is connected to the analog-to-digital converter to hold and manipulate data provided by the analog-to-digital converter on the position of the spot and to compute the linear displacement of the moving object based upon the data from the analog-to-digital converter.

  8. A novel viral thymidylate kinase with dual kinase activity.

    PubMed

    Guevara-Hernandez, Eduardo; Arvizu-Flores, Aldo A; Lugo-Sanchez, Maria E; Velazquez-Contreras, Enrique F; Castillo-Yañez, Francisco J; Brieba, Luis G; Sotelo-Mundo, Rogerio R

    2015-10-01

    Nucleotide phosphorylation is a key step in DNA replication and viral infections, since suitable levels of nucleotide triphosphates pool are required for this process. Deoxythymidine monophosphate (dTMP) is produced either by de novo or salvage pathways, which is further phosphorylated to deoxythymidine triphosphate (dTTP). Thymidyne monophosphate kinase (TMK) is the enzyme in the junction of both pathways, which phosphorylates dTMP to yield deoxythymidine diphosphate (dTDP) using adenosine triphosphate (ATP) as a phosphate donor. White spot syndrome virus (WSSV) genome contains an open reading frame (ORF454) that encodes a thymidine kinase and TMK domains in a single polypeptide. We overexpressed the TMK ORF454 domain (TMKwssv) and its specific activity was measured with dTMP and dTDP as phosphate acceptors. We found that TMKwssv can phosphorylate dTMP to yield dTDP and also is able to use dTDP as a substrate to produce dTTP. Kinetic parameters K M and k cat were calculated for dTMP (110 μM, 3.6 s(-1)), dTDP (251 μM, 0.9 s(-1)) and ATP (92 μM, 3.2 s(-1)) substrates, and TMKwssv showed a sequential ordered bi-bi reaction mechanism. The binding constants K d for dTMP (1.9 μM) and dTDP (10 μM) to TMKwssv were determined by Isothermal Titration Calorimetry. The affinity of the nucleotidic analog stavudine monophosphate was in the same order of magnitude (K d 3.6 μM) to the canonical substrate dTMP. These results suggest that nucleotide analogues such as stavudine could be a suitable antiviral strategy for the WSSV-associated disease.

  9. Dual-histidine kinases in basidiomycete fungi.

    PubMed

    Lavín, José L; Sarasola-Puente, Vanessa; Ramírez, Lucía; Pisabarro, Antonio G; Oguiza, José A

    2014-02-01

    Dual-histidine kinases (HKs) are complex hybrid HKs containing in a single polypeptide two HK transmitter modules (T) and two-response regulator received domains (R) that are combined in a TRTR geometry. In fungi, this protein family is limited to some particular species of the phylum Basidiomycota and absent in the other phyla. This study extends the investigation of dual-HKs to 80 fully sequenced genomes of basidiomycetes, analyzing their distribution, domain architecture and phylogenetic relationships. Moreover, similarly to dual-HKs of basidiomycetes, several species of bacteria were found that contain hybrid HKs with a TRTR domain architecture encoded in a single gene. PMID:24581805

  10. Systematic deletion analysis of fission yeast protein kinases.

    PubMed

    Bimbó, Andrea; Jia, Yonghui; Poh, Siew Lay; Karuturi, R Krishna Murthy; den Elzen, Nicole; Peng, Xu; Zheng, Liling; O'Connell, Matthew; Liu, Edison T; Balasubramanian, Mohan K; Liu, Jianhua

    2005-04-01

    Eukaryotic protein kinases are key molecules mediating signal transduction that play a pivotal role in the regulation of various biological processes, including cell cycle progression, cellular morphogenesis, development, and cellular response to environmental changes. A total of 106 eukaryotic protein kinase catalytic-domain-containing proteins have been found in the entire fission yeast genome, 44% (or 64%) of which possess orthologues (or nearest homologues) in humans, based on sequence similarity within catalytic domains. Systematic deletion analysis of all putative protein kinase-encoding genes have revealed that 17 out of 106 were essential for viability, including three previously uncharacterized putative protein kinases. Although the remaining 89 protein kinase mutants were able to form colonies under optimal growth conditions, 46% of the mutants exhibited hypersensitivity to at least 1 of the 17 different stress factors tested. Phenotypic assessment of these mutants allowed us to arrange kinases into functional groups. Based on the results of this assay, we propose also the existence of four major signaling pathways that are involved in the response to 17 stresses tested. Microarray analysis demonstrated a significant correlation between the expression signature and growth phenotype of kinase mutants tested. Our complete microarray data sets are available at http://giscompute.gis.a-star.edu.sg/~gisljh/kinome. PMID:15821139

  11. Space vehicle onboard command encoder

    NASA Technical Reports Server (NTRS)

    1975-01-01

    A flexible onboard encoder system was designed for the space shuttle. The following areas were covered: (1) implementation of the encoder design into hardware to demonstrate the various encoding algorithms/code formats, (2) modulation techniques in a single hardware package to maintain comparable reliability and link integrity of the existing link systems and to integrate the various techniques into a single design using current technology. The primary function of the command encoder is to accept input commands, generated either locally onboard the space shuttle or remotely from the ground, format and encode the commands in accordance with the payload input requirements and appropriately modulate a subcarrier for transmission by the baseband RF modulator. The following information was provided: command encoder system design, brassboard hardware design, test set hardware and system packaging, and software.

  12. N-Consecutive-Phase Encoder

    NASA Technical Reports Server (NTRS)

    Divsalar, Dariush; Lee, Ho-Kyoung; Weber, Charles

    1995-01-01

    N-consecutive-phase encoder (NCPE) is conceptual encoder for generating alphabet of N consecutive full-response continuous-phase-modulation (CPM) signals. Enables use of binary preencoder of higher rate than used with simple continuous-phase encoder (CPE). NCPE makes possible to achieve power efficiencies and bandwidth efficiencies greater than conventional trellis coders with continuous-phase frequency-shift keying (CPFSK).

  13. Cytoskeletal Signaling: Is Memory Encoded in Microtubule Lattices by CaMKII Phosphorylation?

    PubMed Central

    Craddock, Travis J. A.; Tuszynski, Jack A.; Hameroff, Stuart

    2012-01-01

    Memory is attributed to strengthened synaptic connections among particular brain neurons, yet synaptic membrane components are transient, whereas memories can endure. This suggests synaptic information is encoded and ‘hard-wired’ elsewhere, e.g. at molecular levels within the post-synaptic neuron. In long-term potentiation (LTP), a cellular and molecular model for memory, post-synaptic calcium ion (Ca2+) flux activates the hexagonal Ca2+-calmodulin dependent kinase II (CaMKII), a dodacameric holoenzyme containing 2 hexagonal sets of 6 kinase domains. Each kinase domain can either phosphorylate substrate proteins, or not (i.e. encoding one bit). Thus each set of extended CaMKII kinases can potentially encode synaptic Ca2+ information via phosphorylation as ordered arrays of binary ‘bits’. Candidate sites for CaMKII phosphorylation-encoded molecular memory include microtubules (MTs), cylindrical organelles whose surfaces represent a regular lattice with a pattern of hexagonal polymers of the protein tubulin. Using molecular mechanics modeling and electrostatic profiling, we find that spatial dimensions and geometry of the extended CaMKII kinase domains precisely match those of MT hexagonal lattices. This suggests sets of six CaMKII kinase domains phosphorylate hexagonal MT lattice neighborhoods collectively, e.g. conveying synaptic information as ordered arrays of six “bits”, and thus “bytes”, with 64 to 5,281 possible bit states per CaMKII-MT byte. Signaling and encoding in MTs and other cytoskeletal structures offer rapid, robust solid-state information processing which may reflect a general code for MT-based memory and information processing within neurons and other eukaryotic cells. PMID:22412364

  14. Centrosome-Kinase Fusions Promote Oncogenic Signaling and Disrupt Centrosome Function in Myeloproliferative Neoplasms

    PubMed Central

    Lee, Joanna Y.; Hong, Wan-Jen; Majeti, Ravindra; Stearns, Tim

    2014-01-01

    Chromosomal translocations observed in myeloproliferative neoplasms (MPNs) frequently fuse genes that encode centrosome proteins and tyrosine kinases. This causes constitutive activation of the kinase resulting in aberrant, proliferative signaling. The function of centrosome proteins in these fusions is not well understood. Among others, kinase centrosome localization and constitutive kinase dimerization are possible consequences of centrosome protein-kinase fusions. To test the relative contributions of localization and dimerization on kinase signaling, we targeted inducibly dimerizable FGFR1 to the centrosome and other subcellular locations and generated a mutant of the FOP-FGFR1 MPN fusion defective in centrosome localization. Expression in mammalian cells followed by western blot analysis revealed a significant decrease in kinase signaling upon loss of FOP-FGFR1 centrosome localization. Kinase dimerization alone resulted in phosphorylation of the FGFR1 signaling target PLCγ, however levels comparable to FOP-FGFR1 required subcellular targeting in addition to kinase dimerization. Expression of MPN fusion proteins also resulted in centrosome disruption in epithelial cells and transformed patient cells. Primary human MPN cells showed masses of modified tubulin that colocalized with centrin, Smoothened (Smo), IFT88, and Arl13b. This is distinct from acute myeloid leukemia (AML) cells, which are not associated with centrosome-kinase fusions and had normal centrosomes. Our results suggest that effective proliferative MPN signaling requires both subcellular localization and dimerization of MPN kinases, both of which may be provided by centrosome protein fusion partners. Furthermore, centrosome disruption may contribute to the MPN transformation phenotype. PMID:24658090

  15. The yeast carboxyl-terminal repeat domain kinase CTDK-I is a divergent cyclin-cyclin-dependent kinase complex.

    PubMed Central

    Sterner, D E; Lee, J M; Hardin, S E; Greenleaf, A L

    1995-01-01

    Saccharomyces cerevisiae CTDK-I is a protein kinase complex that specifically and efficiently hyperphosphorylates the carboxyl-terminal repeat domain (CTD) of RNA polymerase II and is composed of three subunits of 58, 38, and 32 kDa. The kinase is essential in vivo for normal phosphorylation of the CTD and for normal growth and differentiation. We have now cloned the genes for the two smaller kinase subunits, CTK2 and CTK3, and found that they form a unique, divergent cyclin-cyclin-dependent kinase complex with the previously characterized largest subunit protein CTK1, a cyclin-dependent kinase homolog. The CTK2 gene encodes a cyclin-related protein with limited homology to cyclin C, while CTK3 shows no similarity to other known proteins. Copurification of the three gene products with each other and CTDK-I activity by means of conventional chromatography and antibody affinity columns has verified their participation in the complex in vitro. In addition, null mutations of each of the genes and all combinations thereof conferred very similar growth-impaired, cold-sensitive phenotypes, consistent with their involvement in the same function in vivo. These characterizations and the availability of all of the genes encoding CTDK-I and reagents derivable from them will facilitate investigations into CTD phosphorylation and its functional consequences both in vivo and in vitro. PMID:7565723

  16. Prosodic Encoding in Silent Reading.

    ERIC Educational Resources Information Center

    Wilkenfeld, Deborah

    In silent reading, short-memory tasks, such as semantic and syntactic processing, require a stage of phonetic encoding between visual representation and the actual extraction of meaning, and this encoding includes prosodic as well as segmental features. To test for this suprasegmental coding, an experiment was conducted in which subjects were…

  17. Information encoder/decoder using chaotic systems

    DOEpatents

    Miller, Samuel Lee; Miller, William Michael; McWhorter, Paul Jackson

    1997-01-01

    The present invention discloses a chaotic system-based information encoder and decoder that operates according to a relationship defining a chaotic system. Encoder input signals modify the dynamics of the chaotic system comprising the encoder. The modifications result in chaotic, encoder output signals that contain the encoder input signals encoded within them. The encoder output signals are then capable of secure transmissions using conventional transmission techniques. A decoder receives the encoder output signals (i.e., decoder input signals) and inverts the dynamics of the encoding system to directly reconstruct the original encoder input signals.

  18. Information encoder/decoder using chaotic systems

    DOEpatents

    Miller, S.L.; Miller, W.M.; McWhorter, P.J.

    1997-10-21

    The present invention discloses a chaotic system-based information encoder and decoder that operates according to a relationship defining a chaotic system. Encoder input signals modify the dynamics of the chaotic system comprising the encoder. The modifications result in chaotic, encoder output signals that contain the encoder input signals encoded within them. The encoder output signals are then capable of secure transmissions using conventional transmission techniques. A decoder receives the encoder output signals (i.e., decoder input signals) and inverts the dynamics of the encoding system to directly reconstruct the original encoder input signals. 32 figs.

  19. Phosphatidylinositol kinase from rabbit reticulocytes

    SciTech Connect

    Tuazon, P.T.; Heng, A.B.W.; Traugh, J.A.

    1986-05-01

    Phosphatidylinositol (PI) kinase was isolated from the postribosomal supernatant of rabbit reticulocytes. This activity was identified by the formation of a product that comigrated with phosphatidylinositol-4-phosphate (PIP) when purified PI was phosphorylated in the presence of (/sup 32/P)ATP and Mg/sup 2 +/. Three major peaks of PI kinase activity were resolved by chromatography on DEAE-cellulose. The first peak eluted at 50-100 mM NaCl together with several serine protein kinases, casein kinase (CK) I and protease activated kinase (PAK) I and II. The PI kinase was subsequently separated from the protein kinases by chromatography on phosphocellulose. The second peak eluted at 125-160 mM NaCl and contained another lipid kinase activity that produced a product which comigrated with phosphatidic acid on thin layer chromatography. The third peak, which eluted at 165-200 mM NaCl, partly comigrated with casein kinase (CK) II and an active protein kinase(s) which phosphorylated mixed histone and histone I. CK II and the histone kinase activities were also separated by chromatography on phosphocelluslose. The different forms of PI kinase were characterized and compared with respect to substrate and salt requirements.

  20. Microbial Protein-tyrosine Kinases*

    PubMed Central

    Chao, Joseph D.; Wong, Dennis; Av-Gay, Yossef

    2014-01-01

    Microbial ester kinases identified in the past 3 decades came as a surprise, as protein phosphorylation on Ser, Thr, and Tyr amino acids was thought to be unique to eukaryotes. Current analysis of available microbial genomes reveals that “eukaryote-like” protein kinases are prevalent in prokaryotes and can converge in the same signaling pathway with the classical microbial “two-component” systems. Most microbial tyrosine kinases lack the “eukaryotic” Hanks domain signature and are designated tyrosine kinases based upon their biochemical activity. These include the tyrosine kinases termed bacterial tyrosine kinases (BY-kinases), which are responsible for the majority of known bacterial tyrosine phosphorylation events. Although termed generally as bacterial tyrosine kinases, BY-kinases can be considered as one family belonging to the superfamily of prokaryotic protein-tyrosine kinases in bacteria. Other members of this superfamily include atypical “odd” tyrosine kinases with diverse mechanisms of protein phosphorylation and the “eukaryote-like” Hanks-type tyrosine kinases. Here, we discuss the distribution, phylogeny, and function of the various prokaryotic protein-tyrosine kinases, focusing on the recently discovered Mycobacterium tuberculosis PtkA and its relationship with other members of this diverse family of proteins. PMID:24554699

  1. The FIKK kinase of Toxoplasma gondii is not essential for the parasite's lytic cycle.

    PubMed

    Skariah, S; Walwyn, O; Engelberg, K; Gubbels, M-J; Gaylets, C; Kim, N; Lynch, B; Sultan, A; Mordue, D G

    2016-05-01

    FIKK kinases are a novel family of kinases unique to the Apicomplexa. While most apicomplexans encode a single FIKK kinase, Plasmodium falciparum expresses 21 and piroplasms do not encode a FIKK kinase. FIKK kinases share a conserved C-terminal catalytic domain, but the N-terminal region is highly variable and contains no known functional domains. To date, FIKK kinases have been primarily studied in P. falciparum and Plasmodium berghei. Those that have been studied are exported from the parasite and associate with diverse locations in the infected erythrocyte cytosol or membrane. Deletion of individual P. falciparum FIKK kinases indicates that they may play a role in modification of the infected erythrocyte. The current study characterises the single FIKK gene in Toxoplasma gondii to evaluate the importance of the FIKK kinase in an apicomplexan that has a single FIKK kinase. The TgFIKK gene encoded a protein of approximately 280kDa. Endogenous tagging of the FIKK protein with Yellow Fluorescent Protein showed that the FIKK protein exclusively localised to the posterior end of tachyzoites. A Yellow Fluorescent Protein-tagged FIKK and a Ty-tagged FIKK both co-localised with T. gondii membrane occupation and recognition nexus protein to the basal complex and were localised apical to inner membrane complex protein-5 and Centrin2. Deletion of TgFIKK, surprisingly, had no detectable effect on the parasite's lytic cycle in vitro in human fibroblast cells or in acute virulence in vivo. Thus, our results clearly show that while the FIKK kinase is expressed in tachyzoites, it is not essential for the lytic cycle of T. gondii.

  2. Molecular cloning and characterization of the pyrB1 and pyrB2 genes encoding aspartate transcarbamoylase in pea (Pisum sativum L.).

    PubMed

    Williamson, C L; Slocum, R D

    1994-05-01

    We cloned cDNAs encoding two different pea (Pisum sativum L.) aspartate transcarbamoylases (ATCases) by complementation of an Escherichia coli delta pyrB mutant. The two cDNAs, designated pyrB1 and pyrB2, encode polypeptides of 386 and 385 amino acid residues, respectively, both of which exhibit typical chloroplast transit peptide sequences. Wheat germ ATCase antibody recognizes a 36.5-kD polypeptide in pea leaf and root tissues that is similar in size to other plant ATCase polypeptides and to the catalytic polypeptides of bacterial ATCases. Northern analyses indicate that the pyrB1 and pyrB2 transcripts are 1.6 kb in size and are differentially expressed in pea tissues. The small transcript size and data from biochemical studies indicate that plant ATCases are simple homotrimers of 36- to 37-kD catalytic subunits, rather than part of a multifunctional enzyme containing glutamine-dependent carbamoylphosphate synthetase and dihydroorotase activities, as is seen in other eukaryotes. In the pea ATCases, the carbamoylphosphate- and aspartate-binding domains are highly homologous to those of other prokaryotic and eukaryotic ATCases and critical active-site residues are completely conserved. The pea ATCases also exhibit a putative pyrimidine-binding site, consistent with the known allosteric regulation of plant ATCases by UMP in vitro. PMID:8029359

  3. PNA-encoded chemical libraries.

    PubMed

    Zambaldo, Claudio; Barluenga, Sofia; Winssinger, Nicolas

    2015-06-01

    Peptide nucleic acid (PNA)-encoded chemical libraries along with DNA-encoded libraries have provided a powerful new paradigm for library synthesis and ligand discovery. PNA-encoding stands out for its compatibility with standard solid phase synthesis and the technology has been used to prepare libraries of peptides, heterocycles and glycoconjugates. Different screening formats have now been reported including selection-based and microarray-based methods that have yielded specific ligands against diverse target classes including membrane receptors, lectins and challenging targets such as Hsp70.

  4. Two digital video encoder circuits

    NASA Astrophysics Data System (ADS)

    Eldon, John A.

    1992-11-01

    Central to `multimedia' image processing is the desire to encode computer graphics data into a standard television signal, complete with line, field, and color subcarrier synchronizing information. The numerous incompatibilities between television and computer display standards render this operation far less trivial than it sounds to anyone who hasn't worked with both types of signals. To simplify the task of encoding computer graphics signals into standard NTSC (North America and Japan) or PAL (most of Europe) television format for display, broadcast, or recording, TRW LSI Products Inc. has introduced the two newest members of it multimedia integrated circuit family, the TMC22090 and TMC22190 digital video encoders.

  5. Protein kinase C is essential for viability of the rice blast fungus M agnaporthe oryzae

    PubMed Central

    Penn, Tina J.; Wood, Mark E.; Soanes, Darren M.; Csukai, Michael; Corran, Andrew John

    2015-01-01

    Summary Protein kinase C constitutes a family of serine–threonine kinases found in all eukaryotes and implicated in a wide range of cellular functions, including regulation of cell growth, cellular differentiation and immunity. Here, we present three independent lines of evidence which indicate that protein kinase C is essential for viability of M agnaporthe oryzae. First, all attempts to generate a target deletion of PKC 1, the single copy protein kinase C‐encoding gene, proved unsuccessful. Secondly, conditional gene silencing of PKC 1 by RNA interference led to severely reduced growth of the fungus, which was reversed by targeted deletion of the Dicer2‐encoding gene, MDL 2. Finally, selective kinase inhibition of protein kinase C by targeted allelic replacement with an analogue‐sensitive PKC 1AS allele led to specific loss of fungal viability in the presence of the PP1 inhibitor. Global transcriptional profiling following selective PKC inhibition identified significant changes in gene expression associated with cell wall re‐modelling, autophagy, signal transduction and secondary metabolism. When considered together, these results suggest protein kinase C is essential for growth and development of M . oryzae with extensive downstream targets in addition to the cell integrity pathway. Targeting protein kinase C signalling may therefore prove an effective means of controlling rice blast disease. PMID:26192090

  6. Visualizing autophosphorylation in histidine kinases.

    PubMed

    Casino, Patricia; Miguel-Romero, Laura; Marina, Alberto

    2014-01-01

    Reversible protein phosphorylation is the most widespread regulatory mechanism in signal transduction. Autophosphorylation in a dimeric sensor histidine kinase is the first step in two-component signalling, the predominant signal-transduction device in bacteria. Despite being the most abundant sensor kinases in nature, the molecular bases of the histidine kinase autophosphorylation mechanism are still unknown. Furthermore, it has been demonstrated that autophosphorylation can occur in two directions, cis (intrasubunit) or trans (intersubunit) within the dimeric histidine kinase. Here, we present the crystal structure of the complete catalytic machinery of a chimeric histidine kinase. The structure shows an asymmetric histidine kinase dimer where one subunit is caught performing the autophosphorylation reaction. A structure-guided functional analysis on HK853 and EnvZ, two prototypical cis- and trans-phosphorylating histidine kinases, has allowed us to decipher the catalytic mechanism of histidine kinase autophosphorylation, which seems to be common independently of the reaction directionality.

  7. Molecular characterization of polyphosphate kinase (ppk) gene from Serratia marcescens.

    PubMed

    Lee, Seung-Jin; Song, Ok-Ryul; Lee, Young-Choon; Choi, Yong-Lark

    2003-02-01

    To understand the mechanism of phosphate accumulation, a gene encoding polyphosphate kinase (PPK) was cloned from the genomic library of Serratia marcescens by Southern hybridization. From the nucleotide sequence of a 4 kb DNA fragment, an open reading frame of 2063 nucleotides was identified encoding a protein of 686 amino acids with molecular mass of 70 kDa. The potential CRP binding site and pho box sequence were found upstream of the putative promoter in the regulatory region. The expression of PPK resulted in the formation of inclusion bodies and the product was active at low temperature. The E. coli strain harboring plasmid pSPK5 with ppk gene increased enzyme activity of polyphosphate kinase, resulting in increased accumulation of polyphosphate in E. coli.

  8. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Linn, Anning

    1996-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK.

  9. Plant 5-Methylthioribose Kinase

    PubMed Central

    Guranowski, Andrzej

    1983-01-01

    Activity of 5-methylthioribose kinase, the enzyme which catalyzes the ATP-dependent formation of 1-phospho-5-methylthioribose, has been revealed in the extracts from various higher plant species. Almost 2,000-fold-purified enzyme has been obtained from yellow lupin (Lupinus luteus L. cv Topaz) seed extract. Molecular weight of the native enzyme is 70,000 as judged by gel filtration. The lupin 5-methylthioribose kinase exhibits a strict requirement for divalent metal ions. Among the ions tested, only Mg2+ and Mn2+ acted as cofactors. The curve of kinase initial velocity versus pH reaches plateau at pH 10 to 10.5. The Km values calculated for 5-methylthioribose and ATP are 4.3 and 8.3 micromolar, respectively. Among nucleoside triphosphates tested as potential phosphate donors, only dATP could substitute in the reaction for ATP. 5-Isobutylthioribose, an analog of 5-methylthioribose, proved to be the γ-ATP-phosphate acceptor, too. The compound inhibits competitively synthesis of 1-phospho-5-methylthioribose (Ki = 1.4 micromolar). Lupin 5-methylthioribose kinase is completely and irreversibly inhibited by the antisulfhydryl reagent, p-hydroxymercuribenzoate. As in bacteria (Ferro, Barrett, Shapiro 1978 J Biol Chem 253: 6021-6025), the enzyme may be involved in a new, alternative pathway of methionine synthesis in plant tissues. PMID:16662931

  10. Plant Raf-like kinase integrates abscisic acid and hyperosmotic stress signaling upstream of SNF1-related protein kinase2.

    PubMed

    Saruhashi, Masashi; Kumar Ghosh, Totan; Arai, Kenta; Ishizaki, Yumiko; Hagiwara, Kazuya; Komatsu, Kenji; Shiwa, Yuh; Izumikawa, Keiichi; Yoshikawa, Harunori; Umezawa, Taishi; Sakata, Yoichi; Takezawa, Daisuke

    2015-11-17

    Plant response to drought and hyperosmosis is mediated by the phytohormone abscisic acid (ABA), a sesquiterpene compound widely distributed in various embryophyte groups. Exogenous ABA as well as hyperosmosis activates the sucrose nonfermenting 1 (SNF1)-related protein kinase2 (SnRK2), which plays a central role in cellular responses against drought and dehydration, although the details of the activation mechanism are not understood. Analysis of a mutant of the moss Physcomitrella patens with reduced ABA sensitivity and reduced hyperosmosis tolerance revealed that a protein kinase designated "ARK" (for "ABA and abiotic stress-responsive Raf-like kinase") plays an essential role in the activation of SnRK2. ARK encoded by a single gene in P. patens belongs to the family of group B3 Raf-like MAP kinase kinase kinases (B3-MAPKKKs) mediating ethylene, disease resistance, and salt and sugar responses in angiosperms. Our findings indicate that ARK, as a novel regulatory component integrating ABA and hyperosmosis signals, represents the ancestral B3-MAPKKKs, which multiplied, diversified, and came to have specific functions in angiosperms. PMID:26540727

  11. Overexpression of the potential kinase serine/ threonine/tyrosine kinase 1 (STYK 1) in castration-resistant prostate cancer.

    PubMed

    Chung, Suyoun; Tamura, Kenji; Furihata, Mutsuo; Uemura, Motohide; Daigo, Yataro; Nasu, Yasutomo; Miki, Tsuneharu; Shuin, Taro; Fujioka, Tomoaki; Nakamura, Yusuke; Nakagawa, Hidewaki

    2009-11-01

    Despite high response rates and clinical benefits, androgen ablation often fails to cure advanced or relapsed prostate cancer because castration-resistant prostate cancer (CRPC) cells inevitably emerge. CRPC cells not only grow under castration, but also behave more aggressively, indicating that a number of malignant signaling pathways are activated in CRPC cells as well as androgen receptor signaling. Based on information from the gene expression profiles of clinical CRPC cells, we here identified one overexpressed gene, serine/threonine/tyrosine kinase 1 (STYK1), encoding a potential kinase, as a molecular target for CRPC. RNA and immunohistochemical analyses validated the overexpression of STYK1 in prostate cancer cells, and its expression was distinct in CRPC cells. Knockdown of STYK1 by siRNA resulted in drastic suppression of prostate cancer cell growth and, concordantly, enforced expression of STYK1 promoted cell proliferation, whereas ectopic expression of a kinase-dead mutant STYK1 did not. An in vitro kinase assay using recombinant STYK1 demonstrated that STYK1 could have some potential as a kinase, although its specific substrates are unknown. These findings suggest that STYK1 could be a possible molecular target for CRPC, and small molecules specifically inhibiting STYK1 kinase could be a possible approach for the development of novel CRPC therapies.

  12. Biochemical and molecular analysis of a transmembrane protein kinase from Arabidopsis thaliana. Progress report, January 1993

    SciTech Connect

    Bleecker, A.B.

    1993-06-01

    We have isolated genomic and cDNA clones encoding a novel receptor-like protein kinase from the higher plant Arabidopsis thaliana. This kinase is being studied by combining biochemical, molecular, and genetic approaches. Domain-specific antibodies immunodecorate a polypeptide with a molecular mass of 120,000 daltons in extracts of Arabidopsis, where it has been found in all portions of the plant examined including root, stem, leaf, flower, and silique. Cytochemical analysis and initial studies using the kinase promoter with the GUS reporter gene system also indicate that the kinase is present throughout the plant. The kinase is glycosylated, like animal receptor kinases, and has been partially purified from Arabidopsis by using lectin columns. The kinase has been expressed in E coli, purified, and found to autophosphorylate on serine and threonine residues, but not on tyrosine residues. As such, it belongs to the small family of receptor-like kinases with serine/threonine specificity. Transgenic plants are now being produced that either overexpress or carry altered forms of the protein kinase gene. These experiments will help determine the natural role the kinase plays in a pathway of signal transduction.

  13. Biochemical and molecular analysis of a transmembrane protein kinase from Arabidopsis thaliana

    SciTech Connect

    Bleecker, A.B.

    1993-01-01

    We have isolated genomic and cDNA clones encoding a novel receptor-like protein kinase from the higher plant Arabidopsis thaliana. This kinase is being studied by combining biochemical, molecular, and genetic approaches. Domain-specific antibodies immunodecorate a polypeptide with a molecular mass of 120,000 daltons in extracts of Arabidopsis, where it has been found in all portions of the plant examined including root, stem, leaf, flower, and silique. Cytochemical analysis and initial studies using the kinase promoter with the GUS reporter gene system also indicate that the kinase is present throughout the plant. The kinase is glycosylated, like animal receptor kinases, and has been partially purified from Arabidopsis by using lectin columns. The kinase has been expressed in E coli, purified, and found to autophosphorylate on serine and threonine residues, but not on tyrosine residues. As such, it belongs to the small family of receptor-like kinases with serine/threonine specificity. Transgenic plants are now being produced that either overexpress or carry altered forms of the protein kinase gene. These experiments will help determine the natural role the kinase plays in a pathway of signal transduction.

  14. Transcriptional regulation through glutamate receptors: Involvement of tyrosine kinases.

    PubMed

    López-Bayghen, Esther; Aguirre, Adán; Ortega, Arturo

    2003-12-01

    Glutamate receptors play a key role in neuronal plasticity, learning and memory, and in several neuropathologies. Short-term and long-term changes in synaptic efficacy are triggered by glutamate. Although an enhanced glutamate-dependent tyrosine phosphorylation has been described in several systems, its role in membrane-to-nuclei signaling is unclear. Taking advantage of the fact that the gene encoding the chick kainate-binding protein undergoes a glutamate-dependent transcriptional regulation via an activator protein-1 (AP-1) site, we evaluated the involvement of tyrosine kinases in this process. We describe here the participation of receptor and non-receptor tyrosine kinases in the signaling cascade triggered by glutamate. Our results suggest that in Bergmann glia cells, glutamate receptors transactivate receptor tyrosine kinases, favoring the idea of a complex network of signals activated by this excitatory neurotransmitter that results in regulation of gene expression.

  15. A novel Toxoplasma gondii calcium-dependent protein kinase.

    PubMed

    Tzen, M; Benarous, R; Dupouy-Camet, J; Roisin, M P

    2007-06-01

    Toxoplasma gondii is an obligate intracellular parasite that infects all types of cells in humans. A family of calcium-dependent protein kinases (CDPKs), previously identified as important in the development of plants and protists, was recently shown to play a role in the infectivity of apicomplexans, and in motility and host cell invasion in particular. We report here the isolation of a new calcium-dependent protein kinase gene from the human toxoplasmosis parasite, Toxoplasma gondii. The gene consists of 12 exons. The encoded protein, TgCDPK4, consists of the four characteristic domains of members of the CDPK family and is most similar to PfCDPK2 from Plasmodium falciparum. We measured TgCDPK4 activity, induced by calcium influx, using a kinase assay. A calcium chelator (EGTA) inhibited this activity. These findings provide evidence of signal transduction involving members of the CDPK family in T. gondii.

  16. Serial position encoding of signs.

    PubMed

    Miozzo, Michele; Petrova, Anna; Fischer-Baum, Simon; Peressotti, Francesca

    2016-09-01

    Reduced short-term memory (STM) capacity has been reported for sign as compared to speech when items have to be recalled in a specific order. This difference has been attributed to a more precise and efficient serial position encoding in verbal STM (used for speech) than visuo-spatial STM (used for sign). We tested in the present investigation whether the reduced STM capacity with signs stems from a lack of positional encoding available in verbal STM. Error analyses reported in prior studies have revealed that positions are defined in verbal STM by distance from both the start and the end of the sequence (both-edges positional encoding scheme). Our analyses of the errors made by deaf participants with finger-spelled letters revealed that the both-edges positional encoding scheme underlies the STM representation of signs. These results indicate that the cause of the STM disadvantage is not the type of positional encoding but rather the difficulties in binding an item in visuo-spatial STM to its specific position in the sequence. Both-edges positional encoding scheme could be specific of sign, since it has not been found in visuo-spatial STM tasks conducted with hearing participants. PMID:27244095

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

    DOEpatents

    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.

  18. Dominant negative selection of vaccinia virus using a thymidine kinase/thymidylate kinase fusion gene and the prodrug azidothymidine

    SciTech Connect

    Holzer, Georg W. . E-mail: falknef@baxter.com

    2005-07-05

    The Escherichia coli thymidine kinase/thymidylate kinase (tk/tmk) fusion gene encodes an enzyme that efficiently converts the prodrug 3'-azido-2',3'-dideoxythymidine (AZT) into its toxic triphosphate derivative, a substance which stops DNA chain elongation. Integration of this marker gene into vaccinia virus that normally is not inhibited by AZT allowed the establishment of a powerful selection procedure for recombinant viruses. In contrast to the conventional vaccinia thymidine kinase (tk) selection that is performed in tk-negative cell lines, AZT selection can be performed in normal (tk-positive) cell lines. The technique is especially useful for the generation of replication-deficient vaccinia viruses and may also be used for gene knock-out studies of essential vaccinia genes.

  19. Escherichia coli O157:H7 lacking qseBC encoded quorum sensing system outcompetes the parent strain in colonization of cattle intestine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The qseBC encoded quorum-sensing system (QS) regulates motility of enterohemorrhagic Escherichia coli (EHEC) O157:H7 in response to bacterial autoinducer-3 (AI-3) and mammalian stress hormones epinephrine (E) and norepinephrine (NE). The qseC gene encodes a sensory kinase that post-autophosphorylati...

  20. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2002-01-29

    The present invention provides an isolated polynucleotide encoding a c-Jun peptide consisting of about amino acid residues 33 to 79 as set fort in SEQ ID NO: 10 or conservative variations thereof. The invention also provides a method for producing a peptide of SEQ ID NO:1 comprising (a) culturing a host cell containing a polynucleotide encoding a c-Jun peptide consisting of about amino acid residues 33 to 79 as set forth in SEQ ID NO: 10 under conditions which allow expression of the polynucleotide; and (b) obtaining the peptide of SEQ ID NO:1.

  1. Cloning and characterization of a 92 kDa soluble phosphatidylinositol 4-kinase.

    PubMed Central

    Nakagawa, T; Goto, K; Kondo, H

    1996-01-01

    A phosphatidylinositol (PtdIns) 4-kinase cDNA cloned from a rat brain cDNA library encoded a protein of 816 amino acids with a calculated molecular mass of 91654 Da. This molecule contained a lipid-kinase-unique domain and a presumed lipid/ protein kinase homology domain that are found in other PtdIns 4-kinases and PtdIns 3-kinases. Furthermore, this kinase molecule had 43.3% shared identity with the presumed catalytic domain of yeast PtdIns 4-kinase, PtdInsK1, and the two molecules had a region of similarity that is not conserved in other lipid kinases. By examining PtdIns kinase activity in transfected COS-7 cells using epitope tag immunoprecipitation as well as conventional methods, the product PtdIns phosphate was identified as phosphatidylinositol 4-phosphate (PtdIns4P), but not phosphatidylinositol 3-phosphate (PtdIns3P). The PtdIns 4-kinase activity was recovered predominantly from the soluble fraction and the activity was markedly enhanced in the presence of Triton X-100 and was relatively insensitive to inhibition by adenosine. In addition, the PtdIns 4-kinase activity was completely inhibited in the presence of 10 microM wortmannin. When examined by epitope tag immunocytochemistry, the immunoreactivity for the PtdIns 4-kinase molecule was dominantly aggregated in a cytoplasmic region juxtaposed to the nuclei and was faintly but widely dispersed in the cytoplasm. By in situ hybridization analysis, the mRNA for PtdIns 4-kinase was expressed ubiquitously and was detected in most neurons throughout the grey matter of the brain, with higher expression intensity found in fetal than in adult brain. PMID:8973579

  2. Oncoprotein protein kinase

    DOEpatents

    Karin, M.; Hibi, M.; Lin, A.

    1997-02-25

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE is disclosed. The polypeptide has serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences. The method of detection of JNK is also provided. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites. 44 figs.

  3. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2004-03-16

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  4. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning; Davis, Roger; Derijard, Benoit

    2003-02-04

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  5. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1998-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  6. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1999-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  7. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning; Davis, Roger; Derijard, Benoit

    2005-03-08

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  8. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1997-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  9. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1997-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  10. Oncoprotein protein kinase

    DOEpatents

    Davis, Roger; Derijard, Benoit; Karin, Michael; Hibi, Masahiko; Lin, Anning

    2005-01-25

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  11. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Lin, Anning

    1999-11-30

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  12. Cyclin-dependent kinases.

    PubMed

    Malumbres, Marcos

    2014-01-01

    Cyclin-dependent kinases (CDKs) are protein kinases characterized by needing a separate subunit - a cyclin - that provides domains essential for enzymatic activity. CDKs play important roles in the control of cell division and modulate transcription in response to several extra- and intracellular cues. The evolutionary expansion of the CDK family in mammals led to the division of CDKs into three cell-cycle-related subfamilies (Cdk1, Cdk4 and Cdk5) and five transcriptional subfamilies (Cdk7, Cdk8, Cdk9, Cdk11 and Cdk20). Unlike the prototypical Cdc28 kinase of budding yeast, most of these CDKs bind one or a few cyclins, consistent with functional specialization during evolution. This review summarizes how, although CDKs are traditionally separated into cell-cycle or transcriptional CDKs, these activities are frequently combined in many family members. Not surprisingly, deregulation of this family of proteins is a hallmark of several diseases, including cancer, and drug-targeted inhibition of specific members has generated very encouraging results in clinical trials. PMID:25180339

  13. Multidimensionally encoded magnetic resonance imaging.

    PubMed

    Lin, Fa-Hsuan

    2013-07-01

    Magnetic resonance imaging (MRI) typically achieves spatial encoding by measuring the projection of a q-dimensional object over q-dimensional spatial bases created by linear spatial encoding magnetic fields (SEMs). Recently, imaging strategies using nonlinear SEMs have demonstrated potential advantages for reconstructing images with higher spatiotemporal resolution and reducing peripheral nerve stimulation. In practice, nonlinear SEMs and linear SEMs can be used jointly to further improve the image reconstruction performance. Here, we propose the multidimensionally encoded (MDE) MRI to map a q-dimensional object onto a p-dimensional encoding space where p > q. MDE MRI is a theoretical framework linking imaging strategies using linear and nonlinear SEMs. Using a system of eight surface SEM coils with an eight-channel radiofrequency coil array, we demonstrate the five-dimensional MDE MRI for a two-dimensional object as a further generalization of PatLoc imaging and O-space imaging. We also present a method of optimizing spatial bases in MDE MRI. Results show that MDE MRI with a higher dimensional encoding space can reconstruct images more efficiently and with a smaller reconstruction error when the k-space sampling distribution and the number of samples are controlled.

  14. [Construction of the flavinogenic yeast Candida famata strains with high riboflavin kinase activity using gene engineering].

    PubMed

    Ishchuk, O P; Iatsyshyn, V Iu; Dmytruk, K V; Voronovs'kyĭ, A Ia; Fedorovych, D V; Sybirnyĭ, A A

    2006-01-01

    The recombinant strains of the flavinogenic yeast Candida famata, which contain the DNA fragment consisting of the FMN1 gene (encoding the riboflavin kinase, enzyme that converts riboflavin to flavinmononucleotide) driven by the strong promoters (the regulated RIB1 or constitutive TEF1 promoter) were isolated. Riboflavin kinase activity in the isolated transformants was tested. The 6-8-fold increase of the riboflavin kinase activity was shown in the recombinant strains containing the integrated Debaryomyces hansenii FMN1 gene under the strong constitutive TEF1 promoter. The recombinant strains can be used for the following construction of flavinmononucleotide overproducers. PMID:17290783

  15. 3pK, a new mitogen-activated protein kinase-activated protein kinase located in the small cell lung cancer tumor suppressor gene region.

    PubMed Central

    Sithanandam, G; Latif, F; Duh, F M; Bernal, R; Smola, U; Li, H; Kuzmin, I; Wixler, V; Geil, L; Shrestha, S

    1996-01-01

    NotI linking clones, localized to the human chromosome 3p21.3 region and homozygously deleted in small cell lung cancer cell lines NCI-H740 and NCI-H1450, were used to search for a putative tumor suppressor gene(s). One of these clones, NL1G210, detected a 2.5-kb mRNA in all examined human tissues, expression being especially high in the heart and skeletal muscle. Two overlapping cDNA clones containing the entire open reading frame were isolated from a human heart cDNA library and fully characterized. Computer analysis and a search of the GenBank database to reveal high sequence identity of the product of this gene to serine-threonine kinases, especially to mitogen-activated protein kinase-activated protein kinase 2, a recently described substrate of mitogen-activated kinases. Sequence identitiy was 72% at the nucleotide level and 75% at the amino acid level, strongly suggesting that this protein is a serine-threonine kinase. Here we demonstrate that the new gene, referred to as 3pK (for chromosome 3p kinase), in fact encodes a mitogen-activated protein kinase-regulated protein serine-threonine kinase with a novel substrate specificity. PMID:8622688

  16. Fly Photoreceptors Encode Phase Congruency

    PubMed Central

    Friederich, Uwe; Billings, Stephen A.; Hardie, Roger C.; Juusola, Mikko; Coca, Daniel

    2016-01-01

    More than five decades ago it was postulated that sensory neurons detect and selectively enhance behaviourally relevant features of natural signals. Although we now know that sensory neurons are tuned to efficiently encode natural stimuli, until now it was not clear what statistical features of the stimuli they encode and how. Here we reverse-engineer the neural code of Drosophila photoreceptors and show for the first time that photoreceptors exploit nonlinear dynamics to selectively enhance and encode phase-related features of temporal stimuli, such as local phase congruency, which are invariant to changes in illumination and contrast. We demonstrate that to mitigate for the inherent sensitivity to noise of the local phase congruency measure, the nonlinear coding mechanisms of the fly photoreceptors are tuned to suppress random phase signals, which explains why photoreceptor responses to naturalistic stimuli are significantly different from their responses to white noise stimuli. PMID:27336733

  17. Fly Photoreceptors Encode Phase Congruency.

    PubMed

    Friederich, Uwe; Billings, Stephen A; Hardie, Roger C; Juusola, Mikko; Coca, Daniel

    2016-01-01

    More than five decades ago it was postulated that sensory neurons detect and selectively enhance behaviourally relevant features of natural signals. Although we now know that sensory neurons are tuned to efficiently encode natural stimuli, until now it was not clear what statistical features of the stimuli they encode and how. Here we reverse-engineer the neural code of Drosophila photoreceptors and show for the first time that photoreceptors exploit nonlinear dynamics to selectively enhance and encode phase-related features of temporal stimuli, such as local phase congruency, which are invariant to changes in illumination and contrast. We demonstrate that to mitigate for the inherent sensitivity to noise of the local phase congruency measure, the nonlinear coding mechanisms of the fly photoreceptors are tuned to suppress random phase signals, which explains why photoreceptor responses to naturalistic stimuli are significantly different from their responses to white noise stimuli. PMID:27336733

  18. Characterization of the fission yeast mcs2 cyclin and its associated protein kinase activity.

    PubMed Central

    Molz, L; Beach, D

    1993-01-01

    We have previously described the isolation of mcs2-75, a mutation obtained as an allele-specific suppressor of a dominant allele of cdc2. mcs2 was cloned and determined to be an essential gene, the product of which shares homology with the cyclin family of proteins. In contrast to the behavior of some, but not all cyclins, the mcs2 protein is constant in its abundance and localization throughout the cell cycle. A kinase activity that co-precipitates with mcs2 can be detected when myelin basic protein (MBP) is provided as an exogenous substrate. This kinase activity is constant throughout the cell cycle. mcs2 does not appear to associate with the cdc2 protein kinase or an antigenically related kinase. Finally, a protein kinase termed csk1 (cyclin suppressing kinase) was isolated as a high copy suppressor of an mcs2 mutation. csk1 is not essential, however, the level of kinase activity that co-precipitates with mcs2 is reduced approximately 3-fold in strains harboring a csk1 null allele. Therefore, csk1 may encode a protein kinase physically associated with mcs2 or alternatively may function as an upstream activator of the mcs2-associated kinase. Images PMID:8467814

  19. Redox Regulation of Protein Kinases

    PubMed Central

    Truong, Thu H.; Carroll, Kate S.

    2015-01-01

    Protein kinases represent one of the largest families of genes found in eukaryotes. Kinases mediate distinct cellular processes ranging from proliferation, differentiation, survival, and apoptosis. Ligand-mediated activation of receptor kinases can lead to the production of endogenous H2O2 by membrane-bound NADPH oxidases. In turn, H2O2 can be utilized as a secondary messenger in signal transduction pathways. This review presents an overview of the molecular mechanisms involved in redox regulation of protein kinases and its effects on signaling cascades. In the first half, we will focus primarily on receptor tyrosine kinases (RTKs), whereas the latter will concentrate on downstream non-receptor kinases involved in relaying stimulant response. Select examples from the literature are used to highlight the functional role of H2O2 regarding kinase activity, as well as the components involved in H2O2 production and regulation during cellular signaling. In addition, studies demonstrating direct modulation of protein kinases by H2O2 through cysteine oxidation will be emphasized. Identification of these redox-sensitive residues may help uncover signaling mechanisms conserved within kinase subfamilies. In some cases, these residues can even be exploited as targets for the development of new therapeutics. Continued efforts in this field will further basic understanding of kinase redox regulation, and delineate the mechanisms involved in physiologic and pathological H2O2 responses. PMID:23639002

  20. Synaptic encoding of temporal contiguity

    PubMed Central

    Ostojic, Srdjan; Fusi, Stefano

    2013-01-01

    Often we need to perform tasks in an environment that changes stochastically. In these situations it is important to learn the statistics of sequences of events in order to predict the future and the outcome of our actions. The statistical description of many of these sequences can be reduced to the set of probabilities that a particular event follows another event (temporal contiguity). Under these conditions, it is important to encode and store in our memory these transition probabilities. Here we show that for a large class of synaptic plasticity models, the distribution of synaptic strengths encodes transitions probabilities. Specifically, when the synaptic dynamics depend on pairs of contiguous events and the synapses can remember multiple instances of the transitions, then the average synaptic weights are a monotonic function of the transition probabilities. The synaptic weights converge to the distribution encoding the probabilities also when the correlations between consecutive synaptic modifications are considered. We studied how this distribution depends on the number of synaptic states for a specific model of a multi-state synapse with hard bounds. In the case of bistable synapses, the average synaptic weights are a smooth function of the transition probabilities and the accuracy of the encoding depends on the learning rate. As the number of synaptic states increases, the average synaptic weights become a step function of the transition probabilities. We finally show that the information stored in the synaptic weights can be read out by a simple rate-based neural network. Our study shows that synapses encode transition probabilities under general assumptions and this indicates that temporal contiguity is likely to be encoded and harnessed in almost every neural circuit in the brain. PMID:23641210

  1. Functions of Aurora kinase C in meiosis and cancer

    PubMed Central

    Quartuccio, Suzanne M.; Schindler, Karen

    2015-01-01

    The mammalian genome encodes three Aurora kinase protein family members: A, B, and C. While Aurora kinase A (AURKA) and B (AURKB) are found in cells throughout the body, significant protein levels of Aurora kinase C (AURKC) are limited to cells that undergo meiosis (sperm and oocyte). Despite its discovery nearly 20 years ago, we know little about the function of AURKC compared to that of the other 2 Aurora kinases. This lack of understanding can be attributed to the high sequence homology between AURKB and AURKC preventing the use of standard approaches to understand non-overlapping and meiosis I (MI)-specific functions of the two kinases. Recent evidence has revealed distinct functions of AURKC in meiosis and may aid in our understanding of why chromosome segregation during MI often goes awry in oocytes. Many cancers aberrantly express AURKC, but because we do not fully understand AURKC function in its normal cellular context, it is difficult to predict the biological significance of this expression on the disease. Here, we consolidate and update what is known about AURKC signaling in meiotic cells to better understand why it has oncogenic potential. PMID:26347867

  2. The MEKK1-MKK1/MKK2-MPK4 Kinase Cascade Negatively Regulates Immunity Mediated by a Mitogen-Activated Protein Kinase Kinase Kinase in Arabidopsis[C][W

    PubMed Central

    Kong, Qing; Qu, Na; Gao, Minghui; Zhang, Zhibin; Ding, Xiaojun; Yang, Fan; Li, Yingzhong; Dong, Oliver X.; Chen, She; Li, Xin; Zhang, Yuelin

    2012-01-01

    In Arabidopsis thaliana, the MEKK1-MKK1/MKK2-MPK4 mitogen-activated protein (MAP) kinase cascade represses cell death and immune responses. In mekk1, mkk1 mkk2, and mpk4 mutants, programmed cell death and defense responses are constitutively activated, but the mechanism by which MEKK1, MKK1/MKK2, and MPK4 negatively regulate cell death and immunity was unknown. From a screen for suppressors of mkk1 mkk2, we found that mutations in suppressor of mkk1 mkk2 1 (summ1) suppress the cell death and defense responses not only in mkk1 mkk2 but also in mekk1 and mpk4. SUMM1 encodes the MAP kinase kinase kinase MEKK2. It interacts with MPK4 and is phosphorylated by MPK4 in vitro. Overexpression of SUMM1 activates cell death and defense responses that are dependent on the nucleotide binding–leucine-rich repeat protein SUMM2. Taken together, our data suggest that the MEKK1-MKK1/MKK2-MPK4 kinase cascade negatively regulates MEKK2 and activation of MEKK2 triggers SUMM2-mediated immune responses. PMID:22643122

  3. Kinase regulation by sulfur and selenium containing compounds.

    PubMed

    Sanmartín, Carmen; Plano, Daniel; Font, María; Palop, Juan Antonio

    2011-05-01

    Kinases are enzymes that are involved in a wide-range of cellular targets such as cell proliferation, metabolism, survival and apoptosis. Aberrations in the activity of the kinases have been linked to many human diseases such as diabetes, inflammation and cancer. The discovery of more than 518 kinases encoded by the human genome has spurred the development of rapid screening techniques for potential drugs against these enzymes and these have been identified as interesting targets for medicinal chemistry programs, especially in cancer therapy. On the other hand, sulfur and selenium have been increasingly recognized as essential elements in biology and medicine. Converging data from epidemiological and clinical studies have highlighted these elements as effective chemopreventive agents, particularly against various types of cancer (prostate, lung, breast, leukemia, colon, skin, lymphome, thyroid, pancreas, liver). These elements act through a wide range of potential mechanisms where one identified signal pathway event is kinase modulation, which is common for the two elements and emerges as a valid target. The kinases modulated by sulfur and selenium derivatives include MAP, ERK, JNK, Akt, Cdc2, Cyclin B1 and Cdc25c amongst others. Although both of the elements in question are in the same group in the periodic table and have similar biochemistries, there are relevant differences related to redox potentials, stabilities, oxidation states and anticancer activity. Literature data suggest that the replacement of sulfur by selenium in established cancer chemopreventive agents results in more effective chemopreventive analogs. In view of the multi-target kinase mechanisms in preventing cellular transformation, as well as the differences and similarities between them, in this review we focus on the development of new structures that contain selenium and/or sulfur and discuss our understanding of the regulation of antitumoral effects with emphasis on kinase modulation

  4. Plant Raf-like kinase integrates abscisic acid and hyperosmotic stress signaling upstream of SNF1-related protein kinase2

    PubMed Central

    Saruhashi, Masashi; Kumar Ghosh, Totan; Arai, Kenta; Ishizaki, Yumiko; Hagiwara, Kazuya; Komatsu, Kenji; Shiwa, Yuh; Izumikawa, Keiichi; Yoshikawa, Harunori; Umezawa, Taishi; Sakata, Yoichi; Takezawa, Daisuke

    2015-01-01

    Plant response to drought and hyperosmosis is mediated by the phytohormone abscisic acid (ABA), a sesquiterpene compound widely distributed in various embryophyte groups. Exogenous ABA as well as hyperosmosis activates the sucrose nonfermenting 1 (SNF1)-related protein kinase2 (SnRK2), which plays a central role in cellular responses against drought and dehydration, although the details of the activation mechanism are not understood. Analysis of a mutant of the moss Physcomitrella patens with reduced ABA sensitivity and reduced hyperosmosis tolerance revealed that a protein kinase designated “ARK” (for “ABA and abiotic stress-responsive Raf-like kinase”) plays an essential role in the activation of SnRK2. ARK encoded by a single gene in P. patens belongs to the family of group B3 Raf-like MAP kinase kinase kinases (B3-MAPKKKs) mediating ethylene, disease resistance, and salt and sugar responses in angiosperms. Our findings indicate that ARK, as a novel regulatory component integrating ABA and hyperosmosis signals, represents the ancestral B3-MAPKKKs, which multiplied, diversified, and came to have specific functions in angiosperms. PMID:26540727

  5. Map kinases in fungal pathogens.

    PubMed

    Xu, J R

    2000-12-01

    MAP kinases in eukaryotic cells are well known for transducing a variety of extracellular signals to regulate cell growth and differentiation. Recently, MAP kinases homologous to the yeast Fus3/Kss1 MAP kinases have been identified in several fungal pathogens and found to be important for appressorium formation, invasive hyphal growth, and fungal pathogenesis. This MAP kinase pathway also controls diverse growth or differentiation processes, including conidiation, conidial germination, and female fertility. MAP kinases homologous to yeast Slt2 and Hog1 have also been characterized in Candida albicans and Magnaporthe grisea. Mutants disrupted of the Slt2 homologues have weak cell walls, altered hyphal growth, and reduced virulence. The Hog1 homologues are dispensable for growth but are essential for regulating responses to hyperosmotic stress in C. albicans and M. grisea. Overall, recent studies have indicated that MAP kinase pathways may play important roles in regulating growth, differentiation, survival, and pathogenesis in fungal pathogens. PMID:11273677

  6. How Infants Encode Spatial Extent

    ERIC Educational Resources Information Center

    Duffy, Sean; Huttenlocher, Janellen; Levine, Susan; Duffy, Renee

    2005-01-01

    This study explores how infants encode an object's spatial extent. We habituated 6.5-month-old infants to a dowel inside a container and then tested whether they dishabituate to a change in absolute size when the relation between dowel and container is held constant (by altering the size of both container and dowel) and when the relation changes…

  7. Encoding Standards for Linguistic Corpora.

    ERIC Educational Resources Information Center

    Ide, Nancy

    The demand for extensive reusability of large language text collections for natural languages processing research requires development of standardized encoding formats. Such formats must be capable of representing different kinds of information across the spectrum of text types and languages, capable of representing different levels of…

  8. AMP-activated Protein Kinase Suppresses Biosynthesis of Glucosylceramide by Reducing Intracellular Sugar Nucleotides*

    PubMed Central

    Ishibashi, Yohei; Hirabayashi, Yoshio

    2015-01-01

    The membrane glycolipid glucosylceramide (GlcCer) plays a critical role in cellular homeostasis. Its intracellular levels are thought to be tightly regulated. How cells regulate GlcCer levels remains to be clarified. AMP-activated protein kinase (AMPK), which is a crucial cellular energy sensor, regulates glucose and lipid metabolism to maintain energy homeostasis. Here, we investigated whether AMPK affects GlcCer metabolism. AMPK activators (5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside and metformin) decreased intracellular GlcCer levels and synthase activity in mouse fibroblasts. AMPK inhibitors or AMPK siRNA reversed these effects, suggesting that GlcCer synthesis is negatively regulated by an AMPK-dependent mechanism. Although AMPK did not affect the phosphorylation or expression of GlcCer synthase, the amount of UDP-glucose, an activated form of glucose required for GlcCer synthesis, decreased under AMPK-activating conditions. Importantly, the UDP-glucose pyrophosphatase Nudt14, which degrades UDP-glucose, generating UMP and glucose 1-phosphate, was phosphorylated and activated by AMPK. On the other hand, suppression of Nudt14 by siRNA had little effect on UDP-glucose levels, indicating that mammalian cells have an alternative UDP-glucose pyrophosphatase that mainly contributes to the reduction of UDP-glucose under AMPK-activating conditions. Because AMPK activators are capable of reducing GlcCer levels in cells from Gaucher disease patients, our findings suggest that reducing GlcCer through AMPK activation may lead to a new strategy for treating diseases caused by abnormal accumulation of GlcCer. PMID:26048992

  9. The nucleotide sequence of the chicken thymidine kinase gene and the relationship of its predicted polypeptide to that of the vaccinia virus thymidine kinase.

    PubMed

    Kwoh, T J; Engler, J A

    1984-05-11

    The entire DNA nucleotide sequence of a 3.0 kilobase pair Hind III fragment containing the chicken cytoplasmic thymidine kinase gene was determined. Oligonucleotide linker insertion mutations distributed throughout this gene and having known effects upon gene activity ( Kwoh , T.J., Zipser , D., and Wigler , M. 1983. J. Mol. Appl. Genet. 2, 191-200), were used to access regions of the Hind III fragment for sequencing reactions. The complete nucleotide sequence, together with the positions of the linker insertion mutations within the sequence, allows us to propose a structure for the chicken thymidine kinase gene. The protein coding sequence of the gene is divided into seven small segments (each less than 160 base pairs) by six small introns (each less than 230 base pairs). The proposed 244 amino acid polypeptide encoded by this gene bears strong homology to the vaccinia virus thymidine kinase. No homology with the thymidine kinases of the herpes simplex viruses was found.

  10. Adenylate kinase complements nucleoside diphosphate kinase deficiency in nucleotide metabolism.

    PubMed Central

    Lu, Q; Inouye, M

    1996-01-01

    Nucleoside diphosphate (NDP) kinase is a ubiquitous nonspecific enzyme that evidently is designed to catalyze in vivo ATP-dependent synthesis of ribo- and deoxyribonucleoside triphosphates from the corresponding diphosphates. Because Escherichia coli contains only one copy of ndk, the structural gene for this enzyme, we were surprised to find that ndk disruption yields bacteria that are still viable. These mutant cells contain a protein with a small amount NDP kinase activity. The protein responsible for this activity was purified and identified as adenylate kinase. This enzyme, also called myokinase, catalyzes the reversible ATP-dependent synthesis of ADP from AMP. We found that this enzyme from E. coli as well as from higher eukaryotes has a broad substrate specificity displaying dual enzymatic functions. Among the nucleoside monophosphate kinases tested, only adenylate kinase was found to have NDP kinase activity. To our knowledge, this is the first report of NDP kinase activity associated with adenylate kinase. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:8650159

  11. Comparative genomics on nemo-like kinase gene.

    PubMed

    Katoh, Masuko; Katoh, Masaru

    2005-06-01

    WNT signals are transduced to the planar cell polarity (PCP) pathway or the beta-catenin pathway. Drosophila Frizzled (Fz), Starry night (Stan), Van Gogh (Vang), Dishevelled (Dsh), Prickle (Pk), Diego (Dgo) and Nemo (Nmo) are implicated in the PCP signaling pathway. Choi and Benzer identified Drosophila Nmo in 1994, and Brott et al identified mouse Nemo-like kinase (Nlk) in 1998. Nlk positively regulates the PCP pathway, and negatively regulates the beta-catenin pathway. Here, we identified and characterized rat Nlk gene, Nlk2 gene and Nlkp pseudogene by using bioinformatics. Nlk gene, consisting of 11 exons, was mapped to rat chromosome 10q25. Rat Nlk gene encoded 515-aa Nlk protein with the serine/threonine kinase domain, poly(His) tracts and poly(Ala) tract, which showed 100, 99.8, 97.1 and 89.5% total-amino-acid identity with mouse Nlk, human NLK, Xenopus nlk and zebrafish nlk, respectively. Rat Nlk2 gene and Nlkp pseudogene were mapped to rat chromosome 13p13 and 2q44, respectively. Nlk2 gene and Nlkp pseudogene, consisting of a single exon, were not evolutionarily conserved. Nlk2 gene and Nlkp pseudogene were predicted as retrotransposed Nlk homologs within the rat genome. Nlk2 gene encoded a 480-aa Nlk2 protein with partial deletion within the kinase domain, which was predicted as the dominant negative Nlk homolog. This is the first report on the Nlk gene and retrotransposed Nlk homologs within the rat genome.

  12. Monolithic-integrated microlaser encoder.

    PubMed

    Sawada, R; Higurashi, E; Ito, T; Ohguchi, O; Tsubamoto, M

    1999-11-20

    We have developed an extremely small integrated microencoder whose sides are less than 1 mm long. It is 1/100 the size of conventional encoders. This microencoder consists of a laser diode, monolithic photodiodes, and fluorinated polyimide waveguides with total internal reflection mirrors. The instrument can measure the relative displacement between a grating scale and the encoder with a resolution of the order of 0.01 microm; it can also determine the direction in which the scale is moving. By using the two beams that were emitted from the two etched mirrors of the laser diode, by monolithic integration of the waveguide and photodiodes, and by fabrication of a step at the edge of the waveguide, we were able to eliminate conventional bulky optical components such as the beam splitter, the quarter-wavelength plate, bulky mirrors, and bulky photodetectors. PMID:18324228

  13. Encoding information into precipitation structures

    NASA Astrophysics Data System (ADS)

    Martens, Kirsten; Bena, Ioana; Droz, Michel; Rácz, Zoltan

    2008-12-01

    Material design at submicron scales would be profoundly affected if the formation of precipitation patterns could be easily controlled. It would allow the direct building of bulk structures, in contrast to traditional techniques which consist of removing material in order to create patterns. Here, we discuss an extension of our recent proposal of using electrical currents to control precipitation bands which emerge in the wake of reaction fronts in A+ + B- → C reaction-diffusion processes. Our main result, based on simulating the reaction-diffusion-precipitation equations, is that the dynamics of the charged agents can be guided by an appropriately designed time-dependent electric current so that, in addition to the control of the band spacing, the width of the precipitation bands can also be tuned. This makes straightforward the encoding of information into precipitation patterns and, as an amusing example, we demonstrate the feasibility by showing how to encode a musical rhythm.

  14. Evolution of phosphagen kinase V. cDNA-derived amino acid sequences of two molluscan arginine kinases from the chiton Liolophura japonica and the turbanshell Battilus cornutus.

    PubMed

    Suzuki, T; Ban, T; Furukohri, T

    1997-06-20

    The cDNAs of arginine kinases from the chiton Liolophura japonica (Polyplacophora) and the turbanshell Battilus cornutus (Gastropoda) were amplified by polymerase chain reaction (PCR), and the complete nucleotide sequences of 1669 and 1624 bp, respectively, were determined. The open reading frame for Liolophura arginine kinase is 1050 nucleotides in length and encodes a protein with 349 amino acid residues, and that for Battilus is 1077 nucleotides and 358 residues. The validity of the cDNA-derived amino acid sequence was supported by chemical sequencing of internal tryptic peptides. The molecular masses were calculated to be 39,057 and 39,795 Da, respectively. The amino acid sequence of Liolophura arginine kinase showed 65-68% identity with those of Battilus and Nordotis (abalone) arginine kinases, and the homology between Battilus and Nordotis was 79%. Molluscan arginine kinases also show lower, but significant homology (38-43%) with rabbit creatine kinase. The sequences of arginine kinases could be used as a molecular clock to elucidate the phylogeny of Mollusca, one of the most diverse animal phyla.

  15. Saccharomyces cerevisiae Env7 is a novel serine/threonine kinase 16-related protein kinase and negatively regulates organelle fusion at the lysosomal vacuole.

    PubMed

    Manandhar, Surya P; Ricarte, Florante; Cocca, Stephanie M; Gharakhanian, Editte

    2013-02-01

    Membrane fusion depends on conserved components and is responsible for organelle biogenesis and vesicular trafficking. Yeast vacuoles are dynamic structures analogous to mammalian lysosomes. We report here that yeast Env7 is a novel palmitoylated protein kinase ortholog that negatively regulates vacuolar membrane fusion. Microscopic and biochemical studies confirmed the localization of tagged Env7 at the vacuolar membrane and implicated membrane association via the palmitoylation of its N-terminal Cys13 to -15. In vitro kinase assays established Env7 as a protein kinase. Site-directed mutagenesis of the Env7 alanine-proline-glutamic acid (APE) motif Glu269 to alanine results in an unstable kinase-dead allele that is stabilized and redistributed to the detergent-resistant fraction by interruption of the proteasome system in vivo. Palmitoylation-deficient Env7C13-15S is also kinase dead and mislocalizes to the cytoplasm. Microscopy studies established that env7Δ is defective in maintaining fragmented vacuoles during hyperosmotic response and in buds. ENV7 function is not redundant with a similar role of vacuolar membrane kinase Yck3, as the two do not share a substrate, and ENV7 is not a suppressor of yck3Δ. Bayesian phylogenetic analyses strongly support ENV7 as an ortholog of the gene encoding human STK16, a Golgi apparatus protein kinase with undefined function. We propose that Env7 function in fusion/fission dynamics may be conserved within the endomembrane system.

  16. Identification of novel pheromone-response regulators through systematic overexpression of 120 protein kinases in yeast.

    PubMed

    Burchett, S A; Scott, A; Errede, B; Dohlman, H G

    2001-07-13

    Protein kinases are well known to transmit and regulate signaling pathways. To identify additional regulators of the pheromone signaling apparatus in yeast, we evaluated an array of 120 likely protein kinases encoded by the yeast genome. Each kinase was fused to glutathione S-transferase, overexpressed, and tested for changes in pheromone responsiveness in vivo. As expected, several known components of the pathway (YCK1, STE7, STE11, FUS3, and KSS1) impaired the growth arrest response. Seven other kinases also interfered with pheromone-induced growth arrest; in rank order they are as follows: YKL116c (renamed PRR1) = YDL214c (renamed PRR2) > YJL141c (YAK1, SRA1) > YNR047w = YCR091w (KIN82) = YIL095w (PRK1) > YCL024w (KCC4). Inhibition of pheromone signaling by PRR1, but not PRR2, required the glutathione S-transferase moiety. Both kinases inhibited gene transcription after stimulation with pheromone, a constitutively active kinase mutant STE11-4, or overexpression of the transcription factor STE12. Neither protein altered the ability of the mitogen-activated protein kinase (MAPK) Fus3 to feedback phosphorylate a known substrate, the MAPK kinase Ste7. These results reveal two new components of the pheromone-signaling cascade in yeast, each acting at a point downstream of the MAPK. PMID:11337509

  17. DNA sequence, structure, and tyrosine kinase activity of the Drosophila melanogaster abelson proto-oncogene homolog

    SciTech Connect

    Henkemeyer, M.J.; Bennett, R.L.; Gertler, F.B.; Hoffmann, F.M.

    1988-02-01

    The authors report their molecular characterization of the Drosophila melanogaster Abelson gene (abl), a gene in which recessive loss-of-function mutations result in lethality at the pupal stage of development. This essential gene consists of 10 exons extending over 26 kilobase pairs of genomic DNA. The DNA sequence encodes a protein of 1,520 amino acids with strong sequence similarity to the human c-abl proto-oncogene beginning in the type 1b 5' exon and extending through the region essential for tyrosine kinase activity. When the tyrosine kinase homologous region was expressed in Escherichia coli, phosphorylation of proteins on tyrosine residues was observed with an antiphosphotyrosine antibody. These results show that the abl gene is highly conserved through evolution and encodes a functional tyrosine protein kinase required for Drosophila development.

  18. Vector Encoding in Biochemical Networks

    NASA Astrophysics Data System (ADS)

    Potter, Garrett; Sun, Bo

    Encoding of environmental cues via biochemical signaling pathways is of vital importance in the transmission of information for cells in a network. The current literature assumes a single cell state is used to encode information, however, recent research suggests the optimal strategy utilizes a vector of cell states sampled at various time points. To elucidate the optimal sampling strategy for vector encoding, we take an information theoretic approach and determine the mutual information of the calcium signaling dynamics obtained from fibroblast cells perturbed with different concentrations of ATP. Specifically, we analyze the sampling strategies under the cases of fixed and non-fixed vector dimension as well as the efficiency of these strategies. Our results show that sampling with greater frequency is optimal in the case of non-fixed vector dimension but that, in general, a lower sampling frequency is best from both a fixed vector dimension and efficiency standpoint. Further, we find the use of a simple modified Ornstein-Uhlenbeck process as a model qualitatively captures many of our experimental results suggesting that sampling in biochemical networks is based on a few basic components.

  19. Lessons from modENCODE.

    PubMed

    Brown, James B; Celniker, Susan E

    2015-01-01

    The modENCODE (Model Organism Encyclopedia of DNA Elements) Consortium aimed to map functional elements-including transcripts, chromatin marks, regulatory factor binding sites, and origins of DNA replication-in the model organisms Drosophila melanogaster and Caenorhabditis elegans. During its five-year span, the consortium conducted more than 2,000 genome-wide assays in developmentally staged animals, dissected tissues, and homogeneous cell lines. Analysis of these data sets provided foundational insights into genome, epigenome, and transcriptome structure and the evolutionary turnover of regulatory pathways. These studies facilitated a comparative analysis with similar data types produced by the ENCODE Consortium for human cells. Genome organization differs drastically in these distant species, and yet quantitative relationships among chromatin state, transcription, and cotranscriptional RNA processing are deeply conserved. Of the many biological discoveries of the modENCODE Consortium, we highlight insights that emerged from integrative studies. We focus on operational and scientific lessons that may aid future projects of similar scale or aims in other, emerging model systems. PMID:26133010

  20. Parallel encoding of sensory history and behavioral preference during Caenorhabditis elegans olfactory learning.

    PubMed

    Cho, Christine E; Brueggemann, Chantal; L'Etoile, Noelle D; Bargmann, Cornelia I

    2016-01-01

    Sensory experience modifies behavior through both associative and non-associative learning. In Caenorhabditis elegans, pairing odor with food deprivation results in aversive olfactory learning, and pairing odor with food results in appetitive learning. Aversive learning requires nuclear translocation of the cGMP-dependent protein kinase EGL-4 in AWC olfactory neurons and an insulin signal from AIA interneurons. Here we show that the activity of neurons including AIA is acutely required during aversive, but not appetitive, learning. The AIA circuit and AGE-1, an insulin-regulated PI3 kinase, signal to AWC to drive nuclear enrichment of EGL-4 during conditioning. Odor exposure shifts the AWC dynamic range to higher odor concentrations regardless of food pairing or the AIA circuit, whereas AWC coupling to motor circuits is oppositely regulated by aversive and appetitive learning. These results suggest that non-associative sensory adaptation in AWC encodes odor history, while associative behavioral preference is encoded by altered AWC synaptic activity.

  1. Design, synthesis and selection of DNA-encoded small-molecule libraries.

    PubMed

    Clark, Matthew A; Acharya, Raksha A; Arico-Muendel, Christopher C; Belyanskaya, Svetlana L; Benjamin, Dennis R; Carlson, Neil R; Centrella, Paolo A; Chiu, Cynthia H; Creaser, Steffen P; Cuozzo, John W; Davie, Christopher P; Ding, Yun; Franklin, G Joseph; Franzen, Kurt D; Gefter, Malcolm L; Hale, Steven P; Hansen, Nils J V; Israel, David I; Jiang, Jinwei; Kavarana, Malcolm J; Kelley, Michael S; Kollmann, Christopher S; Li, Fan; Lind, Kenneth; Mataruse, Sibongile; Medeiros, Patricia F; Messer, Jeffrey A; Myers, Paul; O'Keefe, Heather; Oliff, Matthew C; Rise, Cecil E; Satz, Alexander L; Skinner, Steven R; Svendsen, Jennifer L; Tang, Lujia; van Vloten, Kurt; Wagner, Richard W; Yao, Gang; Zhao, Baoguang; Morgan, Barry A

    2009-09-01

    Biochemical combinatorial techniques such as phage display, RNA display and oligonucleotide aptamers have proven to be reliable methods for generation of ligands to protein targets. Adapting these techniques to small synthetic molecules has been a long-sought goal. We report the synthesis and interrogation of an 800-million-member DNA-encoded library in which small molecules are covalently attached to an encoding oligonucleotide. The library was assembled by a combination of chemical and enzymatic synthesis, and interrogated by affinity selection. We describe methods for the selection and deconvolution of the chemical display library, and the discovery of inhibitors for two enzymes: Aurora A kinase and p38 MAP kinase. PMID:19648931

  2. Focal adhesion kinase

    PubMed Central

    Stone, Rebecca L; Baggerly, Keith A; Armaiz-Pena, Guillermo N; Kang, Yu; Sanguino, Angela M; Thanapprapasr, Duangmani; Dalton, Heather J; Bottsford-Miller, Justin; Zand, Behrouz; Akbani, Rehan; Diao, Lixia; Nick, Alpa M; DeGeest, Koen; Lopez-Berestein, Gabriel; Coleman, Robert L; Lutgendorf, Susan; Sood, Anil K

    2014-01-01

    This investigation describes the clinical significance of phosphorylated focal adhesion kinase (FAK) at the major activating tyrosine site (Y397) in epithelial ovarian cancer (EOC) cells and tumor-associated endothelial cells. FAK gene amplification as a mechanism for FAK overexpression and the effects of FAK tyrosine kinase inhibitor VS-6062 on tumor growth, metastasis, and angiogenesis were examined. FAK and phospho-FAKY397 were quantified in tumor (FAK-T; pFAK-T) and tumor-associated endothelial (FAK-endo; pFAK-endo) cell compartments of EOCs using immunostaining and qRT-PCR. Associations between expression levels and clinical variables were evaluated. Data from The Cancer Genome Atlas were used to correlate FAK gene copy number and expression levels in EOC specimens. The in vitro and in vivo effects of VS-6062 were assayed in preclinical models. FAK-T and pFAK-T overexpression was significantly associated with advanced stage disease and increased microvessel density (MVD). High MVD was observed in tumors with elevated endothelial cell FAK (59%) and pFAK (44%). Survival was adversely affected by FAK-T overexpression (3.03 vs 2.06 y, P = 0.004), pFAK-T (2.83 vs 1.78 y, P < 0.001), and pFAK-endo (2.33 vs 2.17 y, P = 0.005). FAK gene copy number was increased in 34% of tumors and correlated with expression levels (P < 0.001). VS-6062 significantly blocked EOC and endothelial cell migration as well as endothelial cell tube formation in vitro. VS-6062 reduced mean tumor weight by 56% (P = 0.005), tumor MVD by 40% (P = 0.0001), and extraovarian metastasis (P < 0.01) in orthotopic EOC mouse models. FAK may be a unique therapeutic target in EOC given the dual anti-angiogenic and anti-metastatic potential of FAK inhibitors. PMID:24755674

  3. Protein Kinase C δ: a Gatekeeper of Immune Homeostasis.

    PubMed

    Salzer, Elisabeth; Santos-Valente, Elisangela; Keller, Bärbel; Warnatz, Klaus; Boztug, Kaan

    2016-10-01

    Human autoimmune disorders present in various forms and are associated with a life-long burden of high morbidity and mortality. Many different circumstances lead to the loss of immune tolerance and often the origin is suspected to be multifactorial. Recently, patients with autosomal recessive mutations in PRKCD encoding protein kinase c delta (PKCδ) have been identified, representing a monogenic prototype for one of the most prominent forms of humoral systemic autoimmune diseases, systemic lupus erythematosus (SLE). PKCδ is a signaling kinase with multiple downstream target proteins and with functions in various signaling pathways. Interestingly, mouse models have indicated a special role of the ubiquitously expressed protein in the control of B-cell tolerance revealed by the severe autoimmunity in Prkcd (-/-) knockout mice as the major phenotype. As such, the study of PKCδ deficiency in humans has tremendous potential in enhancing our knowledge on the mechanisms of B-cell tolerance. PMID:27541826

  4. Protein Kinase C δ: a Gatekeeper of Immune Homeostasis.

    PubMed

    Salzer, Elisabeth; Santos-Valente, Elisangela; Keller, Bärbel; Warnatz, Klaus; Boztug, Kaan

    2016-10-01

    Human autoimmune disorders present in various forms and are associated with a life-long burden of high morbidity and mortality. Many different circumstances lead to the loss of immune tolerance and often the origin is suspected to be multifactorial. Recently, patients with autosomal recessive mutations in PRKCD encoding protein kinase c delta (PKCδ) have been identified, representing a monogenic prototype for one of the most prominent forms of humoral systemic autoimmune diseases, systemic lupus erythematosus (SLE). PKCδ is a signaling kinase with multiple downstream target proteins and with functions in various signaling pathways. Interestingly, mouse models have indicated a special role of the ubiquitously expressed protein in the control of B-cell tolerance revealed by the severe autoimmunity in Prkcd (-/-) knockout mice as the major phenotype. As such, the study of PKCδ deficiency in humans has tremendous potential in enhancing our knowledge on the mechanisms of B-cell tolerance.

  5. Bivalent Inhibitors of Protein Kinases

    PubMed Central

    Gower, Carrie M.; Chang, Matthew E. K.; Maly, Dustin J.

    2015-01-01

    Protein kinases are key players in a large number of cellular signaling pathways. Dysregulated kinase activity has been implicated in a number of diseases, and members of this enzyme family are of therapeutic interest. However, due to the fact that most inhibitors interact with the highly conserved ATP-binding sites of kinases, it is a significant challenge to develop pharmacological agents that target only one of the greater than 500 kinases present in humans. A potential solution to this problem is the development of bisubstrate and bivalent kinase inhibitors, in which an active site-directed moiety is tethered to another ligand that targets a location outside of the ATP-binding cleft. Because kinase signaling specificity is modulated by regions outside of the ATP-binding site, strategies that exploit these interactions have the potential to provide reagents with high target selectivity. This review highlights examples of kinase interaction sites that can potentially be exploited by bisubstrate and bivalent inhibitors. Furthermore, an overview of efforts to target these interactions with bisubstrate and bivalent inhibitors is provided. Finally, several examples of the successful application of these reagents in a cellular setting are described. PMID:24564382

  6. Novel optical encoder for harsh environments

    NASA Astrophysics Data System (ADS)

    Kress, Bernard; Mueller, Ulrich; Brac-de-la-Perriere, Vincent

    2014-09-01

    We are presenting a new optical encoder architecture for shaft encoding, both in incremental and absolute modes. This encoder is based on a diffractive optics technology platform. We have developed various disk based rotary diffractive encoders previously. This encoder is different in the way it is not a disk composed of successive gratings or computer generated holograms, but rather composed of a single element placed on the shaft. It is thus best suited for hollow shaft or end of shaft applications such as in encoder controlled electrical motors. This new architecture aims at solving some of the problems encountered with previous implementations of diffractive encoders such as disk wobble, disk to shaft centering and also encoding in harsh environments.

  7. Protein kinase A catalytic subunit isoform PRKACA; History, function and physiology.

    PubMed

    Turnham, Rigney E; Scott, John D

    2016-02-15

    Our appreciation of the scope and influence of second messenger signaling has its origins in pioneering work on the cAMP-dependent protein kinase. Also called protein kinase A (PKA), this holoenzyme exists as a tetramer comprised of a regulatory (R) subunit dimer and two catalytic (C) subunits. Upon binding of two molecules of the second messenger cAMP to each R subunit, a conformational change in the PKA holoenzyme occurs to release the C subunits. These active kinases phosphorylate downstream targets to propagate cAMP responsive cell signaling events. This article focuses on the discovery, structure, cellular location and physiological effects of the catalytic subunit alpha of protein kinase A (encoded by the gene PRKACA). We also explore the potential role of this essential gene as a molecular mediator of certain disease states.

  8. Characterization and chromosomal localization of the gene for human rhodopsin kinase

    SciTech Connect

    Khani, S.C.; Yamamoto, S.; Dryja, T.P.

    1996-08-01

    G-protein-dependent receptor kinases (GRKs) play a key role in the adapatation of receptors to persistent stimuli. In rod photoreceptors rhodopsin kinase (RK) mediates rapid densensitization of rod photoreceptors to light by catalyzing phosphorylation of the visual pigment rhodopsin. To study the structure and mechanism of FRKs in human photoreceptors, we have isolated and characterized cDNA and genomic clones derived from the human RK locus using a bovine rhodopsin kinase cDNA fragment as a probe. The RK locus, assigned to chromosome 13 band q34, is composed of seven exons that encode a protein 92% identical in amino acid sequence to bovine rhodopsin kinase. The marked difference between the structure of this gene and that of another recently clone human GRK gene suggests the existence of a wide evolutionary gap between members of the GRK gene family. 39 refs., 3 figs.

  9. Protein kinase A catalytic subunit isoform PRKACA; History, function and physiology.

    PubMed

    Turnham, Rigney E; Scott, John D

    2016-02-15

    Our appreciation of the scope and influence of second messenger signaling has its origins in pioneering work on the cAMP-dependent protein kinase. Also called protein kinase A (PKA), this holoenzyme exists as a tetramer comprised of a regulatory (R) subunit dimer and two catalytic (C) subunits. Upon binding of two molecules of the second messenger cAMP to each R subunit, a conformational change in the PKA holoenzyme occurs to release the C subunits. These active kinases phosphorylate downstream targets to propagate cAMP responsive cell signaling events. This article focuses on the discovery, structure, cellular location and physiological effects of the catalytic subunit alpha of protein kinase A (encoded by the gene PRKACA). We also explore the potential role of this essential gene as a molecular mediator of certain disease states. PMID:26687711

  10. Time Course of Grammatical Encoding in Agrammatism

    ERIC Educational Resources Information Center

    Lee, Jiyeon

    2011-01-01

    Producing a sentence involves encoding a preverbal message into a grammatical structure by retrieving lexical items and integrating them into a functional (semantic-to-grammatical) structure. Individuals with agrammatism are impaired in this grammatical encoding process. However, it is unclear what aspect of grammatical encoding is impaired and…

  11. Isolation of chloroplastic phosphoglycerate kinase

    SciTech Connect

    Macioszek, J.; Anderson, L.E. ); Anderson, J.B. )

    1990-09-01

    We report here a method for the isolation of high specific activity phosphoglycerate kinase (EC 2.7.2.3) from chloroplasts. The enzyme has been purified over 200-fold from pea (Pisum sativum L.) stromal extracts to apparent homogeneity with 23% recovery. Negative cooperativity is observed with the two enzyme phosphoglycerate kinase/glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13) couple restored from the purified enzymes when NADPH is the reducing pyridine nucleotide, consistent with earlier results obtained with crude chloroplastic extracts. Michaelis Menten kinetics are observed when 3-phosphoglycerate is held constant and phosphoglycerate kinase is varied, which suggests that phosphoglycerate kinase-bound 1,3-bisphosphoglycerate may be the preferred substrate for glyceraldehyde-3-P dehydrogenase in the chloroplast.

  12. Protein Crystals of Raf Kinase

    NASA Technical Reports Server (NTRS)

    1995-01-01

    This image shows crystals of the protein raf kinase grown on Earth (photo a) and on USML-2 (photo b). The space-grown crystals are an order of magnitude larger. Principal Investigator: Dan Carter of New Century Pharmaceuticals

  13. Exploring the function of protein kinases in schistosomes: perspectives from the laboratory and from comparative genomics

    PubMed Central

    Walker, Anthony J.; Ressurreição, Margarida; Rothermel, Rolf

    2014-01-01

    Eukaryotic protein kinases are well conserved through evolution. The genome of Schistosoma mansoni, which causes intestinal schistosomiasis, encodes over 250 putative protein kinases with all of the main eukaryotic groups represented. However, unraveling functional roles for these kinases is a considerable endeavor, particularly as protein kinases regulate multiple and sometimes overlapping cell and tissue functions in organisms. In this article, elucidating protein kinase signal transduction and function in schistosomes is considered from the perspective of the state-of-the-art methodologies used and comparative organismal biology, with a focus on current advances and future directions. Using the free-living nematode Caenorhabditis elegans as a comparator we predict roles for various schistosome protein kinases in processes vital for host invasion and successful parasitism such as sensory behavior, growth and development. It is anticipated that the characterization of schistosome protein kinases in the context of parasite function will catalyze cutting edge research into host-parasite interactions and will reveal new targets for developing drug interventions against human schistosomiasis. PMID:25132840

  14. Crystal Structures of Putative Sugar Kinases from Synechococcus Elongatus PCC 7942 and Arabidopsis Thaliana

    PubMed Central

    Xie, Yuan; Li, Mei; Chang, Wenrui

    2016-01-01

    The genome of the Synechococcus elongatus strain PCC 7942 encodes a putative sugar kinase (SePSK), which shares 44.9% sequence identity with the xylulose kinase-1 (AtXK-1) from Arabidopsis thaliana. Sequence alignment suggests that both kinases belong to the ribulokinase-like carbohydrate kinases, a sub-family of FGGY family carbohydrate kinases. However, their exact physiological function and real substrates remain unknown. Here we solved the structures of SePSK and AtXK-1 in both their apo forms and in complex with nucleotide substrates. The two kinases exhibit nearly identical overall architecture, with both kinases possessing ATP hydrolysis activity in the absence of substrates. In addition, our enzymatic assays suggested that SePSK has the capability to phosphorylate D-ribulose. In order to understand the catalytic mechanism of SePSK, we solved the structure of SePSK in complex with D-ribulose and found two potential substrate binding pockets in SePSK. Using mutation and activity analysis, we further verified the key residues important for its catalytic activity. Moreover, our structural comparison with other family members suggests that there are major conformational changes in SePSK upon substrate binding, facilitating the catalytic process. Together, these results provide important information for a more detailed understanding of the cofactor and substrate binding mode as well as the catalytic mechanism of SePSK, and possible similarities with its plant homologue AtXK-1. PMID:27223615

  15. Tyrosine kinase gene rearrangements in epithelial malignancies.

    PubMed

    Shaw, Alice T; Hsu, Peggy P; Awad, Mark M; Engelman, Jeffrey A

    2013-11-01

    Chromosomal rearrangements that lead to oncogenic kinase activation are observed in many epithelial cancers. These cancers express activated fusion kinases that drive the initiation and progression of malignancy, and often have a considerable response to small-molecule kinase inhibitors, which validates these fusion kinases as 'druggable' targets. In this Review, we examine the aetiologic, pathogenic and clinical features that are associated with cancers harbouring oncogenic fusion kinases, including anaplastic lymphoma kinase (ALK), ROS1 and RET. We discuss the clinical outcomes with targeted therapies and explore strategies to discover additional kinases that are activated by chromosomal rearrangements in solid tumours.

  16. Tyrosine kinase gene rearrangements in epithelial malignancies

    PubMed Central

    Shaw, Alice T.; Hsu, Peggy P.; Awad, Mark M.; Engelman, Jeffrey A.

    2014-01-01

    Chromosomal rearrangements that lead to oncogenic kinase activation are observed in many epithelial cancers. These cancers express activated fusion kinases that drive the initiation and progression of malignancy, and often have a considerable response to small-molecule kinase inhibitors, which validates these fusion kinases as ‘druggable’ targets. In this Review, we examine the aetiologic, pathogenic and clinical features that are associated with cancers harbouring oncogenic fusion kinases, including anaplastic lymphoma kinase (ALK), ROS1 and RET. We discuss the clinical outcomes with targeted therapies and explore strategies to discover additional kinases that are activated by chromosomal rearrangements in solid tumours. PMID:24132104

  17. Molecular mechanisms for protein-encoded inheritance

    SciTech Connect

    Wiltzius, Jed J.W.; Landau, Meytal; Nelson, Rebecca; Sawaya, Michael R.; Apostol, Marcin I.; Goldschmidt, Lukasz; Soriaga, Angela B.; Cascio, Duilio; Rajashankar, Kanagalaghatta; Eisenberg, David

    2009-12-01

    In prion inheritance and transmission, strains are phenotypic variants encoded by protein 'conformations'. However, it is unclear how a protein conformation can be stable enough to endure transmission between cells or organisms. Here we describe new polymorphic crystal structures of segments of prion and other amyloid proteins, which offer two structural mechanisms for the encoding of prion strains. In packing polymorphism, prion strains are encoded by alternative packing arrangements (polymorphs) of {beta}-sheets formed by the same segment of a protein; in segmental polymorphism, prion strains are encoded by distinct {beta}-sheets built from different segments of a protein. Both forms of polymorphism can produce enduring conformations capable of encoding strains. These molecular mechanisms for transfer of protein-encoded information into prion strains share features with the familiar mechanism for transfer of nucleic acid-encoded information into microbial strains, including sequence specificity and recognition by noncovalent bonds.

  18. Neuronal Cell Shape and Neurite Initiation Are Regulated by the Ndr Kinase SAX-1, a Member of the Orb6/COT-1/Warts Serine/Threonine Kinase Family

    PubMed Central

    Zallen, Jennifer A.; Peckol, Erin L.; Tobin, David M.; Bargmann, Cornelia I.

    2000-01-01

    The Caenorhabditis elegans sax-1 gene regulates several aspects of neuronal cell shape. sax-1 mutants have expanded cell bodies and ectopic neurites in many classes of neurons, suggesting that SAX-1 functions to restrict cell and neurite growth. The ectopic neurites in sensory neurons of sax-1 mutants resemble the defects caused by decreased sensory activity. However, the activity-dependent pathway, mediated in part by the UNC-43 calcium/calmodulin-dependent kinase II, functions in parallel with SAX-1 to suppress neurite initiation. sax-1 encodes a serine/threonine kinase in the Ndr family that is related to the Orb6 (Schizosaccharomyces pombe), Warts/Lats (Drosophila), and COT-1 (Neurospora) kinases that function in cell shape regulation. These kinases have similarity to Rho kinases but lack consensus Rho-binding domains. Dominant negative mutations in the C. elegans RhoA GTPase cause neuronal cell shape defects similar to those of sax-1 mutants, and genetic interactions between rhoA and sax-1 suggest shared functions. These results suggest that SAX-1/Ndr kinases are endogenous inhibitors of neurite initiation and cell spreading. PMID:10982409

  19. Pho85p, a cyclin-dependent protein kinase, and the Snf1p protein kinase act antagonistically to control glycogen accumulation in Saccharomyces cerevisiae.

    PubMed Central

    Huang, D; Farkas, I; Roach, P J

    1996-01-01

    In Saccharomyces cerevisiae, nutrient levels control multiple cellular processes. Cells lacking the SNF1 gene cannot express glucose-repressible genes and do not accumulate the storage polysaccharide glycogen. The impaired glycogen synthesis is due to maintenance of glycogen synthase in a hyperphosphorylated, inactive state. In a screen for second site suppressors of the glycogen storage defect of snf1 cells, we identified a mutant gene that restored glycogen accumulation and which was allelic with PHO85, which encodes a member of the cyclin-dependent kinase family. In cells with disrupted PHO85 genes, we observed hyperaccumulation of glycogen, activation of glycogen synthase, and impaired glycogen synthase kinase activity. In snf1 cells, glycogen synthase kinase activity was elevated. Partial purification of glycogen synthase kinase activity from yeast extracts resulted in the separation of two fractions by phenyl-Sepharose chromatography, both of which phosphorylated and inactivated glycogen synthase. The activity of one of these, GPK2, was inhibited by olomoucine, which potently inhibits cyclin-dependent protein kinases, and contained an approximately 36-kDa species that reacted with antibodies to Pho85p. Analysis of Ser-to-Ala mutations at the three potential Gsy2p phosphorylation sites in pho85 cells implicated Ser-654 and/or Thr-667 in PHO85 control of glycogen synthase. We propose that Pho85p is a physiological glycogen synthase kinase, possibly acting downstream of Snf1p. PMID:8754836

  20. Role of the Yes and Csk tyrosine kinases in the development of a pathological state in the human retina.

    PubMed

    Baranova, Lyudmila; Emelyanova, Valentina; Volotovski, Igor

    2010-07-01

    Amplification and a cloning of fragments of genes of human retina tyrosine kinases, the nucleotide sequences of which feature a high homology to the gene families of the Yes and Csk tyrosine kinases, and a cloning of the complete coding sequence of the cDNA of the Csk tyrosine kinase gene of the human lymphocytes have been carried out. It has been established that this sequence contains 1,624 bp and encodes a protein that, with a 99% homology, corresponds to the human tyrosine kinase. A comparative analysis of the nucleotide sequences of the full-size cDNA of the Csk tyrosine kinase of the lymphocytes of healthy donors and of patients with an eye choroidal melanoma has shown that a risk of development of an eye choroidal melanoma can be estimated by the frequency of occurrence of a mutant allele in the 10th exon.

  1. Engineering Genetically Encoded FRET Sensors

    PubMed Central

    Lindenburg, Laurens; Merkx, Maarten

    2014-01-01

    Förster Resonance Energy Transfer (FRET) between two fluorescent proteins can be exploited to create fully genetically encoded and thus subcellularly targetable sensors. FRET sensors report changes in energy transfer between a donor and an acceptor fluorescent protein that occur when an attached sensor domain undergoes a change in conformation in response to ligand binding. The design of sensitive FRET sensors remains challenging as there are few generally applicable design rules and each sensor must be optimized anew. In this review we discuss various strategies that address this shortcoming, including rational design approaches that exploit self-associating fluorescent domains and the directed evolution of FRET sensors using high-throughput screening. PMID:24991940

  2. Characterization of a tomato protein kinase gene induced by infection by Potato spindle tuber viroid.

    PubMed

    Hammond, R W; Zhao, Y

    2000-09-01

    Viroids--covalently closed, circular RNA molecules in the size range of 250 to 450 nucleotides-are the smallest known infectious agents and cause a number of diseases of crop plants. Viroids do not encode proteins and replicate within the nucleus without a helper virus. In many cases, viroid infection results in symptoms of stunting, epinasty, and vein clearing. In our study of the molecular basis of the response of tomato cv. Rutgers to infection by Potato spindle tuber viroid (PSTVd), we have identified a specific protein kinase gene, pkv, that is transcriptionally activated in plants infected with either the intermediate or severe strain of PSTVd, at a lower level in plants inoculated with a mild strain, and not detectable in mock-inoculated plants. A full-length copy of the gene encoding the 55-kDa PKV (protein kinase viroid)-induced protein has been isolated and sequence analysis revealed significant homologies to cyclic nucleotide-dependent protein kinases. Although the sequence motifs in the catalytic domain suggest that it is a serine/threonine protein kinase, the recombinant PKV protein autophosphorylates in vitro on serine and tyrosine residues, suggesting that it is a putative member of the class of dual-specificity protein kinases. PMID:10975647

  3. Cell cycle-dependent nuclear accumulation of the p94fer tyrosine kinase is regulated by its NH2 terminus and is affected by kinase domain integrity and ATP binding.

    PubMed

    Ben-Dor, I; Bern, O; Tennenbaum, T; Nir, U

    1999-02-01

    p94fer and p51ferT are two tyrosine kinases that are encoded by differentially spliced transcripts of the FER locus in the mouse. The two tyrosine kinases share identical SH2 and kinase domains but differ in their NH2-terminal amino acid sequence. Unlike p94fer, the presence of which has been demonstrated in most mammalian cell lines analyzed, the expression of p51ferT is restricted to meiotic cells. Here, we show that the two related tyrosine kinases also differ in their subcellular localization profiles. Although p51ferT accumulates constitutively in the cell nucleus, p94fer is cytoplasmic in quiescent cells and enters the nucleus concomitantly with the onset of S phase. The nuclear translocation of the FER proteins is driven by a nuclear localization signal (NLS), which is located within the kinase domain of these enzymes. The functioning of that NLS depends on the integrity of the kinase domain but was not affected by inactivation of the kinase activity. The NH2 terminus of p94fer dictated the cell cycle-dependent functioning of the NLS of FER kinase. This process was governed by coiled-coil forming sequences that are present in the NH2 terminus of the kinase. The regulatory effect of the p94fer NH2-terminal sequences was not affected by kinase activity but was perturbed by mutations in the kinase domain ATP binding site. Ectopic expression of the constitutively nuclear p51ferT in CHO cells interfered with S-phase progression in these cells. This was not seen in p94fer-overexpressing cells. The FER tyrosine kinases seem, thus, to be regulated by novel mechanisms that direct their different subcellular distribution profiles and may, consequently, control their cellular functioning. PMID:10074905

  4. Analysis of Substrates of Protein Kinase C Isoforms in Human Breast Cells By The Traceable Kinase Method

    PubMed Central

    Chen, Xiangyu; Zhao, Xin; Abeyweera, Thushara P.; Rotenberg, Susan A.

    2012-01-01

    A previous report (Biochemistry 46: 2364–2370, 2007) described the application of The Traceable Kinase Method to identify substrates of PKCα in non-transformed human breast MCF-10A cells. Here, a non-radioactive variation of this method compared the phospho-protein profiles of three traceable PKC isoforms (α, δ and ζ) for the purpose of identifying novel, isoform-selective substrates. Each FLAG-tagged traceable kinase was expressed and co-immunoprecipitated along with high affinity substrates. The isolated kinase and its associated substrates were subjected to an in vitro phosphorylation reaction with traceable kinase-specific N6-phenyl-ATP, and the resulting phospho-proteins were analyzed by Western blot with an antibody that recognizes the phosphorylated PKC consensus site. Phospho-protein profiles generated by PKC-α and -δ were similar and differed markedly from that of PKC-ζ. Mass spectrometry of selected bands revealed known PKC substrates and several potential substrates that included the small GTPase-associated effector protein Cdc42 effector protein-4 (CEP4). Of those potential substrates tested, only CEP4 was phosphorylated by pure PKC-α, –δ, and −ζ isoforms in vitro, and by endogenous PKC isoforms in MCF-10A cells treated with DAG-lactone, a membrane permeable PKC activator. Under these conditions, the stoichiometry of CEP4 phosphorylation was 3.2 ± 0.5 (mol phospho-CEP4/mol CEP4). Following knock-down with isoform-specific shRNA-encoding plasmids, phosphorylation of CEP4 was substantially decreased in response to silencing of each of the three isoforms (PKC–α, –δ, or –ζ), whereas testing of kinase-dead mutants supported a role for only PKC-α and –δ in CEP4 phosphorylation. These findings identify CEP4 as a novel intracellular PKC substrate that is phosphorylated by multiple PKC isoforms. PMID:22897107

  5. Structural basis for the mechanism and substrate specificity of glycocyamine kinase, a phosphagen kinase family member

    SciTech Connect

    Lim, Kap; Pullalarevu, Sadhana; Surabian, Karen Talin; Howard, Andrew; Suzuki, Tomohiko; Moult, John; Herzberg, Osnat

    2010-03-12

    Glycocyamine kinase (GK), a member of the phosphagen kinase family, catalyzes the Mg{sup 2+}-dependent reversible phosphoryl group transfer of the N-phosphoryl group of phosphoglycocyamine to ADP to yield glycocyamine and ATP. This reaction helps to maintain the energy homeostasis of the cell in some multicelullar organisms that encounter high and variable energy turnover. GK from the marine worm Namalycastis sp. is heterodimeric, with two homologous polypeptide chains, {alpha} and {beta}, derived from a common pre-mRNA by mutually exclusive N-terminal alternative exons. The N-terminal exon of GK{beta} encodes a peptide that is different in sequence and is 16 amino acids longer than that encoded by the N-terminal exon of GK{alpha}. The crystal structures of recombinant GK{alpha}{beta} and GK{beta}{beta} from Namalycastis sp. were determined at 2.6 and 2.4 {angstrom} resolution, respectively. In addition, the structure of the GK{beta}{beta} was determined at 2.3 {angstrom} resolution in complex with a transition state analogue, Mg{sup 2+}-ADP-NO{sub 3}{sup -}-glycocyamine. Consistent with the sequence homology, the GK subunits adopt the same overall fold as that of other phosphagen kinases of known structure (the homodimeric creatine kinase (CK) and the monomeric arginine kinase (AK)). As with CK, the GK N-termini mediate the dimer interface. In both heterodimeric and homodimeric GK forms, the conformations of the two N-termini are asymmetric, and the asymmetry is different than that reported previously for the homodimeric CKs from several organisms. The entire polypeptide chains of GK{alpha}{beta} are structurally defined, and the longer N-terminus of the {beta} subunit is anchored at the dimer interface. In GK{beta}{beta} the 24 N-terminal residues of one subunit and 11 N-terminal residues of the second subunit are disordered. This observation is consistent with a proposal that the GK{alpha}{beta} amino acids involved in the interface formation were optimized once

  6. NMDA receptors and memory encoding.

    PubMed

    Morris, Richard G M

    2013-11-01

    It is humbling to think that 30 years have passed since the paper by Collingridge, Kehl and McLennan showing that one of Jeff Watkins most interesting compounds, R-2-amino-5-phosphonopentanoate (d-AP5), blocked the induction of long-term potentiation in vitro at synapses from area CA3 of the hippocampus to CA1 without apparent effect on baseline synaptic transmission (Collingridge et al., 1983). This dissociation was one of the key triggers for an explosion of interest in glutamate receptors, and much has been discovered since that collectively contributes to our contemporary understanding of glutamatergic synapses - their biophysics and subunit composition, of the agonists and antagonists acting on them, and their diverse functions in different networks of the brain and spinal cord. It can be fairly said that Collingridge et al.'s (1983) observation was the stimulus that has led, on the one hand, to structural biological work at the atomic scale describing the key features of NMDA receptors that enables their coincidence function to happen; and, on the other, to work with whole animals investigating the contributions that calcium signalling via this receptor can have on rhythmical activities controlled by spinal circuits, memory encoding in the hippocampus (the topic of this article), visual cortical plasticity, sensitization in pain, and other functions. In this article, I lay out how my then interest in long-term potentiation (LTP) as a model of memory enabled me to recognise the importance of Collingridge et al.'s discovery - and how I and my colleagues endeavoured to take things forward in the area of learning and memory. This is in some respects a personal story, and I tell it as such. The idea that NMDA receptor activation is essential for memory encoding, though not for storage, took time to develop and to be accepted. Along the way, there have been confusions, challenges, and surprises surrounding the idea that activation of NMDA receptors can

  7. Structural Bioinformatics-Based Prediction of Exceptional Selectivity of p38 MAP Kinase Inhibitor PH-797804

    SciTech Connect

    Xing, Li; Shieh, Huey S.; Selness, Shaun R.; Devraj, Rajesh V.; Walker, John K.; Devadas, Balekudru; Hope, Heidi R.; Compton, Robert P.; Schindler, John F.; Hirsch, Jeffrey L.; Benson, Alan G.; Kurumbail, Ravi G.; Stegeman, Roderick A.; Williams, Jennifer M.; Broadus, Richard M.; Walden, Zara; Monahan, Joseph B.; Pfizer

    2009-07-24

    PH-797804 is a diarylpyridinone inhibitor of p38{alpha} mitogen-activated protein (MAP) kinase derived from a racemic mixture as the more potent atropisomer (aS), first proposed by molecular modeling and subsequently confirmed by experiments. On the basis of structural comparison with a different biaryl pyrazole template and supported by dozens of high-resolution crystal structures of p38{alpha} inhibitor complexes, PH-797804 is predicted to possess a high level of specificity across the broad human kinase genome. We used a structural bioinformatics approach to identify two selectivity elements encoded by the TXXXG sequence motif on the p38{alpha} kinase hinge: (i) Thr106 that serves as the gatekeeper to the buried hydrophobic pocket occupied by 2,4-difluorophenyl of PH-797804 and (ii) the bidentate hydrogen bonds formed by the pyridinone moiety with the kinase hinge requiring an induced 180{sup o} rotation of the Met109-Gly110 peptide bond. The peptide flip occurs in p38{alpha} kinase due to the critical glycine residue marked by its conformational flexibility. Kinome-wide sequence mining revealed rare presentation of the selectivity motif. Corroboratively, PH-797804 exhibited exceptionally high specificity against MAP kinases and the related kinases. No cross-reactivity was observed in large panels of kinase screens (selectivity ratio of >500-fold). In cellular assays, PH-797804 demonstrated superior potency and selectivity consistent with the biochemical measurements. PH-797804 has met safety criteria in human phase I studies and is under clinical development for several inflammatory conditions. Understanding the rationale for selectivity at the molecular level helps elucidate the biological function and design of specific p38{alpha} kinase inhibitors.

  8. Coordinate regulation of IkappaB kinases by mitogen-activated protein kinase kinase kinase 1 and NF-kappaB-inducing kinase.

    PubMed

    Nemoto, S; DiDonato, J A; Lin, A

    1998-12-01

    IkappaB kinases (IKKalpha and IKKbeta) are key components of the IKK complex that mediates activation of the transcription factor NF-kappaB in response to extracellular stimuli such as inflammatory cytokines, viral and bacterial infection, and UV irradiation. Although NF-kappaB-inducing kinase (NIK) interacts with and activates the IKKs, the upstream kinases for the IKKs still remain obscure. We identified mitogen-activated protein kinase kinase kinase 1 (MEKK1) as an immediate upstream kinase of the IKK complex. MEKK1 is activated by tumor necrosis factor alpha (TNF-alpha) and interleukin-1 and can potentiate the stimulatory effect of TNF-alpha on IKK and NF-kappaB activation. The dominant negative mutant of MEKK1, on the other hand, partially blocks activation of IKK by TNF-alpha. MEKK1 interacts with and stimulates the activities of both IKKalpha and IKKbeta in transfected HeLa and COS-1 cells and directly phosphorylates the IKKs in vitro. Furthermore, MEKK1 appears to act in parallel to NIK, leading to synergistic activation of the IKK complex. The formation of the MEKK1-IKK complex versus the NIK-IKK complex may provide a molecular basis for regulation of the IKK complex by various extracellular signals.

  9. Modelling the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase on adenylate kinase.

    PubMed Central

    Bertrand, L; Vertommen, D; Depiereux, E; Hue, L; Rider, M H; Feytmans, E

    1997-01-01

    Simultaneous multiple alignment of available sequences of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase revealed several segments of conserved residues in the 2-kinase domain. The sequence of the kinase domain was also compared with proteins of known three-dimensional structure. No similarity was found between the kinase domain of 6-phosphofructo-2-kinase and 6-phosphofructo-1-kinase. This questions the modelling of the 2-kinase domain on bacterial 6-phosphofructo-1-kinase that has previously been proposed [Bazan, Fletterick and Pilkis (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646]. However, sequence similarities were found between the 2-kinase domain and several nucleotide-binding proteins, the most similar being adenylate kinase. A structural model of the 2-kinase domain based on adenylate kinase is proposed. It accommodates all the results of site-directed mutagenesis studies carried out to date on residues in the 2-kinase domain. It also allows residues potentially involved in catalysis and/or substrate binding to be predicted. PMID:9032445

  10. Unconscious relational encoding depends on hippocampus

    PubMed Central

    Duss, Simone B.; Reber, Thomas P.; Hänggi, Jürgen; Schwab, Simon; Wiest, Roland; Müri, René M.; Brugger, Peter; Gutbrod, Klemens

    2014-01-01

    Textbooks divide between human memory systems based on consciousness. Hippocampus is thought to support only conscious encoding, while neocortex supports both conscious and unconscious encoding. We tested whether processing modes, not consciousness, divide between memory systems in three neuroimaging experiments with 11 amnesic patients (mean age = 45.55 years, standard deviation = 8.74, range = 23–60) and 11 matched healthy control subjects. Examined processing modes were single item versus relational encoding with only relational encoding hypothesized to depend on hippocampus. Participants encoded and later retrieved either single words or new relations between words. Consciousness of encoding was excluded by subliminal (invisible) word presentation. Amnesic patients and controls performed equally well on the single item task activating prefrontal cortex. But only the controls succeeded on the relational task activating the hippocampus, while amnesic patients failed as a group. Hence, unconscious relational encoding, but not unconscious single item encoding, depended on hippocampus. Yet, three patients performed normally on unconscious relational encoding in spite of amnesia capitalizing on spared hippocampal tissue and connections to language cortex. This pattern of results suggests that processing modes divide between memory systems, while consciousness divides between levels of function within a memory system. PMID:25273998

  11. Encoders for block-circulant LDPC codes

    NASA Technical Reports Server (NTRS)

    Andrews, Kenneth; Dolinar, Sam; Thorpe, Jeremy

    2005-01-01

    In this paper, we present two encoding methods for block-circulant LDPC codes. The first is an iterative encoding method based on the erasure decoding algorithm, and the computations required are well organized due to the block-circulant structure of the parity check matrix. The second method uses block-circulant generator matrices, and the encoders are very similar to those for recursive convolutional codes. Some encoders of the second type have been implemented in a small Field Programmable Gate Array (FPGA) and operate at 100 Msymbols/second.

  12. Evaluation of GOES encoder lamps

    NASA Technical Reports Server (NTRS)

    Viehmann, W.; Helmold, N.

    1983-01-01

    Aging characteristics and life expectancies of flight quality, tungsten filament, encoder lamps are similar to those of 'commercial' grade gas filled lamps of similar construction, filament material and filament temperature. The aging and final failure by filament burnout are caused by single crystal growth over large portions of the filament with the concomitant development of facets and notches resulting in reduction of cross section and mechanical weakening of the filament. The life expectancy of presently produced lamps is about one year at their nominal operating voltage of five volts dc. At 4.5 volts, it is about two years. These life times are considerably shorter, and the degradation rates of lamp current and light flux are considerably higher, than were observed in the laboratory and in orbit on lamps of the same type manufactured more than a decade ago. It is speculated that the filaments of these earlier lamps contained a crystallization retarding dopant, possibly thorium oxide. To obtain the desired life expectancy of or = to four years in present lamps, operating voltages of or = to four volts dc would be required.

  13. Calcium-Dependent Protein Kinase Genes in Corn Roots

    NASA Technical Reports Server (NTRS)

    Takezawa, D.; Patil, S.; Bhatia, A.; Poovaiah, B. W.

    1996-01-01

    Two cDNAs encoding Ca-2(+) - Dependent Protein Kinases (CDPKs), Corn Root Protein Kinase 1 and 2 (CRPK 1, CRPK 2) were isolated from the root tip library of corn (Zea mays L., cv. Merit) and their nucleotide sequences were determined. Deduced amino acid sequences of both the clones have features characteristic of plant CDPKS, including all 11 conserved serine/threonine kinase subdomains, a junction domain and a calmodulin-like domain with four Ca-2(+), -binding sites. Northern analysis revealed that CRPKI mRNA is preferentially expressed in roots, especially in the root tip; whereas, the expression of CRPK2 mRNA was very low in all the tissues tested. In situ hybridization experiments revealed that CRPKI mRNA is highly expressed in the root apex, as compared to other parts of the root. Partially purified CDPK from the root tip phosphorylates syntide-2, a common peptide substrate for plant CDPKs, and the phosphorylation was stimulated 7-fold by the addition of Ca-2(+). Our results show that two CDPK isoforms are expressed in corn roots and they may be involved in the Ca-2(+)-dependent signal transduction process.

  14. Behavioral Analysis of Ste20 Kinase SPAK Knockout Mice

    PubMed Central

    Geng, Yang; Byun, Nellie; Delpire, Eric

    2009-01-01

    SPAK/STK39 is a mammalian protein kinase involved in the regulation of inorganic ion transport mechanisms known to modulate GABAergic neurotransmission in the both central and the peripheral nervous systems. We have previously shown that disruption of the gene encoding SPAK by homologous recombination in mouse embryonic stem cells results in viable mice that lack expression of the kinase [16]. With the exception of reduced fertility, these mice do not exhibit an overt adverse phenotype. In the present study, we examine the neurological phenotype of these mice by subjecting them to an array of behavioral tests. We show that SPAK knockout mice displayed a higher nociceptive threshold than their wild-type counterparts on the hot plate and tail flick assays. SPAK knockout mice also exhibited a strong locomotor phenotype evidenced by significant deficits on the rotarod and decreased activity in open field tests. In contrast, balance and proprioception was not affected. Finally, they demonstrated an increased anxiety-like phenotype, spending significantly longer periods of time in the dark area of the light/dark box and increased thigmotaxis in the open field chamber. These results suggest that the kinase plays an important role in CNS function, consistent with SPAK regulating ion transport mechanisms directly involved in inhibitory neurotransmission. PMID:20006650

  15. The optional E. coli prr locus encodes a latent form of phage T4-induced anticodon nuclease.

    PubMed Central

    Levitz, R; Chapman, D; Amitsur, M; Green, R; Snyder, L; Kaufmann, G

    1990-01-01

    The optional Escherichia coli prr locus restricts phage T4 mutants lacking polynucleotide kinase or RNA ligase. Underlying this restriction is the specific manifestation of the T4-induced anticodon nuclease, an enzyme which triggers the cleavage-ligation of the host tRNALys. We report here the molecular cloning, nucleotide sequence and mutational analysis of prr-associated DNA. The results indicate that prr encodes a latent form of anticodon nuclease consisting of a core enzyme and cognate masking agents. They suggest that the T4-encoded factors of anticodon nuclease counteract the prr-encoded masking agents, thus activating the latent enzyme. The encoding of a tRNA cleavage-ligation pathway by two separate genetic systems which cohabitate E. coli may provide a clue to the evolution of RNA splicing mechanisms mediated by proteins. Images Fig.3. Fig.4. PMID:1691706

  16. Activity of cGMP-Dependent Protein Kinase (PKG) Affects Sucrose Responsiveness and Habituation in "Drosophila melanogaster"

    ERIC Educational Resources Information Center

    Scheiner, Ricarda; Sokolowski, Marla B.; Erber, Joachim

    2004-01-01

    The cGMP-dependent protein kinase (PKG) has many cellular functions in vertebrates and insects that affect complex behaviors such as locomotion and foraging. The "foraging" ("for") gene encodes a PKG in "Drosophila melanogaster." Here, we demonstrate a function for the "for" gene in sensory responsiveness and nonassociative learning. Larvae of the…

  17. Evolutionary Ancestry of Eukaryotic Protein Kinases and Choline Kinases.

    PubMed

    Lai, Shenshen; Safaei, Javad; Pelech, Steven

    2016-03-01

    The reversible phosphorylation of proteins catalyzed by protein kinases in eukaryotes supports an important role for eukaryotic protein kinases (ePKs) in the emergence of nucleated cells in the third superkingdom of life. Choline kinases (ChKs) could also be critical in the early evolution of eukaryotes, because of their function in the biosynthesis of phosphatidylcholine, which is unique to eukaryotic membranes. However, the genomic origins of ePKs and ChKs are unclear. The high degeneracy of protein sequences and broad expansion of ePK families have made this fundamental question difficult to answer. In this study, we identified two class-I aminoacyl-tRNA synthetases with high similarities to consensus amino acid sequences of human protein-serine/threonine kinases. Comparisons of primary and tertiary structures supported that ePKs and ChKs evolved from a common ancestor related to glutaminyl aminoacyl-tRNA synthetases, which may have been one of the key factors in the successful of emergence of ancient eukaryotic cells from bacterial colonies.

  18. Binding site identification and role of permanent water molecule of PIM-3 kinase: A molecular dynamics study.

    PubMed

    Ul-Haq, Zaheer; Gul, Sana; Usmani, Saman; Wadood, Abdul; Khan, Waqasuddin

    2015-11-01

    The kinome is a protein kinase complement of the human genome, categorized as serine/threonine and tyrosine kinases. These kinases catalyze phosphorylation reaction by using ATP as phosphoryl donor. Proviral Integration Site for Moloney Murine Leukemia Virus (PIM) kinase encodes serine/threonine protein kinases that recognized as proto-oncogene, responsible for rapid growth of cancerous cells. It is implicated in cell survival and function via cell cycle progression and its metabolism. PIM-3, sub-member of PIM kinases is a proto-oncogene, its overexpression inhibits apoptosis, and results in progression of hepatocellular carcinoma. PIM-3 is considered as a promising drug target but attempts to develop its specific inhibitors is slowed down due to the lack of 3D structure by any experimental technique. In silico techniques generally facilitate scientist to explore hidden structural features in order to improve drug discovery. In the present study, homology modeling, molecular docking and MD simulation techniques were utilized to explore the structure and dynamics of PIM-3 kinase. Induction of water molecules during molecular docking simulation explored differences in the hinge region between PIM-1 and PIM-3 kinases that may be responsible for specificity. Furthermore, role of water molecules in the active site was also explored via radial distribution function (RDF) after a 10 ns molecular dynamics (MD) simulations. Generated RDF plots exhibited the importance of water for inhibitor binding through their bridging capability that links the ligand with binding site residues.

  19. Pyp1 and Pyp2 PTPases dephosphorylate an osmosensing MAP kinase controlling cell size at division in fission yeast.

    PubMed

    Millar, J B; Buck, V; Wilkinson, M G

    1995-09-01

    Simultaneous inactivation of pyp1 and pyp2 PTPases in fission yeast leads to aberrant cell morphology and growth arrest. Spontaneous recessive mutations that bypass the requirement for pyp1 and pyp2 and reside in two complementation groups were isolated, sty1 and sty2. sty1- and sty2- mutant cells are substantially delayed in the timing of mitotic initiation. We have isolated the sty1 gene, which encodes a MAP kinase that is closely related to a subfamily of MAP kinases regulated by osmotic stress including Saccharomyces cervisiae HOG1 and human CSBP1. We find that sty2 is allelic to the wis1 MAP kinase kinase and that delta sty1 and delta wis1 cells are unable to grow in high osmolarity medium. Osmotic stress induces both tyrosine phosphorylation of Sty1 and a reduction in cell size at division. Pyp2 associates with and tyrosine dephosphorylates Sty1 in vitro. We find that wis1-dependent induction of pyp2 mRNA is responsible for tyrosine dephosphorylation of Sty1 in vivo on prolonged exposure to osmotic stress. We conclude that Pyp1 and Pyp2 are tyrosine-specific MAP kinase phosphatases that inactivate an osmoregulated MAP kinase, Sty1, which acts downstream of the Wis1 MAP kinase kinase to control cell size at division in fission yeast. PMID:7657164

  20. Binding site identification and role of permanent water molecule of PIM-3 kinase: A molecular dynamics study.

    PubMed

    Ul-Haq, Zaheer; Gul, Sana; Usmani, Saman; Wadood, Abdul; Khan, Waqasuddin

    2015-11-01

    The kinome is a protein kinase complement of the human genome, categorized as serine/threonine and tyrosine kinases. These kinases catalyze phosphorylation reaction by using ATP as phosphoryl donor. Proviral Integration Site for Moloney Murine Leukemia Virus (PIM) kinase encodes serine/threonine protein kinases that recognized as proto-oncogene, responsible for rapid growth of cancerous cells. It is implicated in cell survival and function via cell cycle progression and its metabolism. PIM-3, sub-member of PIM kinases is a proto-oncogene, its overexpression inhibits apoptosis, and results in progression of hepatocellular carcinoma. PIM-3 is considered as a promising drug target but attempts to develop its specific inhibitors is slowed down due to the lack of 3D structure by any experimental technique. In silico techniques generally facilitate scientist to explore hidden structural features in order to improve drug discovery. In the present study, homology modeling, molecular docking and MD simulation techniques were utilized to explore the structure and dynamics of PIM-3 kinase. Induction of water molecules during molecular docking simulation explored differences in the hinge region between PIM-1 and PIM-3 kinases that may be responsible for specificity. Furthermore, role of water molecules in the active site was also explored via radial distribution function (RDF) after a 10 ns molecular dynamics (MD) simulations. Generated RDF plots exhibited the importance of water for inhibitor binding through their bridging capability that links the ligand with binding site residues. PMID:26529487

  1. Cloning and expression of two human p70 S6 kinase polypeptides differing only at their amino termini

    SciTech Connect

    Grove, J.R.; Banerjee, P.; Balasubramanyam, A.; Price, D.J.; Avruch, J. ); Coffer, P.J.; Woodgett, J.R. )

    1991-11-01

    Two classes of human cDNA encoding the insulin/mitogen-activated p70 S6 kinase have been isolated; the two classes differ only in the 5{prime} region, such that the longer polypeptide consists of 525 amino acids, of which the last 502 longer residues are identical in sequence to the entire polypeptide encoded by the second cDNA. Both p70 S6 kinase polypeptides predicted by these cDNAs are present in p70 S6 kinase purified from rat liver, and each is thus expressed in vivo. Moreover, both polypeptides are expressed from a single mRNA transcribed from the (longer) p70 S6 kinase {alpha}I cDNA through the utilization of different translational start sites. Transient expression of p70 {alpha}I and {alpha}II S6 kinase cDNA in COA cells results in a 2.5- to 4-fold increase in overall S6 kinase activity. Transfection with the {alpha}II cDNA yields only the smaller set of bands, while transfection with the {alpha}I cDNA generates both sets of bands. Mutation of Met-24 in the {alpha}I cDNA to Leu or Thr suppresses synthesis of the {alpha}II polypeptides. Only the p70 {alpha}I and {alpha}II polypeptides of slowest mobility on SDS-PAGE comigrate with the 70-and 90-kDa proteins observed in purified rat liver S6 kinase. The recombinant p70 S6 kinase undergoes multiple phosphorylation and partial activation in COS cells. Acquisition of S6 protein kinase catalytic function, however, is apparently restricted to the most extensively phosphorylated recombinant polypeptides.

  2. HIV-1 incorporates and proteolytically processes human NDR1 and NDR2 serine-threonine kinases

    SciTech Connect

    Devroe, Eric; Silver, Pamela A.; Engelman, Alan . E-mail: alan_engelman@dfci.harvard.edu

    2005-01-05

    Mammalian genomes encode two related serine-threonine kinases, nuclear Dbf2 related (NDR)1 and NDR2, which are homologous to the Saccharomyces cerevisiae Dbf2 kinase. Recently, a yeast genetic screen implicated the Dbf2 kinase in Ty1 retrotransposition. Since several virion-incorporated kinases regulate the infectivity of human immunodeficiency virus type 1 (HIV-1), we speculated that the human NDR1 and NDR2 kinases might play a role in the HIV-1 life cycle. Here we show that the NDR1 and NDR2 kinases were incorporated into HIV-1 particles. Furthermore, NDR1 and NDR2 were cleaved by the HIV-1 protease (PR), both within virions and within producer cells. Truncation at the PR cleavage site altered NDR2 subcellular localization and inhibited NDR1 and NDR2 enzymatic activity. These studies identify two new virion-associated host cell enzymes and suggest a novel mechanism by which HIV-1 alters the intracellular environment of human cells.

  3. Novel receptor-like kinases in cacao contain PR-1 extracellular domains.

    PubMed

    Teixeira, Paulo José Pereira Lima; Costa, Gustavo Gilson Lacerda; Fiorin, Gabriel Lorencini; Pereira, Gonçalo Amarante Guimarães; Mondego, Jorge Maurício Costa

    2013-08-01

    Members of the pathogenesis-related protein 1 (PR-1) family are well-known markers of plant defence responses, forming part of the arsenal of the secreted proteins produced on pathogen recognition. Here, we report the identification of two cacao (Theobroma cacao L.) PR-1s that are fused to transmembrane regions and serine/threonine kinase domains, in a manner characteristic of receptor-like kinases (RLKs). These proteins (TcPR-1f and TcPR-1g) were named PR-1 receptor kinases (PR-1RKs). Phylogenetic analysis of RLKs and PR-1 proteins from cacao indicated that PR-1RKs originated from a fusion between sequences encoding PR-1 and the kinase domain of a LecRLK (Lectin Receptor-Like Kinase). Retrotransposition marks surround TcPR-1f, suggesting that retrotransposition was involved in the origin of PR-1RKs. Genes with a similar domain architecture to cacao PR-1RKs were found in rice (Oryza sativa), barrel medic (Medicago truncatula) and a nonphototrophic bacterium (Herpetosiphon aurantiacus). However, their kinase domains differed from those found in LecRLKs, indicating the occurrence of convergent evolution. TcPR-1g expression was up-regulated in the biotrophic stage of witches' broom disease, suggesting a role for PR-1RKs during cacao defence responses. We hypothesize that PR-1RKs transduce a defence signal by interacting with a PR-1 ligand.

  4. Molecular and biochemical characterisation of DNA-dependent protein kinase-defective rodent mutant irs-20.

    PubMed Central

    Priestley, A; Beamish, H J; Gell, D; Amatucci, A G; Muhlmann-Diaz, M C; Singleton, B K; Smith, G C; Blunt, T; Schalkwyk, L C; Bedford, J S; Jackson, S P; Jeggo, P A; Taccioli, G E

    1998-01-01

    The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) is a member of a sub-family of phosphatidylinositol (PI) 3-kinases termed PIK-related kinases. A distinguishing feature of this sub-family is the presence of a conserved C-terminal region downstream of a PI 3-kinase domain. Mutants defective in DNA-PKcs are sensitive to ionising radiation and are unable to carry out V(D)J recombination. Irs-20 is a DNA-PKcs-defective cell line with milder gamma-ray sensitivity than two previously characterised mutants, V-3 and mouse scid cells. Here we show that the DNA-PKcs protein from irs-20 cells can bind to DNA but is unable to function as a protein kinase. To verify the defect in irs-20 cells and provide insight into the function and expression of DNA-PKcs in double-strand break repair and V(D)J recombination we introduced YACs encoding human and mouse DNA-PKcs into defective mutants and achieved complementation of the defective phenotypes. Furthermore, in irs-20 we identified a mutation in DNA-PKcs that causes substitution of a lysine for a glutamic acid in the fourth residue from the C-terminus. This represents a strong candidate for the inactivating mutation and provides supportive evidence that the extreme C-terminal motif is important for protein kinase activity. PMID:9518490

  5. HPK1, a hematopoietic protein kinase activating the SAPK/JNK pathway.

    PubMed Central

    Kiefer, F; Tibbles, L A; Anafi, M; Janssen, A; Zanke, B W; Lassam, N; Pawson, T; Woodgett, J R; Iscove, N N

    1996-01-01

    In mammalian cells, a specific stress-activated protein kinase (SAPK/JNK) pathway is activated in response to inflammatory cytokines, injury from heat, chemotherapeutic drugs and UV or ionizing radiation. The mechanisms that link these stimuli to activation of the SAPK/JNK pathway in different tissues remain to be identified. We have developed and applied a PCR-based subtraction strategy to identify novel genes that are differentially expressed at specific developmental points in hematopoiesis. We show that one such gene, hematopoietic progenitor kinase 1 (hpk1), encodes a serine/threonine kinase sharing similarity with the kinase domain of Ste20. HPK1 specifically activates the SAPK/JNK pathway after transfection into COS1 cells, but does not stimulate the p38/RK or mitogen-activated ERK signaling pathways. Activation of SAPK requires a functional HPK1 kinase domain and HPK1 signals via the SH3-containing mixed lineage kinase MLK-3 and the known SAPK activator SEK1. HPK1 therefore provides an example of a cell type-specific input into the SAPK/JNK pathway. The developmental specificity of its expression suggests a potential role in hematopoietic lineage decisions and growth regulation. Images PMID:9003777

  6. Congruity of Encoding in Children's Redintegrative Memory.

    ERIC Educational Resources Information Center

    Hall, Donald M.; Geis, Mary Fulcher

    The mnemonic consequences of semantic, acoustic, and orthographic encoding and the relationships between encoding and retrieval cues were investigated in an incidental-learning experiment involving 24 first-, third-, and fifth-grade pupils. Each child was asked one orienting question for each of 18 words; the questions differed in the type of…

  7. Pseudochromatic encoding fractional Fourier transform rainbow hologram

    NASA Astrophysics Data System (ADS)

    Guo, Yongkang; Huang, Qizhong; Du, Jinglei

    1998-08-01

    The FRTH is presented in this paper and its properties are discussed. Then we make a pseudo chromatic encoding fractional Fourier transform rainbow hologram, based on its specialty in its reconstruction and that the encoding color has relationship with the order of the reconstruction FRT system, a new type of anti-counterfeiting hologram is introduced.

  8. DNA encoding a DNA repair protein

    DOEpatents

    Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

    2006-08-15

    An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

  9. Experiments in encoding multilevel images as quadtrees

    NASA Technical Reports Server (NTRS)

    Lansing, Donald L.

    1987-01-01

    Image storage requirements for several encoding methods are investigated and the use of quadtrees with multigray level or multicolor images are explored. The results of encoding a variety of images having up to 256 gray levels using three schemes (full raster, runlength and quadtree) are presented. Although there is considerable literature on the use of quadtrees to store and manipulate binary images, their application to multilevel images is relatively undeveloped. The potential advantage of quadtree encoding is that an entire area with a uniform gray level may be encoded as a unit. A pointerless quadtree encoding scheme is described. Data are presented on the size of the quadtree required to encode selected images and on the relative storage requirements of the three encoding schemes. A segmentation scheme based on the statistical variation of gray levels within a quadtree quadrant is described. This parametric scheme may be used to control the storage required by an encoded image and to preprocess a scene for feature identification. Several sets of black and white and pseudocolor images obtained by varying the segmentation parameter are shown.

  10. The Acquisition of Syntactically Encoded Evidentiality

    ERIC Educational Resources Information Center

    Rett, Jessica; Hyams, Nina

    2014-01-01

    This article presents several empirical studies of syntactically encoded evidentiality in English. The first part of our study consists of an adult online experiment that confirms claims in Asudeh & Toivonen (2012) that raised Perception Verb Similatives (PVSs; e.g. "John looks like he is sick") encode direct evidentiality. We then…

  11. The small GTP-binding protein Rho binds to and activates a 160 kDa Ser/Thr protein kinase homologous to myotonic dystrophy kinase.

    PubMed Central

    Ishizaki, T; Maekawa, M; Fujisawa, K; Okawa, K; Iwamatsu, A; Fujita, A; Watanabe, N; Saito, Y; Kakizuka, A; Morii, N; Narumiya, S

    1996-01-01

    The small GTP-binding protein Rho functions as a molecular switch in the formation of focal adhesions and stress fibers, cytokinesis and transcriptional activation. The biochemical mechanism underlying these actions remains unknown. Using a ligand overlay assay, we purified a 160 kDa platelet protein that bound specifically to GTP-bound Rho. This protein, p160, underwent autophosphorylation at its serine and threonine residues and showed the kinase activity to exogenous substrates. Both activities were enhanced by the addition of GTP-bound Rho. A cDNA encoding p160 coded for a 1354 amino acid protein. This protein has a Ser/Thr kinase domain in its N-terminus, followed by a coiled-coil structure approximately 600 amino acids long, and a cysteine-rich zinc finger-like motif and a pleckstrin homology region in the C-terminus. The N-terminus region including a kinase domain and a part of coiled-coil structure showed strong homology to myotonic dystrophy kinase over 500 residues. When co-expressed with RhoA in COS cells, p160 was co-precipitated with the expressed Rho and its kinase activity was activated, indicating that p160 can associate physically and functionally with Rho both in vitro and in vivo. Images PMID:8617235

  12. The Interaction between Cyclin B1 and Cytomegalovirus Protein Kinase pUL97 is Determined by an Active Kinase Domain.

    PubMed

    Steingruber, Mirjam; Socher, Eileen; Hutterer, Corina; Webel, Rike; Bergbrede, Tim; Lenac, Tihana; Sticht, Heinrich; Marschall, Manfred

    2015-08-11

    Replication of human cytomegalovirus (HCMV) is characterized by a tight virus-host cell interaction. Cyclin-dependent protein kinases (CDKs) are functionally integrated into viral gene expression and protein modification. The HCMV-encoded protein kinase pUL97 acts as a CDK ortholog showing structural and functional similarities. Recently, we reported an interaction between pUL97 kinase with a subset of host cyclins, in particular with cyclin T1. Here, we describe an interaction of pUL97 at an even higher affinity with cyclin B1. As a striking feature, the interaction between pUL97 and cyclin B1 proved to be strictly dependent on pUL97 activity, as interaction could be abrogated by treatment with pUL97 inhibitors or by inserting mutations into the conserved kinase domain or the nonconserved C-terminus of pUL97, both producing loss of activity. Thus, we postulate that the mechanism of pUL97-cyclin B1 interaction is determined by an active pUL97 kinase domain.

  13. Overexpression of a kinase-deficient form of the EDR1 gene enhances powdery mildew resistance and ethylene-induced senescence in Arabidopsis.

    PubMed

    Tang, Dingzhong; Innes, Roger W

    2002-12-01

    The EDR1 gene of Arabidopsis has previously been reported to encode a Raf-like mitogen-activated protein kinase kinase (MAPKK) kinase, and to function as a negative regulator of disease resistance. A phylogenetic analysis of plant and animal protein kinases revealed, however, that plant Raf-like kinases are more closely related to animal mixed lineage kinases (MLKs) than Raf-like kinases, and are deeply divergent from both classes of animal kinases, making inferences of substrate specificity questionable. We, therefore, assayed the kinase activity of recombinant EDR1 protein in vitro. The EDR1 kinase domain displayed autophosphorylation activity and phosphorylated the common MAP kinase substrate myelin basic protein. The EDR1 kinase domain also phosphorylated a kinase-deficient EDR1 protein, indicating that EDR1 autophosphorylation can occur via an intermolecular mechanism. Overexpression of a kinase-deficient full-length EDR1 gene (35S::dnEDR1) in wild-type Arabidopsis plants caused a dominant negative phenotype, conferring resistance to powdery mildew (Erysiphe cichoracearum) and enhancing ethylene-induced senescence. RNA-gel blot analyses showed that the 35S::dnEDR1 transgene was highly transcribed in transgenic plants. Western blot analysis, however, revealed that neither the wild-type nor mutant EDR1 protein could be detected in these lines, indicating that the dominant negative phenotype may be caused by a translational inhibition mechanism rather than by a protein level effect. Overexpression of orthologous dnEDR1 constructs may provide a novel strategy for controlling powdery mildew disease in crops.

  14. KID, a Kinase Inhibitor Database project.

    PubMed

    Collin, O; Meijer, L

    1999-01-01

    The Kinase Inhibitor Database is a small specialized database dedicated to the gathering of information on protein kinase inhibitors. The database is accessible through the World Wide Web system and gives access to structural and bibliographic information on protein kinase inhibitors. The data in the database will be collected and submitted by researchers working in the kinase inhibitor field. The submitted data will be checked by the curator of the database before entry.

  15. A model for visual memory encoding.

    PubMed

    Nenert, Rodolphe; Allendorfer, Jane B; Szaflarski, Jerzy P

    2014-01-01

    Memory encoding engages multiple concurrent and sequential processes. While the individual processes involved in successful encoding have been examined in many studies, a sequence of events and the importance of modules associated with memory encoding has not been established. For this reason, we sought to perform a comprehensive examination of the network for memory encoding using data driven methods and to determine the directionality of the information flow in order to build a viable model of visual memory encoding. Forty healthy controls ages 19-59 performed a visual scene encoding task. FMRI data were preprocessed using SPM8 and then processed using independent component analysis (ICA) with the reliability of the identified components confirmed using ICASSO as implemented in GIFT. The directionality of the information flow was examined using Granger causality analyses (GCA). All participants performed the fMRI task well above the chance level (>90% correct on both active and control conditions) and the post-fMRI testing recall revealed correct memory encoding at 86.33 ± 5.83%. ICA identified involvement of components of five different networks in the process of memory encoding, and the GCA allowed for the directionality of the information flow to be assessed, from visual cortex via ventral stream to the attention network and then to the default mode network (DMN). Two additional networks involved in this process were the cerebellar and the auditory-insular network. This study provides evidence that successful visual memory encoding is dependent on multiple modules that are part of other networks that are only indirectly related to the main process. This model may help to identify the node(s) of the network that are affected by a specific disease processes and explain the presence of memory encoding difficulties in patients in whom focal or global network dysfunction exists.

  16. A model for visual memory encoding.

    PubMed

    Nenert, Rodolphe; Allendorfer, Jane B; Szaflarski, Jerzy P

    2014-01-01

    Memory encoding engages multiple concurrent and sequential processes. While the individual processes involved in successful encoding have been examined in many studies, a sequence of events and the importance of modules associated with memory encoding has not been established. For this reason, we sought to perform a comprehensive examination of the network for memory encoding using data driven methods and to determine the directionality of the information flow in order to build a viable model of visual memory encoding. Forty healthy controls ages 19-59 performed a visual scene encoding task. FMRI data were preprocessed using SPM8 and then processed using independent component analysis (ICA) with the reliability of the identified components confirmed using ICASSO as implemented in GIFT. The directionality of the information flow was examined using Granger causality analyses (GCA). All participants performed the fMRI task well above the chance level (>90% correct on both active and control conditions) and the post-fMRI testing recall revealed correct memory encoding at 86.33 ± 5.83%. ICA identified involvement of components of five different networks in the process of memory encoding, and the GCA allowed for the directionality of the information flow to be assessed, from visual cortex via ventral stream to the attention network and then to the default mode network (DMN). Two additional networks involved in this process were the cerebellar and the auditory-insular network. This study provides evidence that successful visual memory encoding is dependent on multiple modules that are part of other networks that are only indirectly related to the main process. This model may help to identify the node(s) of the network that are affected by a specific disease processes and explain the presence of memory encoding difficulties in patients in whom focal or global network dysfunction exists. PMID:25272154

  17. Membrane Ig cross-linking regulates phosphatidylinositol 3-kinase in B lymphocytes.

    PubMed

    Gold, M R; Chan, V W; Turck, C W; DeFranco, A L

    1992-04-01

    Cross-linking of the B cell AgR results in activation of mature B cells and tolerization of immature B cells. The initial signaling events stimulated by membrane immunoglobulin (mIg) cross-linking are tyrosine phosphorylation of a number of proteins. Among the targets of mIg-induced tyrosine phosphorylation are the tyrosine kinases encoded by the lyn, blk, fyn, and syk genes, the mIg-associated proteins MB-1 and Ig-beta, phospholipase C-gamma 1 and -gamma 2, as well as many unidentified proteins. In this report we show that mIg cross-linking also regulates phosphatidylinositol 3-kinase (PtdIns 3-kinase), an enzyme that phosphorylates inositol phospholipids and plays a key role in mediating the effects of tyrosine kinases on growth control in fibroblasts. Cross-linking mIg on B lymphocytes greatly increased the amount of PtdIns 3-kinase activity which could be immunoprecipitated with anti-phosphotyrosine (anti-tyr(P) antibodies. This response was observed after mIg cross-linking in mIgM- and mIgG-bearing B cell lines and after cross-linking either mIgM or mIgD in murine splenic B cells. Thus, regulation of PtdIns 3-kinase is a common feature of signaling by several different isotypes of mIg. This response was rapid and peaked 2 to 3 min after the addition of anti-Ig antibodies. The anti-Ig-stimulated increase in PtdIns 3-kinase activity associated with anti-Tyr(P) immunoprecipitates could reflect increased tyrosine phosphorylation of PtdIns 3-kinase, increased activity of the enzyme, or both. In favor of the first possibility, the tyrosine kinase inhibitor herbimycin A blocked the increase in ant-Tyr(P)-immunoprecipitated PtdIns 3-kinase activity as well as the anti-Ig-induced tyrosine phosphorylation. Moreover, this response was not secondary to phospholipase C activation but rather seemed to be a direct consequence of mIg-induced tyrosine phosphorylation. Activation of the phosphoinositide pathway by a transfected M1 muscarinic acetylcholine receptor expressed in

  18. Assessing Kinase Activity in Plants with In-Gel Kinase Assays.

    PubMed

    Wang, Pengcheng; Zhu, Jian-Kang

    2016-01-01

    The in-gel protein kinase assay is a powerful method to measure the protein phosphorylation activity of specific protein kinases. Any protein substrate can be embedded in polyacrylamide gels where they can be phosphorylated by protein kinases that are separated in the gel under denaturing conditions and then renatured. The kinase activity can be visualized in situ in the gels by autoradiography. This method has been used to compare the activities of protein kinases in parallel samples or to identify their potential substrates. Here, we describe in detail an in-gel kinase assay to measure the activity of some protein kinases in plants.

  19. Enzymatic characteristics of the c-Raf-1 protein kinase.

    PubMed

    Force, T; Bonventre, J V; Heidecker, G; Rapp, U; Avruch, J; Kyriakis, J M

    1994-02-15

    The c-Raf-1 protein kinase plays a central role in the mitogenic response of cells to growth factors, cytokines, and many oncogenes. Despite the critical importance of this enzyme, very little is known of its biochemical properties or mechanisms of regulation. In these experiments, we used the only candidate physiologic substrate identified as yet for c-Raf-1, mitogen-activated protein kinase kinase (MAPKK), to examine enzymatic characteristics and candidate modulators of c-Raf-1, c-Raf-1 was purified from Sf9 cells infected with recombinant baculovirus encoding a histidine-tagged c-Raf-1. The Km values of c-Raf-1 for ATP and MAPKK were 11.6 microM and 0.8 microM, respectively, and the stoichiometry of phosphorylation of MAPKK by c-Raf-1 was 1.67 mol of phosphate per mol of MAPKK. In contrast to prior reports, Mg2+ was the preferred cation at Mg2+ and Mn2+ concentrations > 5 mM. c-Raf-1 substrate specificity was extremely restricted, consistent with the identification of only one candidate physiologic substrate to date and highlighting the necessity of using MAPKK rather than artificial substrates in c-Raf-1 activity assays. Of multiple potential substrates tested, the only one phosphorylated to > 20% of the level of MAPKK phosphorylation was myelin basic protein (22%). Heat-denatured MAPKK was phosphorylated at only 2% the level of native MAPKK, indicating that the restricted substrate specificity may be due to tertiary-structural requirements. We also examined whether c-Raf-1 activity is modulated by lipid binding to the cysteine finger region in its regulatory domain. Of multiple mitogen-stimulated or cell-membrane lipids tested, only phosphatidylserine and diacylglycerol in the presence of Ca2+ (2.5 mM) increased c-Raf-1 kinase activity significantly (1.5-fold). The increase is probably not of physiologic significance because it was about two orders of magnitude less than the stimulation of protein kinase C by these lipids. On gel-filtration chromatography, the

  20. Enzymatic characteristics of the c-Raf-1 protein kinase.

    PubMed Central

    Force, T; Bonventre, J V; Heidecker, G; Rapp, U; Avruch, J; Kyriakis, J M

    1994-01-01

    The c-Raf-1 protein kinase plays a central role in the mitogenic response of cells to growth factors, cytokines, and many oncogenes. Despite the critical importance of this enzyme, very little is known of its biochemical properties or mechanisms of regulation. In these experiments, we used the only candidate physiologic substrate identified as yet for c-Raf-1, mitogen-activated protein kinase kinase (MAPKK), to examine enzymatic characteristics and candidate modulators of c-Raf-1, c-Raf-1 was purified from Sf9 cells infected with recombinant baculovirus encoding a histidine-tagged c-Raf-1. The Km values of c-Raf-1 for ATP and MAPKK were 11.6 microM and 0.8 microM, respectively, and the stoichiometry of phosphorylation of MAPKK by c-Raf-1 was 1.67 mol of phosphate per mol of MAPKK. In contrast to prior reports, Mg2+ was the preferred cation at Mg2+ and Mn2+ concentrations > 5 mM. c-Raf-1 substrate specificity was extremely restricted, consistent with the identification of only one candidate physiologic substrate to date and highlighting the necessity of using MAPKK rather than artificial substrates in c-Raf-1 activity assays. Of multiple potential substrates tested, the only one phosphorylated to > 20% of the level of MAPKK phosphorylation was myelin basic protein (22%). Heat-denatured MAPKK was phosphorylated at only 2% the level of native MAPKK, indicating that the restricted substrate specificity may be due to tertiary-structural requirements. We also examined whether c-Raf-1 activity is modulated by lipid binding to the cysteine finger region in its regulatory domain. Of multiple mitogen-stimulated or cell-membrane lipids tested, only phosphatidylserine and diacylglycerol in the presence of Ca2+ (2.5 mM) increased c-Raf-1 kinase activity significantly (1.5-fold). The increase is probably not of physiologic significance because it was about two orders of magnitude less than the stimulation of protein kinase C by these lipids. On gel-filtration chromatography, the

  1. A membrane-anchored protein kinase involved in Brassica self-incompatibility signaling.

    PubMed

    Murase, Kohji; Shiba, Hiroshi; Iwano, Megumi; Che, Fang-Sik; Watanabe, Masao; Isogai, Akira; Takayama, Seiji

    2004-03-01

    Self-incompatibility (SI) response in Brassica is initiated by haplotype-specific interactions between the pollen-borne ligand S locus protein 11/SCR and its stigmatic S receptor kinase, SRK. This binding induces autophosphorylation of SRK, which is then thought to trigger a signaling cascade that leads to self-pollen rejection. A recessive mutation of the modifier (m) gene eliminates the SI response in stigma. Positional cloning of M has revealed that it encodes a membrane-anchored cytoplasmic serine/threonine protein kinase, designated M locus protein kinase (MLPK). Transient expression of MLPK restores the ability of mm papilla cells to reject self-pollen, suggesting that MLPK is a positive mediator of Brassica SI signaling.

  2. The Arabidopsis functional homolog of the p34cdc2 protein kinase.

    PubMed Central

    Ferreira, P C; Hemerly, A S; Villarroel, R; Van Montagu, M; Inzé, D

    1991-01-01

    The p34cdc2 protein kinase is a key component of the eukaryotic cell cycle, which is required for G1 to S-phase transition and for entry into mitosis. Using a 380-base pair DNA fragment obtained by polymerase chain reaction amplification from an Arabidopsis thaliana flower cDNA library as a probe, we isolated and sequenced a cdc2-homologous cDNA from Arabidopsis. The encoded polypeptide has extensive homology with cdc2-like kinases. Furthermore, when expressed in a CDC28ts Saccharomyces strain, it partially restores the capacity to grow at 36 degrees C, indicating that the plant cDNA is a functional homolog of the p34cdc2 kinase. Genomic hybridization demonstrated that there is one copy of the cdc2 gene per Arabidopsis haploid genome. Using RNA gel blot analysis, we found that cdc2 mRNA is present in all plant organs. PMID:1840925

  3. Tobacco serine/threonine protein kinase gene NrSTK enhances black shank resistance.

    PubMed

    Gao, Y-L; Wang, B-W; Xu, Z-L; Li, M-Y; Song, Z-B; Li, W-Z; Li, Y-P

    2015-01-01

    A serine/threonine protein kinase gene (NrSTK) was cloned from Nicotiana repanda based on the sequence of a previously isolated resistance gene analog (RGA). Expression of RGA was induced by challenge with the pathogen black shank. The NrSTK gene was predicted to encode a protein kinase that contained an ATP binding site at residues 41-69 and a serine/threonine protein kinase activation sequence spanning the region 161-173. Overexpression of NrSTK in the susceptible tobacco variety Honghuadajinyuan significantly enhanced resistance to black shank, indicating that NrSTK plays a role in incompatibility reactions between tobacco and the pathogen. Characterization of NrSTK will help elucidate the molecular mechanisms involved in black shank resistance in N. repanda.

  4. Src kinase regulation by phosphorylation and dephosphorylation

    SciTech Connect

    Roskoski, Robert . E-mail: biocrr@lsuhsc.edu

    2005-05-27

    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, PTP{alpha}, PTP{epsilon}, and PTP{lambda}. 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.

  5. Targeting Mitogen-Activated Protein Kinase Signaling in Mouse Models of Cardiomyopathy Caused by Lamin A/C Gene Mutations.

    PubMed

    Muchir, Antoine; Worman, Howard J

    2016-01-01

    The most frequently occurring mutations in the gene encoding nuclear lamin A and nuclear lamin C cause striated muscle diseases virtually always involving the heart. In this review, we describe the approaches and methods used to discover that cardiomyopathy-causing lamin A/C gene mutations increase MAP kinase signaling in the heart and that this plays a role in disease pathogenesis. We review different mouse models of cardiomyopathy caused by lamin A/C gene mutations and how transcriptomic analysis of one model identified increased cardiac activity of the ERK1/2, JNK, and p38α MAP kinases. We describe methods used to measure the activity of these MAP kinases in mouse hearts and then discuss preclinical treatment protocols using pharmacological inhibitors to demonstrate their role in pathogenesis. Several of these kinase inhibitors are in clinical development and could potentially be used to treat human subjects with cardiomyopathy caused by lamin A/C gene mutations.

  6. Phosphorylation of LRRK2 by casein kinase 1α regulates trans-Golgi clustering via differential interaction with ARHGEF7.

    PubMed

    Chia, Ruth; Haddock, Sara; Beilina, Alexandra; Rudenko, Iakov N; Mamais, Adamantios; Kaganovich, Alice; Li, Yan; Kumaran, Ravindran; Nalls, Michael A; Cookson, Mark R

    2014-01-01

    LRRK2, a gene relevant to Parkinson's disease, encodes a scaffolding protein with both GTPase and kinase activities. LRRK2 protein is itself phosphorylated and therefore is subject to regulation by cell signalling; however, the kinase(s) responsible for this event have not been definitively identified. Here using an unbiased siRNA kinome screen, we identify and validate casein kinase 1α (CK1α) as being responsible for LRRK2 phosphorylation, including in the adult mouse striatum. We further show that LRRK2 recruitment to TGN46-positive Golgi-derived vesicles is modulated by constitutive LRRK2 phosphorylation by CK1α. These effects are mediated by differential protein interactions of LRRK2 with a guanine nucleotide exchange factor, ARHGEF7. These pathways are therefore likely involved in the physiological maintenance of the Golgi in cells, which may play a role in the pathogenesis of Parkinson's disease. PMID:25500533

  7. Programmable Pulse-Position-Modulation Encoder

    NASA Technical Reports Server (NTRS)

    Zhu, David; Farr, William

    2006-01-01

    A programmable pulse-position-modulation (PPM) encoder has been designed for use in testing an optical communication link. The encoder includes a programmable state machine and an electronic code book that can be updated to accommodate different PPM coding schemes. The encoder includes a field-programmable gate array (FPGA) that is programmed to step through the stored state machine and code book and that drives a custom high-speed serializer circuit board that is capable of generating subnanosecond pulses. The stored state machine and code book can be updated by means of a simple text interface through the serial port of a personal computer.

  8. Pulse Vector-Excitation Speech Encoder

    NASA Technical Reports Server (NTRS)

    Davidson, Grant; Gersho, Allen

    1989-01-01

    Proposed pulse vector-excitation speech encoder (PVXC) encodes analog speech signals into digital representation for transmission or storage at rates below 5 kilobits per second. Produces high quality of reconstructed speech, but with less computation than required by comparable speech-encoding systems. Has some characteristics of multipulse linear predictive coding (MPLPC) and of code-excited linear prediction (CELP). System uses mathematical model of vocal tract in conjunction with set of excitation vectors and perceptually-based error criterion to synthesize natural-sounding speech.

  9. Protein Kinase A: A Master Kinase of Granulosa Cell Differentiation

    PubMed Central

    Puri, Pawan; Little-Ihrig, Lynda; Chandran, Uma; Law, Nathan C.; Hunzicker-Dunn, Mary; Zeleznik, Anthony J.

    2016-01-01

    Activation of protein kinase A (PKA) by follicle stimulating hormone (FSH) transduces the signal that drives differentiation of ovarian granulosa cells (GCs). An unresolved question is whether PKA is sufficient to initiate the complex program of GC responses to FSH. We compared signaling pathways and gene expression profiles of GCs stimulated with FSH or expressing PKA-CQR, a constitutively active mutant of PKA. Both FSH and PKA-CQR stimulated the phosphorylation of proteins known to be involved in GC differentiation including CREB, ß-catenin, AKT, p42/44 MAPK, GAB2, GSK-3ß, FOXO1, and YAP. In contrast, FSH stimulated the phosphorylation of p38 MAP kinase but PKA-CQR did not. Microarray analysis revealed that 85% of transcripts that were up-regulated by FSH were increased to a comparable extent by PKA-CQR and of the transcripts that were down-regulated by FSH, 76% were also down-regulated by PKA-CQR. Transcripts regulated similarly by FSH and PKA-CQR are involved in steroidogenesis and differentiation, while transcripts more robustly up-regulated by PKA-CQR are involved in ovulation. Thus, PKA, under the conditions of our experimental approach appears to function as a master upstream kinase that is sufficient to initiate the complex pattern of intracellular signaling pathway and gene expression profiles that accompany GC differentiation. PMID:27324437

  10. Biochemical and functional analysis of CTR1, a protein kinase that negatively regulates ethylene signaling in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Huang, Yafan; Li, Hui; Hutchison, Claire E.; Laskey, James; Kieber, Joseph J.

    2003-01-01

    CTR1 encodes a negative regulator of the ethylene response pathway in Arabidopsis thaliana. The C-terminal domain of CTR1 is similar to the Raf family of protein kinases, but its first two-thirds encodes a novel protein domain. We used a variety of approaches to investigate the function of these two CTR1 domains. Recombinant CTR1 protein was purified from a baculoviral expression system, and shown to possess intrinsic Ser/Thr protein kinase activity with enzymatic properties similar to Raf-1. Deletion of the N-terminal domain did not elevate the kinase activity of CTR1, indicating that, at least in vitro, this domain does not autoinhibit kinase function. Molecular analysis of loss-of-function ctr1 alleles indicated that several mutations disrupt the kinase catalytic domain, and in vitro studies confirmed that at least one of these eliminates kinase activity, which indicates that kinase activity is required for CTR1 function. One missense mutation, ctr1-8, was found to result from an amino acid substitution within a new conserved motif within the N-terminal domain. Ctr1-8 has no detectable effect on the kinase activity of CTR1 in vitro, but rather disrupts the interaction with the ethylene receptor ETR1. This mutation also disrupts the dominant negative effect that results from overexpression of the CTR1 amino-terminal domain in transgenic Arabidopsis. These results suggest that CTR1 interacts with ETR1 in vivo, and that this association is required to turn off the ethylene-signaling pathway.

  11. Endocytosis of Receptor Tyrosine Kinases

    PubMed Central

    Goh, Lai Kuan

    2013-01-01

    Endocytosis is the major regulator of signaling from receptor tyrosine kinases (RTKs). The canonical model of RTK endocytosis involves rapid internalization of an RTK activated by ligand binding at the cell surface and subsequent sorting of internalized ligand-RTK complexes to lysosomes for degradation. Activation of the intrinsic tyrosine kinase activity of RTKs results in autophosphorylation, which is mechanistically coupled to the recruitment of adaptor proteins and conjugation of ubiquitin to RTKs. Ubiquitination serves to mediate interactions of RTKs with sorting machineries both at the cell surface and on endosomes. The pathways and kinetics of RTK endocytic trafficking, molecular mechanisms underlying sorting processes, and examples of deviations from the standard trafficking itinerary in the RTK family are discussed in this work. PMID:23637288

  12. An adaptive error-resilient video encoder

    NASA Astrophysics Data System (ADS)

    Cheng, Liang; El Zarki, Magda

    2003-06-01

    When designing an encoder for a real-time video application over a wireless channel, we must take into consideration the unpredictable fluctuation of the quality of the channel and its impact on the transmitted video data. This uncertainty motivates the development of an adaptive video encoding mechanism that can compensate for the infidelity caused either by data loss and/or by the post-processing (error concealment) at the decoder. In this paper, we first explore the major factors that cause quality degradation. We then propose an adaptive progressive replenishment algorithm for a packet loss rate (PLR) feedback enabled system. Assuming the availability of a feedback channel, we discuss a video quality assessment method, which allows the encoder to be aware of the decoder-side perceptual quality. Finally, we present a novel dual-feedback mechanism that guarantees an acceptable level of quality at the receiver side with modest increase in the complexity of the encoder.

  13. Cellobiohydrolase variants and polynucleotides encoding same

    SciTech Connect

    Wogulis, Mark

    2014-10-14

    The present invention relates to variants of a parent cellobiohydrolase II. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

  14. Cellobiohydrolase variants and polynucleotides encoding the same

    SciTech Connect

    Wogulis, Mark

    2014-09-09

    The present invention relates to variants of a parent cellobiohydrolase. The present invention also relates to polynucleotides encoding the cellobiohydrolase variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the cellobiohydrolase variants.

  15. Cellobiohydrolase variants and polynucleotides encoding same

    DOEpatents

    Wogulis, Mark

    2013-09-24

    The present invention relates to variants of a parent cellobiohydrolase II. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

  16. Encoding and reinstatement of threat: recognition potentials.

    PubMed

    Weymar, Mathias; Bradley, Margaret M; Hamm, Alfons O; Lang, Peter J

    2014-01-01

    On a recognition test, stimuli originally encoded in the context of shock threat show an enhanced late parietal positivity during later recognition compared to stimuli encoded during safety, particularly for emotionally arousing stimuli. The present study investigated whether this ERP old/new effect is further influenced when a threat context is reinstated during the recognition test. ERPs were measured in a yes-no recognition test for words rated high or low in emotional arousal that were encoded and recognized in the context of cues that signaled threat of shock or safety. Correct recognition of words encoded under threat, irrespective of reinstatement, was associated with an enhanced old-new ERP difference (500-700ms; centro-parietal), and this difference was only reliable for emotionally arousing words. Taken together, the data suggest that information processed in a stressful context are associated with better recollection on later recognition, an effect that was not modulated by reinstating the stressful context at retrieval.

  17. Oncoprotein protein kinase antibody kit

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2008-12-23

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  18. MAPK feedback encodes a switch and timer for tunable stress adaptation in yeast

    PubMed Central

    English, Justin G.; Shellhammer, James P.; Malahe, Michael; McCarter, Patrick C.; Elston, Timothy C.; Dohlman, Henrik G.

    2015-01-01

    Signaling pathways can behave as switches or rheostats, generating binary or graded responses to a given cell stimulus. We evaluated whether a single signaling pathway can simultaneously encode a switch and a rheostat. We found that the kinase Hog1 mediated a bifurcated cellular response: Activation and commitment to adaptation to osmotic stress are switch-like, whereas protein induction and the resolution of this commitment are graded. Through experimentation, bioinformatics analysis, and computational modeling, we determined that graded recovery is encoded through feedback phosphorylation and a gene induction program that is both temporally staggered and variable across the population. This switch-to-rheostat signaling mechanism represents a versatile stress adaptation system, wherein a broad range of inputs generate an “all-in” response that is later tuned to allow graded recovery of individual cells over time. PMID:25587192

  19. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, David B.; Lao, Guifang

    1998-01-01

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium.

  20. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, D.B.; Lao, G.

    1998-01-06

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium. 3 figs.

  1. Neurally Encoding Time for Olfactory Navigation

    PubMed Central

    Park, In Jun; Hein, Andrew M.; Bobkov, Yuriy V.; Reidenbach, Matthew A.; Ache, Barry W.; Principe, Jose C.

    2016-01-01

    Accurately encoding time is one of the fundamental challenges faced by the nervous system in mediating behavior. We recently reported that some animals have a specialized population of rhythmically active neurons in their olfactory organs with the potential to peripherally encode temporal information about odor encounters. If these neurons do indeed encode the timing of odor arrivals, it should be possible to demonstrate that this capacity has some functional significance. Here we show how this sensory input can profoundly influence an animal’s ability to locate the source of odor cues in realistic turbulent environments—a common task faced by species that rely on olfactory cues for navigation. Using detailed data from a turbulent plume created in the laboratory, we reconstruct the spatiotemporal behavior of a real odor field. We use recurrence theory to show that information about position relative to the source of the odor plume is embedded in the timing between odor pulses. Then, using a parameterized computational model, we show how an animal can use populations of rhythmically active neurons to capture and encode this temporal information in real time, and use it to efficiently navigate to an odor source. Our results demonstrate that the capacity to accurately encode temporal information about sensory cues may be crucial for efficient olfactory navigation. More generally, our results suggest a mechanism for extracting and encoding temporal information from the sensory environment that could have broad utility for neural information processing. PMID:26730727

  2. [The ENCODE project and functional genomics studies].

    PubMed

    Ding, Nan; Qu, Hongzhu; Fang, Xiangdong

    2014-03-01

    Upon the completion of the Human Genome Project, scientists have been trying to interpret the underlying genomic code for human biology. Since 2003, National Human Genome Research Institute (NHGRI) has invested nearly $0.3 billion and gathered over 440 scientists from more than 32 institutions in the United States, China, United Kingdom, Japan, Spain and Singapore to initiate the Encyclopedia of DNA Elements (ENCODE) project, aiming to identify and analyze all regulatory elements in the human genome. Taking advantage of the development of next-generation sequencing technologies and continuous improvement of experimental methods, ENCODE had made remarkable achievements: identified methylation and histone modification of DNA sequences and their regulatory effects on gene expression through altering chromatin structures, categorized binding sites of various transcription factors and constructed their regulatory networks, further revised and updated database for pseudogenes and non-coding RNA, and identified SNPs in regulatory sequences associated with diseases. These findings help to comprehensively understand information embedded in gene and genome sequences, the function of regulatory elements as well as the molecular mechanism underlying the transcriptional regulation by noncoding regions, and provide extensive data resource for life sciences, particularly for translational medicine. We re-viewed the contributions of high-throughput sequencing platform development and bioinformatical technology improve-ment to the ENCODE project, the association between epigenetics studies and the ENCODE project, and the major achievement of the ENCODE project. We also provided our prospective on the role of the ENCODE project in promoting the development of basic and clinical medicine.

  3. Neurally Encoding Time for Olfactory Navigation.

    PubMed

    Park, In Jun; Hein, Andrew M; Bobkov, Yuriy V; Reidenbach, Matthew A; Ache, Barry W; Principe, Jose C

    2016-01-01

    Accurately encoding time is one of the fundamental challenges faced by the nervous system in mediating behavior. We recently reported that some animals have a specialized population of rhythmically active neurons in their olfactory organs with the potential to peripherally encode temporal information about odor encounters. If these neurons do indeed encode the timing of odor arrivals, it should be possible to demonstrate that this capacity has some functional significance. Here we show how this sensory input can profoundly influence an animal's ability to locate the source of odor cues in realistic turbulent environments-a common task faced by species that rely on olfactory cues for navigation. Using detailed data from a turbulent plume created in the laboratory, we reconstruct the spatiotemporal behavior of a real odor field. We use recurrence theory to show that information about position relative to the source of the odor plume is embedded in the timing between odor pulses. Then, using a parameterized computational model, we show how an animal can use populations of rhythmically active neurons to capture and encode this temporal information in real time, and use it to efficiently navigate to an odor source. Our results demonstrate that the capacity to accurately encode temporal information about sensory cues may be crucial for efficient olfactory navigation. More generally, our results suggest a mechanism for extracting and encoding temporal information from the sensory environment that could have broad utility for neural information processing. PMID:26730727

  4. Sequence analysis of the cDNA for the human casein kinase I {delta} (CSNK1D) gene and its chromosomal localization

    SciTech Connect

    Kusuda, Jun; Hidari, Nobuko; Hirai, Momoki; Hashimoto, Katsuyuki

    1996-02-15

    This article reports on the genetic mapping of a cDNA clone encoding human casein kinase I (CK1) using fluorescence in situ hybridization and polymerase chain reaction analysis of human-rodent hybrid cell panels. When compared to the amino acid sequence in the kinase domain of the rat, this cDNA seems to be a human homologue of the CK1 {delta} isoform. Sequence similarity to the kinase domains and function in DNA repair in Saccharomyces cerevisiae and Saccharomyces pombe are discussed. 14 refs., 2 figs.

  5. Cloning of the mitogen-activated S6 kinase from rat liver reveals an enzyme of the second messenger subfamily

    SciTech Connect

    Kozma, S.C.; Ferrari, S. Bassand, P.; Siegmann, M.; Thomas, G. ); Totty, N. )

    1990-10-01

    Recently the authors reported the purification of a mitogen-activated S6 kinase from Swiss mouse 3T3 fibroblasts and rat liver. The rat liver protein was cleaved with cyanogen bromide or trypsin and 17 of the resulting peptides were sequenced. DNA primers were generated from 3 peptides that had homology to sequences of the conserved catalytic domain of protein kinases. These primers were used in the polymerase chain reaction to obtain a 0.4-kilobase DNA fragment. This fragment was either radioactively labeled and hybridized to Northern blots of poly(A){sup {sup plus}} mRNA or used to screen a rat liver cDNA library. Northern blot analysis revealed four transcripts of 2.5, 3.2, 4.0, and 6.0 kilobases, and five S6 kinase clones were obtained by screening the library. Only two of the clones, which were identical, encoded a full-length protein. This protein had a molecular weight of 56,160, which correlated closely to that of the dephosphorylated kinase determined by SDS/PAGE. The catalytic domain of the kinase resembles that of other serine/threonine kinases belonging to the second messenger subfamily of protein kinases.

  6. Differential expression of a novel protein kinase in human B lymphocytes. Preferential localization in the germinal center.

    PubMed

    Katz, P; Whalen, G; Kehrl, J H

    1994-06-17

    B lymphocytes which reside in the germinal center region of lymphoid follicles are functionally and phenotypically distinct from the surrounding mantle zone B cells. We have isolated cDNA clones for several genes that are differentially expressed between these two populations of B lymphocytes. One such gene, BL44, is preferentially expressed in germinal center B cells. The nucleotide sequence of a 2,874-base pair BL44 cDNA was determined and a 2,451-bp open reading frame found that encodes for a 97-kDa serine/threonine protein kinase referred to as GC kinase. It has an NH2-terminal catalytic domain most similar to that of the Drosophila NinaC protein and the yeast STE20 protein. GC kinase mRNA transcripts are not unique to germinal center B cells and are found in several other tissues, including brain, lung, and placenta. The GC kinase protein was immunoprecipitated from transfected COS cells and from the Burkitt cell line RAMOS. GC kinase immunoprecipitated from transfected COS cells phosphorylated the substrates casein and myelin basic protein. In addition, a 97-kDa phosphoprotein likely to be GC kinase itself was detected. GC kinase may participate in an important signal transduction pathway in germinal center B cells.

  7. The protein kinase homologue Ste20p is required to link the yeast pheromone response G-protein beta gamma subunits to downstream signalling components.

    PubMed Central

    Leberer, E; Dignard, D; Harcus, D; Thomas, D Y; Whiteway, M

    1992-01-01

    In the yeast Saccharomyces cerevisiae the G-protein beta gamma subunits have been shown to trigger downstream events of the pheromone response pathway. We have identified a new gene, designated STE20, which encodes a protein kinase homologue with sequence similarity to protein kinase C, which is required to transmit the pheromone signal from G beta gamma to downstream components of the signalling pathway. Overproduction of the kinase suppresses the mating defect of dominant-negative G beta mutations providing genetic evidence for an interaction with G beta, and epistasis experiments show that this kinase functions after or at the same point as G beta gamma, but before any of the other currently identified components of the signalling pathway. This points to a potentially new mechanism of G-protein mediated signal transduction, the activation of a protein kinase through G beta gamma. Images PMID:1464311

  8. Differential regulation of the MAP, SAP and RK/p38 kinases by Pyst1, a novel cytosolic dual-specificity phosphatase.

    PubMed Central

    Groom, L A; Sneddon, A A; Alessi, D R; Dowd, S; Keyse, S M

    1996-01-01

    The Pyst1 and Pyst2 mRNAs encode closely related proteins, which are novel members of a family of dual-specificity MAP kinase phosphatases typified by CL100/MKP-1. Pyst1 is expressed constitutively in human skin fibroblasts and, in contrast to other members of this family of enzymes, its mRNA is not inducible by either stress or mitogens. Furthermore, unlike the nuclear CL100 protein, Pyst1 is localized in the cytoplasm of transfected Cos-1 cells. Like CL100/ MKP-1, Pyst1 dephosphorylates and inactivates MAP kinase in vitro and in vivo. In addition, Pyst1 is able to form a physical complex with endogenous MAP kinase in Cos-1 cells. However, unlike CL100, Pyst1 displays very low activity towards the stress-activated protein kinases (SAPKs) or RK/p38 in vitro, indicating that these kinases are not physiological substrates for Pyst1. This specificity is underlined by the inability of Pyst1 to block either the stress-mediated activation of the JNK-1 SAP kinase or RK/p38 in vivo, or to inhibit nuclear signalling events mediated by the SAP kinases in response to UV radiation. Our results provide the first evidence that the members of the MAP kinase family of enzymes are differentially regulated by dual-specificity phosphatases and also indicate that the MAP kinases may be regulated by different members of this family of enzymes depending on their subcellular location. Images PMID:8670865

  9. Mumps Virus Nucleoprotein Enhances Phosphorylation of the Phosphoprotein by Polo-Like Kinase 1

    PubMed Central

    Pickar, Adrian; Zengel, James; Xu, Pei; Li, Zhuo

    2015-01-01

    ABSTRACT The viral RNA-dependent RNA polymerases (vRdRps) of nonsegmented, negative-sense viruses (NNSVs) consist of the enzymatic large protein (L) and the phosphoprotein (P). P is heavily phosphorylated, and its phosphorylation plays a critical role in viral RNA synthesis. Since NNSVs do not encode kinases, P is phosphorylated by host kinases. In this study, we investigate the roles that viral proteins play in the phosphorylation of mumps virus (MuV) P. We found that nucleoprotein (NP) enhances the phosphorylation of P. We have identified the serine/threonine kinase Polo-like kinase 1 (PLK1) as a host kinase that phosphorylates P and have found that phosphorylation of P by PLK1 is enhanced by NP. The PLK1 binding site in MuV P was mapped to residues 146 to 148 within the S(pS/T)P motif, and the phosphorylation site was identified as residues S292 and S294. IMPORTANCE It has previously been shown that P acts as a chaperone for NP, which encapsidates viral genomic RNA to form the NP-RNA complex, the functional template for viral RNA synthesis. Thus, it is assumed that phosphorylation of P may regulate NP's ability to form the NP-RNA complex, thereby regulating viral RNA synthesis. Our work demonstrates that MuV NP affects phosphorylation of P, suggesting that NP can regulate viral RNA synthesis by regulating phosphorylation of P. PMID:26608325

  10. Evolutionary paths of the cAMP-dependent protein kinase (PKA) catalytic subunits.

    PubMed

    Søberg, Kristoffer; Jahnsen, Tore; Rognes, Torbjørn; Skålhegg, Bjørn S; Laerdahl, Jon K

    2013-01-01

    3',5'-cyclic adenosine monophosphate (cAMP) dependent protein kinase or protein kinase A (PKA) has served as a prototype for the large family of protein kinases that are crucially important for signal transduction in eukaryotic cells. The PKA catalytic subunits Cα and Cβ, encoded by the two genes PRKACA and PRKACB, respectively, are among the best understood and characterized human kinases. Here we have studied the evolution of this gene family in chordates, arthropods, mollusks and other animals employing probabilistic methods and show that Cα and Cβ arose by duplication of an ancestral PKA catalytic subunit in a common ancestor of vertebrates. The two genes have subsequently been duplicated in teleost fishes. The evolution of the PRKACG retroposon in simians was also investigated. Although the degree of sequence conservation in the PKA Cα/Cβ kinase family is exceptionally high, a small set of signature residues defining Cα and Cβ subfamilies were identified. These conserved residues might be important for functions that are unique to the Cα or Cβ clades. This study also provides a good example of a seemingly simple phylogenetic problem which, due to a very high degree of sequence conservation and corresponding weak phylogenetic signals, combined with problematic nonphylogenetic signals, is nontrivial for state-of-the-art probabilistic phylogenetic methods. PMID:23593352

  11. Molecular characterization of WDCP, a novel fusion partner for the anaplastic lymphoma tyrosine kinase ALK

    PubMed Central

    YOKOYAMA, NORIKO; MILLER, W. TODD

    2015-01-01

    Anaplastic lymphoma kinase (ALK) is a member of the receptor tyrosine kinase superfamily. The ALK gene is a site of frequent mutation and chromosomal rearrangement in various types of human cancers. A novel chromosomal translocation was recently identified in human colorectal cancer between the ALK gene and chromosome 2, open reading frame 44 (C2orf44), a gene of unknown function. As a first step in understanding the oncogenic properties of this fusion protein, C2orf44 cDNA was cloned and the encoded protein was characterized, which was designated as WD repeat and coiled coil containing protein (WDCP). A C-terminal proline-rich segment in WDCP was shown to mediate binding to the Src homology 3 domain of the Src family kinase hematopoietic cell kinase (Hck). Co-expression with Hck lead to tyrosine phosphorylation of WDCP. Chromatographic fractionation of WDCP-containing lysates indicates that the protein exists as an oligomer in mammalian cells. These results suggest that, in the context of the ALK-C2orf44 gene fusion, WDCP imposes an oligomeric structure on ALK that results in constitutive kinase activation and signaling. PMID:25469238

  12. Chimeric calcium/calmodulin-dependent protein kinase in tobacco: differential regulation by calmodulin isoforms

    NASA Technical Reports Server (NTRS)

    Liu, Z.; Xia, M.; Poovaiah, B. W.

    1998-01-01

    cDNA clones of chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) from tobacco (TCCaMK-1 and TCCaMK-2) were isolated and characterized. The polypeptides encoded by TCCaMK-1 and TCCaMK-2 have 15 different amino acid substitutions, yet they both contain a total of 517 amino acids. Northern analysis revealed that CCaMK is expressed in a stage-specific manner during anther development. Messenger RNA was detected when tobacco bud sizes were between 0.5 cm and 1.0 cm. The appearance of mRNA coincided with meiosis and became undetectable at later stages of anther development. The reverse polymerase chain reaction (RT-PCR) amplification assay using isoform-specific primers showed that both of the CCaMK mRNAs were expressed in anther with similar expression patterns. The CCaMK protein expressed in Escherichia coli showed Ca2+-dependent autophosphorylation and Ca2+/calmodulin-dependent substrate phosphorylation. Calmodulin isoforms (PCM1 and PCM6) had differential effects on the regulation of autophosphorylation and substrate phosphorylation of tobacco CCaMK, but not lily CCaMK. The evolutionary tree of plant serine/threonine protein kinases revealed that calmodulin-dependent kinases form one subgroup that is distinctly different from Ca2+-dependent protein kinases (CDPKs) and other serine/threonine kinases in plants.

  13. Differentially expressed epigenome modifiers, including Aurora kinase A and B, in immune cells of rheumatoid arthritis

    PubMed Central

    Glant, Tibor T.; Besenyei, Timea; Kádár, András; Kurkó, Júlia; Tryniszewska, Beata; Gál, János; Soós, Györgyi; Szekanecz, Zoltán; Hoffmann, Gyula; Block, Joel A.; Katz, Robert S.; Mikecz, Katalin; Rauch, Tibor A.

    2014-01-01

    Objective The aim of this study was to identify epigenetic factors that are implicated in the pathogenesis of rheumatoid arthritis (RA) and to explore the therapeutic potential of the targeted inhibition of these factors. Methods PCR arrays were utilized to investigate the expression profile of genes that encod key epigenetic regulator enzymes. Mononuclear cells from RA patients and mice were monitored for gene expression changes, in association with arthritis development in murine models of RA. Selected genes were further characterized by quantitative real-time PCR, Western blot and flow cytometry methods. The targeted inhibition of the upregulated enzymes was studied in arthritic mice. Results A set of genes with arthritis-specific expression was identified by the PCR arrays. Aurora kinase A and B, both of which were highly expressed in arthritic mice and treatment naïve RA patients, were selected for detailed analysis. Elevated Aurora kinase expression was accompanied with an increased phosphorylation of histone H3, which promotes proliferation of T lymphocytes. Treatment with VX-680, a pan-Aurora kinase inhibitor, promoted B cell apoptosis, provided significant protection against the onset, and attenuated the inflammatory reactions in arthritic mice. Conclusions Arthritis development is accompanied the changes in the expression of a number of epigenome-modifying enzymes. Drug-induced downregulation of the Aurora kinases, among other targets, seems to be sufficient to treat experimental arthritis. Development of new therapeutics that target the Aurora kinases can potentially improve RA management. PMID:23653330

  14. Mitogen-activated Protein Kinase Kinase Kinase 1 Protects against Nickel-induced Acute Lung Injury

    PubMed Central

    Mongan, Maureen; Tan, Zongqing; Chen, Liang; Peng, Zhimin; Dietsch, Maggie; Su, Bing; Leikauf, George; Xia, Ying

    2008-01-01

    Nickel compounds are environmental and occupational hazards that pose serious health problems and are causative factors of acute lung injury. The c-jun N-terminal kinases (JNKs) are regulated through a mitogen-activated protein (MAP) 3 kinase-MAP2 kinase cascade and have been implicated in nickel toxicity. In this study, we used genetically modified cells and mice to investigate the involvement of two upstream MAP3Ks, MAP3K1 and 2, in nickel-induced JNK activation and acute lung injury. In mouse embryonic fibroblasts, levels of JNK activation and cytotoxicity induced by nickel were similar in the Map3k2-null and wild-type cells but were much lower in the Map3k1/Map3k2 double-null cells. Conversely, the levels of JNK activation and cytotoxicity were unexpectedly much higher in the Map3k1-null cells. In adult mouse tissue, MAP3K1 was widely distributed but was abundantly expressed in the bronchiole epithelium of the lung. Accordingly, MAP3K1 ablation in mice resulted in severe nickel-induced acute lung injury and reduced survival. Based on these findings, we propose a role for MAP3K1 in reducing JNK activation and protecting the mice from nickel-induced acute lung injury. PMID:18467339

  15. An encyclopedia of mouse DNA elements (Mouse ENCODE).

    PubMed

    Stamatoyannopoulos, John A; Snyder, Michael; Hardison, Ross; Ren, Bing; Gingeras, Thomas; Gilbert, David M; Groudine, Mark; Bender, Michael; Kaul, Rajinder; Canfield, Theresa; Giste, Erica; Johnson, Audra; Zhang, Mia; Balasundaram, Gayathri; Byron, Rachel; Roach, Vaughan; Sabo, Peter J; Sandstrom, Richard; Stehling, A Sandra; Thurman, Robert E; Weissman, Sherman M; Cayting, Philip; Hariharan, Manoj; Lian, Jin; Cheng, Yong; Landt, Stephen G; Ma, Zhihai; Wold, Barbara J; Dekker, Job; Crawford, Gregory E; Keller, Cheryl A; Wu, Weisheng; Morrissey, Christopher; Kumar, Swathi A; Mishra, Tejaswini; Jain, Deepti; Byrska-Bishop, Marta; Blankenberg, Daniel; Lajoie, Bryan R; Jain, Gaurav; Sanyal, Amartya; Chen, Kaun-Bei; Denas, Olgert; Taylor, James; Blobel, Gerd A; Weiss, Mitchell J; Pimkin, Max; Deng, Wulan; Marinov, Georgi K; Williams, Brian A; Fisher-Aylor, Katherine I; Desalvo, Gilberto; Kiralusha, Anthony; Trout, Diane; Amrhein, Henry; Mortazavi, Ali; Edsall, Lee; McCleary, David; Kuan, Samantha; Shen, Yin; Yue, Feng; Ye, Zhen; Davis, Carrie A; Zaleski, Chris; Jha, Sonali; Xue, Chenghai; Dobin, Alex; Lin, Wei; Fastuca, Meagan; Wang, Huaien; Guigo, Roderic; Djebali, Sarah; Lagarde, Julien; Ryba, Tyrone; Sasaki, Takayo; Malladi, Venkat S; Cline, Melissa S; Kirkup, Vanessa M; Learned, Katrina; Rosenbloom, Kate R; Kent, W James; Feingold, Elise A; Good, Peter J; Pazin, Michael; Lowdon, Rebecca F; Adams, Leslie B

    2012-08-13

    To complement the human Encyclopedia of DNA Elements (ENCODE) project and to enable a broad range of mouse genomics efforts, the Mouse ENCODE Consortium is applying the same experimental pipelines developed for human ENCODE to annotate the mouse genome.

  16. MET Receptor Tyrosine Kinase as an Autism Genetic Risk Factor

    PubMed Central

    Peng, Yun; Huentelman, Matthew; Smith, Christopher; Qiu, Shenfeng

    2014-01-01

    In this chapter, we will briefly discuss recent literature on the role of MET receptor tyrosine kinase (RTK) in brain development and how perturbation of MET signaling may alter normal neurodevelopmental outcomes. Recent human genetic studies have established MET as a risk factor for autism, and the molecular and cellular underpinnings of this genetic risk are only beginning to emerge from obscurity. Unlike many autism risk genes that encode synaptic proteins, the spatial and temporal expression pattern of MET RTK indicates this signaling system is ideally situated to regulate neuronal growth, functional maturation, and establishment of functional brain circuits, particularly in those brain structures involved in higher levels of cognition, social skills, and executive functions. PMID:24290385

  17. Oscillatory Dynamics of the Extracellular Signal-regulated Kinase Pathway

    SciTech Connect

    Shankaran, Harish; Wiley, H. S.

    2010-12-01

    The extracellular signal-regulated kinase (ERK) pathway is a central signaling pathway in development and disease and is regulated by multiple negative and positive feedback loops. Recent studies have shown negative feedback from ERK to upstream regulators can give rise to biochemical oscillations with a periodicity of between 15-30 minutes. Feedback due to the stimulated transcription of negative regulators of the ERK pathway can also give rise to transcriptional oscillations with a periodicity of 1-2h. The biological significance of these oscillations is not clear, but recent evidence suggests that transcriptional oscillations participate in developmental processes, such as somite formation. Biochemical oscillations are more enigmatic, but could provide a mechanism for encoding different types of inputs into a common signaling pathway.

  18. Multichannel compressive sensing MRI using noiselet encoding.

    PubMed

    Pawar, Kamlesh; Egan, Gary; Zhang, Jingxin

    2015-01-01

    The incoherence between measurement and sparsifying transform matrices and the restricted isometry property (RIP) of measurement matrix are two of the key factors in determining the performance of compressive sensing (CS). In CS-MRI, the randomly under-sampled Fourier matrix is used as the measurement matrix and the wavelet transform is usually used as sparsifying transform matrix. However, the incoherence between the randomly under-sampled Fourier matrix and the wavelet matrix is not optimal, which can deteriorate the performance of CS-MRI. Using the mathematical result that noiselets are maximally incoherent with wavelets, this paper introduces the noiselet unitary bases as the measurement matrix to improve the incoherence and RIP in CS-MRI. Based on an empirical RIP analysis that compares the multichannel noiselet and multichannel Fourier measurement matrices in CS-MRI, we propose a multichannel compressive sensing (MCS) framework to take the advantage of multichannel data acquisition used in MRI scanners. Simulations are presented in the MCS framework to compare the performance of noiselet encoding reconstructions and Fourier encoding reconstructions at different acceleration factors. The comparisons indicate that multichannel noiselet measurement matrix has better RIP than that of its Fourier counterpart, and that noiselet encoded MCS-MRI outperforms Fourier encoded MCS-MRI in preserving image resolution and can achieve higher acceleration factors. To demonstrate the feasibility of the proposed noiselet encoding scheme, a pulse sequences with tailored spatially selective RF excitation pulses was designed and implemented on a 3T scanner to acquire the data in the noiselet domain from a phantom and a human brain. The results indicate that noislet encoding preserves image resolution better than Fouirer encoding. PMID:25965548

  19. Multichannel compressive sensing MRI using noiselet encoding.

    PubMed

    Pawar, Kamlesh; Egan, Gary; Zhang, Jingxin

    2015-01-01

    The incoherence between measurement and sparsifying transform matrices and the restricted isometry property (RIP) of measurement matrix are two of the key factors in determining the performance of compressive sensing (CS). In CS-MRI, the randomly under-sampled Fourier matrix is used as the measurement matrix and the wavelet transform is usually used as sparsifying transform matrix. However, the incoherence between the randomly under-sampled Fourier matrix and the wavelet matrix is not optimal, which can deteriorate the performance of CS-MRI. Using the mathematical result that noiselets are maximally incoherent with wavelets, this paper introduces the noiselet unitary bases as the measurement matrix to improve the incoherence and RIP in CS-MRI. Based on an empirical RIP analysis that compares the multichannel noiselet and multichannel Fourier measurement matrices in CS-MRI, we propose a multichannel compressive sensing (MCS) framework to take the advantage of multichannel data acquisition used in MRI scanners. Simulations are presented in the MCS framework to compare the performance of noiselet encoding reconstructions and Fourier encoding reconstructions at different acceleration factors. The comparisons indicate that multichannel noiselet measurement matrix has better RIP than that of its Fourier counterpart, and that noiselet encoded MCS-MRI outperforms Fourier encoded MCS-MRI in preserving image resolution and can achieve higher acceleration factors. To demonstrate the feasibility of the proposed noiselet encoding scheme, a pulse sequences with tailored spatially selective RF excitation pulses was designed and implemented on a 3T scanner to acquire the data in the noiselet domain from a phantom and a human brain. The results indicate that noislet encoding preserves image resolution better than Fouirer encoding.

  20. Multichannel Compressive Sensing MRI Using Noiselet Encoding

    PubMed Central

    Pawar, Kamlesh; Egan, Gary; Zhang, Jingxin

    2015-01-01

    The incoherence between measurement and sparsifying transform matrices and the restricted isometry property (RIP) of measurement matrix are two of the key factors in determining the performance of compressive sensing (CS). In CS-MRI, the randomly under-sampled Fourier matrix is used as the measurement matrix and the wavelet transform is usually used as sparsifying transform matrix. However, the incoherence between the randomly under-sampled Fourier matrix and the wavelet matrix is not optimal, which can deteriorate the performance of CS-MRI. Using the mathematical result that noiselets are maximally incoherent with wavelets, this paper introduces the noiselet unitary bases as the measurement matrix to improve the incoherence and RIP in CS-MRI. Based on an empirical RIP analysis that compares the multichannel noiselet and multichannel Fourier measurement matrices in CS-MRI, we propose a multichannel compressive sensing (MCS) framework to take the advantage of multichannel data acquisition used in MRI scanners. Simulations are presented in the MCS framework to compare the performance of noiselet encoding reconstructions and Fourier encoding reconstructions at different acceleration factors. The comparisons indicate that multichannel noiselet measurement matrix has better RIP than that of its Fourier counterpart, and that noiselet encoded MCS-MRI outperforms Fourier encoded MCS-MRI in preserving image resolution and can achieve higher acceleration factors. To demonstrate the feasibility of the proposed noiselet encoding scheme, a pulse sequences with tailored spatially selective RF excitation pulses was designed and implemented on a 3T scanner to acquire the data in the noiselet domain from a phantom and a human brain. The results indicate that noislet encoding preserves image resolution better than Fouirer encoding. PMID:25965548

  1. tek, a novel tyrosine kinase gene located on mouse chromosome 4, is expressed in endothelial cells and their presumptive precursors.

    PubMed

    Dumont, D J; Yamaguchi, T P; Conlon, R A; Rossant, J; Breitman, M L

    1992-08-01

    A search for protein tyrosine kinases expressed during murine cardiogenesis resulted in the isolation of a novel tyrosine kinase, designated tek, which maps to mouse chromosome 4 between the brown and pmv-23 loci. The deduced amino acid sequence of tek predicts that it encodes a putative receptor tyrosine kinase that contains a 21 amino acid kinase insert and which is most closely related in its catalytic domains to FGFR1 and the product of the ret proto-oncogene. In situ hybridization analysis of adult tissues, as well as sectioned and whole-mount embryos, showed that tek is specifically expressed in the endocardium, the leptomeninges and the endothelial lining of the vasculature from the earliest stages of their development. Moreover, examination of the morphology of tek-expressing cells, and staging of tek expression relative to that of the endothelial cell marker von Willebrand factor, revealed that tek is expressed prior to von Willebrand factor and appears to mark the embryonic progenitors of mature endothelial cells. tek encodes a novel putative receptor tyrosine kinase that may be critically involved in the determination and/or maintenance of cells of the endothelial lineage.

  2. The Ste20-like kinase SvkA of Dictyostelium discoideum is essential for late stages of cytokinesis.

    PubMed

    Rohlfs, Meino; Arasada, Rajesh; Batsios, Petros; Janzen, Julia; Schleicher, Michael

    2007-12-15

    The genome of the social amoeba Dictyostelium discoideum encodes approximately 285 kinases, which represents approximately 2.6% of the total genome and suggests a signaling complexity similar to that of yeasts and humans. The behavior of D. discoideum as an amoeba and during development relies heavily on fast rearrangements of the actin cytoskeleton. Here, we describe the knockout phenotype of the svkA gene encoding severin kinase, a homolog of the human MST3, MST4 and YSK1 kinases. SvkA-knockout cells show drastic defects in cytokinesis, development and directed slug movement. The defect in cytokinesis is most prominent, leading to multinucleated cells sometimes with >30 nuclei. The defect arises from the frequent inability of svkA-knockout cells to maintain symmetry during formation of the cleavage furrow and to sever the last cytosolic connection. We demonstrate that GFP-SvkA is enriched at the centrosome and localizes to the midzone during the final stage of cell division. This distribution is mediated by the C-terminal half of the kinase, whereas a rescue of the phenotypic changes requires the active N-terminal kinase domain as well. The data suggest that SvkA is part of a regulatory pathway from the centrosome to the midzone, thus regulating the completion of cell division. PMID:18042625

  3. The Ste20-like kinase SvkA of Dictyostelium discoideum is essential for late stages of cytokinesis.

    PubMed

    Rohlfs, Meino; Arasada, Rajesh; Batsios, Petros; Janzen, Julia; Schleicher, Michael

    2007-12-15

    The genome of the social amoeba Dictyostelium discoideum encodes approximately 285 kinases, which represents approximately 2.6% of the total genome and suggests a signaling complexity similar to that of yeasts and humans. The behavior of D. discoideum as an amoeba and during development relies heavily on fast rearrangements of the actin cytoskeleton. Here, we describe the knockout phenotype of the svkA gene encoding severin kinase, a homolog of the human MST3, MST4 and YSK1 kinases. SvkA-knockout cells show drastic defects in cytokinesis, development and directed slug movement. The defect in cytokinesis is most prominent, leading to multinucleated cells sometimes with >30 nuclei. The defect arises from the frequent inability of svkA-knockout cells to maintain symmetry during formation of the cleavage furrow and to sever the last cytosolic connection. We demonstrate that GFP-SvkA is enriched at the centrosome and localizes to the midzone during the final stage of cell division. This distribution is mediated by the C-terminal half of the kinase, whereas a rescue of the phenotypic changes requires the active N-terminal kinase domain as well. The data suggest that SvkA is part of a regulatory pathway from the centrosome to the midzone, thus regulating the completion of cell division.

  4. Large-Scale Analysis of Kinase Signaling in Yeast Pseudohyphal Development Identifies Regulation of Ribonucleoprotein Granules.

    PubMed

    Shively, Christian A; Kweon, Hye Kyong; Norman, Kaitlyn L; Mellacheruvu, Dattatreya; Xu, Tao; Sheidy, Daniel T; Dobry, Craig J; Sabath, Ivan; Cosky, Eric E P; Tran, Elizabeth J; Nesvizhskii, Alexey; Andrews, Philip C; Kumar, Anuj

    2015-10-01

    Yeast pseudohyphal filamentation is a stress-responsive growth transition relevant to processes required for virulence in pathogenic fungi. Pseudohyphal growth is controlled through a regulatory network encompassing conserved MAPK (Ste20p, Ste11p, Ste7p, Kss1p, and Fus3p), protein kinase A (Tpk2p), Elm1p, and Snf1p kinase pathways; however, the scope of these pathways is not fully understood. Here, we implemented quantitative phosphoproteomics to identify each of these signaling networks, generating a kinase-dead mutant in filamentous S. cerevisiae and surveying for differential phosphorylation. By this approach, we identified 439 phosphoproteins dependent upon pseudohyphal growth kinases. We report novel phosphorylation sites in 543 peptides, including phosphorylated residues in Ras2p and Flo8p required for wild-type filamentous growth. Phosphoproteins in these kinase signaling networks were enriched for ribonucleoprotein (RNP) granule components, and we observe co-localization of Kss1p, Fus3p, Ste20p, and Tpk2p with the RNP component Igo1p. These kinases localize in puncta with GFP-visualized mRNA, and KSS1 is required for wild-type levels of mRNA localization in RNPs. Kss1p pathway activity is reduced in lsm1Δ/Δ and pat1Δ/Δ strains, and these genes encoding P-body proteins are epistatic to STE7. The P-body protein Dhh1p is also required for hyphal development in Candida albicans. Collectively, this study presents a wealth of data identifying the yeast phosphoproteome in pseudohyphal growth and regulatory interrelationships between pseudohyphal growth kinases and RNPs.

  5. Large-Scale Analysis of Kinase Signaling in Yeast Pseudohyphal Development Identifies Regulation of Ribonucleoprotein Granules

    PubMed Central

    Mellacheruvu, Dattatreya; Xu, Tao; Sheidy, Daniel T.; Dobry, Craig J.; Sabath, Ivan; Cosky, Eric E. P.; Tran, Elizabeth J.; Nesvizhskii, Alexey; Andrews, Philip C.; Kumar, Anuj

    2015-01-01

    Yeast pseudohyphal filamentation is a stress-responsive growth transition relevant to processes required for virulence in pathogenic fungi. Pseudohyphal growth is controlled through a regulatory network encompassing conserved MAPK (Ste20p, Ste11p, Ste7p, Kss1p, and Fus3p), protein kinase A (Tpk2p), Elm1p, and Snf1p kinase pathways; however, the scope of these pathways is not fully understood. Here, we implemented quantitative phosphoproteomics to identify each of these signaling networks, generating a kinase-dead mutant in filamentous S. cerevisiae and surveying for differential phosphorylation. By this approach, we identified 439 phosphoproteins dependent upon pseudohyphal growth kinases. We report novel phosphorylation sites in 543 peptides, including phosphorylated residues in Ras2p and Flo8p required for wild-type filamentous growth. Phosphoproteins in these kinase signaling networks were enriched for ribonucleoprotein (RNP) granule components, and we observe co-localization of Kss1p, Fus3p, Ste20p, and Tpk2p with the RNP component Igo1p. These kinases localize in puncta with GFP-visualized mRNA, and KSS1 is required for wild-type levels of mRNA localization in RNPs. Kss1p pathway activity is reduced in lsm1Δ/Δ and pat1Δ/Δ strains, and these genes encoding P-body proteins are epistatic to STE7. The P-body protein Dhh1p is also required for hyphal development in Candida albicans. Collectively, this study presents a wealth of data identifying the yeast phosphoproteome in pseudohyphal growth and regulatory interrelationships between pseudohyphal growth kinases and RNPs. PMID:26447709

  6. Phosphorylation of FADD by the kinase CK1α promotes KRASG12D-induced lung cancer

    PubMed Central

    Bowman, Brittany M.; Sebolt, Katrina A.; Hoff, Benjamin A.; Boes, Jennifer L.; Daniels, Danette L.; Heist, Kevin A.; Galbán, Craig J.; Patel, Rajiv M.; Zhang, Jianke; Beer, David G.; Ross, Brian D.; Rehemtulla, Alnawaz; Galbán, Stefanie

    2015-01-01

    Genomic amplification of the gene encoding and phosphorylation of the protein FADD (Fas-associated death domain) is associated with poor clinical outcome in lung cancer and in head and neck cancer. Activating mutations in the guanosine triphosphatase RAS promotes cell proliferation in various cancers. We found that the abundance of phosphorylated FADD correlated with that of mutant KRAS in patient lung cancer tissues. Using immunohistochemistry analysis and in vivo imaging of conditional mouse models of KRASG12D-driven lung cancer, we found that the deletion of the gene encoding FADD suppressed tumor growth, reduced the proliferative index of cells, and decreased the activation of downstream effectors of the RAS–MAPK (mitogen-activated protein kinase) pathway that promote the cell cycle, including retinoblastoma (RB) and cyclin D1. In mouse embryonic fibroblasts, the induction of mitosis upon activation of KRAS required FADD and the phosphorylation of FADD by CK1α (casein kinase 1α). Deleting the gene encoding CK1α in KRAS-mutant mice abrogated the phosphorylation of FADD and suppressed lung cancer development. Phosphorylated FADD was most abundant during the G2/M phase of the cell cycle, and mass spectrometry revealed that phosphorylated FADD interacted with kinases that mediate the G2/M transition, including PLK1 (Polo-like kinase 1), AURKA (Aurora kinase A) and BUB1 (budding uninhibited by benzimidazoles 1). This interaction was decreased in cells treated with a CKI-7, a CK1α inhibitor. Therefore, as the kinase that phosphorylates FADD downstream of RAS, CK1α may be a therapeutic target for KRAS-driven lung cancer. PMID:25628462

  7. Multi-dimensionally encoded magnetic resonance imaging

    PubMed Central

    Lin, Fa-Hsuan

    2013-01-01

    Magnetic resonance imaging typically achieves spatial encoding by measuring the projection of a q-dimensional object over q-dimensional spatial bases created by linear spatial encoding magnetic fields (SEMs). Recently, imaging strategies using nonlinear SEMs have demonstrated potential advantages for reconstructing images with higher spatiotemporal resolution and reducing peripheral nerve stimulation. In practice, nonlinear SEMs and linear SEMs can be used jointly to further improve the image reconstruction performance. Here we propose the multi-dimensionally encoded (MDE) MRI to map a q-dimensional object onto a p-dimensional encoding space where p > q. MDE MRI is a theoretical framework linking imaging strategies using linear and nonlinear SEMs. Using a system of eight surface SEM coils with an eight-channel RF coil array, we demonstrate the five-dimensional MDE MRI for a two-dimensional object as a further generalization of PatLoc imaging and O-space imaging. We also present a method of optimizing spatial bases in MDE MRI. Results show that MDE MRI with a higher dimensional encoding space can reconstruct images more efficiently and with a smaller reconstruction error when the k-space sampling distribution and the number of samples are controlled. PMID:22926830

  8. Cloud-based uniform ChIP-Seq processing tools for modENCODE and ENCODE

    PubMed Central

    2013-01-01

    Background Funded by the National Institutes of Health (NIH), the aim of the Model Organism ENCyclopedia of DNA Elements (modENCODE) project is to provide the biological research community with a comprehensive encyclopedia of functional genomic elements for both model organisms C. elegans (worm) and D. melanogaster (fly). With a total size of just under 10 terabytes of data collected and released to the public, one of the challenges faced by researchers is to extract biologically meaningful knowledge from this large data set. While the basic quality control, pre-processing, and analysis of the data has already been performed by members of the modENCODE consortium, many researchers will wish to reinterpret the data set using modifications and enhancements of the original protocols, or combine modENCODE data with other data sets. Unfortunately this can be a time consuming and logistically challenging proposition. Results In recognition of this challenge, the modENCODE DCC has released uniform computing resources for analyzing modENCODE data on Galaxy (https://github.com/modENCODE-DCC/Galaxy), on the public Amazon Cloud (http://aws.amazon.com), and on the private Bionimbus Cloud for genomic research (http://www.bionimbus.org). In particular, we have released Galaxy workflows for interpreting ChIP-seq data which use the same quality control (QC) and peak calling standards adopted by the modENCODE and ENCODE communities. For convenience of use, we have created Amazon and Bionimbus Cloud machine images containing Galaxy along with all the modENCODE data, software and other dependencies. Conclusions Using these resources provides a framework for running consistent and reproducible analyses on modENCODE data, ultimately allowing researchers to use more of their time using modENCODE data, and less time moving it around. PMID:23875683

  9. Protein Kinase D family kinases: roads start to segregate.

    PubMed

    Wille, Christoph; Seufferlein, Thomas; Eiseler, Tim

    2014-01-01

    Highly invasive pancreatic tumors are often recognized in late stages due to a lack of clear symptoms and pose major challenges for treatment and disease management. Broad-band Protein Kinase D (PKD) inhibitors have recently been proposed as additional treatment option for this disease. PKDs are implicated in the control of cancer cell motility, angiogenesis, proliferation and metastasis. In particular, PKD2 expression is elevated in pancreatic cancer, whereas PKD1 expression is comparably lower. In our recent study we report that both kinases control PDAC cell invasive properties in an isoform-specific, but opposing manner. PKD1 selectively mediates anti-migratory/anti-invasive features by preferential regulation of the actin-regulatory Cofilin-phosphatase Slingshot1L (SSH1L). PKD2, on the other hand enhances invasion and angiogenesis of PDAC cells in 3D-ECM cultures and chorioallantois tumor models by stimulating expression and secretion of matrix-metalloproteinase 7 and 9 (MMP7/9). MMP9 also enhances PKD2-mediated tumor angiogenesis releasing extracellular matrix-bound VEGF-A. We thus suggest high PKD2 expression and loss of PKD1 may be beneficial for tumor cells to enhance their matrix-invading abilities. In our recent study we demonstrate for the first time PKD1 and 2 isoform-selective effects on pancreatic cancer cell invasion, in-vitro and in-vivo, defining isoform-specific regulation of PKDs as a major future issue. PMID:24847910

  10. Structure and developmental expression of the chicken CDC2 kinase.

    PubMed Central

    Krek, W; Nigg, E A

    1989-01-01

    The cdc2 protein kinase plays a key role in controlling the eukaryotic cell cycle. We have isolated a cDNA clone for the chicken homolog of the cdc2 gene, raised antibodies against the corresponding protein, and studied the expression of cdc2 mRNA and protein during chicken embryonic development. The protein encoded by the chicken cdc2 cDNA shares extensive structural homology with cdc2 gene products from other species. Moreover, when expressed in fission yeast, the chicken cdc2 kinase is able to rescue a temperature-sensitive (ts) cdc2 mutant, demonstrating that it is functional as a cell cycle regulator. By Northern analysis and immunoblotting, we found that in total embryos both cdc2 mRNA and protein levels decreased substantially between day 3 and day 11 after egg laying, and no significant amounts of either cdc2 mRNA or protein were detected in adult liver, brain, heart or skeletal muscle. These data indicate the existence of a coarse correlation between the abundance of cdc2 mRNA and the proliferative state of a given tissue. Interestingly, however, when examining individual embryonic tissues, no correlation was observed between levels of cdc2 mRNA and protein, suggesting that cdc2 expression in developing chicken may be regulated at multiple levels. Images PMID:2684635

  11. LAMMER kinase contributes to genome stability in Ustilago maydis.

    PubMed

    de Sena-Tomás, Carmen; Sutherland, Jeanette H; Milisavljevic, Mira; Nikolic, Dragana B; Pérez-Martín, José; Kojic, Milorad; Holloman, William K

    2015-09-01

    Here we report identification of the lkh1 gene encoding a LAMMER kinase homolog (Lkh1) from a screen for DNA repair-deficient mutants in Ustilago maydis. The mutant allele isolated results from a mutation at glutamine codon 488 to a stop codon that would be predicted to lead to truncation of the carboxy-terminal kinase domain of the protein. This mutant (lkh1(Q488*)) is highly sensitive to ultraviolet light, methyl methanesulfonate, and hydroxyurea. In contrast, a null mutant (lkh1Δ) deleted of the entire lkh1 gene has a less severe phenotype. No epistasis was observed when an lkh1(Q488*)rad51Δ double mutant was tested for genotoxin sensitivity. However, overexpressing the gene for Rad51, its regulator Brh2, or the Brh2 regulator Dss1 partially restored genotoxin resistance of the lkh1Δ and lkh1(Q488*) mutants. Deletion of lkh1 in a chk1Δ mutant enabled these double mutant cells to continue to cycle when challenged with hydroxyurea. lkh1Δ and lkh1(Q488*) mutants were able to complete the meiotic process but exhibited reduced heteroallelic recombination and aberrant chromosome segregation. The observations suggest that Lkh1 serves in some aspect of cell cycle regulation after DNA damage or replication stress and that it also contributes to proper chromosome segregation in meiosis.

  12. Identification of pantoate kinase and phosphopantothenate synthetase from Methanospirillum hungatei.

    PubMed

    Katoh, Hiroki; Tamaki, Hideyuki; Tokutake, Yuka; Hanada, Satoshi; Chohnan, Shigeru

    2013-04-01

    Pantothenate synthetase (PanC) and pantothenate kinase which function in the canonical coenzyme A (CoA) biosynthetic pathway cannot be found in most archaea. COG1829 and COG1701 intrinsic to archaea were proposed as the candidate proteins for producing 4'-phosphopantothenate instead, and the COG1701 protein from Methanosarcina mazei was assigned as PanC. Meanwhile, the Thermococcus kodakarensis COG1829 and COG1701 proteins were biochemically identified as novel enzymes, i.e., pantoate kinase (PoK) and phosphopantothenate synthetase (PPS). In this study, the functions of Mhun_0831 (COG1829) and Mhun_0832 (COG1701) from Methanospirillum hungatei were identified, and the recombinant enzymes were partially characterized. Plasmids simultaneously possessing the two genes encoding Mhun_0831 and Mhun_0832 complemented the poor growth of the temperature-sensitive Escherichia coli pantothenate kinase mutant ts9. The recombinant Mhun_0831 and Mhun_0832 expressed in E. coli cells exhibited PoK and PPS activities, respectively, being in accord with the functions of T. kodakarensis proteins. The PoK activity was most active at pH 8.5 and 40°C, and accepted ATP and UTP as a phosphate donor. Although CoA did not affect the PoK activity, the end product considerably accelerated the PPS activity. The homologs of both proteins are widely conserved in most archaeal genomes. Taken together, our findings indicate that archaea can synthesize CoA through the unique pathway involving PoK and PPS, in addition to the canonical one that the order Thermoplasmatales employs.

  13. Genome-wide review of transcriptional complexity in mouse protein kinases and phosphatases

    PubMed Central

    Forrest, Alistair RR; Taylor, Darrin F; Crowe, Mark L; Chalk, Alistair M; Waddell, Nic J; Kolle, Gabriel; Faulkner, Geoffrey J; Kodzius, Rimantas; Katayama, Shintaro; Wells, Christine; Kai, Chikatoshi; Kawai, Jun; Carninci, Piero; Hayashizaki, Yoshihide; Grimmond, Sean M

    2006-01-01

    Background Alternative transcripts of protein kinases and protein phosphatases are known to encode peptides with altered substrate affinities, subcellular localizations, and activities. We undertook a systematic study to catalog the variant transcripts of every protein kinase-like and phosphatase-like locus of mouse . Results By reviewing all available transcript evidence, we found that at least 75% of kinase and phosphatase loci in mouse generate alternative splice forms, and that 44% of these loci have well supported alternative 5' exons. In a further analysis of full-length cDNAs, we identified 69% of loci as generating more than one peptide isoform. The 1,469 peptide isoforms generated from these loci correspond to 1,080 unique Interpro domain combinations, many of which lack catalytic or interaction domains. We also report on the existence of likely dominant negative forms for many of the receptor kinases and phosphatases, including some 26 secreted decoys (seven known and 19 novel: Alk, Csf1r, Egfr, Epha1, 3, 5,7 and 10, Ephb1, Flt1, Flt3, Insr, Insrr, Kdr, Met, Ptk7, Ptprc, Ptprd, Ptprg, Ptprl, Ptprn, Ptprn2, Ptpro, Ptprr, Ptprs, and Ptprz1) and 13 transmembrane forms (four known and nine novel: Axl, Bmpr1a, Csf1r, Epha4, 5, 6 and 7, Ntrk2, Ntrk3, Pdgfra, Ptprk, Ptprm, Ptpru). Finally, by mining public gene expression data (MPSS and microarrays), we confirmed tissue-specific expression of ten of the novel isoforms. Conclusion These findings suggest that alternative transcripts of protein kinases and phosphatases are produced that encode different domain structures, and that these variants are likely to play important roles in phosphorylation-dependent signaling pathways. PMID:16507138

  14. Tec family kinases in inflammation and disease.

    PubMed

    Horwood, Nicole J; Urbaniak, Ania M; Danks, Lynett

    2012-04-01

    Over the last decade, the Tec family of nonreceptor tyrosine kinases (Btk, Tec, Bmx, Itk, and Rlk) have been shown to play a key role in inflammation and bone destruction. Bruton's tyrosine kinase (Btk) has been the most widely studied due to the critical role of this kinase in B-cell development and recent evidence showing that blocking Btk signaling is effective in ameliorating lymphoma progression and experimental arthritis. This review will examine the role of TFK in myeloid cell function and the potential of targeting these kinases as a therapeutic intervention in autoimmune disorders such as rheumatoid arthritis. PMID:22449071

  15. Protein kinase profiling assays: a technology review.

    PubMed

    Wang, Yuren; Ma, Haiching

    2015-11-01

    Protein kinases have become one of the most intensively pursued classes of drug targets for many diseases such as cancers and inflammatory diseases. Kinase profiling work seeks to understand general selectivity trends of lead compounds across the kinome, which help with target selection, compound prioritization, and potential implications in toxicity. Under the current drug discovery process, screening of compounds against comprehensive panels of kinases and their mutants has become the standard approach. Many screening assays and technologies which are compatible for high-throughput screening (HTS) against kinases have been extensively pursued and developed.

  16. Regulation of maltose utilization in Saccharomyces cerevisiae by genes of the RAS/protein kinase A pathway.

    PubMed

    Wanke, V; Vavassori, M; Thevelein, J M; Tortora, P; Vanoni, M

    1997-02-01

    In Saccharomyces cerevisiae maltose utilization requires a functional MAL locus, each composed of three genes: MALR (gene 3) encoding a regulatory protein, MALT (gene 1) encoding maltose permease and MALS (gene 2) encoding maltase. We show that constitutive activation of the RAS/protein kinase A pathway severely reduces growth of MAL1 strains on maltose. This may be a consequence of reduction in MALT mRNA, reduced Vmax and increased catabolite inactivation of the MALT-encoded maltose transporter in the MAL1 strain. Mutations in the GGS1/TPS1 gene, which restricts glucose influx and possibly affects signalling, relieve carbon catabolite repression on both maltase and maltose permease and reduce maltose permease inactivation.

  17. An information theoretic characterisation of auditory encoding.

    PubMed

    Overath, Tobias; Cusack, Rhodri; Kumar, Sukhbinder; von Kriegstein, Katharina; Warren, Jason D; Grube, Manon; Carlyon, Robert P; Griffiths, Timothy D

    2007-10-23

    The entropy metric derived from information theory provides a means to quantify the amount of information transmitted in acoustic streams like speech or music. By systematically varying the entropy of pitch sequences, we sought brain areas where neural activity and energetic demands increase as a function of entropy. Such a relationship is predicted to occur in an efficient encoding mechanism that uses less computational resource when less information is present in the signal: we specifically tested the hypothesis that such a relationship is present in the planum temporale (PT). In two convergent functional MRI studies, we demonstrated this relationship in PT for encoding, while furthermore showing that a distributed fronto-parietal network for retrieval of acoustic information is independent of entropy. The results establish PT as an efficient neural engine that demands less computational resource to encode redundant signals than those with high information content. PMID:17958472

  18. Feature encoding for color image segmentation

    NASA Astrophysics Data System (ADS)

    Li, Ning; Li, Youfu

    2001-09-01

    An approach for color image segmentation is proposed based on the contributions of color features to segmentation rather than the choice of a particular color space. It is different from the pervious methods where SOFM is used for construct the feature encoding so that the feature-encoding can self-organize the effective features for different color images. Fuzzy clustering is applied for the final segmentation when the well-suited color features and the initial parameter are available. The proposed method has been applied in segmenting different types of color images and the experimental results show that it outperforms the classical clustering method. Our study shows that the feature encoding approach offers great promise in automating and optimizing color image segmentation.

  19. Adaptive delta modulation systems for video encoding

    NASA Technical Reports Server (NTRS)

    Lei, T.-L. R.; Scheinberg, N.; Schilling, D. L.

    1977-01-01

    This paper describes several adaptive delta modulators designed to encode video signals. One- and two-dimensional ADM algorithms are discussed and compared. Results are shown for bit rates of 2 bits/pixel, 1 bit/pixel and 0.5 bits/pixel. Pictures showing the difference between the encoded-decoded pictures and the original pictures are presented. Results are also presented to illustrate the effect of channel errors on the reconstructed picture. A two-dimensional ADM using interframe encoding is also presented. This system operates at the rate of 2 bits/pixel and produces excellent quality pictures when there is little motion. We also describe and illustrate the effect of large amounts of motion on the reconstructed picture.

  20. Structure and strategy in encoding simplified graphs

    NASA Technical Reports Server (NTRS)

    Schiano, Diane J.; Tversky, Barbara

    1992-01-01

    Tversky and Schiano (1989) found a systematic bias toward the 45-deg line in memory for the slopes of identical lines when embedded in graphs, but not in maps, suggesting the use of a cognitive reference frame specifically for encoding meaningful graphs. The present experiments explore this issue further using the linear configurations alone as stimuli. Experiments 1 and 2 demonstrate that perception and immediate memory for the slope of a test line within orthogonal 'axes' are predictable from purely structural considerations. In Experiments 3 and 4, subjects were instructed to use a diagonal-reference strategy in viewing the stimuli, which were described as 'graphs' only in Experiment 3. Results for both studies showed the diagonal bias previously found only for graphs. This pattern provides converging evidence for the diagonal as a cognitive reference frame in encoding linear graphs, and demonstrates that even in highly simplified displays, strategic factors can produce encoding biases not predictable solely from stimulus structure alone.

  1. Interoperability in encoded quantum repeater networks

    NASA Astrophysics Data System (ADS)

    Nagayama, Shota; Choi, Byung-Soo; Devitt, Simon; Suzuki, Shigeya; Van Meter, Rodney

    2016-04-01

    The future of quantum repeater networking will require interoperability between various error-correcting codes. A few specific code conversions and even a generalized method are known, however, no detailed analysis of these techniques in the context of quantum networking has been performed. In this paper we analyze a generalized procedure to create Bell pairs encoded heterogeneously between two separate codes used often in error-corrected quantum repeater network designs. We begin with a physical Bell pair and then encode each qubit in a different error-correcting code, using entanglement purification to increase the fidelity. We investigate three separate protocols for preparing the purified encoded Bell pair. We calculate the error probability of those schemes between the Steane [[7,1,3

  2. Quantum repeater with continuous variable encoding

    NASA Astrophysics Data System (ADS)

    Li, Linshu; Albert, Victor V.; Michael, Marios; Muralidharan, Sreraman; Zou, Changling; Jiang, Liang

    2016-05-01

    Quantum communication enables faithful quantum state transfer between different parties and protocols for cryptographic purposes. However, quantum communication over long distances (>1000km) remains challenging due to optical channel attenuation. This calls for investigation on developing novel encoding schemes that correct photon loss errors efficiently. In this talk, we introduce the generalization of multi-component Schrödinger cat states and propose to encode quantum information in these cat states for ultrafast quantum repeaters. We detail the quantum error correction procedures at each repeater station and characterize the performance of this novel encoding scheme given practical imperfections, such as coupling loss. A comparison with other quantum error correcting codes for bosonic modes will be discussed.

  3. Parallel encoding of sensory history and behavioral preference during Caenorhabditis elegans olfactory learning.

    PubMed

    Cho, Christine E; Brueggemann, Chantal; L'Etoile, Noelle D; Bargmann, Cornelia I

    2016-01-01

    Sensory experience modifies behavior through both associative and non-associative learning. In Caenorhabditis elegans, pairing odor with food deprivation results in aversive olfactory learning, and pairing odor with food results in appetitive learning. Aversive learning requires nuclear translocation of the cGMP-dependent protein kinase EGL-4 in AWC olfactory neurons and an insulin signal from AIA interneurons. Here we show that the activity of neurons including AIA is acutely required during aversive, but not appetitive, learning. The AIA circuit and AGE-1, an insulin-regulated PI3 kinase, signal to AWC to drive nuclear enrichment of EGL-4 during conditioning. Odor exposure shifts the AWC dynamic range to higher odor concentrations regardless of food pairing or the AIA circuit, whereas AWC coupling to motor circuits is oppositely regulated by aversive and appetitive learning. These results suggest that non-associative sensory adaptation in AWC encodes odor history, while associative behavioral preference is encoded by altered AWC synaptic activity. PMID:27383131

  4. Human brain n-chimaerin cDNA encodes a novel phorbol ester receptor.

    PubMed Central

    Ahmed, S; Kozma, R; Monfries, C; Hall, C; Lim, H H; Smith, P; Lim, L

    1990-01-01

    A human brain-specific cDNA encoding n-chimaerin, a protein of predicted molecular mass 34 kDa, has sequence identity with two different proteins: protein kinase C (PKC) at the N-terminus and BCR protein [product of the breakpoint-cluster-region (BCR) gene, involved in Philadelphia chromosome translocation] at the C-terminus [Hall, Monfries, Smith, Lim, Kozma, Ahmed, Vannaisungham, Leung & Lim (1990) J. Mol. Biol. 211, 11-16]. The sequence identity of n-chimaerin with PKC includes the cysteine-rich motif CX2CX13CX2CX7CX7C, and amino acids upstream of the first cysteine residue, but not the kinase domain. This region of PKC has been implicated in the binding of diacylglycerol and phorbol esters in a phospholipid-dependent fashion. Part of this cysteine-rich motif (CX2CX13CX2C) has the potential of forming a 'Zn-finger' structure. Phorbol esters cause a variety of physiological changes and are among the most potent tumour promoters that have been described. PKC is the only known protein target for these compounds. We now report that n-chimaerin cDNA encodes a novel phospholipid-dependent phorbol ester receptor, with the cysteine-rich region being responsible for this activity. This finding has wide implications for previous studies equating phorbol ester binding with the presence of PKC in the brain. Images Fig. 4. PMID:2268301

  5. Parallel encoding of sensory history and behavioral preference during Caenorhabditis elegans olfactory learning

    PubMed Central

    Cho, Christine E; Brueggemann, Chantal; L'Etoile, Noelle D; Bargmann, Cornelia I

    2016-01-01

    Sensory experience modifies behavior through both associative and non-associative learning. In Caenorhabditis elegans, pairing odor with food deprivation results in aversive olfactory learning, and pairing odor with food results in appetitive learning. Aversive learning requires nuclear translocation of the cGMP-dependent protein kinase EGL-4 in AWC olfactory neurons and an insulin signal from AIA interneurons. Here we show that the activity of neurons including AIA is acutely required during aversive, but not appetitive, learning. The AIA circuit and AGE-1, an insulin-regulated PI3 kinase, signal to AWC to drive nuclear enrichment of EGL-4 during conditioning. Odor exposure shifts the AWC dynamic range to higher odor concentrations regardless of food pairing or the AIA circuit, whereas AWC coupling to motor circuits is oppositely regulated by aversive and appetitive learning. These results suggest that non-associative sensory adaptation in AWC encodes odor history, while associative behavioral preference is encoded by altered AWC synaptic activity. DOI: http://dx.doi.org/10.7554/eLife.14000.001 PMID:27383131

  6. A murine fer testis-specific transcript (ferT) encodes a truncated Fer protein.

    PubMed Central

    Fischman, K; Edman, J C; Shackleford, G M; Turner, J A; Rutter, W J; Nir, U

    1990-01-01

    A cDNA for a potential tyrosine kinase-encoding mRNA was isolated from a mouse testis cDNA library. In a survey of eight mouse tissues, a transcript of 2.4 kilobases restricted to testis tissue was found. The mRNA encodes a 453-amino-acid protein of 51,383 daltons, the smallest tyrosine kinase protein ever described. RNA synthesized from the cDNA template directs the synthesis of a 51,000-Mr protein in a cell-free translation system. The carboxy-terminal 409 amino acids are 98 and 90% identical to the carboxy halves of the rat and human Fer proteins, respectively. This suggests that the cDNA represents an alternatively spliced testis-specific fer mRNA and is therefore termed by us ferT. On the basis of the appearance time of the fer mRNA in the testis of maturing neonatal mice, we speculate on the role played by this protein in the development of this organ. Images PMID:2294399

  7. Tissue- and cell-specific expression of Ins(1,4,5)P3 3-kinase isoenzymes.

    PubMed Central

    Vanweyenberg, V; Communi, D; D'Santos, C S; Erneux, C

    1995-01-01

    The phosphorylation of Ins(1,4,5)P3 (InsP3) to Ins(1,3,4,5)P4 (InsP4) is catalysed by InsP3 3-kinase. Molecular-biological data have shown the presence of two human isoenzymes of InsP3 3-kinase, namely InsP3 3-kinases A and B. We have isolated from a rat thymus cDNA library a 2235 bp cDNA (clone B15) encoding rat InsP3 3-kinase B. Northern-blot analysis of mRNA isolated from rat tissues (thymus, testis, brain, spleen, liver, kidney, heart, lung and intestine) revealed that a rat InsP3 3-kinase B probe hybridized to a 6 kb mRNA in lung, thymus, testis, brain and heart. In contrast, Northern-blot analysis of the same tissues probed under stringent conditions with a rat InsP3 3-kinase A probe hybridized to a 2 kb mRNA only in brain and a 1.8-2.0 kb mRNA species in testis. Northern-blot analysis of three human cell lines (HL-60, SH-SY5Y and HTB-138) probed with a human InsP3 3-kinase B probe showed the presence of a 6 kb mRNA in all cell lines, except in the human neuroblastoma cell line (SH-SY5Y), where two mRNA species of 5.7 and 6 kb were detected. Using the same blot, no hybridization signal could be seen with a human InsP3 3-kinase A probe. Altogether, our data are consistent with the notion that the two InsP3 3-kinase isoenzymes, A and B, are specifically expressed in different tissues and cells. Images Figure 3 Figure 4 PMID:7887896

  8. Fission yeast LAMMER kinase Lkh1 regulates the cell cycle by phosphorylating the CDK-inhibitor Rum1

    SciTech Connect

    Yu, Eun-Young; Lee, Ju-Hee; Kang, Won-Hwa; Park, Yun-Hee; Kim, Lila; Park, Hee-Moon

    2013-03-01

    Highlights: ► Deletion of lkh1{sup +} made cells pass the G1/S phase faster than the wild type. ► Lkh1 can interact with a cyclin-dependent kinase inhibitor (CKI) Rum1. ► Lkh1 can phosphorylate Rum1 to activate its CKI activity. ► Thr110 was confirmed as the Lkh1-dependent phosphorylation site of Rum1. ► Positive acting mechanism for the Rum1 activation is reported for the first time. - Abstract: In eukaryotes, LAMMER kinases are involved in various cellular events, including the cell cycle. However, no attempt has been made to investigate the mechanisms that underlie the involvement of LAMMER kinase. In this study, we performed a functional analysis of LAMMER kinase using the fission yeast, Schizosaccharomyces pombe. FACS analyses revealed that deletion of the gene that encodes the LAMMER kinase Lkh1 made mutant cells pass through the G1/S phase faster than their wild-type counterparts. Co-immunoprecipitation and an in vitro kinase assay also revealed that Lkh1 can interact with and phosphorylate Rum1 to activate this molecule as a cyclin-dependent kinase inhibitor, which blocks cell cycle progression from the G1 phase to the S phase. Peptide mass fingerprinting and kinase assay with Rum1{sup T110A} confirmed T110 as the Lkh1-dependent phosphorylation residue. In this report we present for the first time a positive acting mechanism that is responsible for the CKI activity of Rum1, in which the LAMMER kinase-mediated phosphorylation of Rum1 is involved.

  9. Encoding many qubits in a rotor

    SciTech Connect

    Raynal, Philippe; Kalev, Amir; Suzuki, Jun; Englert, Berthold-Georg

    2010-05-15

    We propose a scheme for encoding many qubits in a single rotor, that is, a continuous and periodic degree of freedom. A key feature of this scheme is its ability to manipulate and entangle the encoded qubits with a single operation on the system. We also show, using quantum error-correcting codes, how to protect the qubits against small errors in angular position and momentum which may affect the rotor. We then discuss the feasibility of this scheme and suggest several candidates for its implementation. The proposed scheme is immediately generalizable to qudits of any finite dimension.

  10. Vector Adaptive/Predictive Encoding Of Speech

    NASA Technical Reports Server (NTRS)

    Chen, Juin-Hwey; Gersho, Allen

    1989-01-01

    Vector adaptive/predictive technique for digital encoding of speech signals yields decoded speech of very good quality after transmission at coding rate of 9.6 kb/s and of reasonably good quality at 4.8 kb/s. Requires 3 to 4 million multiplications and additions per second. Combines advantages of adaptive/predictive coding, and code-excited linear prediction, yielding speech of high quality but requires 600 million multiplications and additions per second at encoding rate of 4.8 kb/s. Vector adaptive/predictive coding technique bridges gaps in performance and complexity between adaptive/predictive coding and code-excited linear prediction.

  11. Preparing encoded states in an oscillator

    SciTech Connect

    Travaglione, B.C.; Milburn, G.J.

    2002-11-01

    Recently a scheme has been proposed for constructing quantum error-correcting codes that embed a finite-dimensional code space in the infinite-dimensional Hilbert space of a system described by continuous quantum variables. One of the difficult steps in this scheme is the preparation of the encoded states. We show how these states can be generated by coupling a continuous quantum variable to a single qubit. An ion trap quantum computer provides a natural setting for a continuous system coupled to a qubit. We discuss how encoded states may be generated in an ion trap.

  12. Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates.

    PubMed

    Xue, Liang; Wang, Wen-Horng; Iliuk, Anton; Hu, Lianghai; Galan, Jacob A; Yu, Shuai; Hans, Michael; Geahlen, Robert L; Tao, W Andy

    2012-04-10

    Our understanding of the molecular control of many disease pathologies requires the identification of direct substrates targeted by specific protein kinases. Here we describe an integrated proteomic strategy, termed kinase assay linked with phosphoproteomics, which combines a sensitive kinase reaction with endogenous kinase-dependent phosphoproteomics to identify direct substrates of protein kinases. The unique in vitro kinase reaction is carried out in a highly efficient manner using a pool of peptides derived directly from cellular kinase substrates and then dephosphorylated as substrate candidates. The resulting newly phosphorylated peptides are then isolated and identified by mass spectrometry. A further comparison of these in vitro phosphorylated peptides with phosphopeptides derived from endogenous proteins isolated from cells in which the kinase is either active or inhibited reveals new candidate protein substrates. The kinase assay linked with phosphoproteomics strategy was applied to identify unique substrates of spleen tyrosine kinase (Syk), a protein-tyrosine kinase with duel properties of an oncogene and a tumor suppressor in distinctive cell types. We identified 64 and 23 direct substrates of Syk specific to B cells and breast cancer cells, respectively. Both known and unique substrates, including multiple centrosomal substrates for Syk, were identified, supporting a unique mechanism that Syk negatively affects cell division through its centrosomal kinase activity. PMID:22451900

  13. The myxoma virus thymidine kinase gene: sequence and transcriptional mapping.

    PubMed

    Jackson, R J; Bults, H G

    1992-02-01

    The myxoma virus thymidine kinase (TK) gene is encoded on a 1.6 kb SacI-SalI restriction fragment located between 57.7 and 59.3 kb on the 163 kb genomic map. The nucleotide sequence of this fragment as well as 228 bp from the adjacent SalI-AA2 fragment was determined and found to encode four major open reading frames (ORFs). Three of these ORFs are similar in nucleotide sequence to ORFs L5R and J1R, and the TK gene of vaccinia virus (VV). The fourth ORF, MF8a, shows similarity to the ORFs found in the same position relative to the TK genes of Shope fibroma virus, Kenya sheep-1 virus and swine-pox virus. A search of the complete VV nucleotide sequence for regions of similarity to MF8a identified the host specificity gene C7L. Northern blot analysis of early viral RNA identified transcripts of approximately 700 nucleotides for both the TK gene and ORF MF8a. The 5' ends of the TK gene and ORF MF8a early mRNAs were mapped by primer extension to initiation sites 13 nucleotides downstream of sequences with similarity to the VV early promoter consensus. The sizes of the TK and MF8a mRNAs are consistent with transcription termination and polyadenylation occurring downstream of the sequence TTTTTNT, which is identical to the consensus sequence for the VV transcription termination signal.

  14. v-mos proteins encoded by myeloproliferative sarcoma virus and its ts159 mutant.

    PubMed Central

    Singh, B; Stocking, C; Walker, R; Yang, Y D; Ostertag, W; Arlinghaus, R B

    1992-01-01

    The myeloproliferative sarcoma virus (MPSV) v-mos protein was predicted to be identical in size to p39c-mos because of an observed one-base deletion in the seventh codon of the env-mos open reading frame, which would allow translation to initiate at the methionine equivalent to codon 32 of the env-mos gene. On the basis of published results, p39c-mos is known to have greatly reduced in vitro protein kinase activity compared with p37env-mos encoded by Moloney murine sarcoma virus. Unexpectedly, the relative activity of the MPSV v-mos protein kinase was comparable to that of p37env-mos. Consistent with this finding, the size of MPSV v-mos protein was found to be similar to the size of p37env-mos. Moreover, the pattern and sizes of phosphorylated bands produced by autophosphorylation of the MPSV v-mos protein were similar to those of p37env-mos. These results were confirmed by in vitro transcription-translation of the MPSV v-mos gene. Resequencing portions of the MPSV mos gene failed to show the deletion within codon 7. Except for the codon 262 deletion, other mutations characteristic of MPSV and temperature-sensitive MPSV v-mos genes were confirmed. A glycine-to-arginine mutation at residue 338 of the MPSV env-mos sequence, previously shown to cause thermosensitivity of the mutant virus (termed ts159) transforming function, yielded a v-mos protein that had significantly reduced protein kinase activity in vitro. These findings indicate that MPSV, like other Moloney murine sarcoma virus strains, also encodes a functional env-mos protein. Images PMID:1309903

  15. Retrieval during Learning Facilitates Subsequent Memory Encoding

    ERIC Educational Resources Information Center

    Pastotter, Bernhard; Schicker, Sabine; Niedernhuber, Julia; Bauml, Karl-Heinz T.

    2011-01-01

    In multiple-list learning, retrieval during learning has been suggested to improve recall of the single lists by enhancing list discrimination and, at test, reducing interference. Using electrophysiological, oscillatory measures of brain activity, we examined to what extent retrieval during learning facilitates list encoding. Subjects studied 5…

  16. Encoded Archival Description as a Halfway Technology

    ERIC Educational Resources Information Center

    Dow, Elizabeth H.

    2009-01-01

    In the mid 1990s, Encoded Archival Description (EAD) appeared as a revolutionary technology for publishing archival finding aids on the Web. The author explores whether or not, given the advent of Web 2.0, the archival community should abandon EAD and look for something to replace it. (Contains 18 notes.)

  17. Gene encoding acetyl-coenzyme A carboxylase

    DOEpatents

    Roessler, P.G.; Ohlrogge, J.B.

    1996-09-24

    A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives are disclosed which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides. 5 figs.

  18. Encoding of Others' Beliefs without Overt Instruction

    ERIC Educational Resources Information Center

    Cohen, Adam S.; German, Tamsin C.

    2009-01-01

    Under what conditions do people automatically encode and track the mental states of others? A recent investigation showed that when subjects are instructed to track the location of an object but are not instructed to track a belief about that location in a non-verbal false-belief task, they respond more slowly to questions about an agent's belief,…

  19. 47 CFR 11.32 - EAS Encoder.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... levels of the mark or space frequencies. (9) Attention Signal generator. The encoder must provide an attention signal that complies with the following: (i) Tone Frequencies. The audio tones shall have... period of not less than 8 nor longer than 25 seconds. NOTE: Prior to July 1, 1995, the Attention...

  20. 47 CFR 11.32 - EAS Encoder.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... or space frequencies. (9) Attention Signal generator. The encoder must provide an attention signal...) Inadvertent activation. The switch used for initiating the automatic generation of the simultaneous tones... provided with a visual and/or aural indicator which clearly shows that the Attention Signal is...

  1. 47 CFR 11.32 - EAS Encoder.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... levels of the mark or space frequencies. (9) Attention Signal generator. The encoder must provide an attention signal that complies with the following: (i) Tone Frequencies. The audio tones shall have... period of not less than 8 nor longer than 25 seconds. NOTE: Prior to July 1, 1995, the Attention...

  2. 47 CFR 11.32 - EAS Encoder.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... or space frequencies. (9) Attention Signal generator. The encoder must provide an attention signal...) Inadvertent activation. The switch used for initiating the automatic generation of the simultaneous tones... provided with a visual and/or aural indicator which clearly shows that the Attention Signal is...

  3. 47 CFR 11.32 - EAS Encoder.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... or space frequencies. (9) Attention Signal generator. The encoder must provide an attention signal...) Inadvertent activation. The switch used for initiating the automatic generation of the simultaneous tones... provided with a visual and/or aural indicator which clearly shows that the Attention Signal is...

  4. Gene encoding acetyl-coenzyme A carboxylase

    DOEpatents

    Roessler, Paul G.; Ohlrogge, John B.

    1996-01-01

    A DNA encoding an acetyl-coenzyme A carboxylase (ACCase) from a photosynthetic organism and functional derivatives thereof which are resistant to inhibition from certain herbicides. This gene can be placed in organisms to increase their fatty acid content or to render them resistant to certain herbicides.

  5. Encoding attentional states during visuomotor adaptation

    PubMed Central

    Im, Hee Yeon; Bédard, Patrick; Song, Joo-Hyun

    2015-01-01

    We recently showed that visuomotor adaptation acquired under attentional distraction is better recalled under a similar level of distraction compared to no distraction. This paradoxical effect suggests that attentional state (e.g., divided or undivided) is encoded as an internal context during visuomotor learning and should be reinstated for successful recall (Song & Bédard, 2015). To investigate if there is a critical temporal window for encoding attentional state in visuomotor memory, we manipulated whether participants performed the secondary attention-demanding task concurrently in the early or late phase of visuomotor learning. Recall performance was enhanced when the attentional states between recall and the early phase of visuomotor learning were consistent. However, it reverted to untrained levels when tested under the attentional state of the late-phase learning. This suggests that attentional state is primarily encoded during the early phase of learning before motor errors decrease and reach an asymptote. Furthermore, we demonstrate that when divided and undivided attentional states were mixed during visuomotor adaptation, only divided attention was encoded as an internal cue for memory retrieval. Therefore, a single attentional state appears to be primarily integrated with visuomotor memory while motor error reduction is in progress during learning. PMID:26114683

  6. Recombinant DNA encoding a desulfurization biocatalyst

    DOEpatents

    Rambosek, John; Piddington, Chris S.; Kovacevich, Brian R.; Young, Kevin D.; Denome, Sylvia A.

    1994-01-01

    This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous.

  7. Recombinant DNA encoding a desulfurization biocatalyst

    DOEpatents

    Rambosek, J.; Piddington, C.S.; Kovacevich, B.R.; Young, K.D.; Denome, S.A.

    1994-10-18

    This invention relates to a recombinant DNA molecule containing a gene or genes which encode a biocatalyst capable of desulfurizing a fossil fuel which contains organic sulfur molecules. For example, the present invention encompasses a recombinant DNA molecule containing a gene or genes of a strain of Rhodococcus rhodochrous. 13 figs.

  8. Encoders for block-circulant LDPC codes

    NASA Technical Reports Server (NTRS)

    Divsalar, Dariush (Inventor); Abbasfar, Aliazam (Inventor); Jones, Christopher R. (Inventor); Dolinar, Samuel J. (Inventor); Thorpe, Jeremy C. (Inventor); Andrews, Kenneth S. (Inventor); Yao, Kung (Inventor)

    2009-01-01

    Methods and apparatus to encode message input symbols in accordance with an accumulate-repeat-accumulate code with repetition three or four are disclosed. Block circulant matrices are used. A first method and apparatus make use of the block-circulant structure of the parity check matrix. A second method and apparatus use block-circulant generator matrices.

  9. Typicality as a Dimension of Encoding

    ERIC Educational Resources Information Center

    Keller, Dennis; Kellas, George

    1978-01-01

    The salience of encoding attributes in instances of differing levels of category membership was examined using the release from proactive interference (PI) task with college students. Results are discussed in terms of providing converging evidence for Rosch's (1973,1975) theory of semantic category structure. (Editor/RK)

  10. Encoding and Retrieval During Bimanual Rhythmic Coordination

    ERIC Educational Resources Information Center

    Shockley, Kevin; Turvey, Michael T.

    2005-01-01

    In 2 experiments, bimanual 1:1 rhythmic coordination was performed concurrently with encoding or retrieval of word lists. Effects of divided attention (DA) on coordination were indexed by changes in mean relative phase and recurrence measures of shared activity between the 2 limbs. Effects of DA on memory were indexed by deficits in recall…

  11. Encoding and Retrieval in Visual Memory Tasks

    ERIC Educational Resources Information Center

    Frost, Nancy

    1972-01-01

    It was concluded that pictures are encoded differently depending on task expectation. Parallel access of visual and semantic memory codes occurs; but when recognition is expected, a visual cue provides faster access, and when expecting recall, verbal access is more efficient. (Author)

  12. Young Children's Automatic Encoding of Social Categories

    ERIC Educational Resources Information Center

    Weisman, Kara; Johnson, Marissa V.; Shutts, Kristin

    2015-01-01

    The present research investigated young children's automatic encoding of two social categories that are highly relevant to adults: gender and race. Three- to 6-year-old participants learned facts about unfamiliar target children who varied in either gender or race and were asked to remember which facts went with which targets. When participants…

  13. Measuring the Activity of Leucine-Rich Repeat Kinase 2: A Kinase Involved in Parkinson's Disease

    PubMed Central

    Lee, Byoung Dae; Li, Xiaojie; Dawson, Ted M.; Dawson, Valina L.

    2015-01-01

    Mutations in the LRRK2 (Leucine-Rich Repeat Kinase 2) gene are the most common cause of autosomal dominant Parkinson's disease. LRRK2 has multiple functional domains including a kinase domain. The kinase activity of LRRK2 is implicated in the pathogenesis of Parkinson's disease. Developing an assay to understand the mechanisms of LRRK2 kinase activity is important for the development of pharmacologic and therapeutic applications. Here, we describe how to measure in vitro LRRK2 kinase activity and its inhibition. PMID:21960214

  14. How Attention Modulates Encoding of Dynamic Stimuli

    PubMed Central

    Oren, Noga; Shapira-Lichter, Irit; Lerner, Yulia; Tarrasch, Ricardo; Hendler, Talma; Giladi, Nir; Ash, Elissa L.

    2016-01-01

    When encoding a real-life, continuous stimulus, the same neural circuits support processing and integration of prior as well as new incoming information. This ongoing interplay is modulated by attention, and is evident in regions such as the prefrontal cortex section of the task positive network (TPN), and in the posterior cingulate cortex (PCC), a hub of the default mode network (DMN). Yet the exact nature of such modulation is still unclear. To investigate this issue, we utilized an fMRI task that employed movies as the encoded stimuli and manipulated attentional load via an easy or hard secondary task that was performed simultaneously with encoding. Results showed increased intersubject correlation (inter-SC) levels when encoding movies in a condition of high, as compared to low attentional load. This was evident in bilateral ventrolateral and dorsomedial prefrontal cortices and the dorsal PCC (dPCC). These regions became more attuned to the combination of the movie and the secondary task as the attentional demand of the latter increased. Activation analyses revealed that at higher load the prefrontal TPN regions were more activated, whereas the dPCC was more deactivated. Attentional load also influenced connectivity within and between the networks. At high load the dPCC was anti-correlated to the prefrontal regions, which were more functionally coherent amongst themselves. Finally and critically, greater inter-SC in the dPCC at high load during encoding predicted lower memory strength when that information was retrieved. This association between inter-SC levels and memory strength suggest that as attentional demands increased, the dPCC was more attuned to the secondary task at the expense of the encoded stimulus, thus weakening memory for the encoded stimulus. Together, our findings show that attentional load modulated the function of core TPN and DMN regions. Furthermore, the observed relationship between memory strength and the modulation of the dPCC points

  15. JPEG 2000 Encoding with Perceptual Distortion Control

    NASA Technical Reports Server (NTRS)

    Watson, Andrew B.; Liu, Zhen; Karam, Lina J.

    2008-01-01

    An alternative approach has been devised for encoding image data in compliance with JPEG 2000, the most recent still-image data-compression standard of the Joint Photographic Experts Group. Heretofore, JPEG 2000 encoding has been implemented by several related schemes classified as rate-based distortion-minimization encoding. In each of these schemes, the end user specifies a desired bit rate and the encoding algorithm strives to attain that rate while minimizing a mean squared error (MSE). While rate-based distortion minimization is appropriate for transmitting data over a limited-bandwidth channel, it is not the best approach for applications in which the perceptual quality of reconstructed images is a major consideration. A better approach for such applications is the present alternative one, denoted perceptual distortion control, in which the encoding algorithm strives to compress data to the lowest bit rate that yields at least a specified level of perceptual image quality. Some additional background information on JPEG 2000 is prerequisite to a meaningful summary of JPEG encoding with perceptual distortion control. The JPEG 2000 encoding process includes two subprocesses known as tier-1 and tier-2 coding. In order to minimize the MSE for the desired bit rate, a rate-distortion- optimization subprocess is introduced between the tier-1 and tier-2 subprocesses. In tier-1 coding, each coding block is independently bit-plane coded from the most-significant-bit (MSB) plane to the least-significant-bit (LSB) plane, using three coding passes (except for the MSB plane, which is coded using only one "clean up" coding pass). For M bit planes, this subprocess involves a total number of (3M - 2) coding passes. An embedded bit stream is then generated for each coding block. Information on the reduction in distortion and the increase in the bit rate associated with each coding pass is collected. This information is then used in a rate-control procedure to determine the

  16. The protein tyrosine kinases EpsB and PtkA differentially affect biofilm formation in Bacillus subtilis

    PubMed Central

    Gerwig, Jan; Kiley, Taryn B.; Gunka, Katrin; Stanley-Wall, Nicola

    2014-01-01

    The Gram-positive soil bacterium Bacillus subtilis is able to choose between motile and sessile lifestyles. The sessile way of life, also referred to as biofilm, depends on the formation of an extracellular polysaccharide matrix and some extracellular proteins. Moreover, a significant proportion of cells in a biofilm form spores. The first two genes of the 15-gene operon for extracellular polysaccharide synthesis, epsA and epsB, encode a putative transmembrane modulator protein and a putative protein tyrosine kinase, respectively, with similarity to the TkmA/PtkA modulator/kinase couple. Here we show that the putative kinase EpsB is required for the formation of structured biofilms. However, an epsB mutant is still able to form biofilms. As shown previously, a ptkA mutant is also partially defective in biofilm formation, but this defect is related to spore formation in the biofilm. The absence of both kinases resulted in a complete loss of biofilm formation. Thus, EpsB and PtkA fulfil complementary functions in biofilm formation. The activity of bacterial protein tyrosine kinases depends on their interaction with modulator proteins. Our results demonstrate the specific interaction between the putative kinase EpsB and its modulator protein EpsA and suggest that EpsB activity is stimulated by its modulator EpsA. PMID:24493247

  17. Expression analysis of a novel pyridoxal kinase messenger RNA splice variant, PKL, in oil rape suffering abiotic stress and phytohormones.

    PubMed

    Yu, Shunwu; Luo, Lijun

    2008-12-01

    Pyridoxal kinase is key enzyme for the biosynthesis of pyridoxal 5'-phosphate, the biologically active form of vitamin B6, in the salvage pathway. A pyridoxal kinase gene, BnPKL (GenBank accession No. DQ463962), was isolated from oilseed rape (Brassica napus L.) following water stress through rapid amplification of complementary DNA (cDNA) ends. The results showed that the gene had two splice variants: PKL and PKL2. PKL, the long cDNA, encodes a 334 amino acid protein with a complete ATP-binding site, pyridoxal kinase-binding site and dimer interface site of a pyridoxal kinase, while PKL2, the short cDNA, lacked a partial domain. Southern blot showed that there were two copies in Brassica napus. The expression of BnPKL cDNA could rescue the mutant phenotype of Escherichia coli defective in pyridoxal kinase. Real-time reverse transcription-polymerase chain reaction revealed that the relative abundance of two transcripts are modulated by development and environmental stresses. Abscisic acid and NaCl were inclined to decrease PKL expression, but H2O2 and cold temperatures induced the PKL expression. In addition, the PKL expression could be transiently induced by jasmonate acid at an early stage, abscisic acid, salicylic acid and jasmonate acid enhanced the PKL expression in roots. Our results demonstrated that BnPKL was a pyridoxal kinase involved in responses to biotic and abiotic stresses.

  18. Drosophila Wee1 kinase rescues fission yeast from mitotic catastrophe and phosphorylates Drosophila Cdc2 in vitro.

    PubMed Central

    Campbell, S D; Sprenger, F; Edgar, B A; O'Farrell, P H

    1995-01-01

    Cdc2 kinase activity is required for triggering entry into mitosis in all known eukaryotes. Elaborate mechanisms have evolved for regulating Cdc2 activity so that mitosis occurs in a timely manner, when preparations for its execution are complete. In Schizosaccharomyces pombe, Wee1 and a related Mik1 kinase are Cdc2-inhibitory kinases that are required for preventing premature activation of the mitotic program. To identify Cdc2-inhibitory kinases in Drosophila, we screened for cDNA clones that rescue S. pombe wee1- mik1- mutants from lethal mitotic catastrophe. One of the genes identified in this screen, Drosophila wee1 (Dwee1), encodes a new Wee1 homologue. Dwee1 kinase is closely related to human and Xenopus Wee1 homologues, and can inhibit Cdc2 activity by phosphorylating a critical tyrosine residue. Dwee1 mRNA is maternally provided to embryos, and is zygotically expressed during the postblastoderm divisions of embryogenesis. Expression remains high in the proliferating cells of the central nervous system well after cells in the rest of the embryo have ceased dividing. The loss of zygotically expressed Dwee1 does not lead to mitotic catastrophe during postblastoderm cycles 14 to 16. This result may indicate that maternally provided Dwee1 is sufficient for regulating Cdc2 during embryogenesis, or it may reflect the presence of a redundant Cdc2 inhibitory kinase, as in fission yeast. Images PMID:8573790

  19. Impact of overexpressing NADH kinase on glucose and xylose metabolism in recombinant xylose-utilizing Saccharomyces cerevisiae.

    PubMed

    Hou, Jin; Vemuri, Goutham N; Bao, Xiaoming; Olsson, Lisbeth

    2009-04-01

    During growth of Saccharomyces cerevisiae on glucose, the redox cofactors NADH and NADPH are predominantly involved in catabolism and biosynthesis, respectively. A deviation from the optimal level of these cofactors often results in major changes in the substrate uptake and biomass formation. However, the metabolism of xylose by recombinant S. cerevisiae carrying xylose reductase and xylitol dehydrogenase from the fungal pathway requires both NADH and NADPH and creates cofactor imbalance during growth on xylose. As one possible solution to overcoming this imbalance, the effect of overexpressing the native NADH kinase (encoded by the POS5 gene) in xylose-consuming recombinant S. cerevisiae directed either into the cytosol or to the mitochondria was evaluated. The physiology of the NADH kinase containing strains was also evaluated during growth on glucose. Overexpressing NADH kinase in the cytosol redirected carbon flow from CO(2) to ethanol during aerobic growth on glucose and to ethanol and acetate during anaerobic growth on glucose. However, cytosolic NADH kinase has an opposite effect during anaerobic metabolism of xylose consumption by channeling carbon flow from ethanol to xylitol. In contrast, overexpressing NADH kinase in the mitochondria did not affect the physiology to a large extent. Overall, although NADH kinase did not increase the rate of xylose consumption, we believe that it can provide an important source of NADPH in yeast, which can be useful for metabolic engineering strategies where the redox fluxes are manipulated.

  20. Pyridopyrimidine analogues as novel adenosine kinase inhibitors.

    PubMed

    Zheng, G Z; Lee, C; Pratt, J K; Perner, R J; Jiang, M Q; Gomtsyan, A; Matulenko, M A; Mao, Y; Koenig, J R; Kim, K H; Muchmore, S; Yu, H; Kohlhaas, K; Alexander, K M; McGaraughty, S; Chu, K L; Wismer, C T; Mikusa, J; Jarvis, M F; Marsh, K; Kowaluk, E A; Bhagwat, S S; Stewart, A O

    2001-08-20

    A novel series of pyridopyrimidine analogues 9 was identified as potent adenosine kinase inhibitors based on the SAR and computational studies. Substitution of the C7 position of the pyridopyrimidino core with C2' substituted pyridino moiety increased the in vivo potency and enhanced oral bioavailability of these adenosine kinase inhibitors.

  1. Multifunctional Abl kinases in health and disease.

    PubMed

    Khatri, Aaditya; Wang, Jun; Pendergast, Ann Marie

    2016-01-01

    The Abelson tyrosine kinases were initially identified as drivers of leukemia in mice and humans. The Abl family kinases Abl1 and Abl2 regulate diverse cellular processes during development and normal homeostasis, and their functions are subverted during inflammation, cancer and other pathologies. Abl kinases can be activated by multiple stimuli leading to cytoskeletal reorganization required for cell morphogenesis, motility, adhesion and polarity. Depending on the cellular context, Abl kinases regulate cell survival and proliferation. Emerging data support important roles for Abl kinases in pathologies linked to inflammation. Among these are neurodegenerative diseases and inflammatory pathologies. Unexpectedly, Abl kinases have also been identified as important players in mammalian host cells during microbial pathogenesis. Thus, the use of Abl kinase inhibitors might prove to be effective in the treatment of pathologies beyond leukemia and solid tumors. In this Cell Science at a Glance article and in the accompanying poster, we highlight the emerging roles of Abl kinases in the regulation of cellular processes in normal cells and diverse pathologies ranging from cancer to microbial pathogenesis.

  2. Genetics Home Reference: pyruvate kinase deficiency

    MedlinePlus

    ... National (UK) Information Centre for Metabolic Diseases National Organization for Rare Disorders (NORD): Pyruvate Kinase Deficiency Genetic Testing Registry (1 link) Pyruvate kinase deficiency of red cells Scientific articles on PubMed (1 link) PubMed OMIM (1 link) ...

  3. Selective regulation of MAP kinase signaling by an endomembrane phosphatidylinositol 4-kinase.

    PubMed

    Cappell, Steven D; Dohlman, Henrik G

    2011-04-29

    Multiple MAP kinase pathways share components yet initiate distinct biological processes. Signaling fidelity can be maintained by scaffold proteins and restriction of signaling complexes to discreet subcellular locations. For example, the yeast MAP kinase scaffold Ste5 binds to phospholipids produced at the plasma membrane and promotes selective MAP kinase activation. Here we show that Pik1, a phosphatidylinositol 4-kinase that localizes primarily to the Golgi, also regulates MAP kinase specificity but does so independently of Ste5. Pik1 is required for full activation of the MAP kinases Fus3 and Hog1 and represses activation of Kss1. Further, we show by genetic epistasis analysis that Pik1 likely regulates Ste11 and Ste50, components shared by all three MAP kinase pathways, through their interaction with the scaffold protein Opy2. These findings reveal a new regulator of signaling specificity functioning at endomembranes rather than at the plasma membrane. PMID:21388955

  4. Tyrosine Kinase Inhibitors and Pregnancy

    PubMed Central

    Abruzzese, Elisabetta; Trawinska, Malgorzata Monika; Perrotti, Alessio Pio; De Fabritiis, Paolo

    2014-01-01

    The management of patients with chronic myeloid leukemia (CML) during pregnancy has become recently a matter of continuous debate. The introduction of the Tyrosine Kinase Inhibitors (TKIs) in clinical practice has dramatically changed the prognosis of CML patients; in fact, patients diagnosed in chronic phase can reasonably expect many years of excellent disease control and good quality of life, as well as a normal life expectancy, including the necessity to address issues relating to fertility and pregnancy. Physicians are frequently being asked for advice regarding the need for, and/or the appropriateness of, stopping treatment in order to conceive. In this report, we will review the data published in terms of fertility, conception, pregnancy, pregnancy outcome and illness control for TKI treated CML patients, as well as how to manage a planned and/or unplanned pregnancy. PMID:24804001

  5. Endothelial Bmx tyrosine kinase activity is essential for myocardial hypertrophy and remodeling.

    PubMed

    Holopainen, Tanja; Räsänen, Markus; Anisimov, Andrey; Tuomainen, Tomi; Zheng, Wei; Tvorogov, Denis; Hulmi, Juha J; Andersson, Leif C; Cenni, Bruno; Tavi, Pasi; Mervaala, Eero; Kivelä, Riikka; Alitalo, Kari

    2015-10-20

    Cardiac hypertrophy accompanies many forms of heart disease, including ischemic disease, hypertension, heart failure, and valvular disease, and it is a strong predictor of increased cardiovascular morbidity and mortality. Deletion of bone marrow kinase in chromosome X (Bmx), an arterial nonreceptor tyrosine kinase, has been shown to inhibit cardiac hypertrophy in mice. This finding raised the possibility of therapeutic use of Bmx tyrosine kinase inhibitors, which we have addressed here by analyzing cardiac hypertrophy in gene-targeted mice deficient in Bmx tyrosine kinase activity. We found that angiotensin II (Ang II)-induced cardiac hypertrophy is significantly reduced in mice deficient in Bmx and in mice with inactivated Bmx tyrosine kinase compared with WT mice. Genome-wide transcriptomic profiling showed that Bmx inactivation suppresses myocardial expression of genes related to Ang II-induced inflammatory and extracellular matrix responses whereas expression of RNAs encoding mitochondrial proteins after Ang II administration was maintained in Bmx-inactivated hearts. Very little or no Bmx mRNA was expressed in human cardiomyocytes whereas human cardiac endothelial cells expressed abundant amounts. Ang II stimulation of endothelial cells increased Bmx phosphorylation, and Bmx gene silencing inhibited downstream STAT3 signaling, which has been implicated in cardiac hypertrophy. Furthermore, activation of the mechanistic target of rapamycin complex 1 pathway by Ang II treatment was decreased in the Bmx-deficient hearts. Our results demonstrate that inhibition of the cross-talk between endothelial cells and cardiomyocytes by Bmx inactivation suppresses Ang II-induced signals for cardiac hypertrophy. These results suggest that the endothelial Bmx tyrosine kinase could provide a target to attenuate the development of cardiac hypertrophy. PMID:26430242

  6. Cloning, purification and preliminary crystallographic analysis of a putative pyridoxal kinase from Bacillus subtilis

    SciTech Connect

    Newman, Joseph A.; Das, Sanjan K.; Sedelnikova, Svetlana E.; Rice, David W.

    2006-10-01

    A putative pyridoxal kinase from B. subtilis has been cloned, overexpressed, purified and crystallized and data have been collected to 2.8 Å resolution. Pyridoxal kinases (PdxK) are able to catalyse the phosphorylation of three vitamin B{sub 6} precursors, pyridoxal, pyridoxine and pyridoxamine, to their 5′-phosphates and play an important role in the vitamin B{sub 6} salvage pathway. Recently, the thiD gene of Bacillus subtilis was found to encode an enzyme which has the activity expected of a pyridoxal kinase despite its previous assignment as an HMPP kinase owing to higher sequence similarity. As such, this enzyme would appear to represent a new class of ‘HMPP kinase-like’ pyridoxal kinases. B. subtilis thiD has been cloned and the protein has been overexpressed in Escherichia coli, purified and subsequently crystallized in a binary complex with ADP and Mg{sup 2+}. X-ray diffraction data have been collected from crystals to 2.8 Å resolution at 100 K. The crystals belong to a primitive tetragonal system, point group 422, and analysis of the systematic absences suggest that they belong to one of the enantiomorphic pair of space groups P4{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2. Consideration of the space-group symmetry and unit-cell parameters (a = b = 102.9, c = 252.6 Å, α = β = γ = 90°) suggest that the crystals contain between three and six molecules in the asymmetric unit. A full structure determination is under way to provide insights into aspects of the enzyme mechanism and substrate specificity.

  7. HCMV pUS28 initiates pro-migratory signaling via activation of Pyk2 kinase

    SciTech Connect

    Vomaske, Jennifer; Varnum, Susan M.; Melnychuk, Ryan; Smith, Patricia; Pasa-Tolic, Ljiljana; Shutthanandan, Janani I.; Streblow, Daniel N.

    2010-12-10

    The HCMV-encoded chemokine receptor US28 mediates smooth muscle cell (SMC) and macrophage motility and this activity has been implicated in the acceleration of vascular disease. US28 induced SMC migration involves the activation of the protein tyrosine kinases (PTKs) Src and Focal adhesion kinase as well as the small GTPase RhoA. In the current study, we examined the involvement of the PTK Pyk2 in US28-induced cellular motility. Expression of a Pyk2 lacking the autophosphorylation site (Tyr-402) blocks US28-mediated SMC migration in response to RANTES, while the kinase-inactive mutant failed to elicit the same negative effect on migration. US28 stimulation with RANTES results in ligand-dependent and calcium-dependent phosphorylation of Pyk2 Tyr-402 and induced the formation of an active Pyk2 kinase complex containing several novel Pyk2 binding proteins. Interestingly, expression of the autophosphorylation site mutant Pyk2 F402Y did not abrogate the formation of an active Pyk2 kinase complex, but instead prevented US28-mediated activation of RhoA. These findings represent the first demonstration that US28 signals through Pyk2 and that this PTK participates in US28-mediated cellular motility via activation of RhoA. Additionally, US28 activated RhoA via Pyk2 in the U373 glioblastoma cells. Interestingly, the Pyk2 kinase complex in U373 contained several proteins known to participate in glioma tumorigenesis. These results provide a potential mechanistic link between HCMV-US28 and glioblastoma cell activation and motility.

  8. Endothelial Bmx tyrosine kinase activity is essential for myocardial hypertrophy and remodeling

    PubMed Central

    Holopainen, Tanja; Räsänen, Markus; Anisimov, Andrey; Tuomainen, Tomi; Zheng, Wei; Tvorogov, Denis; Hulmi, Juha J.; Andersson, Leif C.; Cenni, Bruno; Tavi, Pasi; Mervaala, Eero; Kivelä, Riikka; Alitalo, Kari

    2015-01-01

    Cardiac hypertrophy accompanies many forms of heart disease, including ischemic disease, hypertension, heart failure, and valvular disease, and it is a strong predictor of increased cardiovascular morbidity and mortality. Deletion of bone marrow kinase in chromosome X (Bmx), an arterial nonreceptor tyrosine kinase, has been shown to inhibit cardiac hypertrophy in mice. This finding raised the possibility of therapeutic use of Bmx tyrosine kinase inhibitors, which we have addressed here by analyzing cardiac hypertrophy in gene-targeted mice deficient in Bmx tyrosine kinase activity. We found that angiotensin II (Ang II)-induced cardiac hypertrophy is significantly reduced in mice deficient in Bmx and in mice with inactivated Bmx tyrosine kinase compared with WT mice. Genome-wide transcriptomic profiling showed that Bmx inactivation suppresses myocardial expression of genes related to Ang II-induced inflammatory and extracellular matrix responses whereas expression of RNAs encoding mitochondrial proteins after Ang II administration was maintained in Bmx-inactivated hearts. Very little or no Bmx mRNA was expressed in human cardiomyocytes whereas human cardiac endothelial cells expressed abundant amounts. Ang II stimulation of endothelial cells increased Bmx phosphorylation, and Bmx gene silencing inhibited downstream STAT3 signaling, which has been implicated in cardiac hypertrophy. Furthermore, activation of the mechanistic target of rapamycin complex 1 pathway by Ang II treatment was decreased in the Bmx-deficient hearts. Our results demonstrate that inhibition of the cross-talk between endothelial cells and cardiomyocytes by Bmx inactivation suppresses Ang II-induced signals for cardiac hypertrophy. These results suggest that the endothelial Bmx tyrosine kinase could provide a target to attenuate the development of cardiac hypertrophy. PMID:26430242

  9. Metabolic Dysfunction Consistent with Premature Aging Results from Deletion of Pim Kinases

    PubMed Central

    Din, Shabana; Konstandin, Mathias H; Johnson, Bevan; Emathinger, Jacqueline; Völkers, Mirko; Toko, Haruhiro; Collins, Brett; Ormachea, Lucy; Samse, Kaitlen; Kubli, Dieter A; De La Torre, Andrea; Kraft, Andrew S; Gustafsson, Asa B; Kelly, Daniel P; Sussman, Mark A

    2014-01-01

    Rationale The senescent cardiac phenotype is accompanied by changes in mitochondrial function and biogenesis causing impairment in energy provision. The relationship between myocardial senescence and Pim kinases deserves attention since Pim-1 kinase is cardioprotective, in part, by preservation of mitochondrial integrity. Study of the pathological effects resulting from genetic deletion of all Pim kinase family members could provide important insight regarding cardiac mitochondrial biology and the aging phenotype. Objective Demonstrate myocardial senescence is promoted by loss of Pim leading to premature aging and aberrant mitochondrial function. Methods and Results Cardiac myocyte senescence was evident at three months of age in Pim Triple KnockOut (PTKO) mice, where all three isoforms of Pim kinase family members are genetically deleted. Cellular hypertrophic remodeling and fetal gene program activation was followed by heart failure at six months in PTKO mice. Metabolic dysfunction is an underlying cause of cardiac senescence and instigates a decline in cardiac function. Altered mitochondrial morphology is evident consequential to Pim deletion together with decreased ATP levels and increased phosphorylated AMPK, exposing an energy deficiency in PTKO mice. Expression of the genes encoding master regulators of mitochondrial biogenesis, PPARγ coactivator-1 (PGC-1) α and β were diminished in PTKO hearts, as were downstream targets included in mitochondrial energy transduction, including fatty acid oxidation. Reversal of the dysregulated metabolic phenotype was observed by overexpressing c-Myc, a downstream target of Pim kinases. Conclusion Pim kinases prevent premature cardiac aging and maintain a healthy pool of functional mitochondria leading to efficient cellular energetics. PMID:24916111

  10. Genome-Wide Identification, Evolution, and Co-expression Network Analysis of Mitogen-Activated Protein Kinase Kinase Kinases in Brachypodium distachyon

    PubMed Central

    Feng, Kewei; Liu, Fuyan; Zou, Jinwei; Xing, Guangwei; Deng, Pingchuan; Song, Weining; Tong, Wei; Nie, Xiaojun

    2016-01-01

    Mitogen-activated protein kinase (MAPK) cascades are the conserved and universal signal transduction modules in all eukaryotes, which play the vital roles in plant growth, development, and in response to multiple stresses. In this study, we used bioinformatics methods to identify 86 MAPKKK protein encoded by 73 MAPKKK genes in Brachypodium. Phylogenetic analysis of MAPKKK family from Arabidopsis, rice, and Brachypodium has classified them into three subfamilies, of which 28 belonged to MEKK, 52 to Raf, and 6 to ZIK subfamily, respectively. Conserved protein motif, exon-intron organization, and splicing intron phase in kinase domains supported the evolutionary relationships inferred from the phylogenetic analysis. And gene duplication analysis suggested the chromosomal segment duplication happened before the divergence of the rice and Brachypodium, while all of three tandem duplicated gene pairs happened after their divergence. We further demonstrated that the MAPKKKs have evolved under strong purifying selection, implying the conservation of them. The splicing transcripts expression analysis showed that the splicesome translating longest protein tended to be adopted. Furthermore, the expression analysis of BdMAPKKKs in different organs and development stages as well as heat, virus and drought stresses revealed that the MAPKKK genes were involved in various signaling pathways. And the circadian analysis suggested there were 41 MAPKKK genes in Brachypodium showing cycled expression in at least one condition, of which seven MAPKKK genes expressed in all conditions and the promoter analysis indicated these genes possessed many cis-acting regulatory elements involved in circadian and light response. Finally, the co-expression network of MAPK, MAPKK, and MAPKKK in Brachypodium was constructed using 144 microarray and RNA-seq datasets, and ten potential MAPK cascades pathway were predicted. To conclude, our study provided the important information for evolutionary and

  11. Genome-Wide Identification, Evolution, and Co-expression Network Analysis of Mitogen-Activated Protein Kinase Kinase Kinases in Brachypodium distachyon

    PubMed Central

    Feng, Kewei; Liu, Fuyan; Zou, Jinwei; Xing, Guangwei; Deng, Pingchuan; Song, Weining; Tong, Wei; Nie, Xiaojun

    2016-01-01

    Mitogen-activated protein kinase (MAPK) cascades are the conserved and universal signal transduction modules in all eukaryotes, which play the vital roles in plant growth, development, and in response to multiple stresses. In this study, we used bioinformatics methods to identify 86 MAPKKK protein encoded by 73 MAPKKK genes in Brachypodium. Phylogenetic analysis of MAPKKK family from Arabidopsis, rice, and Brachypodium has classified them into three subfamilies, of which 28 belonged to MEKK, 52 to Raf, and 6 to ZIK subfamily, respectively. Conserved protein motif, exon-intron organization, and splicing intron phase in kinase domains supported the evolutionary relationships inferred from the phylogenetic analysis. And gene duplication analysis suggested the chromosomal segment duplication happened before the divergence of the rice and Brachypodium, while all of three tandem duplicated gene pairs happened after their divergence. We further demonstrated that the MAPKKKs have evolved under strong purifying selection, implying the conservation of them. The splicing transcripts expression analysis showed that the splicesome translating longest protein tended to be adopted. Furthermore, the expression analysis of BdMAPKKKs in different organs and development stages as well as heat, virus and drought stresses revealed that the MAPKKK genes were involved in various signaling pathways. And the circadian analysis suggested there were 41 MAPKKK genes in Brachypodium showing cycled expression in at least one condition, of which seven MAPKKK genes expressed in all conditions and the promoter analysis indicated these genes possessed many cis-acting regulatory elements involved in circadian and light response. Finally, the co-expression network of MAPK, MAPKK, and MAPKKK in Brachypodium was constructed using 144 microarray and RNA-seq datasets, and ten potential MAPK cascades pathway were predicted. To conclude, our study provided the important information for evolutionary and

  12. PRO40 is a scaffold protein of the cell wall integrity pathway, linking the MAP kinase module to the upstream activator protein kinase C.

    PubMed

    Teichert, Ines; Steffens, Eva Katharina; Schnaß, Nicole; Fränzel, Benjamin; Krisp, Christoph; Wolters, Dirk A; Kück, Ulrich

    2014-09-01

    Mitogen-activated protein kinase (MAPK) pathways are crucial signaling instruments in eukaryotes. Most ascomycetes possess three MAPK modules that are involved in key developmental processes like sexual propagation or pathogenesis. However, the regulation of these modules by adapters or scaffolds is largely unknown. Here, we studied the function of the cell wall integrity (CWI) MAPK module in the model fungus Sordaria macrospora. Using a forward genetic approach, we found that sterile mutant pro30 has a mutated mik1 gene that encodes the MAPK kinase kinase (MAPKKK) of the proposed CWI pathway. We generated single deletion mutants lacking MAPKKK MIK1, MAPK kinase (MAPKK) MEK1, or MAPK MAK1 and found them all to be sterile, cell fusion-deficient and highly impaired in vegetative growth and cell wall stress response. By searching for MEK1 interaction partners via tandem affinity purification and mass spectrometry, we identified previously characterized developmental protein PRO40 as a MEK1 interaction partner. Although fungal PRO40 homologs have been implicated in diverse developmental processes, their molecular function is currently unknown. Extensive affinity purification, mass spectrometry, and yeast two-hybrid experiments showed that PRO40 is able to bind MIK1, MEK1, and the upstream activator protein kinase C (PKC1). We further found that the PRO40 N-terminal disordered region and the central region encompassing a WW interaction domain are sufficient to govern interaction with MEK1. Most importantly, time- and stress-dependent phosphorylation studies showed that PRO40 is required for MAK1 activity. The sum of our results implies that PRO40 is a scaffold protein for the CWI pathway, linking the MAPK module to the upstream activator PKC1. Our data provide important insights into the mechanistic role of a protein that has been implicated in sexual and asexual development, cell fusion, symbiosis, and pathogenicity in different fungal systems.

  13. Members of a Novel Family of Mammalian Protein Kinases Complement the DNA-Negative Phenotype of a Vaccinia Virus ts Mutant Defective in the B1 Kinase

    PubMed Central

    Boyle, Kathleen A.; Traktman, Paula

    2004-01-01

    Temperature-sensitive (ts) mutants of vaccinia virus defective in the B1 kinase demonstrate a conditionally lethal defect in DNA synthesis. B1 is the prototypic member of a new family of protein kinases (vaccinia virus-related kinases, or VRK) that possess distinctive B1-like sequence features within their catalytic motifs (R. J. Nichols and P. Traktman, J. Biol. Chem., in press). Given the striking sequence similarity between B1 and the VRK enzymes, we proposed that they might share overlapping substrate specificity. We therefore sought to determine whether the human and mouse VRK1 enzymes (hVRK1 and mVRK1, respectively) could complement a B1 deficiency in vivo. Recombinant ts2 viruses expressing hVRK1, mVRK1, or wild-type B1 were able to synthesize viral DNA at high temperature, but those expressing the more distantly related human casein kinase 1α2 could not. Complementation required the enzymatic activity of hVRK1, since a catalytically inactive allele of hVRK1 was unable to confer a temperature-insensitive phenotype. Interestingly, rescue of viral DNA synthesis was not coupled to the ability to phosphorylate H5, the only virus-encoded protein shown to be a B1 substrate in vivo. Expression of hVRK1 during nonpermissive ts2 infections restored virus production and plaque formation, whereas expression of mVRK1 resulted in an intermediate level of rescue. Taken together, these observations indicate that enzymatically active cellular VRK1 kinases can perform the function(s) of B1 required for genome replication, most likely due to overlapping specificity for cellular and/or viral substrates. PMID:14747564

  14. Multitasking in signal transduction by a promiscuous human Ins(3,4,5,6)P(4) 1-kinase/Ins(1,3,4)P(3) 5/6-kinase.

    PubMed

    Yang, X; Shears, S B

    2000-11-01

    We describe a human cDNA encoding 1-kinase activity that inactivates Ins(3,4,5,6)P(4), an inhibitor of chloride-channel conductance that regulates epithelial salt and fluid secretion, as well as membrane excitability. Unexpectedly, we further discovered that this enzyme has alternative positional specificity (5/6-kinase activity) towards a different substrate, namely Ins(1,3,4)P(3). Kinetic data from a recombinant enzyme indicate that Ins(1,3,4)P(3) (K(m)=0.3 microM; V(max)=320 pmol/min per microg) and Ins(3,4,5,6)P(4) (K(m)=0.1 microM; V(max)=780 pmol/min per microg) actively compete for phosphorylation in vivo. This competition empowers the kinase with multitasking capability in several key aspects of inositol phosphate signalling.

  15. Optochemical Activation of Kinase Function in Live Cells

    PubMed Central

    Karginov, Andrei V.; Hahn, Klaus M.; Deiters, Alexander

    2015-01-01

    Summary Manipulation of protein kinase activity is widely used to dissect signaling pathways controlling physiological and pathological processes. Common methods often cannot provide the desired spatial and temporal resolution in control of kinase activity. Regulation of kinase activity by photocaged kinase inhibitors has been successfully used to achieve tight temporal and local control, but inhibitors are limited to inactivation of kinases, and often do not provide the desired specificity. Here we report detailed methods for light-mediated activation of kinases in living cells using engineered rapamycin-regulated kinases (RapR-kinases) in conjunction with a photocaged analog of rapamycin. PMID:24718793

  16. A MAP kinase gene, BMK1, is required for conidiation and pathogenicity in the rice leaf spot pathogen Bipolaris oryzae.

    PubMed

    Moriwaki, Akihiro; Kihara, Junichi; Mori, Chie; Arase, Sakae

    2007-01-01

    We isolated and characterized BMK1, a gene encoding a mitogen-activated protein kinase (MAPK), from the rice leaf spot pathogen Bipolaris oryzae. The deduced amino acid sequence showed significant homology with Fus3/Kss1 MAPK homologues from other phytopathogenic fungi. The BMK1 disruptants showed impaired hyphal growth, no conidial production, and loss of virulence against rice leaves, indicating that the BMK1 is essential for conidiation and pathogenicity in B. oryzae. PMID:16546358

  17. Insulin-induced Drosophila S6 kinase activation requires phosphoinositide 3-kinase and protein kinase B.

    PubMed Central

    Lizcano, Jose M; Alrubaie, Saif; Kieloch, Agnieszka; Deak, Maria; Leevers, Sally J; Alessi, Dario R

    2003-01-01

    An important mechanism by which insulin regulates cell growth and protein synthesis is through activation of the p70 ribosomal S6 protein kinase (S6K). In mammalian cells, insulin-induced PI3K (phosphoinositide 3-kinase) activation, generates the lipid second messenger PtdIns(3,4,5) P (3), which is thought to play a key role in triggering the activation of S6K. Although the major components of the insulin-signalling pathway are conserved in Drosophila, recent studies suggested that S6K activation does not require PI3K in this system. To investigate further the role of dPI3K (Drosophila PI3K) in dS6K (Drosophila S6K) activation, we examined the effect of two structurally distinct PI3K inhibitors on insulin-induced dS6K activation in Kc167 and S2 Drosophila cell lines. We found that both inhibitors prevented insulin-stimulated phosphorylation and activation of dS6K. To investigate further the role of the dPI3K pathway in regulating dS6K activation, we also used dsRNAi (double-stranded RNA-mediated interference) to decrease expression of dPI3K and the PtdIns(3,4,5) P (3) phosphatase dPTEN ( Drosophila phosphatase and tensin homologue deleted on chromosome 10) in Kc167 and S2 cells. Knock-down of dPI3K prevented dS6K activation, whereas knock-down of dPTEN, which would be expected to increase PtdIns(3,4,5) P (3) levels, stimulated dS6K activity. Moreover, when the expression of the dPI3K target, dPKB (Drosophila protein kinase B), was decreased to undetectable levels, we found that insulin could no longer trigger dS6K activation. This observation provides the first direct demonstration that dPKB is required for insulin-stimulated dS6K activation. We also present evidence that the amino-acid-induced activation of dS6K in the absence of insulin, thought to be mediated by dTOR (Drosophila target of rapamycin), which is unaffected by the inhibition of dPI3K by wortmannin. The results of the present study support the view that, in Drosophila cells, dPI3K and dPKB, as well d

  18. GABAergic mechanisms regulated by miR-33 encode state-dependent fear

    PubMed Central

    Jovasevic, Vladimir; Corcoran, Kevin A; Leaderbrand, Katherine; Yamawaki, Naoki; Guedea, Anita L; Chen, Helen J; Shepherd, Gordon M G; Radulovic, Jelena

    2016-01-01

    Fear-inducing memories can be state dependent, meaning that they can best be retrieved if the brain states at encoding and retrieval are similar. Restricted access to such memories can present a risk for psychiatric disorders and hamper their treatment. To better understand the mechanisms underlying state-dependent fear, we used a mouse model of contextual fear conditioning. We found that heightened activity of hippocampal extrasynaptic GABAA receptors, believed to impair fear and memory, actually enabled their state-dependent encoding and retrieval. This effect required protein kinase C-βII and was influenced by miR-33, a microRNA that regulates several GABA-related proteins. In the extended hippocampal circuit, extrasynaptic GABAA receptors promoted subcortical, but impaired cortical, activation during memory encoding of context fear. Moreover, suppression of retrosplenial cortical activity, which normally impairs retrieval, had an enhancing effect on the retrieval of state-dependent fear. These mechanisms can serve as treatment targets for managing access to state-dependent memories of stressful experiences. PMID:26280760

  19. Saccharomyces Cerevisiae Hoc1, a Suppressor of Pkc1, Encodes a Putative Glycosyltransferase

    PubMed Central

    Neiman, A. M.; Mhaiskar, V.; Manus, V.; Galibert, F.; Dean, N.

    1997-01-01

    The Saccharomyces cerevisiae gene PKC1 encodes a protein kinase C isozyme that regulates cell wall synthesis. Here we describe the characterization of HOC1, a gene identified by its ability to suppress the cell lysis phenotype of pkc1-371 cells. The HOC1 gene (Homologous to OCH1) is predicted to encode a type II integral membrane protein that strongly resembles Och1p, an α-1,6-mannosyltransferase. Immunofluorescence studies localized Hoc1p to the Golgi apparatus. While overexpression of HOC1 rescued the pkc1-371 temperature-sensitive cell lysis phenotype, disruption of HOC1 lowered the restrictive temperature of the pkc1-371 allele. Disruption of HOC1 also resulted in hypersensitivity to Calcofluor White and hygromycin B, phenotypes characteristic of defects in cell wall integrity and protein glycosylation, respectively. The function of HOC1 appears to be distinct from that of OCH1. Taken together, these results suggest that HOC1 encodes a Golgi-localized putative mannosyltransferase required for the proper construction of the cell wall. PMID:9055074

  20. The lin-15 locus encodes two negative regulators of Caenorhabditis elegans vulval development.

    PubMed Central

    Huang, L S; Tzou, P; Sternberg, P W

    1994-01-01

    During Caenorhabditis elegans vulval development, an inductive signal from the anchor cell stimulates three of the six vulval precursor cells (VPCs) to adopt vulval rather than nonvulval epidermal fates. Genes necessary for this induction include the lin-3 growth factor, the let-23 receptor tyrosine kinase, and let-60 ras. lin-15 is a negative regulator of this inductive pathway. In lin-15 mutant animals, all six VPCs adopt vulval fates, even in the absence of inductive signal. Previous genetic studies suggested that lin-15 is a complex locus with two independently mutable activities, A and B. We have cloned the lin-15 locus by germline transformation and find that it encodes two nonoverlapping transcripts that are transcribed in the same direction. The downstream transcript encodes the lin-15A function; the upstream transcript encodes the lin-15B function. The predicted lin-15A and lin-15B proteins are novel and hydrophilic. We have identified a molecular null allele of lin-15 and have used it to analyze the role of lin-15 in the signaling pathway. We find that lin-15 acts upstream of let-23 and in parallel to the inductive signal. Images PMID:8054684

  1. Cell cycle-dependent localization of casein kinase I to mitotic spindles.

    PubMed Central

    Brockman, J L; Gross, S D; Sussman, M R; Anderson, R A

    1992-01-01

    Casein kinase I (CKI) is a class of protein kinases ubiquitous to all eukaryotic cells. Recently, cDNA clones encoding several bovine CKI isoforms have been sequenced that show high sequence identity to the HRR25 gene product of the budding yeast Saccharomyces cerevisiae; HRR25 is required for normal cellular growth, nuclear segregation, DNA repair, and meiosis. We have raised polyclonal antibodies to a human erythroid 34-kDa CKI and have sequenced a portion of this kinase. The amino acid sequence identifies the CKI as the alpha-CKI isoform, which is 62% identical to the HRR25 protein kinase. By use of immunofluorescence, the alpha-CKI has been localized to vesicular cytosolic structures and to the centrosome in interphase cells. As cells progress into mitosis, centrospheric staining increases and, in mitosis, alpha-CKI associates with kinetochore fibers. This localization suggests that alpha-CKI, like HRR25, plays a role in the segregation of chromosomes during mitosis and may be cell cycle-regulated both in humans and in yeast. Images PMID:1409656

  2. Kinase-KCC2 coupling: Cl- rheostasis, disease susceptibility, therapeutic target.

    PubMed

    Kahle, Kristopher T; Delpire, Eric

    2016-01-01

    The intracellular concentration of Cl(-) ([Cl(-)]i) in neurons is a highly regulated variable that is established and modulated by the finely tuned activity of the KCC2 cotransporter. Despite the importance of KCC2 for neurophysiology and its role in multiple neuropsychiatric diseases, our knowledge of the transporter's regulatory mechanisms is incomplete. Recent studies suggest that the phosphorylation state of KCC2 at specific residues in its cytoplasmic COOH terminus, such as Ser940 and Thr906/Thr1007, encodes discrete levels of transporter activity that elicit graded changes in neuronal Cl(-) extrusion to modulate the strength of synaptic inhibition via Cl(-)-permeable GABAA receptors. In this review, we propose that the functional and physical coupling of KCC2 to Cl(-)-sensitive kinase(s), such as the WNK1-SPAK kinase complex, constitutes a molecular "rheostat" that regulates [Cl(-)]i and thereby influences the functional plasticity of GABA. The rapid reversibility of (de)phosphorylation facilitates regulatory precision, and multisite phosphorylation allows for the control of KCC2 activity by different inputs via distinct or partially overlapping upstream signaling cascades that may become more or less important depending on the physiological context. While this adaptation mechanism is highly suited to maintaining homeostasis, its adjustable set points may render it vulnerable to perturbation and dysregulation. Finally, we suggest that pharmacological modulation of this kinase-KCC2 rheostat might be a particularly efficacious strategy to enhance Cl(-) extrusion and therapeutically restore GABA inhibition. PMID:26510764

  3. Arabidopsis MAP kinase 4 regulates gene expression through transcription factor release in the nucleus.

    PubMed

    Qiu, Jin-Long; Fiil, Berthe Katrine; Petersen, Klaus; Nielsen, Henrik Bjørn; Botanga, Christopher J; Thorgrimsen, Stephan; Palma, Kristoffer; Suarez-Rodriguez, Maria Cristina; Sandbech-Clausen, Signe; Lichota, Jacek; Brodersen, Peter; Grasser, Klaus D; Mattsson, Ole; Glazebrook, Jane; Mundy, John; Petersen, Morten

    2008-08-20

    Plant and animal perception of microbes through pathogen surveillance proteins leads to MAP kinase signalling and the expression of defence genes. However, little is known about how plant MAP kinases regulate specific gene expression. We report that, in the absence of pathogens, Arabidopsis MAP kinase 4 (MPK4) exists in nuclear complexes with the WRKY33 transcription factor. This complex depends on the MPK4 substrate MKS1. Challenge with Pseudomonas syringae or flagellin leads to the activation of MPK4 and phosphorylation of MKS1. Subsequently, complexes with MKS1 and WRKY33 are released from MPK4, and WRKY33 targets the promoter of PHYTOALEXIN DEFICIENT3 (PAD3) encoding an enzyme required for the synthesis of antimicrobial camalexin. Hence, wrky33 mutants are impaired in the accumulation of PAD3 mRNA and camalexin production upon infection. That WRKY33 is an effector of MPK4 is further supported by the suppression of PAD3 expression in mpk4-wrky33 double mutant backgrounds. Our data establish direct links between MPK4 and innate immunity and provide an example of how a plant MAP kinase can regulate gene expression by releasing transcription factors in the nucleus upon activation.

  4. Perspectives for the use of structural information and chemical genetics to develop inhibitors of Janus kinases

    PubMed Central

    Haan, Claude; Behrmann, Iris; Haan, Serge

    2010-01-01

    Abstract Gain-of-function mutations in the genes encoding Janus kinases have been discovered in various haematologic diseases. Jaks are composed of a FERM domain, an SH2 domain, a pseudokinase domain and a kinase domain, and a complex interplay of the Jak domains is involved in regulation of catalytic activity and association to cytokine receptors. Most activating mutations are found in the pseudokinase domain. Here we present recently discovered mutations in the context of our structural models of the respective domains. We describe two structural hotspots in the pseudokinase domain of Jak2 that seem to be associated either to myeloproliferation or to lymphoblastic leukaemia, pointing at the involvement of distinct signalling complexes in these disease settings. The different domains of Jaks are discussed as potential drug targets. We present currently available inhibitors targeting Jaks and indicate structural differences in the kinase domains of the different Jaks that may be exploited in the development of specific inhibitors. Moreover, we discuss recent chemical genetic approaches which can be applied to Jaks to better understand the role of these kinases in their biological settings and as drug targets. PMID:20132407

  5. Specific factors are required for kinase-dependent endocytosis of insulin receptors.

    PubMed Central

    Welsh, J B; Worthylake, R; Wiley, H S; Gill, G N

    1994-01-01

    Mouse B82 cells that support high affinity saturable endocytosis of epidermal growth factor receptors (EGFR) exhibited only low rates of nonsaturable internalization of insulin receptors (InsR). To investigate the defect in endocytosis of InsR in B82 cells, we examined the role of sequence motifs and tyrosine kinase, the two receptor components shown to be required for efficient saturable endocytosis of InsR in Rat 1 cells. Placement of residues encoded by exon 16 of the InsR onto an EGFR truncated to residue 958 restored EGF-induced internalization of this mutant receptor indicating that the sequence codes in exon 16 are recognized by B82 cells. To determine whether the kinase function could be provided in trans, a B82 cell expressing both receptors was established. EGF-activated EGFR kinase was not able to restore insulin-dependent rapid endocytosis to InsR. However, fusion of untransfected Rat1 cells with InsR-expressing B82 cells enabled rapid endocytosis of InsR, indicating that the internalization defect can be complemented. These results indicate that, although internalization codes can function in the context of other receptors, activation of tyrosine kinase receptors requires an additional specific component. Images PMID:7919536

  6. Targeting substrate-site in Jak2 kinase prevents emergence of genetic resistance

    PubMed Central

    Kesarwani, Meenu; Huber, Erika; Kincaid, Zachary; Evelyn, Chris R.; Biesiada, Jacek; Rance, Mark; Thapa, Mahendra B.; Shah, Neil P.; Meller, Jarek; Zheng, Yi; Azam, Mohammad

    2015-01-01

    Emergence of genetic resistance against kinase inhibitors poses a great challenge for durable therapeutic response. Here, we report a novel mechanism of JAK2 kinase inhibition by fedratinib (TG101348) that prevents emergence of genetic resistance. Using in vitro drug screening, we identified 211 amino-acid substitutions conferring resistance to ruxolitinib (INCB018424) and cross-resistance to the JAK2 inhibitors AZD1480, CYT-387 and lestaurtinib. In contrast, these resistant variants were fully sensitive to fedratinib. Structural modeling, coupled with mutagenesis and biochemical studies, revealed dual binding sites for fedratinib. In vitro binding assays using purified proteins showed strong affinity for the substrate-binding site (Kd = 20 nM) while affinity for the ATP site was poor (Kd = ~8 μM). Our studies demonstrate that mutations affecting the substrate-binding pocket encode a catalytically incompetent kinase, thereby preventing emergence of resistant variants. Most importantly, our data suggest that in order to develop resistance-free kinase inhibitors, the next-generation drug design should target the substrate-binding site. PMID:26419724

  7. Trypanosoma cruzi arginine kinase characterization and cloning. A novel energetic pathway in protozoan parasites.

    PubMed

    Pereira, C A; Alonso, G D; Paveto, M C; Iribarren, A; Cabanas, M L; Torres, H N; Flawiá, M M

    2000-01-14

    This work contains the first description of a guanidino kinase in a flagellar unicellular parasite. The enzyme phosphorylates L-arginine and was characterized in preparations from Trypanosoma cruzi, the ethiological agent of Chagas' disease. The activity requires ATP and a divalent cation. Under standard assay conditions (1 mM L-arginine), the presence of 5-fold higher concentrations of canavanine or histidine produced a greater than 50% enzyme inhibition. The base sequence of this enzyme revealed an open reading frame of 357 amino acids and a molecular weight of 40,201. The amino acid sequence shows all of the characteristic consensus blocks of the ATP:guanidino phosphotransferase family and a putative "actinin-type" actin-binding domain. The highest amino acid identities of the T. cruzi sequence, about 70%, were with arginine kinases from Arthropoda. Southern and chromosome blots revealed that the kinase is encoded by a single-copy gene. Moreover, Northern blot analysis showed an mRNA subpopulation of about 2.0 kilobases, and Western blotting of T. cruzi-soluble polypeptides revealed a 40-kDa band. The finding in the parasite of a phosphagen and its biosynthetic pathway, which are totally different from those in mammalian host tissues, points out this arginine kinase as a possible chemotherapy target for Chagas' disease. PMID:10625703

  8. Leishmania donovani Encodes a Functional Selenocysteinyl-tRNA Synthase.

    PubMed

    Manhas, Reetika; Gowri, Venkatraman Subramanian; Madhubala, Rentala

    2016-01-15

    The synthesis of selenocysteine, the 21st amino acid, occurs on its transfer RNA (tRNA), tRNA(Sec). tRNA(Sec) is initially aminoacylated with serine by seryl-tRNA synthetase and the resulting seryl moiety is converted to phosphoserine by O-phosphoseryl-tRNA kinase (PSTK) in eukaryotes. The selenium donor, selenophosphate is synthesized from selenide and ATP by selenophosphate synthetase. Selenocysteinyl-tRNA synthase (SepSecS) then uses the O-phosphoseryl-tRNA(Sec) and selenophosphate to form Sec-tRNA(Sec) in eukaryotes. Here, we report the characterization of selenocysteinyl-tRNA synthase from Leishmania donovani. Kinetoplastid SepSecS enzymes are phylogenetically closer to worm SepSecS. LdSepSecS was found to exist as a tetramer. Leishmania SepSecS enzyme was found to be active and able to complement the ΔselA deletion in Escherichia coli JS1 strain only in the presence of archaeal PSTK, indicating the conserved nature of the PSTK-SepSecS pathway. LdSepSecS was found to localize in the cytoplasm of the parasite. Gene deletion studies indicate that Leishmania SepSecS is dispensable for the parasite survival. The parasite was found to encode three selenoproteins, which were only expressed in the presence of SepSecS. Selenoproteins of L. donovani are not required for the growth of the promastigotes. Auranofin, a known inhibitor of selenoprotein synthesis showed the same sensitivity toward the wild-type and null mutants suggesting its effect is not through binding to selenoproteins. The three-dimensional structural comparison indicates that human and Leishmania homologs are structurally highly similar but their association modes leading to tetramerization seem different. PMID:26586914

  9. Identification of a dual-specificity protein phosphatase that inactivates a MAP kinase from Arabidopsis

    NASA Technical Reports Server (NTRS)

    Gupta, R.; Huang, Y.; Kieber, J.; Luan, S.; Evans, M. L. (Principal Investigator)

    1998-01-01

    Mitogen-activated protein kinases (MAPKs) play a key role in plant responses to stress and pathogens. Activation and inactivation of MAPKs involve phosphorylation and dephosphorylation on both threonine and tyrosine residues in the kinase domain. Here we report the identification of an Arabidopsis gene encoding a dual-specificity protein phosphatase capable of hydrolysing both phosphoserine/threonine and phosphotyrosine in protein substrates. This enzyme, designated AtDsPTP1 (Arabidopsis thaliana dual-specificity protein tyrosine phosphatase), dephosphorylated and inactivated AtMPK4, a MAPK member from the same plant. Replacement of a highly conserved cysteine by serine abolished phosphatase activity of AtDsPTP1, indicating a conserved catalytic mechanism of dual-specificity protein phosphatases from all eukaryotes.

  10. Anaplastic Lymphoma Kinase Acts in the Drosophila Mushroom Body to Negatively Regulate Sleep.

    PubMed

    Bai, Lei; Sehgal, Amita

    2015-11-01

    Though evidence is mounting that a major function of sleep is to maintain brain plasticity and consolidate memory, little is known about the molecular pathways by which learning and sleep processes intercept. Anaplastic lymphoma kinase (Alk), the gene encoding a tyrosine receptor kinase whose inadvertent activation is the cause of many cancers, is implicated in synapse formation and cognitive functions. In particular, Alk genetically interacts with Neurofibromatosis 1 (Nf1) to regulate growth and associative learning in flies. We show that Alk mutants have increased sleep. Using a targeted RNAi screen we localized the negative effects of Alk on sleep to the mushroom body, a structure important for both sleep and memory. We also report that mutations in Nf1 produce a sexually dimorphic short sleep phenotype, and suppress the long sleep phenotype of Alk. Thus Alk and Nf1 interact in both learning and sleep regulation, highlighting a common pathway in these two processes. PMID:26536237

  11. Function and evolution of 'green' GSK3/Shaggy-like kinases.

    PubMed

    Saidi, Younousse; Hearn, Timothy J; Coates, Juliet C

    2012-01-01

    Glycogen synthase kinase 3 (GSK3) proteins, also known as SHAGGY-like kinases, have many important cell signalling roles in animals, fungi and amoebae. In particular, GSK3s participate in key developmental signalling pathways and also regulate the cytoskeleton. GSK3-encoding genes are also present in all land plants and in algae and protists, raising questions about possible ancestral functions in eukaryotes. Recent studies have revealed that plant GSK3 proteins are actively implicated in hormonal signalling networks during development as well as in biotic and abiotic stress responses. In this review, we outline the mechanisms of Arabidopsis GSK3 action, summarize GSK3 functions in dicot and monocot flowering plants, and speculate on the possible functions of GSK3s in the earliest-evolving land plants.

  12. A cloned ATP:guanidino kinase in the trematode Schistosoma mansoni has a novel duplicated structure.

    PubMed

    Stein, L D; Harn, D A; David, J R

    1990-04-25

    Creatine kinase (CK) is part of a conserved family of ATP:guanidino phosphotransferases whose members play important roles in intracellular energy flow. Previously characterized members of this family are approximately 80-kDa dimers of two related 40-kDa subunits. We have cloned a gene from the parasitic trematode Schistosoma mansoni which has substantial amino acid sequence similarities to CK. Like the genes for vertebrate CKs, this gene is developmentally regulated; mRNA levels are high in the infective cercarial stage but rapidly decrease upon transformation to the parasitic schistosomulum stage. In contrast to members of the guanidino phosphotransferase family characterized previously, however, the schistosome gene appears to be a direct fusion of two CK-like domains that encode a single 74-kDa polypeptide. Correlative evidence from enzyme assays of crude parasite homogenates suggests that the cloned gene is a creatine kinase. This represents the first molecular cloning of an invertebrate ATP:guanidino phosphotransferase.

  13. GRID2 a novel gene possibly associated with mevalonate kinase deficiency.

    PubMed

    Moura, Ronald; Tricarico, Paola Maura; Campos Coelho, Antonio Victor; Crovella, Sergio

    2015-04-01

    Mevalonate kinase deficiency (MKD) is a rare autosomal disease caused by mutations in the mevalonate kinase gene (MVK). The genotype-phenotype correlation is sometimes problematic due to the great genetic and clinical heterogeneity; so we hypothesize that genes other than MVK are able to modulate MKD clinical phenotypes. This hypothesis was tested by analyzing the exome of 22 patients with MKD all carrying MVK gene mutations, and 20 patients with recurrent fevers (RF) not carrying MVK mutations. Our preliminary findings suggest a possible role of GRID2 in the susceptibility to develop MKD. GRID2 gene (4q22.2), encoding for human glutamate receptor delta-2, associated with MKD: The rs1450500 SNP was differently distributed in patients with MKD with respect to those with RF. Being aware of the small number of patients analyzed, we hypothesized a possible role for GRID2 as possible phenotype modifier in MKD patients, especially in those with severe phenotypes.

  14. Positional signaling mediated by a receptor-like kinase in Arabidopsis.

    PubMed

    Kwak, Su-Hwan; Shen, Ronglai; Schiefelbein, John

    2005-02-18

    The position-dependent specification of root epidermal cells in Arabidopsis provides an elegant paradigm for cell patterning during development. Here, we describe a new gene, SCRAMBLED (SCM), required for cells to appropriately interpret their location within the developing root epidermis. SCM encodes a receptor-like kinase protein with a predicted extracellular domain of six leucine-rich repeats and an intracellular serine-threonine kinase domain. SCM regulates the expression of the GLABRA2, CAPRICE, WEREWOLF, and ENHANCER OF GLABRA3 transcription factor genes that define the cell fates. Further, the SCM gene is expressed throughout the developing root. Therefore, SCM likely enables developing epidermal cells to detect positional cues and establish an appropriate cell-type pattern.

  15. Temporal information encoding in dynamic memristive devices

    NASA Astrophysics Data System (ADS)

    Ma, Wen; Chen, Lin; Du, Chao; Lu, Wei D.

    2015-11-01

    We show temporal and frequency information can be effectively encoded in memristive devices with inherent short-term dynamics. Ag/Ag2S/Pd based memristive devices with low programming voltage (˜100 mV) were fabricated and tested. At weak programming conditions, the devices exhibit inherent decay due to spontaneous diffusion of the Ag atoms. When the devices were subjected to pulse train inputs emulating different spiking patterns, the switching probability distribution function diverges from the standard Poisson distribution and evolves according to the input pattern. The experimentally observed switching probability distributions and the associated cumulative probability functions can be well-explained using a model accounting for the short-term decay effects. Such devices offer an intriguing opportunity to directly encode neural signals for neural information storage and analysis.

  16. Storing data encoded DNA in living organisms

    DOEpatents

    Wong; Pak C. , Wong; Kwong K. , Foote; Harlan P.

    2006-06-06

    Current technologies allow the generation of artificial DNA molecules and/or the ability to alter the DNA sequences of existing DNA molecules. With a careful coding scheme and arrangement, it is possible to encode important information as an artificial DNA strand and store it in a living host safely and permanently. This inventive technology can be used to identify origins and protect R&D investments. It can also be used in environmental research to track generations of organisms and observe the ecological impact of pollutants. Today, there are microorganisms that can survive under extreme conditions. As well, it is advantageous to consider multicellular organisms as hosts for stored information. These living organisms can provide as memory housing and protection for stored data or information. The present invention provides well for data storage in a living organism wherein at least one DNA sequence is encoded to represent data and incorporated into a living organism.

  17. Genetically encoded fluorescent sensors of membrane potential

    PubMed Central

    Baker, B. J.; Mutoh, H.; Dimitrov, D.; Akemann, W.; Perron, A.; Iwamoto, Y.; Jin, L.; Cohen, L. B.; Isacoff, E. Y.; Pieribone, V. A.; Hughes, T.; Knöpfel, T.

    2009-01-01

    Imaging activity of neurons in intact brain tissue was conceived several decades ago and, after many years of development, voltage-sensitive dyes now offer the highest spatial and temporal resolution for imaging neuronal functions in the living brain. Further progress in this field is expected from the emergent development of genetically encoded fluorescent sensors of membrane potential. These fluorescent protein (FP) voltage sensors overcome the drawbacks of organic voltage sensitive dyes such as non-specificity of cell staining and the low accessibility of the dye to some cell types. In a transgenic animal, a genetically encoded sensor could in principle be expressed specifically in any cell type and would have the advantage of staining only the cell population determined by the specificity of the promoter used to drive expression. Here we critically review the current status of these developments. PMID:18679801

  18. Asymmetric synthesis using chiral-encoded metal.

    PubMed

    Yutthalekha, Thittaya; Wattanakit, Chularat; Lapeyre, Veronique; Nokbin, Somkiat; Warakulwit, Chompunuch; Limtrakul, Jumras; Kuhn, Alexander

    2016-08-26

    The synthesis of chiral compounds is of crucial importance in many areas of society and science, including medicine, biology, chemistry, biotechnology and agriculture. Thus, there is a fundamental interest in developing new approaches for the selective production of enantiomers. Here we report the use of mesoporous metal structures with encoded geometric chiral information for inducing asymmetry in the electrochemical synthesis of mandelic acid as a model molecule. The chiral-encoded mesoporous metal, obtained by the electrochemical reduction of platinum salts in the presence of a liquid crystal phase and the chiral template molecule, perfectly retains the chiral information after removal of the template. Starting from a prochiral compound we demonstrate enantiomeric excess of the (R)-enantiomer when using (R)-imprinted electrodes and vice versa for the (S)-imprinted ones. Moreover, changing the amount of chiral cavities in the material allows tuning the enantioselectivity.

  19. Nucleic acid compositions and the encoding proteins

    SciTech Connect

    Preston, III, James F.; Chow, Virginia; Nong, Guang; Rice, John D.; St. John, Franz J.

    2014-09-02

    The subject invention provides at least one nucleic acid sequence encoding an aldouronate-utilization regulon isolated from Paenibacillus sp. strain JDR-2, a bacterium which efficiently utilizes xylan and metabolizes aldouronates (methylglucuronoxylosaccharides). The subject invention also provides a means for providing a coordinately regulated process in which xylan depolymerization and product assimilation are coupled in Paenibacillus sp. strain JDR-2 to provide a favorable system for the conversion of lignocellulosic biomass to biobased products. Additionally, the nucleic acid sequences encoding the aldouronate-utilization regulon can be used to transform other bacteria to form organisms capable of producing a desired product (e.g., ethanol, 1-butanol, acetoin, 2,3-butanediol, 1,3-propanediol, succinate, lactate, acetate, malate or alanine) from lignocellulosic biomass.

  20. Asymmetric synthesis using chiral-encoded metal.

    PubMed

    Yutthalekha, Thittaya; Wattanakit, Chularat; Lapeyre, Veronique; Nokbin, Somkiat; Warakulwit, Chompunuch; Limtrakul, Jumras; Kuhn, Alexander

    2016-01-01

    The synthesis of chiral compounds is of crucial importance in many areas of society and science, including medicine, biology, chemistry, biotechnology and agriculture. Thus, there is a fundamental interest in developing new approaches for the selective production of enantiomers. Here we report the use of mesoporous metal structures with encoded geometric chiral information for inducing asymmetry in the electrochemical synthesis of mandelic acid as a model molecule. The chiral-encoded mesoporous metal, obtained by the electrochemical reduction of platinum salts in the presence of a liquid crystal phase and the chiral template molecule, perfectly retains the chiral information after removal of the template. Starting from a prochiral compound we demonstrate enantiomeric excess of the (R)-enantiomer when using (R)-imprinted electrodes and vice versa for the (S)-imprinted ones. Moreover, changing the amount of chiral cavities in the material allows tuning the enantioselectivity. PMID:27562028

  1. DNA-Encoded Dynamic Combinatorial Chemical Libraries.

    PubMed

    Reddavide, Francesco V; Lin, Weilin; Lehnert, Sarah; Zhang, Yixin

    2015-06-26

    Dynamic combinatorial chemistry (DCC) explores the thermodynamic equilibrium of reversible reactions. Its application in the discovery of protein binders is largely limited by difficulties in the analysis of complex reaction mixtures. DNA-encoded chemical library (DECL) technology allows the selection of binders from a mixture of up to billions of different compounds; however, experimental results often show low a signal-to-noise ratio and poor correlation between enrichment factor and binding affinity. Herein we describe the design and application of DNA-encoded dynamic combinatorial chemical libraries (EDCCLs). Our experiments have shown that the EDCCL approach can be used not only to convert monovalent binders into high-affinity bivalent binders, but also to cause remarkably enhanced enrichment of potent bivalent binders by driving their in situ synthesis. We also demonstrate the application of EDCCLs in DNA-templated chemical reactions.

  2. Genetically Encoded Voltage Indicators in Circulation Research

    PubMed Central

    Kaestner, Lars; Tian, Qinghai; Kaiser, Elisabeth; Xian, Wenying; Müller, Andreas; Oberhofer, Martin; Ruppenthal, Sandra; Sinnecker, Daniel; Tsutsui, Hidekazu; Miyawaki, Atsushi; Moretti, Alessandra; Lipp, Peter

    2015-01-01

    Membrane potentials display the cellular status of non-excitable cells and mediate communication between excitable cells via action potentials. The use of genetically encoded biosensors employing fluorescent proteins allows a non-invasive biocompatible way to read out the membrane potential in cardiac myocytes and other cells of the circulation system. Although the approaches to design such biosensors date back to the time when the first fluorescent-protein based Förster Resonance Energy Transfer (FRET) sensors were constructed, it took 15 years before reliable sensors became readily available. Here, we review different developments of genetically encoded membrane potential sensors. Furthermore, it is shown how such sensors can be used in pharmacological screening applications as well as in circulation related basic biomedical research. Potentials and limitations will be discussed and perspectives of possible future developments will be provided. PMID:26370981

  3. Asymmetric synthesis using chiral-encoded metal

    PubMed Central

    Yutthalekha, Thittaya; Wattanakit, Chularat; Lapeyre, Veronique; Nokbin, Somkiat; Warakulwit, Chompunuch; Limtrakul, Jumras; Kuhn, Alexander

    2016-01-01

    The synthesis of chiral compounds is of crucial importance in many areas of society and science, including medicine, biology, chemistry, biotechnology and agriculture. Thus, there is a fundamental interest in developing new approaches for the selective production of enantiomers. Here we report the use of mesoporous metal structures with encoded geometric chiral information for inducing asymmetry in the electrochemical synthesis of mandelic acid as a model molecule. The chiral-encoded mesoporous metal, obtained by the electrochemical reduction of platinum salts in the presence of a liquid crystal phase and the chiral template molecule, perfectly retains the chiral information after removal of the template. Starting from a prochiral compound we demonstrate enantiomeric excess of the (R)-enantiomer when using (R)-imprinted electrodes and vice versa for the (S)-imprinted ones. Moreover, changing the amount of chiral cavities in the material allows tuning the enantioselectivity. PMID:27562028

  4. Genetically Encoded Voltage Indicators in Circulation Research.

    PubMed

    Kaestner, Lars; Tian, Qinghai; Kaiser, Elisabeth; Xian, Wenying; Müller, Andreas; Oberhofer, Martin; Ruppenthal, Sandra; Sinnecker, Daniel; Tsutsui, Hidekazu; Miyawaki, Atsushi; Moretti, Alessandra; Lipp, Peter

    2015-09-08

    Membrane potentials display the cellular status of non-excitable cells and mediate communication between excitable cells via action potentials. The use of genetically encoded biosensors employing fluorescent proteins allows a non-invasive biocompatible way to read out the membrane potential in cardiac myocytes and other cells of the circulation system. Although the approaches to design such biosensors date back to the time when the first fluorescent-protein based Förster Resonance Energy Transfer (FRET) sensors were constructed, it took 15 years before reliable sensors became readily available. Here, we review different developments of genetically encoded membrane potential sensors. Furthermore, it is shown how such sensors can be used in pharmacological screening applications as well as in circulation related basic biomedical research. Potentials and limitations will be discussed and perspectives of possible future developments will be provided.

  5. Asymmetric synthesis using chiral-encoded metal

    NASA Astrophysics Data System (ADS)

    Yutthalekha, Thittaya; Wattanakit, Chularat; Lapeyre, Veronique; Nokbin, Somkiat; Warakulwit, Chompunuch; Limtrakul, Jumras; Kuhn, Alexander

    2016-08-01

    The synthesis of chiral compounds is of crucial importance in many areas of society and science, including medicine, biology, chemistry, biotechnology and agriculture. Thus, there is a fundamental interest in developing new approaches for the selective production of enantiomers. Here we report the use of mesoporous metal structures with encoded geometric chiral information for inducing asymmetry in the electrochemical synthesis of mandelic acid as a model molecule. The chiral-encoded mesoporous metal, obtained by the electrochemical reduction of platinum salts in the presence of a liquid crystal phase and the chiral template molecule, perfectly retains the chiral information after removal of the template. Starting from a prochiral compound we demonstrate enantiomeric excess of the (R)-enantiomer when using (R)-imprinted electrodes and vice versa for the (S)-imprinted ones. Moreover, changing the amount of chiral cavities in the material allows tuning the enantioselectivity.

  6. Temporal information encoding in dynamic memristive devices

    SciTech Connect

    Ma, Wen; Chen, Lin; Du, Chao; Lu, Wei D.

    2015-11-09

    We show temporal and frequency information can be effectively encoded in memristive devices with inherent short-term dynamics. Ag/Ag{sub 2}S/Pd based memristive devices with low programming voltage (∼100 mV) were fabricated and tested. At weak programming conditions, the devices exhibit inherent decay due to spontaneous diffusion of the Ag atoms. When the devices were subjected to pulse train inputs emulating different spiking patterns, the switching probability distribution function diverges from the standard Poisson distribution and evolves according to the input pattern. The experimentally observed switching probability distributions and the associated cumulative probability functions can be well-explained using a model accounting for the short-term decay effects. Such devices offer an intriguing opportunity to directly encode neural signals for neural information storage and analysis.

  7. Phosphorylation of AfsR by multiple serine/threonine kinases in Streptomyces coelicolor A3(2).

    PubMed

    Sawai, Reiko; Suzuki, Ayano; Takano, Yuji; Lee, Ping-Chin; Horinouchi, Sueharu

    2004-06-01

    AfsK, a protein serine/threonine kinase, autophosphorylates on serine and threonine residues and phosphorylates serine and threonine residues of AfsR, a transcriptional activator for afsS involved in secondary metabolism in Streptomyces coelicolor A3(2). pkaG encoding a 592-amino-acid protein and SCD10.09 (named afsL) encoding a 580-amino-acid protein, both of which encode an AfsK-like protein, were transcribed throughout growth. PkaG with a histidine-tag and the kinase catalytic domain of PkaG, produced in Escherichia coli, autophosphorylated dominantly on threonine and slightly on serine residues. In addition, these proteins phosphorylated AfsR on threonine and serine residues. The catalytic domain of AfsL also autophosphorylated and phosphorylated AfsR, on threonine and serine residues in both cases. AfsR was thus found to be phosphorylated by multiple kinases. Disruption of the chromosomal pkaG gene resulted in slightly reduced production of the pigmented antibiotic actinorhodin. These findings, together with the presence of about 40 AfsK homologues and at least five AfsR homologues in S. coelicolor A3(2), suggest that the regulatory networks via eukaryotic-type protein phosphorylation are more diverse and versatile than we have expected.

  8. A fungal cell wall integrity-associated MAP kinase cascade in Coniothyrium minitans is required for conidiation and mycoparasitism.

    PubMed

    Zeng, Fanyun; Gong, Xiaoyan; Hamid, Mahammad Imran; Fu, Yanping; Jiatao, Xie; Cheng, Jiasen; Li, Guoqing; Jiang, Daohong

    2012-05-01

    Coniothyrium minitans is an important biocontrol agent against Sclerotinia diseases. Previously, a conidiation-deficient mutant ZS-1T1000 was screened out from a T-DNA insertional library of C. minitans. CmBCK1, encoding MAP kinase kinase kinase and homologous to BCK1 of Saccharomyces cerevisiae, was disrupted by T-DNA insertion in this mutant. Targeted disruption of CmBCK1 led to the mutants undergoing autolysis and displaying hypersensitivity to the cell wall-degrading enzymes. The △CmBCK1 mutants lost the ability to produce pycnidia and conidia compared to the wild-type strain ZS-1. △CmBCK1 mutants could grow on the surface of sclerotia of Sclerotinia sclerotiorum but not form conidia, which resulted in much lower ability to reduce the viability of sclerotia of S. sclerotiorum. Furthermore, CmSlt2, a homolog of Slt2 encoding cell wall integrity-related MAP kinase and up-regulated by BCK1 in S. cerevisiae, was identified and targeted disrupted. The △CmSlt2 mutants had a similar phenotype to the △CmBCK1 mutants. The △CmSlt2 mutants also had autolytic aerial hyphae, hypersensitivity to cell wall-degrading enzymes, lack of conidiation and reduction of sclerotial mycoparasitism. Taken together, our results suggest that CmBCK1 and CmSlt2 are involved in conidiation and the hyperparasitic activities of C. minitans. PMID:22426009

  9. An Encoding of XQuery in Prolog

    NASA Astrophysics Data System (ADS)

    Almendros-Jiménez, Jesús M.

    In this paper we describe the implementation of (a subset of) the XQuery language using logic programming (in particular, by means of Prolog). Such implementation has been developed using the Prolog interpreter SWI-Prolog. XML files are handled by means of the XML Library of SWI-Prolog. XPath/XQuery are encoded by means of Prolog rules. Such Prolog rules are executed in order to obtain the answer of the query.

  10. Gene encoding herbicide safener binding protein

    SciTech Connect

    Walton, J.D.; Scott-Craig, J.S.

    1999-10-26

    The cDNA encoding safener binding protein (SafBP), also referred to as SBP1, is presented. The deduced amino acid sequence is provided. Methods of making and using SBP1 and SafBP to alter a plant's sensitivity to certain herbicides or a plant's responsiveness to certain safeners are also provided, as well as expression vectors, transgenic plants or other organisms transfected with vectors and seeds from the plants.

  11. Population Encoding With Hodgkin–Huxley Neurons

    PubMed Central

    Lazar, Aurel A.

    2013-01-01

    The recovery of (weak) stimuli encoded with a population of Hodgkin–Huxley neurons is investigated. In the absence of a stimulus, the Hodgkin–Huxley neurons are assumed to be tonically spiking. The methodology employed calls for 1) finding an input–output (I/O) equivalent description of the Hodgkin–Huxley neuron and 2) devising a recovery algorithm for stimuli encoded with the I/O equivalent neuron(s). A Hodgkin–Huxley neuron with multiplicative coupling is I/O equivalent with an Integrate-and-Fire neuron with a variable threshold sequence. For bandlimited stimuli a perfect recovery of the stimulus can be achieved provided that a Nyquist-type rate condition is satisfied. A Hodgkin–Huxley neuron with additive coupling and deterministic conductances is first-order I/O equivalent with a Project-Integrate-and-Fire neuron that integrates a projection of the stimulus on the phase response curve. The stimulus recovery is formulated as a spline interpolation problem in the space of finite length bounded energy signals. A Hodgkin–Huxley neuron with additive coupling and stochastic conductances is shown to be first-order I/O equivalent with a Project-Integrate-and-Fire neuron with random thresholds. For stimuli modeled as elements of Sobolev spaces the reconstruction algorithm minimizes a regularized quadratic optimality criterion. Finally, all previous recovery results of stimuli encoded with Hodgkin–Huxley neurons with multiplicative and additive coupling, and deterministic and stochastic conductances are extended to stimuli encoded with a population of Hodgkin–Huxley neurons. PMID:24194625

  12. Encoded libraries of chemically modified peptides.

    PubMed

    Heinis, Christian; Winter, Greg

    2015-06-01

    The use of powerful technologies for generating and screening DNA-encoded protein libraries has helped drive the development of proteins as pharmaceutical ligands. However the development of peptides as pharmaceutical ligands has been more limited. Although encoded peptide libraries are typically several orders of magnitude larger than classical chemical libraries, can be more readily screened, and can give rise to higher affinity ligands, their use as pharmaceutical ligands is limited by their intrinsic properties. Two of the intrinsic limitations include the rotational flexibility of the peptide backbone and the limited number (20) of natural amino acids. However these limitations can be overcome by use of chemical modification. For example, the libraries can be modified to introduce topological constraints such as cyclization linkers, or to introduce new chemical entities such as small molecule ligands, fluorophores and photo-switchable compounds. This article reviews the chemistry involved, the properties of the peptide ligands, and the new opportunities offered by chemical modification of DNA-encoded peptide libraries.

  13. Absolute Position Encoders With Vertical Image Binning

    NASA Technical Reports Server (NTRS)

    Leviton, Douglas B.

    2005-01-01

    Improved optoelectronic patternrecognition encoders that measure rotary and linear 1-dimensional positions at conversion rates (numbers of readings per unit time) exceeding 20 kHz have been invented. Heretofore, optoelectronic pattern-recognition absoluteposition encoders have been limited to conversion rates <15 Hz -- too low for emerging industrial applications in which conversion rates ranging from 1 kHz to as much as 100 kHz are required. The high conversion rates of the improved encoders are made possible, in part, by use of vertically compressible or binnable (as described below) scale patterns in combination with modified readout sequences of the image sensors [charge-coupled devices (CCDs)] used to read the scale patterns. The modified readout sequences and the processing of the images thus read out are amenable to implementation by use of modern, high-speed, ultra-compact microprocessors and digital signal processors or field-programmable gate arrays. This combination of improvements makes it possible to greatly increase conversion rates through substantial reductions in all three components of conversion time: exposure time, image-readout time, and image-processing time.

  14. Neural signals encoding shifts in beliefs

    PubMed Central

    Schwartenbeck, Philipp; FitzGerald, Thomas H.B.; Dolan, Ray

    2016-01-01

    Dopamine is implicated in a diverse range of cognitive functions including cognitive flexibility, task switching, signalling novel or unexpected stimuli as well as advance information. There is also longstanding line of thought that links dopamine with belief formation and, crucially, aberrant belief formation in psychosis. Integrating these strands of evidence would suggest that dopamine plays a central role in belief updating and more specifically in encoding of meaningful information content in observations. The precise nature of this relationship has remained unclear. To directly address this question we developed a paradigm that allowed us to decompose two distinct types of information content, information-theoretic surprise that reflects the unexpectedness of an observation, and epistemic value that induces shifts in beliefs or, more formally, Bayesian surprise. Using functional magnetic-resonance imaging in humans we show that dopamine-rich midbrain regions encode shifts in beliefs whereas surprise is encoded in prefrontal regions, including the pre-supplementary motor area and dorsal cingulate cortex. By linking putative dopaminergic activity to belief updating these data provide a link to false belief formation that characterises hyperdopaminergic states associated with idiopathic and drug induced psychosis. PMID:26520774

  15. Dual-channel spectrally encoded endoscopic probe

    PubMed Central

    Engel, Guy; Genish, Hadar; Rosenbluh, Michael; Yelin, Dvir

    2012-01-01

    High quality imaging through sub-millimeter endoscopic probes provides clinicians with valuable diagnostics capabilities in hard to reach locations within the body. Spectrally encoded endoscopy (SEE) has been shown promising for such task; however, challenging probe fabrication and high speckle noise had prevented its testing in in vivo studies. Here we demonstrate a novel miniature SEE probe which incorporates some of the recent progress in spectrally encoded technology into a compact and robust endoscopic system. A high-quality miniature diffraction grating was fabricated using automated femtosecond laser cutting from a large bulk grating. Using one spectrally encoded channel for imaging and a separate channel for incoherent illumination, the new system has large depth of field, negligible back reflections and well controlled speckle noise which depends on the core diameter of the illumination fiber. Moreover, by using a larger imaging channel, higher groove density grating, shorter wavelength and broader spectrum, the new endoscopic system now allow significant improvements in almost all imaging parameter compared to previous systems, through an ultra-miniature endoscopic probe. PMID:22876349

  16. Virus-encoded microRNAs

    PubMed Central

    Grundhoff, Adam; Sullivan, Christopher S.

    2011-01-01

    microRNAs (miRNAs) are the subject of enormous interest. They are small non-coding RNAs that play a regulatory role in numerous and diverse cellular processes such as immune function, apoptosis and tumorigenesis. Several virus families have been shown to encode miRNAs, and an appreciation for their roles in the viral infectious cycle continues to grow. Despite the identification of numerous (>225) viral miRNAs, an in depth functional understanding of most virus-encoded miRNAs is lacking. Here we focus on a few viral miRNAs with well-defined functions. We use these examples to extrapolate general themes of viral miRNA activities including autoregulation of gene expression, avoidance of host defenses, and a likely important role in maintaining latent and persistent infections. We hypothesize that although the molecular mechanisms and machinery are similar, the majority of viral miRNAs may utilize a target strategy that differs from host miRNAs. That is, many viral miRNAs may have evolved to regulate viral-encoded transcripts or networks of host genes that are unique to viral miRNAs. Included in this latter category are a likely abundant class of viral miRNAs that may regulate only one or a few principal host genes. Key steps forward for the field are discussed, including the need for additional functional studies that utilize surgical viral miRNA mutants combined with relevant models of infection. PMID:21277611

  17. Nucleic acids encoding human trithorax protein

    DOEpatents

    Evans, Glen A.; Djabali, Malek; Selleri, Licia; Parry, Pauline

    2001-01-01

    In accordance with the present invention, there is provided an isolated peptide having the characteristics of human trithorax protein (as well as DNA encoding same, antisense DNA derived therefrom and antagonists therefor). The invention peptide is characterized by having a DNA binding domain comprising multiple zinc fingers and at least 40% amino acid identity with respect to the DNA binding domain of Drosophila trithorax protein and at least 70% conserved sequence with respect to the DNA binding domain of Drosophila trithorax protein, and wherein said peptide is encoded by a gene located at chromosome 11 of the human genome at q23. Also provided are methods for the treatment of subject(s) suffering from immunodeficiency, developmental abnormality, inherited disease, or cancer by administering to said subject a therapeutically effective amount of one of the above-described agents (i.e., peptide, antagonist therefor, DNA encoding said peptide or antisense DNA derived therefrom). Also provided is a method for the diagnosis, in a subject, of immunodeficiency, developmental abnormality, inherited disease, or cancer associated with disruption of chromosome 11 at q23.

  18. Encoding and decoding messages with chaotic lasers

    SciTech Connect

    Alsing, P.M.; Gavrielides, A.; Kovanis, V.; Roy, R.; Thornburg, K.S. Jr.

    1997-12-01

    We investigate the structure of the strange attractor of a chaotic loss-modulated solid-state laser utilizing return maps based on a combination of intensity maxima and interspike intervals, as opposed to those utilizing Poincar{acute e} sections defined by the intensity maxima of the laser ({dot I}=0,{umlt I}{lt}0) alone. We find both experimentally and numerically that a simple, intrinsic relationship exists between an intensity maximum and the pair of preceding and succeeding interspike intervals. In addition, we numerically investigate encoding messages on the output of a chaotic transmitter laser and its subsequent decoding by a similar receiver laser. By exploiting the relationship between the intensity maxima and the interspike intervals, we demonstrate that the method utilized to encode the message is vital to the system{close_quote}s ability to hide the signal from unwanted deciphering. In this work alternative methods are studied in order to encode messages by modulating the magnitude of pumping of the transmitter laser and also by driving its loss modulation with more than one frequency. {copyright} {ital 1997} {ital The American Physical Society}

  19. AtVPS34, a phosphatidylinositol 3-kinase of Arabidopsis thaliana, is an essential protein with homology to a calcium-dependent lipid binding domain.

    PubMed

    Welters, P; Takegawa, K; Emr, S D; Chrispeels, M J

    1994-11-22

    The cDNA encoding phosphatidylinositol (PI) 3-kinase was cloned from Arabidopsis thaliana, and the derived amino acid sequence (AtVPS34) has a significantly higher homology to yeast PI 3-kinase (VPS34) than to the mammalian (p110). The protein has two conserved domains: a catalytic site with the ATP-binding site near the C terminus and a calcium-dependent lipid-binding domain near the N terminus. The plant cDNA does not rescue a yeast vps34 deletion mutant, but a chimeric gene in which the coding sequence for the C-terminal third of VPS34 is replaced by the corresponding sequence from the plant gene does rescue the yeast mutant. PI 3-kinase activity is detectable in extracts from plants that overexpress the plant PI 3-kinase. Expression of antisense constructs gives rise to second-generation transformed plants severely inhibited in growth and development.

  20. Neonatal hyperbilirubinemia caused by pyruvate kinase deficiency.

    PubMed

    Hammer, S G; Lewan, R B

    1988-01-01

    We report an infant with neonatal hyperbilirubinemia due to pyruvate kinase deficiency. The initial approach involved rapid evaluation, phototherapy, and close monitoring of serum bilirubin levels. Follow-up included maintenance on folic acid, monitoring blood counts, and educating the parents about the course of pyruvate kinase deficiency, especially aplastic crisis. We suggest that the informed family practitioner can manage neonatal hyperbilirubinemia and pyruvate kinase deficiency with referrals at critical times to pediatric or surgical specialists. The practitioner must be able to recognize quickly the need for exchange transfusion for severe jaundice and for blood transfusions or splenectomy when significant anemia or aplastic crisis occurs.

  1. Functional analysis of anomeric sugar kinases.

    PubMed

    Conway, Louis P; Voglmeir, Josef

    2016-09-01

    Anomeric sugar kinases perform fundamental roles in the metabolism of carbohydrates. Under- or overexpression of these enzymes, or mutations causing functional impairments can give rise to diseases such as galactosaemia and so the study of this class of kinase is of critical importance. In addition, anomeric sugar kinases which are naturally promiscuous, or have been artificially made so, may find application in the synthesis of libraries of drug candidates (for example, antibiotics), and natural or unnatural oligosaccharides and glycoconjugates. In this review, we provide an overview of the biological functions of these enzymes, the tools which have been developed to investigate them, and the current frontiers in their study. PMID:27351442

  2. Architecture for VLSI design of Reed-Solomon encoders

    NASA Technical Reports Server (NTRS)

    Liu, K. Y.

    1981-01-01

    The logic structure of a universal VLSI chip called the symbol-slice Reed-Solomon (RS) encoder chip is discussed. An RS encoder can be constructed by cascading and properly interconnecting a group of such VLSI chips. As a design example, it is shown that a (255,223) RD encoder requiring around 40 discrete CMOS ICs may be replaced by an RS encoder consisting of four identical interconnected VLSI RS encoder chips. Besides the size advantage, the VLSI RS encoder also has the potential advantages of requiring less power and having a higher reliability.

  3. A Syndromic Intellectual Disability Disorder Caused by Variants in TELO2, a Gene Encoding a Component of the TTT Complex.

    PubMed

    You, Jing; Sobreira, Nara L; Gable, Dustin L; Jurgens, Julie; Grange, Dorothy K; Belnap, Newell; Siniard, Ashley; Szelinger, Szabolcs; Schrauwen, Isabelle; Richholt, Ryan F; Vallee, Stephanie E; Dinulos, Mary Beth P; Valle, David; Armanios, Mary; Hoover-Fong, Julie

    2016-05-01

    The proteins encoded by TELO2, TTI1, and TTI2 interact to form the TTT complex, a co-chaperone for maturation of the phosphatidylinositol 3-kinase-related protein kinases (PIKKs). Here we report six affected individuals from four families with intellectual disability (ID) and neurological and other congenital abnormalities associated with compound heterozygous variants in TELO2. Although their fibroblasts showed reduced steady-state levels of TELO2 and the other components of the TTT complex, PIKK functions were normal in cellular assays. Our results suggest that these TELO2 missense variants result in loss of function, perturb TTT complex stability, and cause an autosomal-recessive syndromic form of ID. PMID:27132593

  4. Genome-wide identification and expression analysis of calcium-dependent protein kinase and its closely related kinase genes in Capsicum annuum.

    PubMed

    Cai, Hanyang; Cheng, Junbin; Yan, Yan; Xiao, Zhuoli; Li, Jiazhi; Mou, Shaoliang; Qiu, Ailian; Lai, Yan; Guan, Deyi; He, Shuilin

    2015-01-01

    As Ca2+ sensors and effectors, calcium-dependent protein kinases (CDPKs) play important roles in plant growth, development, and response to environmental cues. However, no CDPKs have been characterized in Capsicum annuum thus far. Herein, a genome wide comprehensive analysis of genes encoding CDPKs and CDPK-related protein kinases (CRKs) was performed in pepper, a total of 31 CDPK genes and five closely related kinase genes were identified, which were phylogenetically divided into four distinct subfamilies and unevenly distributed across nine chromosomes. Conserved sequence and exon-intron structures were found to be shared by pepper CDPKs within the same subfamily, and the expansion of the CDPK family in pepper was found to be due to segmental duplication events. Five CDPKs in the C. annuum variety CM334 were found to be mutated in the Chiltepin variety, and one CDPK present in CM334 was lost in Chiltepin. The majority of CDPK and CRK genes were expressed in different pepper tissues and developmental stages, and 10, 12, and 8 CDPK genes were transcriptionally modified by salt, heat, and Ralstonia solanacearum stresses, respectively. Furthermore, these genes were found to respond specifically to one stress as well as respond synergistically to two stresses or three stresses, suggesting that these CDPK genes might be involved in the specific or synergistic response of pepper to salt, heat, and R. solanacearum. Our results lay the foundation for future functional characterization of pepper CDPK and its closely related gene families.

  5. Genome-wide identification and expression analysis of calcium-dependent protein kinase and its closely related kinase genes in Capsicum annuum

    PubMed Central

    Cai, Hanyang; Cheng, Junbin; Yan, Yan; Xiao, Zhuoli; Li, Jiazhi; Mou, Shaoliang; Qiu, Ailian; Lai, Yan; Guan, Deyi; He, Shuilin

    2015-01-01

    As Ca2+ sensors and effectors, calcium-dependent protein kinases (CDPKs) play important roles in plant growth, development, and response to environmental cues. However, no CDPKs have been characterized in Capsicum annuum thus far. Herein, a genome wide comprehensive analysis of genes encoding CDPKs and CDPK-related protein kinases (CRKs) was performed in pepper, a total of 31 CDPK genes and five closely related kinase genes were identified, which were phylogenetically divided into four distinct subfamilies and unevenly distributed across nine chromosomes. Conserved sequence and exon-intron structures were found to be shared by pepper CDPKs within the same subfamily, and the expansion of the CDPK family in pepper was found to be due to segmental duplication events. Five CDPKs in the C. annuum variety CM334 were found to be mutated in the Chiltepin variety, and one CDPK present in CM334 was lost in Chiltepin. The majority of CDPK and CRK genes were expressed in different pepper tissues and developmental stages, and 10, 12, and 8 CDPK genes were transcriptionally modified by salt, heat, and Ralstonia solanacearum stresses, respectively. Furthermore, these genes were found to respond specifically to one stress as well as respond synergistically to two stresses or three stresses, suggesting that these CDPK genes might be involved in the specific or synergistic response of pepper to salt, heat, and R. solanacearum. Our results lay the foundation for future functional characterization of pepper CDPK and its closely related gene families. PMID:26442050

  6. Genome-wide identification and expression analysis of calcium-dependent protein kinase and its closely related kinase genes in Capsicum annuum.

    PubMed

    Cai, Hanyang; Cheng, Junbin; Yan, Yan; Xiao, Zhuoli; Li, Jiazhi; Mou, Shaoliang; Qiu, Ailian; Lai, Yan; Guan, Deyi; He, Shuilin

    2015-01-01

    As Ca2+ sensors and effectors, calcium-dependent protein kinases (CDPKs) play important roles in plant growth, development, and response to environmental cues. However, no CDPKs have been characterized in Capsicum annuum thus far. Herein, a genome wide comprehensive analysis of genes encoding CDPKs and CDPK-related protein kinases (CRKs) was performed in pepper, a total of 31 CDPK genes and five closely related kinase genes were identified, which were phylogenetically divided into four distinct subfamilies and unevenly distributed across nine chromosomes. Conserved sequence and exon-intron structures were found to be shared by pepper CDPKs within the same subfamily, and the expansion of the CDPK family in pepper was found to be due to segmental duplication events. Five CDPKs in the C. annuum variety CM334 were found to be mutated in the Chiltepin variety, and one CDPK present in CM334 was lost in Chiltepin. The majority of CDPK and CRK genes were expressed in different pepper tissues and developmental stages, and 10, 12, and 8 CDPK genes were transcriptionally modified by salt, heat, and Ralstonia solanacearum stresses, respectively. Furthermore, these genes were found to respond specifically to one stress as well as respond synergistically to two stresses or three stresses, suggesting that these CDPK genes might be involved in the specific or synergistic response of pepper to salt, heat, and R. solanacearum. Our results lay the foundation for future functional characterization of pepper CDPK and its closely related gene families. PMID:26442050

  7. The Large Binocular Telescope azimuth and elevation encoder system

    NASA Astrophysics Data System (ADS)

    Ashby, David S.; Sargent, Tom; Cox, Dan; Rosato, Jerry; Brynnel, Joar G.

    2008-08-01

    A typical high-resolution encoder interpolator relies on careful mechanical alignment of the encoder read-heads and tight electrical tolerances of the signal processing electronics to ensure linearity. As the interpolation factor increases, maintaining these tight mechanical and electrical tolerances becomes impractical. The Large Binocular Telescope (LBT) is designed to utilize strip-type encoders on the main axes. Because of the very large scale of the telescope, the accumulative length of the azimuth and elevation encoder strips exceeds 80 meters, making optical tape prohibitively expensive. Consequently, the designers of the LBT incorporated the far less expensive Farrand Controls Inductosyn® linear strip encoder to encode the positions of the main axes and the instrument rotators. Since the cycle pitch of these encoders is very large compared to that of optical strip encoders, the interpolation factor must also be large in order to achieve the 0.005 arcsecond encoder resolution as specified. The authors present a description of the innovative DSP-based hardware / software solution that adaptively characterizes and removes common systematic cycle-to-cycle encoder interpolation errors. These errors can be caused by mechanical misalignment, encoder manufacturing flaws, variations in electrical gain, signal offset or cross-coupling of the encoder signals. Simulation data are presented to illustrate the performance of the interpolation algorithm, and telemetry data are presented to demonstrate the actual performance of the LBT main-axis encoder system.

  8. The Crystal Structure of Cancer Osaka Thyroid Kinase Reveals an Unexpected Kinase Domain Fold*

    PubMed Central

    Gutmann, Sascha; Hinniger, Alexandra; Fendrich, Gabriele; Drückes, Peter; Antz, Sylvie; Mattes, Henri; Möbitz, Henrik; Ofner, Silvio; Schmiedeberg, Niko; Stojanovic, Aleksandar; Rieffel, Sebastien; Strauss, André; Troxler, Thomas; Glatthar, Ralf; Sparrer, Helmut

    2015-01-01

    Macrophages are important cellular effectors in innate immune responses and play a major role in autoimmune diseases such as rheumatoid arthritis. Cancer Osaka thyroid (COT) kinase, also known as mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and tumor progression locus 2 (Tpl-2), is a serine-threonine (ST) kinase and is a key regulator in the production of pro-inflammatory cytokines in macrophages. Due to its pivotal role in immune biology, COT kinase has been identified as an attractive target for pharmaceutical research that is directed at the discovery of orally available, selective, and potent inhibitors for the treatment of autoimmune disorders and cancer. The production of monomeric, recombinant COT kinase has proven to be very difficult, and issues with solubility and stability of the enzyme have hampered the discovery and optimization of potent and selective inhibitors. We developed a protocol for the production of recombinant human COT kinase that yields pure and highly active enzyme in sufficient yields for biochemical and structural studies. The quality of the enzyme allowed us to establish a robust in vitro phosphorylation assay for the efficient biochemical characterization of COT kinase inhibitors and to determine the x-ray co-crystal structures of the COT kinase domain in complex with two ATP-binding site inhibitors. The structures presented in this study reveal two distinct ligand binding modes and a unique kinase domain architecture that has not been observed previously. The structurally versatile active site significantly impacts the design of potent, low molecular weight COT kinase inhibitors. PMID:25918157

  9. The Crystal Structure of Cancer Osaka Thyroid Kinase Reveals an Unexpected Kinase Domain Fold.

    PubMed

    Gutmann, Sascha; Hinniger, Alexandra; Fendrich, Gabriele; Drückes, Peter; Antz, Sylvie; Mattes, Henri; Möbitz, Henrik; Ofner, Silvio; Schmiedeberg, Niko; Stojanovic, Aleksandar; Rieffel, Sebastien; Strauss, André; Troxler, Thomas; Glatthar, Ralf; Sparrer, Helmut

    2015-06-12

    Macrophages are important cellular effectors in innate immune responses and play a major role in autoimmune diseases such as rheumatoid arthritis. Cancer Osaka thyroid (COT) kinase, also known as mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and tumor progression locus 2 (Tpl-2), is a serine-threonine (ST) kinase and is a key regulator in the production of pro-inflammatory cytokines in macrophages. Due to its pivotal role in immune biology, COT kinase has been identified as an attractive target for pharmaceutical research that is directed at the discovery of orally available, selective, and potent inhibitors for the treatment of autoimmune disorders and cancer. The production of monomeric, recombinant COT kinase has proven to be very difficult, and issues with solubility and stability of the enzyme have hampered the discovery and optimization of potent and selective inhibitors. We developed a protocol for the production of recombinant human COT kinase that yields pure and highly active enzyme in sufficient yields for biochemical and structural studies. The quality of the enzyme allowed us to establish a robust in vitro phosphorylation assay for the efficient biochemical characterization of COT kinase inhibitors and to determine the x-ray co-crystal structures of the COT kinase domain in complex with two ATP-binding site inhibitors. The structures presented in this study reveal two distinct ligand binding modes and a unique kinase domain architecture that has not been observed previously. The structurally versatile active site significantly impacts the design of potent, low molecular weight COT kinase inhibitors.

  10. Crystal structures of two aminoglycoside kinases bound with a eukaryotic protein kinase inhibitor.

    PubMed

    Fong, Desiree H; Xiong, Bing; Hwang, Jiyoung; Berghuis, Albert M

    2011-05-09

    Antibiotic resistance is recognized as a growing healthcare problem. To address this issue, one strategy is to thwart the causal mechanism using an adjuvant in partner with the antibiotic. Aminoglycosides are a class of clinically important antibiotics used for the treatment of serious infections. Their usefulness has been compromised predominantly due to drug inactivation by aminoglycoside-modifying enzymes, such as aminoglycoside phosphotransferases or kinases. These kinases are structurally homologous to eukaryotic Ser/Thr and Tyr protein kinases and it has been shown that some can be inhibited by select protein kinase inhibitors. The aminoglycoside kinase, APH(3')-IIIa, can be inhibited by CKI-7, an ATP-competitive inhibitor for the casein kinase 1. We have determined that CKI-7 is also a moderate inhibitor for the atypical APH(9)-Ia. Here we present the crystal structures of CKI-7-bound APH(3')-IIIa and APH(9)-Ia, the first structures of a eukaryotic protein kinase inhibitor in complex with bacterial kinases. CKI-7 binds to the nucleotide-binding pocket of the enzymes and its binding alters the conformation of the nucleotide-binding loop, the segment homologous to the glycine-rich loop in eukaryotic protein kinases. Comparison of these structures with the CKI-7-bound casein kinase 1 reveals features in the binding pockets that are distinct in the bacterial kinases and could be exploited for the design of a bacterial kinase specific inhibitor. Our results provide evidence that an inhibitor for a subset of APHs can be developed in order to curtail resistance to aminoglycosides.

  11. Nonnucleoside inhibitors of adenosine kinase.

    PubMed

    Gomtsyan, Arthur; Lee, Chih-Hung

    2004-01-01

    Adenosine (ADO) is an endogenous inhibitory neuromodulator that increases nociceptive thresholds in response to tissue trauma and inflammation. Adenosine kinase (AK) is a key intracellular enzyme regulating intra- and extracellular concentrations of ADO. AK inhibition selectively amplifies extracellular ADO levels at cell and tissue sites where accelerated release of ADO occurs. AK inhibitors have been shown to provide effective antinociceptive, antiinflammatory and anticonvulsant activity in animal models, thus suggesting their potential therapeutic utility for pain, inflammation, epilepsy and possibly other central and peripheral nervous system diseases associated with cellular trauma and inflammation. This beneficial outcome may potentially lack nonspecific effects associated with the systemic administration of ADO receptor agonists. Until recently all of the reported AK inhibitors contained adenosine-like structural motif. The present review will discuss design, synthesis and analgesic and antiinflammatory properties of the novel nonnucleoside AK inhibitors that do not have close structural resemblance with the natural substrate ADO. Two classes of the nonnucleoside AK inhibitors are built on pyridopyrimidine and alkynylpyrimidine cores.

  12. Human CDC2-like kinase 1 (CLK1): a novel target for Alzheimer's disease.

    PubMed

    Jain, Princi; Karthikeyan, Chandrabose; Moorthy, N S Hari Narayana; Waiker, Digambar Kumar; Jain, Arvind Kumar; Trivedi, Piyush

    2014-05-01

    The cdc2-like kinases (CLKs) are an evolutionarily conserved group of dual specificity kinases belonging to the CMGC (cyclin-dependent kinases (CDKs), mitogen-activated protein kinases (MAP kinases), glycogen synthase kinases (GSK) and CDK-like kinases). The CLK family consists of four isoforms namely CLK1, CLK2, CLK3 and CLK4. The human CLK1 encoded protein comprises 454 amino acids and the catalytic domain of CLK1 exhibits the typical protein kinase fold. CLK1 has been shown to autophosphorylate on serine, threonine and tyrosine residues and phosphorylate exogenous substrates on serine and threonine residues. CLK1 plays an important role in the regulation of RNA splicing through phosphorylation of members of the serine and arginine-rich (SR) family of splicing factors. CLK1 is involved in the pathophysiology of Alzheimer's disease by phosphorylating the serine residue in SR proteins. Nuclear speckles of the nucleoplasm contain the stored form of SR proteins and are moderately responsible for the choice of splicing sites during pre-mRNA splicing. Hence, the inhibition of CLK1 can be used as a therapeutic strategy for Alzheimer's disease. Many natural and synthetic molecules are reported to possess CLK1 inhibitory activity. Some specific examples are Marine alkaloid Leucettamine B and KH-CB19. Leucettamine B is a potent inhibitor of CLK1 (15 nM), Dyrk1A (40 nM), and Dyrk2 (35 nM) and a moderate inhibitor of CLK3 (4.5 µM) whereas KH-CB19 is a highly specific and potent inhibitor of the CLK1/CLK4. X-ray crystallographic studies have revealed the binding mode of marine sponge metabolite hymenialdisine and a dichloroindolyl enamino nitrile (KH-CB19) to CLK1. This review focuses on the role of CLKs in the pathophysiology of Alzheimer's disease and therapeutic potential of targeting CLK1 in Alzheimer's disease drug discovery and development. In addition, the recent developments in drug discovery efforts targeting human CLK1 are also highlighted. PMID:24568585

  13. Ocular Toxicity of Tyrosine Kinase Inhibitors

    PubMed Central

    Davis, Mary Elizabeth

    2016-01-01

    Purpose/Objectives To review common tyrosine kinase inhibitors, as well as their ocular side effects and management. Data Sources A comprehensive literature search was conducted using cINahl®, Pubmed, and cochrane databases for articles published since 2004 with the following search terms: ocular toxicities, tyrosine kinase inhibitors, ophthalmology, adverse events, eye, and vision. Data Synthesis Tyrosine kinase inhibitors can cause significant eye toxicity. Conclusions Given the prevalence of new tyrosine kinase inhibitor therapies and the complexity of possible pathogenesis of ocular pathology, oncology nurses can appreciate the occurrence of ocular toxicities and the role of nursing in the management of these problems. Implications for Nursing Knowledge of the risk factors and etiology of ocular toxicity of targeted cancer therapies can guide nursing assessment, enhance patient education, and improve care management. Including a review of eye symptoms and vision issues in nursing assessment can enhance early detection and treatment of ocular toxicity. PMID:26906134

  14. Characterization of protein kinases from Blepharisma intermedium.

    PubMed

    Beyer, J

    1975-12-01

    Three protein kinases (EC 2.7.1.37) were detected in Blepharisma and partially purified. The enzymes were most active with histone as substrate protein. The stability of the bond between phosphate and protein acceptor showed the characteristics of seryl- or threonylphosphate. Protein kinase I was solubilized by ultrasonication or freezing and thawing, while the enzymes II and III were readily solubilized by mild homogenization. Protein II and III were noticeably activated by cAMP and cGMP, while protein kinase I was inhibited by cAMP. Associated with protein kinase II and III activity was the ability to bind labeled cAMP. The following molecular weights were determined: 90000 for enzyme I, 280000 for enzyme II, and 95000 for enzyme III. Various apparent Michaelis constants were estimated.

  15. Genetics Home Reference: mevalonate kinase deficiency

    MedlinePlus

    ... cytoskeleton), gene activity (expression), and protein production and modification. Most MVK gene mutations that cause mevalonate kinase ... What are the different ways in which a genetic condition can be inherited? More about Inheriting Genetic ...

  16. A Molecular Brake in the Kinase Hinge Region Regulates the Activity of Receptor Tyrosine Kinases

    SciTech Connect

    Chen,H.; Ma, J.; Li, W.; Eliseenkova, A.; Xu, C.; Neubert, T.; Miller, W.; Mohammadi, M.

    2007-01-01

    Activating mutations in the tyrosine kinase domain of receptor tyrosine kinases (RTKs) cause cancer and skeletal disorders. Comparison of the crystal structures of unphosphorylated and phosphorylated wild-type FGFR2 kinase domains with those of seven unphosphorylated pathogenic mutants reveals an autoinhibitory 'molecular brake' mediated by a triad of residues in the kinase hinge region of all FGFRs. Structural analysis shows that many other RTKs, including PDGFRs, VEGFRs, KIT, CSF1R, FLT3, TEK, and TIE, are also subject to regulation by this brake. Pathogenic mutations activate FGFRs and other RTKs by disengaging the brake either directly or indirectly.

  17. Identifying Kinase Substrates via a Heavy ATP Kinase Assay and Quantitative Mass Spectrometry

    PubMed Central

    Müller, André C.; Giambruno, Roberto; Weißer, Juliane; Májek, Peter; Hofer, Alexandre; Bigenzahn, Johannes W.; Superti-Furga, Giulio; Jessen, Henning J.; Bennett, Keiryn L.

    2016-01-01

    Mass spectrometry-based in vitro kinase screens play an essential role in the discovery of kinase substrates, however, many suffer from biological and technical noise or necessitate genetically-altered enzyme-cofactor systems. We describe a method that combines stable γ-[18O2]-ATP with classical in vitro kinase assays within a contemporary quantitative proteomic workflow. Our approach improved detection of known substrates of the non-receptor tyrosine kinase ABL1; and identified potential, new in vitro substrates. PMID:27346722

  18. Kinase inhibitor profiling reveals unexpected opportunities to inhibit disease-associated mutant kinases

    PubMed Central

    Duong-Ly, Krisna C.; Devarajan, Karthik; Liang, Shuguang; Horiuchi, Kurumi Y.; Wang, Yuren; Ma, Haiching; Peterson, Jeffrey R.

    2016-01-01

    Summary Small-molecule kinase inhibitors have typically been designed to inhibit wild-type kinases rather than the mutant forms that frequently arise in diseases such as cancer. Mutations can have serious clinical implications by increasing kinase catalytic activity or conferring therapeutic resistance. To identify opportunities to repurpose inhibitors against disease-associated mutant kinases, we conducted a large-scale functional screen of 183 known kinase inhibitors against 76 recombinant, mutant kinases. The results revealed lead compounds with activity against clinically important mutant kinases including ALK, LRRK2, RET, and EGFR as well as unexpected opportunities for repurposing FDA-approved kinase inhibitors as leads for additional indications. Furthermore, using T674I PDGFRα as an example, we show how single-dose screening data can provide predictive structure-activity data to guide subsequent inhibitor optimization. This study provides a resource for the development of inhibitors against numerous disease-associated mutant kinases and illustrates the potential of unbiased profiling as an approach to compound-centric inhibitor development. PMID:26776524

  19. Twitchin kinase interacts with MAPKAP kinase 2 in Caenorhabditis elegans striated muscle

    PubMed Central

    Matsunaga, Yohei; Qadota, Hiroshi; Furukawa, Miho; Choe, Heejoo (Helen); Benian, Guy M.

    2015-01-01

    In Caenorhabditis elegans, twitchin is a giant polypeptide located in muscle A-bands. The protein kinase of twitchin is autoinhibited by 45 residues upstream (NL) and 60 residues downstream (CRD) of the kinase catalytic core. Molecular dynamics simulation on a twitchin fragment revealed that the NL is released by pulling force. However, it is unclear how the CRD is removed. To identify proteins that may remove the CRD, we performed a yeast two-hybrid screen using twitchin kinase as bait. One interactor is MAK-1, C. elegans orthologue of MAPKAP kinase 2. MAPKAP kinase 2 is phosphorylated and activated by p38 MAP kinase. We demonstrate that the CRD of twitchin is important for binding to MAK-1. mak-1 is expressed in nematode body wall muscle, and antibodies to MAK-1 localize between and around Z-disk analogues and to the edge of A-bands. Whereas unc-22 mutants are completely resistant, mak-1 mutants are partially resistant to nicotine. MAK-1 can phosphorylate twitchin NL-Kin-CRD in vitro. Genetic data suggest the involvement of two other mak-1 paralogues and two orthologues of p38 MAP kinase. These results suggest that MAK-1 is an activator of twitchin kinase and that the p38 MAP kinase pathway may be involved in the regulation of twitchin. PMID:25851606

  20. Kinase-interacting substrate screening is a novel method to identify kinase substrates

    PubMed Central

    Amano, Mutsuki; Hamaguchi, Tomonari; Shohag, Md. Hasanuzzaman; Kozawa, Kei; Kato, Katsuhiro; Zhang, Xinjian; Yura, Yoshimitsu; Matsuura, Yoshiharu; Kataoka, Chikako; Nishioka, Tomoki

    2015-01-01

    Protein kinases play pivotal roles in numerous cellular functions; however, the specific substrates of each protein kinase have not been fully elucidated. We have developed a novel method called kinase-interacting substrate screening (KISS). Using this method, 356 phosphorylation sites of 140 proteins were identified as candidate substrates for Rho-associated kinase (Rho-kinase/ROCK2), including known substrates. The KISS method was also applied to additional kinases, including PKA, MAPK1, CDK5, CaMK1, PAK7, PKN, LYN, and FYN, and a lot of candidate substrates and their phosphorylation sites were determined, most of which have not been reported previously. Among the candidate substrates for Rho-kinase, several functional clusters were identified, including the polarity-associated proteins, such as Scrib. We found that Scrib plays a crucial role in the regulation of subcellular contractility by assembling into a ternary complex with Rho-kinase and Shroom2 in a phosphorylation-dependent manner. We propose that the KISS method is a comprehensive and useful substrate screen for various kinases. PMID:26101221

  1. Redundant kinase activation and resistance of EGFR-tyrosine kinase inhibitors

    PubMed Central

    Luo, Min; Fu, Li-Wu

    2014-01-01

    Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown dramatic effects against that tumors harboring EGFR activating mutations in the EGFR intracytoplasmic tyrosine kinase domain and resulted in cell apoptosis. Unfortunately, a number of patients ultimately developed resistance by multiple mechanisms. Thus, elucidation of the mechanism of resistance to EGFR-TKIs can provide strategies for blocking or reversing the situation. Recent studies suggested that redundant kinase activation plays pivotal roles in escaping from the effects of EGFR-TKIs. Herein, we aimed to characterize several molecular events involved in the resistance to EGFR-TKIs mediated by redundant kinase activation. PMID:25520855

  2. Structure of the two-domain hexameric APS kinase from Thiobacillus denitrificans: structural basis for the absence of ATP sulfurylase activity

    SciTech Connect

    Gay, Sean C.; Segel, Irwin H.; Fisher, Andrew J.

    2009-10-01

    APS kinase from Thiobacillus denitrificans contains an inactive N-terminal ATP sulfurylase domain. The structure presented unveils the first hexameric assembly for an APS kinase, and reveals that structural changes in the N-terminal domain disrupt the ATP sulfurylase active site thus prohibiting activity. The Tbd-0210 gene of the chemolithotrophic bacterium Thiobacillus denitrificans is annotated to encode a 60.5 kDa bifunctional enzyme with ATP sulfurylase and APS kinase activity. This putative bifunctional enzyme was cloned, expressed and structurally characterized. The 2.95 Å resolution X-ray crystal structure reported here revealed a hexameric assembly with D{sub 3} symmetry. Each subunit contains a large N-terminal sulfurylase-like domain and a C-terminal APS kinase domain reminiscent of the two-domain fungal ATP sulfurylases of Penicillium chrysogenum and Saccharomyces cerevisiae, which also exhibit a hexameric assembly. However, the T. denitrificans enzyme exhibits numerous structural and sequence differences in the N-terminal domain that render it inactive with respect to ATP sulfurylase activity. Surprisingly, the C-terminal domain does indeed display APS kinase activity, indicating that this gene product is a true APS kinase. Therefore, these results provide the first structural insights into a unique hexameric APS kinase that contains a nonfunctional ATP sulfurylase-like domain of unknown function.

  3. ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia

    PubMed Central

    Landt, Stephen G.; Marinov, Georgi K.; Kundaje, Anshul; Kheradpour, Pouya; Pauli, Florencia; Batzoglou, Serafim; Bernstein, Bradley E.; Bickel, Peter; Brown, James B.; Cayting, Philip; Chen, Yiwen; DeSalvo, Gilberto; Epstein, Charles; Fisher-Aylor, Katherine I.; Euskirchen, Ghia; Gerstein, Mark; Gertz, Jason; Hartemink, Alexander J.; Hoffman, Michael M.; Iyer, Vishwanath R.; Jung, Youngsook L.; Karmakar, Subhradip; Kellis, Manolis; Kharchenko, Peter V.; Li, Qunhua; Liu, Tao; Liu, X. Shirley; Ma, Lijia; Milosavljevic, Aleksandar; Myers, Richard M.; Park, Peter J.; Pazin, Michael J.; Perry, Marc D.; Raha, Debasish; Reddy, Timothy E.; Rozowsky, Joel; Shoresh, Noam; Sidow, Arend; Slattery, Matthew; Stamatoyannopoulos, John A.; Tolstorukov, Michael Y.; White, Kevin P.; Xi, Simon; Farnham, Peggy J.; Lieb, Jason D.; Wold, Barbara J.; Snyder, Michael

    2012-01-01

    Chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) has become a valuable and widely used approach for mapping the genomic location of transcription-factor binding and histone modifications in living cells. Despite its widespread use, there are considerable differences in how these experiments are conducted, how the results are scored and evaluated for quality, and how the data and metadata are archived for public use. These practices affect the quality and utility of any global ChIP experiment. Through our experience in performing ChIP-seq experiments, the ENCODE and modENCODE consortia have developed a set of working standards and guidelines for ChIP experiments that are updated routinely. The current guidelines address antibody validation, experimental replication, sequencing depth, data and metadata reporting, and data quality assessment. We discuss how ChIP quality, assessed in these ways, affects different uses of ChIP-seq data. All data sets used in the analysis have been deposited for public viewing and downloading at the ENCODE (http://encodeproject.org/ENCODE/) and modENCODE (http://www.modencode.org/) portals. PMID:22955991

  4. Evolutionary Diversification of Plant Shikimate Kinase Gene Duplicates

    PubMed Central

    Fucile, Geoffrey; Falconer, Shannon; Christendat, Dinesh

    2008-01-01

    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. PMID:19057671

  5. The Collaborative Encoding Deficit is Attenuated with Specific Warnings

    PubMed Central

    Barber, Sarah J.; Rajaram, Suparna; Paneerselvam, Bavani

    2012-01-01

    Individuals learning together do so less effectively than individuals learning alone, an effect known as the collaborative encoding deficit (Barber, Rajaram, & Aron, 2010). In the present studies we examined whether providing participants with a warning about the collaborative encoding deficit would increase their encoding task performance, and reduce subsequent memory deficits. Across two experiments, specific warnings were beneficial for memory. Collaborating participants who were told about the collaborative encoding deficit, and who received suggestions for how to complete the encoding task, had superior memory than participants who received no warning. This benefit was not due to qualitative changes in encoding task performance, was unrelated to the type of collaboration utilized, was absent when a more general warning was utilized, and was unrelated to self-reported task motivation. Rather, specific warnings appear to protect against the collaborative encoding deficit by increasing time spent on, and attention directed to, the encoding task. PMID:23296389

  6. Non-ATP competitive protein kinase inhibitors.

    PubMed

    Garuti, L; Roberti, M; Bottegoni, G

    2010-01-01

    Protein kinases represent an attractive target in oncology drug discovery. Most of kinase inhibitors are ATP-competitive and are called type I inhibitors. The ATP-binding pocket is highly conserved among members of the kinase family and it is difficult to find selective agents. Moreover, the ATP-competitive inhibitors must compete with high intracellular ATP levels leading to a discrepancy between IC50s measured by biochemical versus cellular assays. The non-ATP competitive inhibitors, called type II and type III inhibitors, offer the possibility to overcome these problems. These inhibitors act by inducing a conformational shift in the target enzyme such that the kinase is no longer able to function. In the DFG-out form, the phenylalanine side chain moves to a new position. This movement creates a hydrophobic pocket available for occupation by the inhibitor. Some common features are present in these inhibitors. They contain a heterocyclic system that forms one or two hydrogen bonds with the kinase hinge residue. They also contain a hydrophobic moiety that occupies the pocket formed by the shift of phenylalanine from the DFG motif. Moreover, all the inhibitors bear a hydrogen bond donor-acceptor pair, usually urea or amide, that links the hinge-binding portion to the hydrophobic moiety and interacts with the allosteric site. Examples of non ATP-competitive inhibitors are available for various kinases. In this review small molecules capable of inducing the DFG-out conformation are reported, especially focusing on structural feature, SAR and biological properties.

  7. Mutations in PLK4, encoding a master regulator of centriole biogenesis, cause microcephaly, growth failure and retinopathy.

    PubMed

    Martin, Carol-Anne; Ahmad, Ilyas; Klingseisen, Anna; Hussain, Muhammad Sajid; Bicknell, Louise S; Leitch, Andrea; Nürnberg, Gudrun; Toliat, Mohammad Reza; Murray, Jennie E; Hunt, David; Khan, Fawad; Ali, Zafar; Tinschert, Sigrid; Ding, James; Keith, Charlotte; Harley, Margaret E; Heyn, Patricia; Müller, Rolf; Hoffmann, Ingrid; Daire, Valérie Cormier; Dollfus, Hélène; Dupuis, Lucie; Bashamboo, Anu; McElreavey, Kenneth; Kariminejad, Ariana; Mendoza-Londono, Roberto; Moore, Anthony T; Saggar, Anand; Schlechter, Catie; Weleber, Richard; Thiele, Holger; Altmüller, Janine; Höhne, Wolfgang; Hurles, Matthew E; Noegel, Angelika Anna; Baig, Shahid Mahmood; Nürnberg, Peter; Jackson, Andrew P

    2014-12-01

    Centrioles are essential for ciliogenesis. However, mutations in centriole biogenesis genes have been reported in primary microcephaly and Seckel syndrome, disorders without the hallmark clinical features of ciliopathies. Here we identify mutations in the genes encoding PLK4 kinase, a master regulator of centriole duplication, and its substrate TUBGCP6 in individuals with microcephalic primordial dwarfism and additional congenital anomalies, including retinopathy, thereby extending the human phenotypic spectrum associated with centriole dysfunction. Furthermore, we establish that different levels of impaired PLK4 activity result in growth and cilia phenotypes, providing a mechanism by which microcephaly disorders can occur with or without ciliopathic features.

  8. Evaluating standard terminologies for encoding allergy information

    PubMed Central

    Goss, Foster R; Zhou, Li; Plasek, Joseph M; Broverman, Carol; Robinson, George; Middleton, Blackford; Rocha, Roberto A

    2013-01-01

    Objective Allergy documentation and exchange are vital to ensuring patient safety. This study aims to analyze and compare various existing standard terminologies for representing allergy information. Methods Five terminologies were identified, including the Systemized Nomenclature of Medical Clinical Terms (SNOMED CT), National Drug File–Reference Terminology (NDF-RT), Medication Dictionary for Regulatory Activities (MedDRA), Unique Ingredient Identifier (UNII), and RxNorm. A qualitative analysis was conducted to compare desirable characteristics of each terminology, including content coverage, concept orientation, formal definitions, multiple granularities, vocabulary structure, subset capability, and maintainability. A quantitative analysis was also performed to compare the content coverage of each terminology for (1) common food, drug, and environmental allergens and (2) descriptive concepts for common drug allergies, adverse reactions (AR), and no known allergies. Results Our qualitative results show that SNOMED CT fulfilled the greatest number of desirable characteristics, followed by NDF-RT, RxNorm, UNII, and MedDRA. Our quantitative results demonstrate that RxNorm had the highest concept coverage for representing drug allergens, followed by UNII, SNOMED CT, NDF-RT, and MedDRA. For food and environmental allergens, UNII demonstrated the highest concept coverage, followed by SNOMED CT. For representing descriptive allergy concepts and adverse reactions, SNOMED CT and NDF-RT showed the highest coverage. Only SNOMED CT was capable of representing unique concepts for encoding no known allergies. Conclusions The proper terminology for encoding a patient's allergy is complex, as multiple elements need to be captured to form a fully structured clinical finding. Our results suggest that while gaps still exist, a combination of SNOMED CT and RxNorm can satisfy most criteria for encoding common allergies and provide sufficient content coverage. PMID:23396542

  9. Novel encoding methods for DNA-templated chemical libraries.

    PubMed

    Li, Gang; Zheng, Wenlu; Liu, Ying; Li, Xiaoyu

    2015-06-01

    Among various types of DNA-encoded chemical libraries, DNA-templated library takes advantage of the sequence-specificity of DNA hybridization, enabling not only highly effective DNA-templated chemical reactions, but also high fidelity in library encoding. This brief review summarizes recent advances that have been made on the encoding strategies for DNA-templated libraries, and it also highlights their respective advantages and limitations for the preparation of DNA-encoded libraries.

  10. Doppler imaging using spectrally-encoded endoscopy

    PubMed Central

    Yelin, Dvir; Bouma, B. E.; Rosowsky, J. J.; Tearney, G. J.

    2009-01-01

    The capability to image tissue motion such as blood flow through an endoscope could have many applications in medicine. Spectrally encoded endoscopy (SEE) is a recently introduced technique that utilizes a single optical fiber and miniature diffractive optics to obtain endoscopic images through small diameter probes. Using spectral-domain interferometry, SEE is furthermore capable of three-dimensional volume imaging at video rates. Here we show that by measuring relative spectral phases, this technology can additionally measure Doppler shifts. Doppler SEE is demonstrated in flowing Intralipid phantoms and vibrating middle ear ossicles. PMID:18795020

  11. Error correction for encoded quantum annealing

    NASA Astrophysics Data System (ADS)

    Pastawski, Fernando; Preskill, John

    2016-05-01

    Recently, W. Lechner, P. Hauke, and P. Zoller [Sci. Adv. 1, e1500838 (2015), 10.1126/sciadv.1500838] have proposed a quantum annealing architecture, in which a classical spin glass with all-to-all pairwise connectivity is simulated by a spin glass with geometrically local interactions. We interpret this architecture as a classical error-correcting code, which is highly robust against weakly correlated bit-flip noise, and we analyze the code's performance using a belief-propagation decoding algorithm. Our observations may also apply to more general encoding schemes and noise models.

  12. Polynucleotides encoding TRF1 binding proteins

    DOEpatents

    Campisi, Judith; Kim, Sahn-Ho

    2002-01-01

    The present invention provides a novel telomere associated protein (Trf1-interacting nuclear protein 2 "Tin2") that hinders the binding of Trf1 to its specific telomere repeat sequence and mediates the formation of a Tin2-Trf1-telomeric DNA complex that limits telomerase access to the telomere. Also included are the corresponding nucleic acids that encode the Tin2 of the present invention, as well as mutants of Tin2. Methods of making, purifying and using Tin2 of the present invention are described. In addition, drug screening assays to identify drugs that mimic and/or complement the effect of Tin2 are presented.

  13. Encoding of Memory in Sheared Amorphous Solids

    NASA Astrophysics Data System (ADS)

    Fiocco, Davide; Foffi, Giuseppe; Sastry, Srikanth

    2014-01-01

    We show that memory can be encoded in a model amorphous solid subjected to athermal oscillatory shear deformations, and in an analogous spin model with disordered interactions, sharing the feature of a deformable energy landscape. When these systems are subjected to oscillatory shear deformation, they retain memory of the deformation amplitude imposed in the training phase, when the amplitude is below a "localization" threshold. Remarkably, multiple persistent memories can be stored using such an athermal, noise-free, protocol. The possibility of such memory is shown to be linked to the presence of plastic deformations and associated limit cycles traversed by the system, which exhibit avalanche statistics also seen in related contexts.

  14. The ENCODE (ENCyclopedia Of DNA Elements) Project.

    PubMed

    2004-10-22

    The ENCyclopedia Of DNA Elements (ENCODE) Project aims to identify all functional elements in the human genome sequence. The pilot phase of the Project is focused on a specified 30 megabases (approximately 1%) of the human genome sequence and is organized as an international consortium of computational and laboratory-based scientists working to develop and apply high-throughput approaches for detecting all sequence elements that confer biological function. The results of this pilot phase will guide future efforts to analyze the entire human genome.

  15. Gene encoding herbicide safener binding protein

    DOEpatents

    Walton, Jonathan D.; Scott-Craig, John S.

    1999-01-01

    The cDNA encoding safener binding protein (SafBP), also referred to as SBP1, is set forth in FIG. 5 and SEQ ID No. 1. The deduced amino acid sequence is provided in FIG. 5 and SEQ ID No. 2. Methods of making and using SBP1 and SafBP to alter a plant's sensitivity to certain herbicides or a plant's responsiveness to certain safeners are also provided, as well as expression vectors, transgenic plants or other organisms transfected with said vectors and seeds from said plants.

  16. Information propagation from IP 3 to target protein: A combined model for encoding and decoding of Ca 2+ signal

    NASA Astrophysics Data System (ADS)

    Zhao, Qi; Yi, Ming; Xia, Kelin; Zhan, Meng

    2009-10-01

    It is well known that information is encoded in the frequency or amplitude of Ca 2+ signal and then decoded by target protein. However, few models considered both the encoding and decoding procedures of Ca 2+ signal. In this work, a minimal Li-Rinzel model is coupled with a phosphorylation-dephosphorylation cycle model, which is used to investigate information transmissions from inositol 1,4,5-trisphosphate (IP 3) to target proteins and their relations. We found that the mean target protein activity increases with the IP 3 concentration, and at a low level of stimulation, the target protein can be more efficiently activated by an oscillatory signal than a constant signal of the same average calcium if Ca 2+ acts cooperatively on the kinase. The internal noise resulting from the finite system size is also taken into account in the combined model.

  17. Methods to Purify and Assay Secretory Pathway Kinases.

    PubMed

    Tagliabracci, Vincent S; Wen, Jianzhong; Xiao, Junyu

    2016-01-01

    Members of the four-jointed and VLK families of secretory pathway kinases appear to be responsible for the phosphorylation of secreted proteins and proteoglycans. These enzymes have been implicated in many biological processes and mutations in several of these kinases cause human diseases. Here, we describe methods to purify and assay two members of the four-jointed family of secretory kinases: the Fam20C protein kinase and the Fam20B proteoglycan kinase. PMID:27632012

  18. ABL kinases promote breast cancer osteolytic metastasis by modulating tumor-bone interactions through TAZ and STAT5 signaling

    PubMed Central

    Wang, Jun; Rouse, Clay; Jasper, Jeff S.; Pendergast, Ann Marie

    2016-01-01

    Bone metastases occur in up to 70% of advanced breast cancer. For most patients with breast cancer, bone metastases are predominantly osteolytic. Interactions between tumor cells and stromal cells in the bone microenvironment drive osteolytic bone metastasis, a process that requires the activation of osteoclasts, cells that break down bone. Here, we report that ABL kinases promoted metastasis of breast cancer cells to bone by regulating the crosstalk between tumor and the bone microenvironment. ABL kinases protected tumor cells from apoptosis induced by TRAIL (TNF-related apoptosis-inducing ligand), activated the transcription factor STAT5, and promoted osteolysis through the STAT5-dependent expression of genes encoding the osteoclast activating factors interleukin 6 (IL6) and matrix metalloproteinase-1 (MMP1). Furthermore, ABL kinases increased the abundance of the Hippo pathway mediator TAZ and the expression of TAZ-dependent target genes that promote bone metastasis. Knockdown of ABL kinases or treatment with ABL-specific allosteric inhibitor impaired osteolytic metastasis of breast cancer cells in mice. These findings revealed a role for ABL kinases in regulating tumor-bone interactions and provide a rationale for targeting both tumor and the bone microenvironment with ABL-specific inhibitors. PMID:26838548

  19. ABL kinases promote breast cancer osteolytic metastasis by modulating tumor-bone interactions through TAZ and STAT5 signaling.

    PubMed

    Wang, Jun; Rouse, Clay; Jasper, Jeff S; Pendergast, Ann Marie

    2016-02-01

    Bone metastases occur in up to 70% of advanced breast cancer. For most patients with breast cancer, bone metastases are predominantly osteolytic. Interactions between tumor cells and stromal cells in the bone microenvironment drive osteolytic bone metastasis, a process that requires the activation of osteoclasts, cells that break down bone. We report that ABL kinases promoted metastasis of breast cancer cells to bone by regulating the crosstalk between tumor cells and the bone microenvironment. ABL kinases protected tumor cells from apoptosis induced by TRAIL (TNF-related apoptosis-inducing ligand), activated the transcription factor STAT5, and promoted osteolysis through the STAT5-dependent expression of genes encoding the osteoclast-activating factors interleukin-6 (IL-6) and matrix metalloproteinase 1 (MMP1). Furthermore, in breast cancer cells, ABL kinases increased the abundance of the Hippo pathway mediator TAZ and the expression of TAZ-dependent target genes that promote bone metastasis. Knockdown of ABL kinases or treatment with ABL-specific allosteric inhibitor impaired osteolytic metastasis of breast cancer cells in mice. These findings revealed a role for ABL kinases in regulating tumor-bone interactions and provide a rationale for using ABL-specific inhibitors to limit breast cancer metastasis to bone. PMID:26838548

  20. Snf1-related protein kinase 1 is needed for growth in a normal day-night light cycle.

    PubMed

    Thelander, Mattias; Olsson, Tina; Ronne, Hans

    2004-04-21

    The yeast Snf1 protein kinase and its animal homologue, the AMP-activated protein kinase, play important roles in metabolic regulation, by serving as energy gauges that turn off energy-consuming processes and mobilize energy reserves during low-energy conditions. The closest homologue of these kinases in plants is Snf1-related protein kinase 1 (SnRK1). We have cloned two SnRK1-encoding genes, PpSNF1a and PpSNF1b, in the moss Physcomitrella patens, where gene function can be studied directly by gene targeting in the haploid gametophyte. A snf1a snf1b double knockout mutant is viable, but lacks all Snf1-like protein kinase activity. The mutant has a complex phenotype that includes developmental abnormalities, premature senescence and altered sensitivities to plant hormones. Remarkably, the double knockout mutant also requires continuous light, and is unable to grow in a normal day-night light cycle. This suggests that SnRK1 is needed for metabolic changes that help the plant cope with the dark hours of the night.

  1. Visual Encoding Mechanisms and Their Relationship to Text Presentation Preference

    ERIC Educational Resources Information Center

    Pammer, Kristen; Lavis, Ruth; Cornelissen, Piers

    2004-01-01

    This study was designed to investigate the importance of spatial encoding in reading, with particular emphasis on visuo-spatial encoding mechanisms. Thirty one school children participated in the first study in which they were measured on their ability to solve a centrally presented spatial encoding task, as well as their sensitivity to the…

  2. Optical Pseudocolor Encoding Of Gray-Scale Image

    NASA Technical Reports Server (NTRS)

    Chao, Tien-Hsin; Liu, Hua-Kuang

    1990-01-01

    Optical encoding much faster than digital electronic encoding. In optical pseudocolor-encoding apparatus brightness modulation in image from television camera transformed into polarization modulation in LCTV, and then into pseudocolor modulation in image on projection screen. Advantageous for such purposes as thermography, inspection of circuit boards, mammography, and mapping.

  3. The Phosphoinositide 3-Kinase Pathway in Human Cancer: Genetic Alterations and Therapeutic Implications

    PubMed Central

    Arcaro, Alexandre; Guerreiro, Ana S

    2007-01-01

    The phosphoinositide 3-kinase (PI3K) pathway is frequently activated in human cancer and represents an attractive target for therapies based on small molecule inhibitors. PI3K isoforms play an essential role in the signal transduction events activated by cell surface receptors including receptor tyrosine kinases (RTKs) and G-protein-coupled receptors (GPCRs). There are eight known PI3K isoforms in humans, which have been subdivided into three classes (I-III). Therefore PI3Ks show considerable diversity and it remains unclear which kinases in this family should be targeted in cancer. The class IA of PI3K comprises the p110α, p110β and p110δ isoforms, which associate with activated RTKs. In human cancer, recent reports have described activating mutations in the PIK3CA gene encoding p110α, and inactivating mutations in the phosphatase and tensin homologue (PTEN) gene, a tumour suppressor and antagonist of the PI3K pathway. The PIK3CA mutations described in cancer constitutively activate p110α and, when expressed in cells drive oncogenic transformation. Moreover, these mutations cause the constitutive activation of downstream signaling molecules such as Akt/protein kinase B (PKB), mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase (S6K) that is commonly observed in cancer cells. In addition to p110α, the other isoforms of the PI3K family may also play a role in human cancer, although their individual functions remain to be precisely identified. In this review we will discuss the evidence implicating individual PI3K isoforms in human cancer and their potential as drug targets in this context. PMID:19384426

  4. Ubiquitin-Mediated Degradation of Aurora Kinases

    PubMed Central

    Lindon, Catherine; Grant, Rhys; Min, Mingwei

    2016-01-01

    The Aurora kinases are essential regulators of mitosis in eukaryotes. In somatic cell divisions of higher eukaryotes, the paralogs Aurora kinase A (AurA) and Aurora kinase B (AurB) play non-overlapping roles that depend on their distinct spatiotemporal activities. These mitotic roles of Aurora kinases depend on their interactions with different partners that direct them to different mitotic destinations and different substrates: AurB is a component of the chromosome passenger complex that orchestrates the tasks of chromosome segregation and cytokinesis, while AurA has many known binding partners and mitotic roles, including a well-characterized interaction with TPX2 that mediates its role in mitotic spindle assembly. Beyond the spatial control conferred by different binding partners, Aurora kinases are subject to temporal control of their activation and inactivation. Ubiquitin-mediated proteolysis is a critical route to irreversible inactivation of these kinases, which must occur for ordered transition from mitosis back to interphase. Both AurA and AurB undergo targeted proteolysis after anaphase onset as substrates of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase, even while they continue to regulate steps during mitotic exit. Temporal control of Aurora kinase destruction ensures that AurB remains active at the midbody during cytokinesis long after AurA activity has been largely eliminated from the cell. Differential destruction of Aurora kinases is achieved despite the fact that they are targeted at the same time and by the same ubiquitin ligase, making these substrates an interesting case study for investigating molecular determinants of ubiquitin-mediated proteolysis in higher eukaryotes. The prevalence of Aurora overexpression in cancers and their potential as therapeutic targets add importance to the task of understanding the molecular determinants of Aurora kinase stability. Here, we review what is known about ubiquitin-mediated targeting

  5. Dual Role of Jun N-Terminal Kinase Activity in Bone Morphogenetic Protein-Mediated Drosophila Ventral Head Development.

    PubMed

    Park, Sung Yeon; Stultz, Brian G; Hursh, Deborah A

    2015-12-01

    The Drosophila bone morphogenetic protein encoded by decapentaplegic (dpp) controls ventral head morphogenesis by expression in the head primordia, eye-antennal imaginal discs. These are epithelial sacs made of two layers: columnar disc proper cells and squamous cells of the peripodial epithelium. dpp expression related to head formation occurs in the peripodial epithelium; cis-regulatory mutations disrupting this expression display defects in sensory vibrissae, rostral membrane, gena, and maxillary palps. Here we document that disruption of this dpp expression causes apoptosis in peripodial cells and underlying disc proper cells. We further show that peripodial Dpp acts directly on the disc proper, indicating that Dpp must cross the disc lumen to act. We demonstrate that palp defects are mechanistically separable from the other mutant phenotypes; both are affected by the c-Jun N-terminal kinase pathway but in opposite ways. Slight reduction of both Jun N-terminal kinase and Dpp activity in peripodial cells causes stronger vibrissae, rostral membrane, and gena defects than Dpp alone; additionally, strong reduction of Jun N-terminal kinase activity alone causes identical defects. A more severe reduction of dpp results in similar vibrissae, rostral membrane, and gena defects, but also causes mutant maxillary palps. This latter defect is correlated with increased peripodial Jun N-terminal kinase activity and can be caused solely by ectopic activation of Jun N-terminal kinase. We conclude that formation of sensory vibrissae, rostral membrane, and gena tissue in head morphogenesis requires the action of Jun N-terminal kinase in peripodial cells, while excessive Jun N-terminal kinase signaling in these same cells inhibits the formation of maxillary palps.

  6. Ras-Driven Transcriptome Analysis Identifies Aurora Kinase A as a Potential Malignant Peripheral Nerve Sheath Tumor Therapeutic Target

    PubMed Central

    Patel, Ami V.; Eaves, David; Jessen, Walter J.; Rizvi, Tilat A.; Ecsedy, Jeffrey A.; Qian, Mark G.; Aronow, Bruce J.; Perentesis, John P.; Serra, Eduard; Cripe, Timothy P.; Miller, Shyra J.; Ratner, Nancy

    2013-01-01

    Purpose Patients with Neurofibromatosis Type 1 (NF1) develop malignant peripheral nerve sheath tumors (MPNST) which are often inoperable and do not respond well to current chemotherapies or radiation. The goal of this study was to utilize comprehensive gene expression analysis to identify novel therapeutic targets. Experimental Design Nerve Schwann cells and/or their precursors are the tumorigenic cell types in MPNST due to the loss of the NF1 gene, which encodes the RasGAP protein neurofibromin. Therefore, we created a transgenic mouse model, CNP-HRas12V, expressing constitutively-active HRas in Schwann cells and defined a Ras-induced gene expression signature to drive a Bayesian factor regression model analysis of differentially expressed genes in mouse and human neurofibromas and MPNSTs. We tested functional significance of Aurora kinase over-expression in MPNST in vitro and in vivo using Aurora kinase shRNAs and compounds that inhibit Aurora kinase. Results We identified 2000 genes with probability of linkage to nerve Ras signaling of which 339 were significantly differentially expressed in mouse and human NF1-related tumor samples relative to normal nerves, including Aurora kinase A (AURKA). AURKA was dramatically over-expressed and genomically amplified in MPNSTs but not neurofibromas. Aurora kinase shRNAs and Aurora kinase inhibitors blocked MPNST cell growth in vitro. Furthermore, an AURKA selective inhibitor, MLN8237, stabilized tumor volume and significantly increased survival of mice with MPNST xenografts. Conclusion Integrative cross-species transcriptome analyses combined with preclinical testing has provided an effective method for identifying candidates for molecular-targeted therapeutics. Blocking Aurora kinases may be a viable treatment platform for MPNST. PMID:22811580

  7. Optical Security System with Fourier Plane encoding.

    PubMed

    Javidi, B; Ahouzi, E

    1998-09-10

    We propose a new technique for security verification of personal documents and other forms of personal identifications such as ID cards, passports, or credit cards. In this technique a primary pattern that might be a phase-encoded image is convolved by a random code. The information is phase encoded on the personal document. Therefore the information cannot be reproduced by an intensity detector such as a CCD camera. An optical processor based on the nonlinear joint transform correlator is used to perform the verification and the validation of documents with this technique. By verification of the biometrics information and the random code simultaneously, the proposed optical system determines whether a card is authentic or is being used by an authorized person. We tested the performance of the optical system for security and validation in the presence of input noise and in the presence of distortion of the information on the card. The performance of the proposed method is evaluated by use of a number of metrics. Statistical analysis of the system is performed to investigate the noise tolerance and the discrimination against false inputs for security verification. PMID:18286124

  8. V123 BEAM SYNCHRONOUS ENCODER MODULE.

    SciTech Connect

    KERNER,T.; CONKLING,C.R.; OERTER,B.

    1999-03-29

    The V123 Synchronous Encoder Module transmits events to distributed trigger modules and embedded decoders around the RHIC rings where they are used to provide beam instrumentation triggers [1,2,3]. The RHIC beam synchronous event link hardware is mainly comprised of three VMEbus board designs, the central input modules (V201), and encoder modules (V123), and the distributed trigger modules (V124). Two beam synchronous links, one for each ring, are distributed via fiber optics and fanned out via twisted wire pair cables. The V123 synchronizes with the RF system clock derived from the beam bucket frequency and a revolution fiducial pulse. The RF system clock is used to create the beam synchronous event link carrier and events are synchronized with the rotation fiducial. A low jitter RF clock is later recovered from this carrier by phase lock loops in the trigger modules. Prioritized hardware and software triggers fill up to 15 beam event code transmission slots per revolution while tracking the ramping RF acceleration frequency and storage frequency. The revolution fiducial event is always the first event transmitted which is used to synchronize the firing of the abort kicker and to locate the first bucket for decoders distributed about the ring.

  9. Intensity encoding in unsupervised neural nets.

    PubMed

    Parkinson, Alan M.; Parpia, Dawood Y.

    1998-06-01

    The requirement of input vector normalisation in unsupervised neural nets results in a loss of information about the intensity of the signal contained in the input datastream. We show through a simple algebraic analysis that the introduction of an additional input channel encoding the root-mean-square intensity in the signals cannot restore this information if the input vectors have to be, nevertheless, all of the same length. We suggest an alternative method of encoding the input vectors where each of the input channels is split into two components in such a way that the resultant input vector is then of fixed length and retains information of the intensity in the signals. We further demonstrate, by using synthetic data, that a Kohonen Net is capable of forming topological maps of signals of different intensity, where an adjacency relationship is maintained both among the signals of the same frequency composition at different intensities and between signals of different frequency compositions at the same intensity. A second experiment reported here shows the same behaviour for less artificial inputs (based on a cochlear model) and additionally demonstrates that the trained network can respond appropriately to signals not previously encountered.

  10. Local encoding of computationally designed enzyme activity

    PubMed Central

    Allert, Malin; Dwyer, Mary A.; Hellinga, Homme W.

    2007-01-01

    One aim of computational protein design is to introduce novel enzyme activity into proteins of known structure by predicting mutations that stabilize transition states. Previously we have shown that it is possible to introduce triose phosphate isomerase activity into the ribose-binding protein of Escherichia coli by constructing 17 mutations in the first two layers of residues that surround the wild-type ligand-binding site. Here we report that these mutations can be “transplanted” into a homologous ribose-binding protein, isolated from the hyperthermophilic bacterium Thermoanaerobacter tengcongensis, with retention of catalytic activity, substrate affinity, and reaction pH dependence. The observed 105–106-fold rate enhancement corresponds to 70% of the maximally known transition-state binding energy. The wild-type sequences in these two homologues are almost perfectly conserved in the vicinity of their ribose-binding sites, but diverge significantly at increasing distance from these sites. The results demonstrate that the computationally designed mutations are sufficient to encode the observed enzyme activity, that all the observed activity is locally encoded within the layer of residues directly in contact with the substrate, and that in this case at least 70% of transition state stabilization energy can be achieved using straightforward considerations of stereochemical complementarity between enzyme and reactants. PMID:17196220

  11. Directed forgetting benefits motor sequence encoding.

    PubMed

    Tempel, Tobias; Frings, Christian

    2016-04-01

    Two experiments investigated directed forgetting of newly learned motor sequences. Concurrently with the list method of directed forgetting, participants successively learned two lists of motor sequences. Each sequence consisted of four consecutive finger movements. After a short distractor task, a recall test was given. Both experiments compared a forget group that was instructed to forget list-1 items with a remember group not receiving a forget instruction. We found that the instruction to forget list 1 enhanced recall of subsequently learned motor sequences. This benefit of directed forgetting occurred independently of costs for list 1. A mediation analysis showed that the encoding accuracy of list 2 was a mediator of the recall benefit, that is, the more accurate execution of motor sequences of list 2 after receiving a forget instruction for list 1 accounted for better recall of list 2. Thus, the adaptation of the list method to motor action provided more direct evidence on the effect of directed forgetting on subsequent learning. The results corroborate the assumption of a reset of encoding as a consequence of directed forgetting. PMID:26471189

  12. Malaria Parasite-Infected Erythrocytes Secrete PfCK1, the Plasmodium Homologue of the Pleiotropic Protein Kinase Casein Kinase 1.

    PubMed

    Dorin-Semblat, Dominique; Demarta-Gatsi, Claudia; Hamelin, Romain; Armand, Florence; Carvalho, Teresa Gil; Moniatte, Marc; Doerig, Christian

    2015-01-01

    Casein kinase 1 (CK1) is a pleiotropic protein kinase implicated in several fundamental processes of eukaryotic cell biology. Plasmodium falciparum encodes a single CK1 isoform, PfCK1, that is expressed at all stages of the parasite's life cycle. We have previously shown that the pfck1 gene cannot be disrupted, but that the locus can be modified if no loss-of-function is incurred, suggesting an important role for this kinase in intra-erythrocytic asexual proliferation. Here, we report on the use of parasite lines expressing GFP- or His-tagged PfCK1 from the endogenous locus to investigate (i) the dynamics of PfCK1 localisation during the asexual cycle in red blood cells, and (ii) potential interactors of PfCK1, so as to gain insight into the involvement of the enzyme in specific cellular processes. Immunofluorescence analysis reveals a dynamic localisation of PfCK1, with evidence for a pool of the enzyme being directed to the membrane of the host erythrocyte in the early stages of infection, followed by a predominantly intra-parasite localisation in trophozoites and schizonts and association with micronemes in merozoites. Furthermore, we present strong evidence that a pool of enzymatically active PfCK1 is secreted into the culture supernatant, demonstrating that PfCK1 is an ectokinase. Our interactome experiments and ensuing kinase assays using recombinant PfCK1 to phosphorylate putative interactors in vitro suggest an involvement of PfCK1 in many cellular processes such as mRNA splicing, protein trafficking, ribosomal, and host cell invasion. PMID:26629826

  13. Malaria Parasite-Infected Erythrocytes Secrete PfCK1, the Plasmodium Homologue of the Pleiotropic Protein Kinase Casein Kinase 1

    PubMed Central

    Dorin-Semblat, Dominique; Demarta-Gatsi, Claudia; Hamelin, Romain; Armand, Florence; Carvalho, Teresa Gil; Moniatte, Marc; Doerig, Christian

    2015-01-01

    Casein kinase 1 (CK1) is a pleiotropic protein kinase implicated in several fundamental processes of eukaryotic cell biology. Plasmodium falciparum encodes a single CK1 isoform, PfCK1, that is expressed at all stages of the parasite’s life cycle. We have previously shown that the pfck1 gene cannot be disrupted, but that the locus can be modified if no loss-of-function is incurred, suggesting an important role for this kinase in intra-erythrocytic asexual proliferation. Here, we report on the use of parasite lines expressing GFP- or His-tagged PfCK1 from the endogenous locus to investigate (i) the dynamics of PfCK1 localisation during the asexual cycle in red blood cells, and (ii) potential interactors of PfCK1, so as to gain insight into the involvement of the enzyme in specific cellular processes. Immunofluorescence analysis reveals a dynamic localisation of PfCK1, with evidence for a pool of the enzyme being directed to the membrane of the host erythrocyte in the early stages of infection, followed by a predominantly intra-parasite localisation in trophozoites and schizonts and association with micronemes in merozoites. Furthermore, we present strong evidence that a pool of enzymatically active PfCK1 is secreted into the culture supernatant, demonstrating that PfCK1 is an ectokinase. Our interactome experiments and ensuing kinase assays using recombinant PfCK1 to phosphorylate putative interactors in vitro suggest an involvement of PfCK1 in many cellular processes such as mRNA splicing, protein trafficking, ribosomal, and host cell invasion. PMID:26629826

  14. Functional specificity of cytoplasmic and transmembrane tyrosine kinases: identification of 130- and 75-kilodalton substrates of c-fps/fes tyrosine kinase in macrophages.

    PubMed Central

    Areces, L B; Dello Sbarba, P; Jücker, M; Stanley, E R; Feldman, R A

    1994-01-01

    c-fps/fes encodes a 92-kDa protein-tyrosine kinase (NCP92) that is expressed at the highest levels in macrophages. To determine if c-fps/fes can mediate the action of the colony-stimulating factor 1 (CSF-1) receptor (CSF-1R) and to identify potential targets of c-fps/fes in macrophages, we have overexpressed c-fps/fes in a CSF-1-dependent macrophage cell line. A 30- to 50-fold overexpression of c-fps/fes partially released these cells from their factor dependence by a nonautocrine mechanism, and this correlated with the tyrosine phosphorylation of two proteins of 130 and 75 kDa (P130 and P75). c-fps/fes did not cause tyrosine phosphorylation or activation of CSF-1 dependent targets, including CSF-1R, Shc, and phosphatidylinositol 3-kinase, and conversely, CSF-1 did not induce tyrosine phosphorylation of P130 and P75. P75 appears to be a novel phosphotyrosyl protein, whereas P130 cross-reacts with a known substrate of v-src. P130 and P75 may be direct substrates of c-fps/fes: P130 was tightly associated with NCP92, and the src homology 2 domain of NCP92 specifically bound phosphorylated P130 and P75 but not the CSF-1-induced phosphotyrosyl proteins, consistent with the possibility that P130 and P75 are physiological targets of c-fps/fes. We conclude that although c-fps/fes can functionally substitute for CSF-1R to a certain extent, these tyrosine kinases act largely independently of each other and that P130 and P75 are novel targets whose mechanisms of action may be unrelated to the signalling pathways utilized by receptor tyrosine kinases. Images PMID:8007965

  15. Overexpression of human selenoprotein H in neuronal cells enhances mitochondrial biogenesis and function through activation of protein kinase A, protein kinase B, and cyclic adenosine monophosphate response element-binding protein pathway.

    PubMed

    Mehta, Suresh L; Mendelev, Natalia; Kumari, Santosh; Andy Li, P

    2013-03-01

    Mitochondrial biogenesis is activated by nuclear encoded transcription co-activator peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), which is regulated by several upstream factors including protein kinase A and Akt/protein kinase B. We have previously shown that selenoprotein H enhances the levels of nuclear regulators for mitochondrial biogenesis, increases mitochondrial mass and improves mitochondrial respiratory rate, under physiological condition. Furthermore, overexpression of selenoprotein H protects neuronal HT22 cells from ultraviolet B irradiation-induced cell damage by lowering reactive oxygen species production, and inhibiting activation of caspase-3 and -9, as well as p53. The objective of this study is to identify the cell signaling pathways by which selenoprotein H initiates mitochondrial biogenesis. We first confirmed our previous observation that selenoprotein H transfected HT22 cells increased the protein levels of nuclear-encoded mitochondrial biogenesis factors, peroxisome proliferator-activated receptor γ coactivator-1α, nuclear respiratory factor 1 and mitochondrial transcription factor A. We then observed that total and phosphorylation of protein kinase A, Akt/protein kinase B and cyclic adenosine monophosphate response element-binding protein (CREB) were significantly increased in selenoprotein H transfected cells compared to vector transfected HT22 cells. To verify whether the observed stimulating effects on mitochondrial biogenesis pathways are caused by selenoprotein H and mediated through CREB, we knocked down selenoprotein H mRNA level using siRNA and inhibited CREB with napthol AS-E phosphate in selenoprotein H transfected cells and repeated the measurements of the aforementioned biomarkers. Our results revealed that silencing of selenoprotein H not only decreased the protein levels of PGC-1α, nuclear respiratory factor 1 and mitochondrial transcription factor A, but also decreased the total and

  16. Mediator kinase module and human tumorigenesis

    PubMed Central

    Clark, Alison D.; Oldenbroek, Marieke; Boyer, Thomas G.

    2016-01-01

    Mediator is a conserved multi-subunit signal processor through which regulatory informatiosn conveyed by gene-specific transcription factors is transduced to RNA Polymerase II (Pol II). In humans, MED13, MED12, CDK8 and Cyclin C (CycC) comprise a four-subunit “kinase” module that exists in variable association with a 26-subunit Mediator core. Genetic and biochemical studies have established the Mediator kinase module as a major ingress of developmental and oncogenic signaling through Mediator, and much of its function in signal-dependent gene regulation derives from its resident CDK8 kinase activity. For example, CDK8-targeted substrate phosphorylation impacts transcription factor half-life, Pol II activity and chromatin chemistry and functional status. Recent structural and biochemical studies have revealed a precise network of physical and functional subunit interactions required for proper kinase module activity. Accordingly, pathologic change in this activity through altered expression or mutation of constituent kinase module subunits can have profound consequences for altered signaling and tumor formation. Herein, we review the structural organization, biological function and oncogenic potential of the Mediator kinase module. We focus principally on tumor-associated alterations in kinase module subunits for which mechanistic relationships as opposed to strictly correlative associations are established. These considerations point to an emerging picture of the Mediator kinase module as an oncogenic unit, one in which pathogenic activation/deactivation through component change drives tumor formation through perturbation of signal-dependent gene regulation. It follows that therapeutic strategies to combat CDK8-driven tumors will involve targeted modulation of CDK8 activity or pharmacologic manipulation of dysregulated CDK8-dependent signaling pathways. PMID:26182352

  17. Non-degradative Ubiquitination of Protein Kinases

    PubMed Central

    Ball, K. Aurelia; Johnson, Jeffrey R.; Lewinski, Mary K.; Guatelli, John; Verschueren, Erik; Krogan, Nevan J.; Jacobson, Matthew P.

    2016-01-01

    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. PMID:27253329

  18. Non-degradative Ubiquitination of Protein Kinases.

    PubMed

    Ball, K Aurelia; Johnson, Jeffrey R; Lewinski, Mary K; Guatelli, John; Verschueren, Erik; Krogan, Nevan J; Jacobson, Matthew P

    2016-06-01

    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.

  19. KinasePA: Phosphoproteomics data annotation using hypothesis driven kinase perturbation analysis

    PubMed Central

    Yang, Pengyi; Patrick, Ellis; Humphrey, Sean J.; Ghazanfar, Shila; James, David E.; Jothi, Raja; Yang, Jean Yee Hwa

    2016-01-01

    Mass spectrometry (MS)-based quantitative phosphoproteomics has become a key approach for proteome-wide profiling of phosphorylation in tissues and cells. Traditional experimental design often compares a single treatment with a control, whereas increasingly more experiments are designed to compare multiple treatments with respect to a control. To this end, the development of bioinformatic tools that can integrate multiple treatments and visualise kinases and substrates under combinatorial perturbations is vital for dissecting concordant and/or independent effects of each treatment. Here, we propose a hypothesis driven kinase perturbation analysis (KinasePA) to annotate and visualise kinases and their substrates that are perturbed by various combinatorial effects of treatments in phosphoproteomics experiments. We demonstrate the utility of KinasePA through its application to two large-scale phosphoproteomics datasets and show its effectiveness in dissecting kinases and substrates within signalling pathways driven by unique combinations of cellular stimuli and inhibitors. We implemented and incorporated KinasePA as part of the “directPA” R package available from the comprehensive R archive network (CRAN). Furthermore, KinasePA also has an interactive web interface that can be readily applied to annotate user provided phosphoproteomics data (http://kinasepa.pengyiyang.org). PMID:27145998

  20. Co-inhibition of polo-like kinase 1 and Aurora kinases promotes mitotic catastrophe

    PubMed Central

    Li, Jingjing; Hong, Myung Jin; Chow, Jeremy P.H.; Man, Wing Yu; Mak, Joyce P.Y.; Ma, Hoi Tang; Poon, Randy Y.C.

    2015-01-01

    Mitosis is choreographed by a number of protein kinases including polo-like kinases and Aurora kinases. As these kinases are frequently dysregulated in cancers, small-molecule inhibitors have been developed for targeted anticancer therapies. Given that PLK1 and Aurora kinases possess both unique functions as well as co-regulate multiple mitotic events, whether pharmacological inhibition of these kinases together can enhance mitotic catastrophe remains an outstanding issue to be determined. Using concentrations of inhibitors that did not induce severe mitotic defects on their own, we found that both the metaphase arrest and mitotic slippage induced by inhibitors targeting Aurora A and Aurora B (MK-5108 and Barasertib respectively) were enhanced by a PLK1 inhibitor (BI 2536). We found that PLK1 is overexpressed in cells from nasopharyngeal carcinoma, a highly invasive cancer with poor prognosis, in comparison to normal nasopharyngeal epithelial cells. Nasopharyngeal carcinoma cells were more sensitive to BI 2536 as a single agent and co-inhibition with Aurora kinases than normal cells. These observations underscore the mechanism and potential benefits of targeting PLK1 and Aurora kinases to induce mitotic catastrophe in cancer cells. PMID:25871386