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Sample records for catalytically inactive kinase

  1. First inactive conformation of CK2 alpha, the catalytic subunit of protein kinase CK2

    DEFF Research Database (Denmark)

    Raaf, Jennifer; Issinger, Olaf-Georg; Niefind, Karsten

    2009-01-01

    (EPKs). To function as regulatory key components, EPKs normally exist in inactive ground states and are activated only upon specific signals. Typically, this activation is accompanied by large conformational changes in helix alpha C and in the activation segment, leading to a characteristic arrangement......The Ser/Thr kinase casein kinase 2 (CK2) is a heterotetrameric enzyme composed of two catalytic chains (CK2alpha, catalytic subunit of CK2) attached to a dimer of two noncatalytic subunits (CK2beta, noncatalytic subunit of CK2). CK2alpha belongs to the superfamily of eukaryotic protein kinases...... of catalytic key elements. For CK2alpha, however, no strict physiological control of activity is known. Accordingly, CK2alpha was found so far exclusively in the characteristic conformation of active EPKs, which is, in this case, additionally stabilized by a unique intramolecular contact between the N...

  2. A Src-like inactive conformation in the abl tyrosine kinase domain.

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    Nicholas M Levinson

    2006-05-01

    Full Text Available The improper activation of the Abl tyrosine kinase results in chronic myeloid leukemia (CML. The recognition of an inactive conformation of Abl, in which a catalytically important Asp-Phe-Gly (DFG motif is flipped by approximately 180 degrees with respect to the active conformation, underlies the specificity of the cancer drug imatinib, which is used to treat CML. The DFG motif is not flipped in crystal structures of inactive forms of the closely related Src kinases, and imatinib does not inhibit c-Src. We present a structure of the kinase domain of Abl, determined in complex with an ATP-peptide conjugate, in which the protein adopts an inactive conformation that resembles closely that of the Src kinases. An interesting aspect of the Src-like inactive structure, suggested by molecular dynamics simulations and additional crystal structures, is the presence of features that might facilitate the flip of the DFG motif by providing room for the phenylalanine to move and by coordinating the aspartate side chain as it leaves the active site. One class of mutations in BCR-Abl that confers resistance to imatinib appears more likely to destabilize the inactive Src-like conformation than the active or imatinib-bound conformations. Our results suggest that interconversion between distinctly different inactive conformations is a characteristic feature of the Abl kinase domain.

  3. LOXL2 catalytically inactive mutants mediate epithelial-to-mesenchymal transition

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    Eva P. Cuevas

    2014-01-01

    Lysyl-oxidase-like 2 (LOXL2 is a member of the lysyl oxidase family that catalyzes the cross-linking of collagens or elastins in the extracellular matrix, thus regulating the tensile strength of tissues. However, many reports have suggested different intracellular roles for LOXL2, including the ability to regulate gene transcription and tumor progression. We previously reported that LOXL2 mediates epithelial-to-mesenchymal transition (EMT by Snail1-dependent and independent mechanisms, related to E-cadherin silencing and downregulation of epidermal differentiation and cell polarity components, respectively. Whether or not the catalytic activity of LOXL2 is required to induce/sustain EMT is actually unknown. Here we show that LOXL2 catalytic inactive mutants collaborate with Snail1 in E-cadherin gene repression to trigger EMT and, in addition, promote FAK/Src pathway activation to support EMT. These findings reveal a non-conventional role of LOXL2 on regulating epithelial cell plasticity.

  4. Phospholipase C-related catalytically inactive protein (PRIP controls KIF5B-mediated insulin secretion

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    Satoshi Asano

    2014-05-01

    Full Text Available We previously reported that phospholipase C-related catalytically inactive protein (PRIP-knockout mice exhibited hyperinsulinemia. Here, we investigated the role of PRIP in insulin granule exocytosis using Prip-knockdown mouse insulinoma (MIN6 cells. Insulin release from Prip-knockdown MIN6 cells was higher than that from control cells, and Prip knockdown facilitated movement of GFP-phogrin-labeled insulin secretory vesicles. Double-immunofluorescent staining and density step-gradient analyses showed that the KIF5B motor protein co-localized with insulin vesicles in Prip-knockdown MIN6 cells. Knockdown of GABAA-receptor-associated protein (GABARAP, a microtubule-associated PRIP-binding partner, by Gabarap silencing in MIN6 cells reduced the co-localization of insulin vesicles with KIF5B and the movement of vesicles, resulting in decreased insulin secretion. However, the co-localization of KIF5B with microtubules was not altered in Prip- and Gabarap-knockdown cells. The presence of unbound GABARAP, freed either by an interference peptide or by Prip silencing, in MIN6 cells enhanced the co-localization of insulin vesicles with microtubules and promoted vesicle mobility. Taken together, these data demonstrate that PRIP and GABARAP function in a complex to regulate KIF5B-mediated insulin secretion, providing new insights into insulin exocytic mechanisms.

  5. Antibiotic Binding Drives Catalytic Activation of Aminoglycoside Kinase APH(2″)-Ia.

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    Caldwell, Shane J; Huang, Yue; Berghuis, Albert M

    2016-06-01

    APH(2″)-Ia is a widely disseminated resistance factor frequently found in clinical isolates of Staphylococcus aureus and pathogenic enterococci, where it is constitutively expressed. APH(2″)-Ia confers high-level resistance to gentamicin and related aminoglycosides through phosphorylation of the antibiotic using guanosine triphosphate (GTP) as phosphate donor. We have determined crystal structures of the APH(2″)-Ia in complex with GTP analogs, guanosine diphosphate, and aminoglycosides. These structures collectively demonstrate that aminoglycoside binding to the GTP-bound kinase drives conformational changes that bring distant regions of the protein into contact. These changes in turn drive a switch of the triphosphate cofactor from an inactive, stabilized conformation to a catalytically competent active conformation. This switch has not been previously reported for antibiotic kinases or for the structurally related eukaryotic protein kinases. This catalytic triphosphate switch presents a means by which the enzyme can curtail wasteful hydrolysis of GTP in the absence of aminoglycosides, providing an evolutionary advantage to this enzyme.

  6. Redox-inactive metal ions promoted the catalytic reactivity of non-heme manganese complexes towards oxygen atom transfer.

    Science.gov (United States)

    Choe, Cholho; Yang, Ling; Lv, Zhanao; Mo, Wanling; Chen, Zhuqi; Li, Guangxin; Yin, Guochuan

    2015-05-21

    Redox-inactive metal ions can modulate the reactivity of redox-active metal ions in a variety of biological and chemical oxidations. Many synthetic models have been developed to help address the elusive roles of these redox-inactive metal ions. Using a non-heme manganese(II) complex as the model, the influence of redox-inactive metal ions as a Lewis acid on its catalytic efficiency in oxygen atom transfer was investigated. In the absence of redox-inactive metal ions, the manganese(II) catalyst is very sluggish, for example, in cyclooctene epoxidation, providing only 9.9% conversion with 4.1% yield of epoxide. However, addition of 2 equiv. of Al(3+) to the manganese(II) catalyst sharply improves the epoxidation, providing up to 97.8% conversion with 91.4% yield of epoxide. EPR studies of the manganese(II) catalyst in the presence of an oxidant reveal a 16-line hyperfine structure centered at g = 2.0, clearly indicating the formation of a mixed valent di-μ-oxo-bridged diamond core, Mn(III)-(μ-O)2-Mn(IV). The presence of a Lewis acid like Al(3+) causes the dissociation of this diamond Mn(III)-(μ-O)2-Mn(IV) core to form monomeric manganese(iv) species which is responsible for improved epoxidation efficiency. This promotional effect has also been observed in other manganese complexes bearing various non-heme ligands. The findings presented here have provided a promising strategy to explore the catalytic reactivity of some di-μ-oxo-bridged complexes by adding non-redox metal ions to in situ dissociate those dimeric cores and may also provide clues to understand the mechanism of methane monooxygenase which has a similar diiron diamond core as the intermediate.

  7. RIPK3 deficiency or catalytically inactive RIPK1 provides greater benefit than MLKL deficiency in mouse models of inflammation and tissue injury

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    Newton, K; Dugger, D L; Maltzman, A; Greve, J M; Hedehus, M; Martin-McNulty, B; Carano, R A D; Cao, T C; van Bruggen, N; Bernstein, L; Lee, W P; Wu, X; DeVoss, J; Zhang, J; Jeet, S; Peng, I; McKenzie, B S; Roose-Girma, M; Caplazi, P; Diehl, L; Webster, J D; Vucic, D

    2016-01-01

    Necroptosis is a caspase-independent form of cell death that is triggered by activation of the receptor interacting serine/threonine kinase 3 (RIPK3) and phosphorylation of its pseudokinase substrate mixed lineage kinase-like (MLKL), which then translocates to membranes and promotes cell lysis. Activation of RIPK3 is regulated by the kinase RIPK1. Here we analyze the contribution of RIPK1, RIPK3, or MLKL to several mouse disease models. Loss of RIPK3 had no effect on lipopolysaccharide-induced sepsis, dextran sodium sulfate-induced colitis, cerulein-induced pancreatitis, hypoxia-induced cerebral edema, or the major cerebral artery occlusion stroke model. However, kidney ischemia–reperfusion injury, myocardial infarction, and systemic inflammation associated with A20 deficiency or high-dose tumor necrosis factor (TNF) were ameliorated by RIPK3 deficiency. Catalytically inactive RIPK1 was also beneficial in the kidney ischemia–reperfusion injury model, the high-dose TNF model, and in A20−/− mice. Interestingly, MLKL deficiency offered less protection in the kidney ischemia–reperfusion injury model and no benefit in A20−/− mice, consistent with necroptosis-independent functions for RIPK1 and RIPK3. Combined loss of RIPK3 (or MLKL) and caspase-8 largely prevented the cytokine storm, hypothermia, and morbidity induced by TNF, suggesting that the triggering event in this model is a combination of apoptosis and necroptosis. Tissue-specific RIPK3 deletion identified intestinal epithelial cells as the major target organ. Together these data emphasize that MLKL deficiency rather than RIPK1 inactivation or RIPK3 deficiency must be examined to implicate a role for necroptosis in disease. PMID:27177019

  8. Molecular basis for the catalytic inactivity of a naturally occurring near-null variant of human ALOX15.

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    Horn, Thomas; Ivanov, Igor; Di Venere, Almerinda; Kakularam, Kumar Reddy; Reddanna, Pallu; Conrad, Melanie L; Richter, Constanze; Scheerer, Patrick; Kuhn, Hartmut

    2013-12-01

    Mammalian lipoxygenases belong to a family of lipid-peroxidizing enzymes, which have been implicated in cardiovascular, hyperproliferative and neurodegenerative diseases. Here we report that a naturally occurring mutation in the hALOX15 gene leads to expression of a catalytically near-null enzyme variant (hGly422Glu). The inactivity may be related to severe misfolding of the enzyme protein, which was concluded from CD-spectra as well as from thermal and chemical stability assays. In silico mutagenesis experiments suggest that most mutations at hGly422 have the potential to induce sterical clash, which might be considered a reason for protein misfolding. hGly422 is conserved among ALOX5, ALOX12 and ALOX15 isoforms and corresponding hALOX12 and hALOX5 mutants also exhibited a reduced catalytic activity. Interestingly, in the hALOX5 Gly429Glu mutants the reaction specificity of arachidonic acid oxygenation was shifted from 5S- to 8S- and 12R-H(p)ETE formation. Taken together, our data indicate that the conserved glycine is of functional importance for these enzyme variants and most mutants at this position lose catalytic activity.

  9. Novel Catalytically-Inactive PII Metalloproteinases from a Viperid Snake Venom with Substitutions in the Canonical Zinc-Binding Motif

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    Camacho, Erika; Sanz, Libia; Escalante, Teresa; Pérez, Alicia; Villalta, Fabián; Lomonte, Bruno; Neves-Ferreira, Ana Gisele C.; Feoli, Andrés; Calvete, Juan J.; Gutiérrez, José María; Rucavado, Alexandra

    2016-01-01

    Snake venom metalloproteinases (SVMPs) play key biological roles in prey immobilization and digestion. The majority of these activities depend on the hydrolysis of relevant protein substrates in the tissues. Hereby, we describe several isoforms and a cDNA clone sequence, corresponding to PII SVMP homologues from the venom of the Central American pit viper Bothriechis lateralis, which have modifications in the residues of the canonical sequence of the zinc-binding motif HEXXHXXGXXH. As a consequence, the proteolytic activity of the isolated proteins was undetectable when tested on azocasein and gelatin. These PII isoforms comprise metalloproteinase and disintegrin domains in the mature protein, thus belonging to the subclass PIIb of SVMPs. PII SVMP homologues were devoid of hemorrhagic and in vitro coagulant activities, effects attributed to the enzymatic activity of SVMPs, but induced a mild edema. One of the isoforms presents the characteristic RGD sequence in the disintegrin domain and inhibits ADP- and collagen-induced platelet aggregation. Catalytically-inactive SVMP homologues may have been hitherto missed in the characterization of snake venoms. The presence of such enzymatically-inactive homologues in snake venoms and their possible toxic and adaptive roles deserve further investigation. PMID:27754342

  10. Cu,Zn-superoxide dismutase without Zn is folded but catalytically inactive.

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    Nedd, Sean; Redler, Rachel L; Proctor, Elizabeth A; Dokholyan, Nikolay V; Alexandrova, Anastassia N

    2014-12-12

    Amyotrophic lateral sclerosis has been linked to the gain of aberrant function of superoxide dismutase, Cu,Zn-SOD1 upon protein misfolding. The mechanism of SOD1 misfolding is thought to involve mutations leading to the loss of Zn, followed by protein unfolding and aggregation. We show that the removal of Zn from SOD1 may not lead to an immediate unfolding but immediately deactivates the enzyme through a combination of subtle structural and electronic effects. Using quantum mechanics/discrete molecular dynamics, we showed that both Zn-less wild-type (WT)-SOD1 and its D124N mutant that does not bind Zn have at least metastable folded states. In those states, the reduction potential of Cu increases, leading to the presence of detectable amounts of Cu(I) instead of Cu(II) in the active site, as confirmed experimentally. The Cu(I) protein cannot participate in the catalytic Cu(I)-Cu(II) cycle. However, even without the full reduction to Cu(I), the Cu site in the Zn-less variants of SOD1 is shown to be catalytically incompetent: unable to bind superoxide in a way comparable to the WT-SOD1. The changes are more radical and different in the D124N Zn-less mutant than in the Zn-less WT-SOD1, suggesting D124N being perhaps not the most adequate model for Zn-less SOD1. Overall, Zn in SOD1 appears to be influencing the Cu site directly by adjusting its reduction potential and geometry. Thus, the role of Zn in SOD1 is not just structural, as was previously thought; it is a vital part of the catalytic machinery.

  11. Catalytic inactive heme oxygenase-1 protein regulates its own expression in oxidative stress.

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    Lin, Qing S; Weis, Sebastian; Yang, Guang; Zhuang, Tiangang; Abate, Aida; Dennery, Phyllis A

    2008-03-01

    Heme oxygenase-1 (HO-1) catalyzes the degradation of heme and forms antioxidant bile pigments as well as the signaling molecule carbon monoxide. HO-1 is inducible in response to a variety of chemical and physical stress conditions to function as a cytoprotective molecule. Therefore, it is important to maintain the basal level of HO-1 expression even when substrate availability is limited. We hypothesized that the HO-1 protein itself could regulate its own expression in a positive feedback manner, and that this positive feedback was important in the HO-1 gene induction in response to oxidative stress. In cultured NIH 3T3 cells, transfection of HO-1 cDNA or intracellular delivery of pure HO-1 protein resulted in activation of a 15-kb HO-1 promoter upstream of luciferase as visualized by bioluminescent technology and increased HO-1 mRNA and protein levels. These effects were independent of HO activity because an enzymatically inactive mutant form of HO-1 similarly activated the HO-1 promoter and incubation with HO inhibitor metalloporphyrin SnPP did not affect the promoter activation. In addition, HO-1-specific siRNA significantly reduced hemin and cadmium chloride-mediated HO-1 induction. Furthermore, deletion analyses demonstrated that the E1 and E2 distal enhancers of the HO-1 promoter are required for this HO-1 autoregulation. These experiments document feed-forward autoregulation of HO-1 in oxidative stress and suggest that HO-1 protein has a role in the induction process. We speculate that this mechanism may be useful for maintaining HO-1 expression when substrate is limited and may also serve to up-regulate other genes to promote cytoprotection and to modulate cell proliferation.

  12. Extending Thymidine Kinase Activity to the Catalytic Repertoire of Human Deoxycytidine Kinase

    Energy Technology Data Exchange (ETDEWEB)

    Hazra, Saugata; Sabini, Eliszbetta; Ort, Stephan; Konrad, Manfred; Lavie, Arnon; (UIC); (MXPL-G)

    2009-03-04

    Salvage of nucleosides in the cytosol of human cells is carried out by deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1). Whereas TK1 is only responsible for thymidine phosphorylation, dCK is capable of converting dC, dA, and dG into their monophosphate forms. Using structural data on dCK, we predicted that select mutations at the active site would, in addition to making the enzyme faster, expand the catalytic repertoire of dCK to include thymidine. Specifically, we hypothesized that steric repulsion between the methyl group of the thymine base and Arg104 is the main factor preventing the phosphorylation of thymidine by wild-type dCK. Here we present kinetic data on several dCK variants where Arg104 has been replaced by select residues, all performed in combination with the mutation of Asp133 to an alanine. We show that several hydrophobic residues at position 104 endow dCK with thymidine kinase activity. Depending on the exact nature of the mutations, the enzyme's substrate preference is modified. The R104M-D133A double mutant is a pyrimidine-specific enzyme due to large K{sub m} values with purines. The crystal structure of the double mutant R104M-D133A in complex with the L-form of thymidine supplies a structural explanation for the ability of this variant to phosphorylate thymidine and thymidine analogs. The replacement of Arg104 by a smaller residue allows L-dT to bind deeper into the active site, making space for the C5-methyl group of the thymine base. The unique catalytic properties of several of the mutants make them good candidates for suicide-gene/protein-therapy applications.

  13. Phospholipase C-related catalytically inactive protein participates in the autophagic elimination of Staphylococcus aureus infecting mouse embryonic fibroblasts.

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    Kae Harada-Hada

    Full Text Available Autophagy is an intrinsic host defense system that recognizes and eliminates invading bacterial pathogens. We have identified microtubule-associated protein 1 light chain 3 (LC3, a hallmark of autophagy, as a binding partner of phospholipase C-related catalytically inactive protein (PRIP that was originally identified as an inositol trisphosphate-binding protein. Here, we investigated the involvement of PRIP in the autophagic elimination of Staphylococcus aureus in infected mouse embryonic fibroblasts (MEFs. We observed significantly more LC3-positive autophagosome-like vacuoles enclosing an increased number of S. aureus cells in PRIP-deficient MEFs than control MEFs, 3 h and 4.5 h post infection, suggesting that S. aureus proliferates in LC3-positive autophagosome-like vacuoles in PRIP-deficient MEFs. We performed autophagic flux analysis using an mRFP-GFP-tagged LC3 plasmid and found that autophagosome maturation is significantly inhibited in PRIP-deficient MEFs. Furthermore, acidification of autophagosomes was significantly inhibited in PRIP-deficient MEFs compared to the wild-type MEFs, as determined by LysoTracker staining and time-lapse image analysis performed using mRFP-GFP-tagged LC3. Taken together, our data show that PRIP is required for the fusion of S. aureus-containing autophagosome-like vacuoles with lysosomes, indicating that PRIP is a novel modulator in the regulation of the innate immune system in non-professional phagocytic host cells.

  14. Phospholipase C-related catalytically inactive protein (PRIP regulates lipolysis in adipose tissue by modulating the phosphorylation of hormone-sensitive lipase.

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    Toshiya Okumura

    Full Text Available Phosphorylation of hormone-sensitive lipase (HSL and perilipin by protein kinase A (PKA promotes the hydrolysis of lipids in adipocytes. Although activation of lipolysis by PKA has been well studied, inactivation via protein phosphatases is poorly understood. Here, we investigated whether phospholipase C-related catalytically inactive protein (PRIP, a binding partner for protein phosphatase 1 and protein phosphatase 2A (PP2A, is involved in lipolysis by regulating phosphatase activity. PRIP knockout (PRIP-KO mice displayed reduced body-fat mass as compared with wild-type mice fed with standard chow ad libitum. Most other organs appeared normal, suggesting that mutant mice had aberrant fat metabolism in adipocytes. HSL in PRIP-KO adipose tissue was highly phosphorylated compared to that in wild-type mice. Starvation of wild-type mice or stimulation of adipose tissue explants with the catabolic hormone, adrenaline, translocated both PRIP and PP2A from the cytosol to lipid droplets, but the translocation of PP2A was significantly reduced in PRIP-KO adipocytes. Consistently, the phosphatase activity associated with lipid droplet fraction in PRIP-KO adipocytes was significantly reduced and was independent of adrenaline stimulation. Lipolysis activity, as assessed by measurement of non-esterified fatty acids and glycerol, was higher in PRIP-KO adipocytes. When wild-type adipocytes were treated with a phosphatase inhibitor, they showed a high lipolysis activity at the similar level to PRIP-KO adipocytes. Collectively, these results suggest that PRIP promotes the translocation of phosphatases to lipid droplets to trigger the dephosphorylation of HSL and perilipin A, thus reducing PKA-mediated lipolysis.

  15. Structural and functional insights into the catalytic inactivity of the major fraction of buffalo milk xanthine oxidoreductase.

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    Kaustubh S Gadave

    Full Text Available BACKGROUND: Xanthine oxidoreductase (XOR existing in two interconvertible forms, xanthine dehydrogenase (XDH and xanthine oxidase (XO, catabolises xanthine to uric acid that is further broken down to antioxidative agent allantoin. XOR also produces free radicals serving as second messenger and microbicidal agent. Large variation in the XO activity has been observed among various species. Both hypo and hyper activity of XOR leads to pathophysiological conditions. Given the important nutritional role of buffalo milk in human health especially in south Asia, it is crucial to understand the functional properties of buffalo XOR and the underlying structural basis of variations in comparison to other species. METHODS AND FINDINGS: Buffalo XO activity of 0.75 U/mg was almost half of cattle XO activity. Enzymatic efficiency (k cat/K m of 0.11 sec(-1 µM(-1 of buffalo XO was 8-10 times smaller than that of cattle XO. Buffalo XOR also showed lower antibacterial activity than cattle XOR. A CD value (Δε430 nm of 46,000 M(-1 cm(-1 suggested occupancy of 77.4% at Fe/S I centre. Buffalo XOR contained 0.31 molybdenum atom/subunit of which 48% existed in active sulfo form. The active form of XO in buffalo was only 16% in comparison to ∼30% in cattle. Sequencing revealed 97.4% similarity between buffalo and cattle XOR. FAD domain was least conserved, while metal binding domains (Fe/S and Molybdenum were highly conserved. Homology modelling of buffalo XOR showed several variations occurring in clusters, especially close to FAD binding pocket which could affect NAD(+ entry in the FAD centre. The difference in XO activity seems to be originating from cofactor deficiency, especially molybdenum. CONCLUSION: A major fraction of buffalo milk XOR exists in a catalytically inactive form due to high content of demolybdo and desulfo forms. Lower Fe/S content and structural factors might be contributing to lower enzymatic efficiency of buffalo XOR in a minor way.

  16. Cyclic AMP analog blocks kinase activation by stabilizing inactive conformation: conformational selection highlights a new concept in allosteric inhibitor design.

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    Badireddy, Suguna; Yunfeng, Gao; Ritchie, Mark; Akamine, Pearl; Wu, Jian; Kim, Choel W; Taylor, Susan S; Qingsong, Lin; Swaminathan, Kunchithapadam; Anand, Ganesh S

    2011-03-01

    The regulatory (R) subunit of protein kinase A serves to modulate the activity of protein kinase A in a cAMP-dependent manner and exists in two distinct and structurally dissimilar, end point cAMP-bound "B" and C-subunit-bound "H"-conformations. Here we report mechanistic details of cAMP action as yet unknown through a unique approach combining x-ray crystallography with structural proteomics approaches, amide hydrogen/deuterium exchange and ion mobility mass spectrometry, applied to the study of a stereospecific cAMP phosphorothioate analog and antagonist((Rp)-cAMPS). X-ray crystallography shows cAMP-bound R-subunit in the B form but surprisingly the antagonist Rp-cAMPS-bound R-subunit crystallized in the H conformation, which was previously assumed to be induced only by C-subunit-binding. Apo R-subunit crystallized in the B form as well but amide exchange mass spectrometry showed large differences between apo, agonist and antagonist-bound states of the R-subunit. Further ion mobility reveals the apo R-subunit as an ensemble of multiple conformations with collisional cross-sectional areas spanning both the agonist and antagonist-bound states. Thus contrary to earlier studies that explained the basis for cAMP action through "induced fit" alone, we report evidence for conformational selection, where the ligand-free apo form of the R-subunit exists as an ensemble of both B and H conformations. Although cAMP preferentially binds the B conformation, Rp-cAMPS interestingly binds the H conformation. This reveals the unique importance of the equatorial oxygen of the cyclic phosphate in mediating conformational transitions from H to B forms highlighting a novel approach for rational structure-based drug design. Ideal inhibitors such as Rp-cAMPS are those that preferentially "select" inactive conformations of target proteins by satisfying all "binding" constraints alone without inducing conformational changes necessary for activation.

  17. Comprehensive Characterization of AMP-Activated Protein Kinase Catalytic Domain by Top-Down Mass Spectrometry

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    Yu, Deyang; Peng, Ying; Ayaz-Guner, Serife; Gregorich, Zachery R.; Ge, Ying

    2016-02-01

    AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that is essential in regulating energy metabolism in all eukaryotic cells. It is a heterotrimeric protein complex composed of a catalytic subunit (α) and two regulatory subunits (β and γ). C-terminal truncation of AMPKα at residue 312 yielded a protein that is active upon phosphorylation of Thr172 in the absence of β and γ subunits, which is refered to as the AMPK catalytic domain and commonly used to substitute for the AMPK heterotrimeric complex in in vitro kinase assays. However, a comprehensive characterization of the AMPK catalytic domain is lacking. Herein, we expressed a His-tagged human AMPK catalytic domin (denoted as AMPKΔ) in E. coli, comprehensively characterized AMPKΔ in its basal state and after in vitro phosphorylation using top-down mass spectrometry (MS), and assessed how phosphorylation of AMPKΔ affects its activity. Unexpectedly, we found that bacterially-expressed AMPKΔ was basally phosphorylated and localized the phosphorylation site to the His-tag. We found that AMPKΔ had noticeable basal activity and was capable of phosphorylating itself and its substrates without activating phosphorylation at Thr172. Moreover, our data suggested that Thr172 is the only site phosphorylated by its upstream kinase, liver kinase B1, and that this phosphorylation dramatically increases the kinase activity of AMPKΔ. Importantly, we demonstrated that top-down MS in conjunction with in vitro phosphorylation assay is a powerful approach for monitoring phosphorylation reaction and determining sequential order of phosphorylation events in kinase-substrate systems.

  18. Presenilins mutated at Asp-257 or Asp-385 restore Pen-2 expression and Nicastrin glycosylation but remain catalytically inactive in the absence of wild type Presenilin.

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    Nyabi, Omar; Bentahir, Mostafa; Horré, Katrien; Herreman, An; Gottardi-Littell, Numa; Van Broeckhoven, Christine; Merchiers, Pascal; Spittaels, Kurt; Annaert, Wim; De Strooper, Bart

    2003-10-31

    The Presenilins are part of the gamma-secretase complex that is involved in the regulated intramembrane proteolysis of amyloid precursor protein and other type I integral membrane proteins. Nicastrin, Pen-2, and Aph1 are the other proteins of this complex. The Presenilins probably contribute the catalytic activity to the protease complex. However, several investigators reported normal Abeta-peptide generation in cells expressing Presenilins mutated at the putative catalytic site residue Asp-257, contradicting this hypothesis. Because endogenously expressed wild type Presenilin could contribute to residual gamma-secretase activity in these experiments, we have reinvestigated the problem by expressing mutated Presenilins in a Presenilin-negative cell line. We confirm that Presenilins with mutated Asp residues are catalytically inactive. Unexpectedly, these mutated Presenilins are still partially processed into amino- and carboxyl-terminal fragments by a "Presenilinase"-like activity. They are also able to rescue Pen-2 expression and Nicastrin glycosylation in Presenilin-negative cells and become incorporated into large approximately 440-kDa complexes as assessed by blue native gel electrophoresis. Our study demonstrates that the catalytic activity of Presenilin and its other functions in the generation, stabilization, and transport of the gamma-secretase complex can be separated and extends the concept that Presenilins are multifunctional proteins.

  19. Effects of FGFR2 kinase activation loop dynamics on catalytic activity.

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    Karp, Jerome M; Sparks, Samuel; Cowburn, David

    2017-02-01

    The structural mechanisms by which receptor tyrosine kinases (RTKs) regulate catalytic activity are diverse and often based on subtle changes in conformational dynamics. The regulatory mechanism of one such RTK, fibroblast growth factor receptor 2 (FGFR2) kinase, is still unknown, as the numerous crystal structures of the unphosphorylated and phosphorylated forms of the kinase domains show no apparent structural change that could explain how phosphorylation could enable catalytic activity. In this study, we use several enhanced sampling molecular dynamics (MD) methods to elucidate the structural changes to the kinase's activation loop that occur upon phosphorylation. We show that phosphorylation favors inward motion of Arg664, while simultaneously favoring outward motion of Leu665 and Pro666. The latter structural change enables the substrate to bind leading to its resultant phosphorylation. Inward motion of Arg664 allows it to interact with the γ-phosphate of ATP as well as the substrate tyrosine. We show that this stabilizes the tyrosine and primes it for the catalytic phosphotransfer, and it may lower the activation barrier of the phosphotransfer reaction. Our work demonstrates the value of including dynamic information gleaned from computer simulation in deciphering RTK regulatory function.

  20. IRBIT Interacts with the Catalytic Core of Phosphatidylinositol Phosphate Kinase Type Iα and IIα through Conserved Catalytic Aspartate Residues.

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    Hideaki Ando

    Full Text Available Phosphatidylinositol phosphate kinases (PIPKs are lipid kinases that generate phosphatidylinositol 4,5-bisphosphate (PI(4,5P2, a critical lipid signaling molecule that regulates diverse cellular functions, including the activities of membrane channels and transporters. IRBIT (IP3R-binding protein released with inositol 1,4,5-trisphosphate is a multifunctional protein that regulates diverse target proteins. Here, we report that IRBIT forms signaling complexes with members of the PIPK family. IRBIT bound to all PIPK isoforms in heterologous expression systems and specifically interacted with PIPK type Iα (PIPKIα and type IIα (PIPKIIα in mouse cerebellum. Site-directed mutagenesis revealed that two conserved catalytic aspartate residues of PIPKIα and PIPKIIα are involved in the interaction with IRBIT. Furthermore, phosphatidylinositol 4-phosphate, Mg2+, and/or ATP interfered with the interaction, suggesting that IRBIT interacts with catalytic cores of PIPKs. Mutations of phosphorylation sites in the serine-rich region of IRBIT affected the selectivity of its interaction with PIPKIα and PIPKIIα. The structural flexibility of the serine-rich region, located in the intrinsically disordered protein region, is assumed to underlie the mechanism of this interaction. Furthermore, in vitro binding experiments and immunocytochemistry suggest that IRBIT and PIPKIα interact with the Na+/HCO3- cotransporter NBCe1-B. These results suggest that IRBIT forms signaling complexes with PIPKIα and NBCe1-B, whose activity is regulated by PI(4,5P2.

  1. Unique kinase catalytic mechanism of AceK with a single magnesium ion.

    Directory of Open Access Journals (Sweden)

    Quanjie Li

    Full Text Available Isocitrate dehydrogenase kinase/phosphatase (AceK is the founding member of the protein phosphorylation system in prokaryotes. Based on the novel and unique structural characteristics of AceK recently uncovered, we sought to understand its kinase reaction mechanism, along with other features involved in the phosphotransfer process. Herein we report density functional theory QM calculations of the mechanism of the phosphotransfer reaction catalysed by AceK. The transition states located by the QM calculations indicate that the phosphorylation reaction, catalysed by AceK, follows a dissociative mechanism with Asp457 serving as the catalytic base to accept the proton delivered by the substrate. Our results also revealed that AceK prefers a single Mg(2+-containing active site in the phosphotransfer reaction. The catalytic roles of conserved residues in the active site are discussed.

  2. Effects of FGFR2 kinase activation loop dynamics on catalytic activity

    Science.gov (United States)

    2017-01-01

    The structural mechanisms by which receptor tyrosine kinases (RTKs) regulate catalytic activity are diverse and often based on subtle changes in conformational dynamics. The regulatory mechanism of one such RTK, fibroblast growth factor receptor 2 (FGFR2) kinase, is still unknown, as the numerous crystal structures of the unphosphorylated and phosphorylated forms of the kinase domains show no apparent structural change that could explain how phosphorylation could enable catalytic activity. In this study, we use several enhanced sampling molecular dynamics (MD) methods to elucidate the structural changes to the kinase’s activation loop that occur upon phosphorylation. We show that phosphorylation favors inward motion of Arg664, while simultaneously favoring outward motion of Leu665 and Pro666. The latter structural change enables the substrate to bind leading to its resultant phosphorylation. Inward motion of Arg664 allows it to interact with the γ-phosphate of ATP as well as the substrate tyrosine. We show that this stabilizes the tyrosine and primes it for the catalytic phosphotransfer, and it may lower the activation barrier of the phosphotransfer reaction. Our work demonstrates the value of including dynamic information gleaned from computer simulation in deciphering RTK regulatory function. PMID:28151998

  3. Structures of human Bruton's tyrosine kinase in active and inactive conformations suggest a mechanism of activation for TEC family kinases

    Energy Technology Data Exchange (ETDEWEB)

    Marcotte, Douglas J.; Liu, Yu-Ting; Arduini, Robert M.; Hession, Catherine A.; Miatkowski, Konrad; Wildes, Craig P.; Cullen, Patrick F.; Hong, Victor; Hopkins, Brian T.; Mertsching, Elisabeth; Jenkins, Tracy J.; Romanowski, Michael J.; Baker, Darren P.; Silvian, Laura F. (Sunesis); (Biogen)

    2010-11-15

    Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, plays a crucial role in B-cell maturation and mast cell activation. Although the structures of the unphosphorylated mouse BTK kinase domain and the unphosphorylated and phosphorylated kinase domains of human ITK are known, understanding the kinase selectivity profiles of BTK inhibitors has been hampered by the lack of availability of a high resolution, ligand-bound BTK structure. Here, we report the crystal structures of the human BTK kinase domain bound to either Dasatinib (BMS-354825) at 1.9 {angstrom} resolution or to 4-amino-5-(4-phenoxyphenyl)-7H-pyrrolospyrimidin- 7-yl-cyclopentane at 1.6 {angstrom} resolution. This data provides information relevant to the development of small molecule inhibitors targeting BTK and the TEC family of nonreceptor tyrosine kinases. Analysis of the structural differences between the TEC and Src families of kinases near the Trp-Glu-Ile motif in the N-terminal region of the kinase domain suggests a mechanism of regulation of the TEC family members.

  4. The Catalytic Subunit of DNA-Dependent Protein Kinase Coordinates with Polo-Like Kinase 1 to Facilitate Mitotic Entry.

    Science.gov (United States)

    Lee, Kyung-Jong; Shang, Zeng-Fu; Lin, Yu-Fen; Sun, Jingxin; Morotomi-Yano, Keiko; Saha, Debabrata; Chen, Benjamin P C

    2015-04-01

    DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is the key regulator of the non-homologous end joining pathway of DNA double-strand break repair. We have previously reported that DNA-PKcs is required for maintaining chromosomal stability and mitosis progression. Our further investigations reveal that deficiency in DNA-PKcs activity caused a delay in mitotic entry due to dysregulation of cyclin-dependent kinase 1 (Cdk1), the key driving force for cell cycle progression through G2/M transition. Timely activation of Cdk1 requires polo-like kinase 1 (Plk1), which affects modulators of Cdk1. We found that DNA-PKcs physically interacts with Plk1 and could facilitate Plk1 activation both in vitro and in vivo. Further, DNA-PKcs-deficient cells are highly sensitive to Plk1 inhibitor BI2536, suggesting that the coordination between DNA-PKcs and Plk1 is not only crucial to ensure normal cell cycle progression through G2/M phases but also required for cellular resistance to mitotic stress. On the basis of the current study, it is predictable that combined inhibition of DNA-PKcs and Plk1 can be employed in cancer therapy strategy for synthetic lethality.

  5. The Catalytic Subunit of DNA-Dependent Protein Kinase Coordinates with Polo-Like Kinase 1 to Facilitate Mitotic Entry

    Directory of Open Access Journals (Sweden)

    Kyung-Jong Lee

    2015-04-01

    Full Text Available DNA-dependent protein kinase catalytic subunit (DNA-PKcs is the key regulator of the non-homologous end joining pathway of DNA double-strand break repair. We have previously reported that DNA-PKcs is required for maintaining chromosomal stability and mitosis progression. Our further investigations reveal that deficiency in DNA-PKcs activity caused a delay in mitotic entry due to dysregulation of cyclin-dependent kinase 1 (Cdk1, the key driving force for cell cycle progression through G2/M transition. Timely activation of Cdk1 requires polo-like kinase 1 (Plk1, which affects modulators of Cdk1. We found that DNA-PKcs physically interacts with Plk1 and could facilitate Plk1 activation both in vitro and in vivo. Further, DNA-PKcs–deficient cells are highly sensitive to Plk1 inhibitor BI2536, suggesting that the coordination between DNA-PKcs and Plk1 is not only crucial to ensure normal cell cycle progression through G2/M phases but also required for cellular resistance to mitotic stress. On the basis of the current study, it is predictable that combined inhibition of DNA-PKcs and Plk1 can be employed in cancer therapy strategy for synthetic lethality.

  6. The assembly of a GTPase–kinase signalling complex by a bacterial catalytic scaffold

    Science.gov (United States)

    Selyunin, Andrey S.; Sutton, Sarah E.; Weigele, Bethany A.; Reddick, L. Evan; Orchard, Robert C.; Bresson, Stefan M.; Tomchick, Diana R.; Alto, Neal M.

    2011-01-01

    The fidelity and specificity of information flow within a cell is controlled by scaffolding proteins that assemble and link enzymes into signalling circuits1,2. These circuits can be inhibited by bacterial effector proteins that post-translationally modify individual pathway components3–6. However, there is emerging evidence that pathogens directly organize higher-order signalling networks through enzyme scaffolding7,8, and the identity of the effectors and their mechanisms of action are poorly understood. Here we identify the enterohaemorrhagic Escherichia coli O157:H7 type III effector EspG as a regulator of endomembrane trafficking using a functional screen, and report ADP-ribosylation factor (ARF) GTPases and p21-activated kinases (PAKs) as its relevant host substrates. The 2.5 Å crystal structure of EspG in complex with ARF6 shows how EspG blocks GTPase-activating-protein-assisted GTP hydrolysis, revealing a potent mechanism of GTPase signalling inhibition at organelle membranes. In addition, the 2.8 Å crystal structure of EspG in complex with the autoinhibitory Iα3-helix of PAK2 defines a previously unknown catalytic site in EspG and provides an allosteric mechanism of kinase activation by a bacterial effector. Unexpectedly, ARF and PAKs are organized on adjacent surfaces of EspG, indicating its role as a ‘catalytic scaffold’ that effectively reprograms cellular events through the functional assembly of GTPase-kinase signalling complex. PMID:21170023

  7. From Kinase to Cyclase: An Unusual Example of Catalytic Promiscuity Modulated by Metal Switching

    OpenAIRE

    Sánchez-Moreno, Israel; Iturrate Montoya, Laura; Martín-Hoyos, Rocio; Jimeno, M. Luisa; Mena, Montaña; Bastida, Ágatha; García-Junceda, Eduardo

    2009-01-01

    “This is the pre-peer reviewed version of the following article: Sánchez-Moreno, I., Iturrate, L., Martín-Hoyos, R., Jimeno, M. L., Mena, M., Bastida, A. and García-Junceda, E. (2009) From Kinase to Cyclase: An Unusual Example of Catalytic Promiscuity Modulated by Metal Switching. ChemBioChem. 10, 225-229, which has been published in final form at http://www3.interscience.wiley.com/journal/121544668/abstract?CRETRY=1&SRETRY=0.”

  8. Cloning and expression of catalytic domain of Abl protein tyrosine kinase gene in E. coli

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Protein tyrosine kinases (PTKs) regulate cell proliferation, differentiation and are involved in signal transduction. Uncontrolled signaling from receptor tyrosine kinases to intracellular tyrosine kinases can lead to inflamma tory responses and diseases such as cancer and atherosclerosis. Thus, inhibitors that block the activity of tyrosine kinases or the signaling pathways of PTKs activation could be assumed as the potential candidate for drug development. On this assumption, we cloned and expressed the Abl PTK gene in E. coli, and purified the PTK, which was used to screen the PTK inhibitors from the extracts of Chinese herbs. The catalytic domain sequence of PTK gene was amplified by PCR us ing the cDNA of abl from Abelson murine leukemia virus as template. The amplified fragment was then cloned into the GST-tagged expression vector pGEX2T. The recombinant plasmid was transformed into host cell E. coli DH5α and was induced to express PTK protein. The expression of the protein was detected using SDS-PAGE. The result showed that a specific protein was induced to express after 12 min induction, and reached peak level about 40% of the host total pro tein after 4 h induction. The molecular weight of the fusion protein was about 58 kD. The purified GST-PTK fusion pro tein presented higher activity for tyrosine phosphorylation.

  9. Structure prediction of the EcoRV DNA methyltransferase based on mutant profiling, secondary structure analysis, comparison with known structures of methyltransferases and isolation of catalytically inactive single mutants.

    Science.gov (United States)

    Jeltsch, A; Sobotta, T; Pingoud, A

    1996-05-01

    The EcoRV DNA methyltransferase (M.EcoRV) is an alpha-adenine methyltransferase. We have used two different programs to predict the secondary structure of M.EcoRV. The resulting consensus prediction was tested by a mutant profiling analysis. 29 neutral mutations of M.EcoRV were generated by five cycles of random mutagenesis and selection for active variants to increase the reliability of the prediction and to get a secondary structure prediction for some ambiguously predicted regions. The predicted consensus secondary structure elements could be aligned to the common topology of the structures of the catalytic domains of M.HhaI and M.TaqI. In a complementary approach we have isolated nine catalytically inactive single mutants. Five of these mutants contain an amino acid exchange within the catalytic domain of M.EcoRV (Val2-Ala, Lys81Arg, Cys192Arg, Asp193Gly, Trp231Arg). The Trp231Arg mutant binds DNA similarly to wild-type M.EcoRV, but is catalytically inactive. Hence this mutant behaves like a bona fide active site mutant. According to the structure prediction, Trp231 is located in a loop at the putative active site of M.EcoRV. The other inactive mutants were insoluble. They contain amino acid exchanges within the conserved amino acid motifs X, III or IV in M.EcoRV confirming the importance of these regions.

  10. Analysis of the Staphylococcus aureus DgkB structure reveals a common catalytic mechanism for the soluble diacylglycerol kinases.

    Science.gov (United States)

    Miller, Darcie J; Jerga, Agoston; Rock, Charles O; White, Stephen W

    2008-07-01

    Soluble diacylglycerol (DAG) kinases function as regulators of diacylglycerol metabolism in cell signaling and intermediary metabolism. We report the structure of a DAG kinase, DgkB from Staphylococcus aureus, both as the free enzyme and in complex with ADP. The molecule is a tight homodimer, and each monomer comprises two domains with the catalytic center located within the interdomain cleft. Two distinctive features of DkgB are a structural Mg2+ site and an associated Asp*water*Mg2+ network that extends toward the active site locale. Site-directed mutagenesis revealed that these features play important roles in the catalytic mechanism. The key active site residues and the components of the Asp*water*Mg2+ network are conserved in the catalytic cores of the mammalian signaling DAG kinases, indicating that these enzymes use the same mechanism and have similar structures as DgkB.

  11. Analysis of the Staphylococcus aureus DgkB Structure Reveals a Common Catalytic Mechanism for the Soluble Diacylglycerol Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Miller, Darcie J.; Jerga, Agoston; Rock, Charles O.; White, Stephen W. (SJCH)

    2008-08-11

    Soluble diacylglycerol (DAG) kinases function as regulators of diacylglycerol metabolism in cell signaling and intermediary metabolism. We report the structure of a DAG kinase, DgkB from Staphylococcus aureus, both as the free enzyme and in complex with ADP. The molecule is a tight homodimer, and each monomer comprises two domains with the catalytic center located within the interdomain cleft. Two distinctive features of DkgB are a structural Mg{sup 2+} site and an associated Asp{center_dot}water{center_dot}Mg{sup 2+} network that extends toward the active site locale. Site-directed mutagenesis revealed that these features play important roles in the catalytic mechanism. The key active site residues and the components of the Asp{center_dot}water{center_dot}Mg{sup 2+} network are conserved in the catalytic cores of the mammalian signaling DAG kinases, indicating that these enzymes use the same mechanism and have similar structures as DgkB.

  12. Intracellular catalytic domain of symbiosis receptor kinase hyperactivates spontaneous nodulation in absence of rhizobia.

    Science.gov (United States)

    Saha, Sudip; Dutta, Ayan; Bhattacharya, Avisek; DasGupta, Maitrayee

    2014-12-01

    Symbiosis Receptor Kinase (SYMRK), a member of the Nod factor signaling pathway, is indispensible for both nodule organogenesis and intracellular colonization of symbionts in rhizobia-legume symbiosis. Here, we show that the intracellular kinase domain of a SYMRK (SYMRK-kd) but not its inactive or full-length version leads to hyperactivation of the nodule organogenic program in Medicago truncatula TR25 (symrk knockout mutant) in the absence of rhizobia. Spontaneous nodulation in TR25/SYMRK-kd was 6-fold higher than rhizobia-induced nodulation in TR25/SYMRK roots. The merged clusters of spontaneous nodules indicated that TR25 roots in the presence of SYMRK-kd have overcome the control over both nodule numbers and their spatial position. In the presence of rhizobia, SYMRK-kd could rescue the epidermal infection processes in TR25, but colonization of symbionts in the nodule interior was significantly compromised. In summary, ligand-independent deregulated activation of SYMRK hyperactivates nodule organogenesis in the absence of rhizobia, but its ectodomain is required for proper symbiont colonization.

  13. The Alpha Catalytic Subunit of Protein Kinase CK2 Is Required for Mouse Embryonic Development▿

    Science.gov (United States)

    Lou, David Y.; Dominguez, Isabel; Toselli, Paul; Landesman-Bollag, Esther; O'Brien, Conor; Seldin, David C.

    2008-01-01

    Protein kinase CK2 (formerly casein kinase II) is a highly conserved and ubiquitous serine/threonine kinase that is composed of two catalytic subunits (CK2α and/or CK2α′) and two CK2β regulatory subunits. CK2 has many substrates in cells, and key roles in yeast cell physiology have been uncovered by introducing subunit mutations. Gene-targeting experiments have demonstrated that in mice, the CK2β gene is required for early embryonic development, while the CK2α′ subunit appears to be essential only for normal spermatogenesis. We have used homologous recombination to disrupt the CK2α gene in the mouse germ line. Embryos lacking CK2α have a marked reduction in CK2 activity in spite of the presence of the CK2α′ subunit. CK2α−/− embryos die in mid-gestation, with abnormalities including open neural tubes and reductions in the branchial arches. Defects in the formation of the heart lead to hydrops fetalis and are likely the cause of embryonic lethality. Thus, CK2α appears to play an essential and uncompensated role in mammalian development. PMID:17954558

  14. Adenylate kinase from Streptococcus pneumoniae is essential for growth through its catalytic activity

    Directory of Open Access Journals (Sweden)

    Trung Thanh Thach

    2014-01-01

    Full Text Available Streptococcus pneumoniae (pneumococcus infection causes more than 1.6 million deaths worldwide. Pneumococcal growth is a prerequisite for its virulence and requires an appropriate supply of cellular energy. Adenylate kinases constitute a major family of enzymes that regulate cellular ATP levels. Some bacterial adenylate kinases (AdKs are known to be critical for growth, but the physiological effects of AdKs in pneumococci have been poorly understood at the molecular level. Here, by crystallographic and functional studies, we report that the catalytic activity of adenylate kinase from S. pneumoniae (SpAdK serotype 2 D39 is essential for growth. We determined the crystal structure of SpAdK in two conformations: ligand-free open form and closed in complex with a two-substrate mimic inhibitor adenosine pentaphosphate (Ap5A. Crystallographic analysis of SpAdK reveals Arg-89 as a key active site residue. We generated a conditional expression mutant of pneumococcus in which the expression of the adk gene is tightly regulated by fucose. The expression level of adk correlates with growth rate. Expression of the wild-type adk gene in fucose-inducible strains rescued a growth defect, but expression of the Arg-89 mutation did not. SpAdK increased total cellular ATP levels. Furthermore, lack of functional SpAdK caused a growth defect in vivo. Taken together, our results demonstrate that SpAdK is essential for pneumococcal growth in vitro and in vivo.

  15. Expression of a Catalytically Inactive Mutant Form of Glutathione Peroxidase 4 (Gpx4) Confers a Dominant-negative Effect in Male Fertility.

    Science.gov (United States)

    Ingold, Irina; Aichler, Michaela; Yefremova, Elena; Roveri, Antonella; Buday, Katalin; Doll, Sebastian; Tasdemir, Adrianne; Hoffard, Nils; Wurst, Wolfgang; Walch, Axel; Ursini, Fulvio; Friedmann Angeli, José Pedro; Conrad, Marcus

    2015-06-05

    The selenoenzyme Gpx4 is essential for early embryogenesis and cell viability for its unique function to prevent phospholipid oxidation. Recently, the cytosolic form of Gpx4 was identified as an upstream regulator of a novel form of non-apoptotic cell death, called ferroptosis, whereas the mitochondrial isoform of Gpx4 was previously shown to be crucial for male fertility. Here, we generated and analyzed mice with a targeted mutation of the active site selenocysteine of Gpx4 (Gpx4_U46S). Mice homozygous for Gpx4_U46S died at the same embryonic stage (E7.5) as Gpx4(-/-) embryos as expected. Surprisingly, male mice heterozygous for Gpx4_U46S presented subfertility. Subfertility was manifested in a reduced number of litters from heterozygous breeding and an impairment of spermatozoa to fertilize oocytes in vitro. Morphologically, sperm isolated from heterozygous Gpx4_U46S mice revealed many structural abnormalities particularly in the spermatozoa midpiece due to improper oxidation and polymerization of sperm capsular proteins and malformation of the mitochondrial capsule surrounding and stabilizing sperm mitochondria. These findings are reminiscent of sperm isolated from selenium-deprived rodents or from mice specifically lacking mitochondrial Gpx4. Due to a strongly facilitated incorporation of Ser in the polypeptide chain as compared with selenocysteine at the UGA codon, expression of the catalytically inactive Gpx4_U46S was found to be strongly increased. Because the stability of the mitochondrial capsule of mature spermatozoa depends on the moonlighting function of Gpx4 both as an enzyme oxidizing capsular protein thiols and as a structural protein, tightly controlled expression of functional Gpx4 emerges as a key for full male fertility.

  16. Expression, purification and crystallization of the catalytic subunit of protein kinase CK2 from Zea mays

    DEFF Research Database (Denmark)

    Guerra, B; Niefind, K; Pinna, L A

    1998-01-01

    a = 142.6, b = 61.3, c = 45.6 A, beta = 103.3 degrees and diffract X-rays to at least 2.0 A resolution. The calculated crystal packing parameter is Vm = 2.47 A3 Da-1 suggesting that one CK2alpha molecule is contained in the asymmetric unit and that the solvent content of the unit cell is 50%.......The catalytic (alpha) subunit of protein kinase CK2 (CK2alpha) was originally cloned and overexpressed in the Escherichia coli strain pT7-7/BL21(DE3). The protein has been purified to homogeneity and crystallized. The crystals belong to the monoclinic space group C2, they have unit-cell parameters...

  17. Evolutionary divergence in the catalytic activity of the CAM-1, ROR1 and ROR2 kinase domains.

    Directory of Open Access Journals (Sweden)

    Travis W Bainbridge

    Full Text Available Receptor tyrosine kinase-like orphan receptors (ROR 1 and 2 are atypical members of the receptor tyrosine kinase (RTK family and have been associated with several human diseases. The vertebrate RORs contain an ATP binding domain that deviates from the consensus amino acid sequence, although the impact of this deviation on catalytic activity is not known and the kinase function of these receptors remains controversial. Recently, ROR2 was shown to signal through a Wnt responsive, β-catenin independent pathway and suppress a canonical Wnt/β-catenin signal. In this work we demonstrate that both ROR1 and ROR2 kinase domains are catalytically deficient while CAM-1, the C. elegans homolog of ROR, has an active tyrosine kinase domain, suggesting a divergence in the signaling processes of the ROR family during evolution. In addition, we show that substitution of the non-consensus residues from ROR1 or ROR2 into CAM-1 and MuSK markedly reduce kinase activity, while restoration of the consensus residues in ROR does not restore robust kinase function. We further demonstrate that the membrane-bound extracellular domain alone of either ROR1 or ROR2 is sufficient for suppression of canonical Wnt3a signaling, and that this domain can also enhance Wnt5a suppression of Wnt3a signaling. Based on these data, we conclude that human ROR1 and ROR2 are RTK-like pseudokinases.

  18. Functional Role of Histidine in the Conserved His-x-Asp Motif in the Catalytic Core of Protein Kinases.

    Science.gov (United States)

    Zhang, Lun; Wang, Jian-Chuan; Hou, Li; Cao, Peng-Rong; Wu, Li; Zhang, Qian-Sen; Yang, Huai-Yu; Zang, Yi; Ding, Jian-Ping; Li, Jia

    2015-05-11

    The His-x-Asp (HxD) motif is one of the most conserved structural components of the catalytic core of protein kinases; however, the functional role of the conserved histidine is unclear. Here we report that replacement of the HxD-histidine with Arginine or Phenylalanine in Aurora A abolishes both the catalytic activity and auto-phosphorylation, whereas the Histidine-to-tyrosine impairs the catalytic activity without affecting its auto-phosphorylation. Comparisons of the crystal structures of wild-type (WT) and mutant Aurora A demonstrate that the impairment of the kinase activity is accounted for by (1) disruption of the regulatory spine in the His-to-Arg mutant, and (2) change in the geometry of backbones of the Asp-Phe-Gly (DFG) motif and the DFG-1 residue in the His-to-Tyr mutant. In addition, bioinformatics analyses show that the HxD-histidine is a mutational hotspot in tumor tissues. Moreover, the H174R mutation of the HxD-histidine, in the tumor suppressor LKB1 abrogates the inhibition of anchorage-independent growth of A549 cells by WT LKB1. Based on these data, we propose that the HxD-histidine is involved in a conserved inflexible organization of the catalytic core that is required for the kinase activity. Mutation of the HxD-histidine may also be involved in the pathogenesis of some diseases including cancer.

  19. Advances in the research of tageting DFG-out allosteric binding site of inactive kinases%靶向非活性激酶DFG-out变构结合位点的研究进展

    Institute of Scientific and Technical Information of China (English)

    彭文; 张小猛; 张仓; 王芳; 尤启冬

    2012-01-01

    目前大多数激酶抑制剂是通过模拟ATP的结构,以识别激酶的活性构象来竞争性结合于ATP结合位点,从而抑制激酶的自磷酸化和下游的信号传导.然而,最近人们对已上市药物甲磺酸伊马替尼、尼罗替尼及对甲苯磺酸索拉非尼的晶体结构研究发现,在非活性激酶中ATP结合位点的相邻位置存在着第二个能与激酶抑制剂结合的位点——DFG-out变构结合位点.该位点的发现为以蛋白激酶为靶标的小分子激酶抑制剂的设计与开发指明了新的方向,成为抗肿瘤研究领域的新热点之一.因此,本文对非活性激酶的DFG-out变构结合位点的发现、非活性激酶与其抑制剂的结合方式及处于临床研究阶段的非活性激酶抑制剂进行了综述.%Up to nowadays, a majority of kinase inhibitors identify the activity conformation of protein ki-nase to integrate competitively with ATP binding site by simulating the structure of ATP. In this way, kinase inhibitors can inhibit kinase autophosphorylation and restrain signal transduction of downstream. However, the crystal structures of imatinib mesylate, nilotnib and sorafenib tosylate have revealed a secondary binding site adjacent to the ATP binding site, which is also bound by kinase inhibitors, known as the DFG-out allosteric binding site, in the inactive conformation of protein kinase. The discovery of the site has pointed out a new direction for the design and development of small molecule kinase inhibitors, which lakes protein kinase as a target. This becomes a new hotspot in antineoplastic research field. In this paper, we reviewed the discovery and inhibitors of the DFG-out allosteric binding site, and the binding mode between inactive kinases, as well as the inactive kinase inhibitors in clinical studies.

  20. Functional analysis of Trichoderma reesei CKIIα2, a catalytic subunit of casein kinase II.

    Science.gov (United States)

    Wang, Mingyu; Yang, Hui; Zhang, Meiling; Liu, Kuimei; Wang, Hanbin; Luo, Yi; Fang, Xu

    2015-07-01

    Trichoderma reesei is the most important industrial cellulase-producing filamentous fungus. Although its molecular physiology has been investigated, the signal transduction pathways are not fully understood. In particular, the role of casein kinase II (CKII) is not yet clear. In this work, we carried out functional investigations on a catalytic subunit of CKII, CKIIα2. Comparison of the phenotypic features of T. reesei parent and Δck2α2 strains showed significant changes following ck2α2 disruption. T. reesei Δck2α2 form significantly smaller mycelial pellets in glucose-containing liquid minimum media, have shorter and fewer branch hyphae, produce smaller amounts of chitinases, produce more spores, show more robust growth on glucose-containing agar plates, and consume glucose at a significantly higher rate. Suggestions can be made that CKIIα2 governs chitinase expression, and the disruption of ck2α2 results in lower levels of chitinase production, leading to a weaker cell wall disruption capability, further resulting in weaker hyphal branching, which eventually leads to smaller mycelial pellets in liquid media. Further conclusions can be made that CKIIα2 is involved in repression of sporulation and glucose metabolism, which is consistent with the proposal that CKIIα2 represses global metabolism. These observations make the deletion of ck2α2 a potentially beneficial genetic disruption for T. reesei during industrial applications, as smaller mycelial pellets, more spores and more robust glucose metabolism are all desired traits for industrial fermentation. This work reports novel unique functions of a CKII catalytic subunit and is also the first genetic and physiological investigation on CKII in T. reesei.

  1. Crystal structures of catalytic and regulatory subunits of rat protein kinase CK2

    Institute of Scientific and Technical Information of China (English)

    ZHOU WeiHong; SHEN YueQuan; QIN XiaoHong; YAN XiaoJie; XIE XingQiao; LI Liang; FANG ShaSha; LONG JiaFu; ADELMAN John; TANG Wei-Jen

    2009-01-01

    Protein kinase CK2 consists of two catalytic subunits (CK2α) and two regulatory subunits (CK2β). Here,we report the crystal structures of rat CK2α mutant (rCK2α-△C, 1-335) and CK2β(rCK2β). The overall topology of rCK2α-△C and rCK2βare very similar to the human enzyme, although large structural dif-ferences could be observed in the N-terminal domain of rCK2α-△C. Our reported structure of rCK2α-△C is in the close conformation state while the counterpart hCK2α is in the open conformation state, indi-cating the conformation of CK2α molecule has high plasticity. The structure of rCK2β represents the conformation of free CK2β. Upon CK2α binding, the C-terminal region undergoes a drastic conforma-tional change. The major region of interaction within the interface of CK2αCK2β may serve as a bridge to transmit the conformational change and thus regulate the activity of CK2α.

  2. Simultaneous blockade of NFkappaB, JNK, and p38 MAPK by a kinase-inactive mutant of the protein kinase TAK1 sensitizes cells to apoptosis and affects a distinct spectrum of tumor necrosis factor [corrected] target genes.

    Science.gov (United States)

    Thiefes, Axel; Wolter, Sabine; Mushinski, J Frederic; Hoffmann, Elke; Dittrich-Breiholz, Oliver; Graue, Nadine; Dörrie, Anneke; Schneider, Heike; Wirth, Dagmar; Luckow, Bruno; Resch, Klaus; Kracht, Michael

    2005-07-29

    The inflammatory response is characterized by the induction (or repression) of hundreds of genes. The activity of many of these genes is controlled by MAPKs and the IkappaB kinase-NFkappaB pathway. To reveal the effects of blocking these pathways simultaneously, fibroblasts were infected with retroviruses encoding TAK1K63W, an inactive mutant of the protein kinase TAK1. Expression of this protein inhibited tumor necrosis factor (TNF)-induced activation of NFkappaB, JNK, and p38 MAPK and sensitized the cells to TNF-induced apoptosis. 23 different microarray experiments were used to analyze the expression of >7000 genes in these cells. We identified 518 genes that were regulated by TNF in both TAK1K63W-expressing cells and control cells, 37 genes induced by TNF only when TAK1K63W was present, and 48 TNF-induced genes that were suppressed by TAK1K63W. The TNF-inducible genes that were most strongly suppressed by TAK1K63W, ccl2, ccl7, ccl5, cxcl1, cxcl5, cxcl10, saa3, and slpi also had much lower basal levels of expression, indicating that TAK1 also played a role in their normal expression. Chromatin immunoprecipitation studies on four of these genes suggested that inactivation of TAK1 activity led to direct suppression of expression at the transcriptional level because of impaired recruitment of RNA polymerase II to their promoters. ccl2 induction by TNF or interleukin-1 was also suppressed in cells that expressed TAK1 antisense RNA or that were genetically deficient in JNK1/2 or p65 NFkappaB. These data suggest that regulation of the expression of a selected group of inflammation-related genes is funneled through TAK1, making it a potentially useful target for more specific anti-inflammatory drug development.

  3. Structural and catalytic properties of CMP kinase from Bacillus subtilis: a comparative analysis with the homologous enzyme from Escherichia coli.

    Science.gov (United States)

    Schultz, C P; Ylisastigui-Pons, L; Serina, L; Sakamoto, H; Mantsch, H H; Neuhard, J; Bârzu, O; Gilles, A M

    1997-04-01

    CMP kinases from Bacillus subtilis and from Escherichia coli are encoded by the cmk gene (formerly known as jofC in B. subtilis and as mssA in E. coli). Similar in their primary structure (43% identity and 67% similarity in amino acid sequence), the two proteins exhibit significant differences in nucleotide binding and catalysis. ATP, dATP, and GTP are equally effective as phosphate donors with E. coli CMP kinase whereas GTP is a poor substrate with B. subtilis CMP kinase. While CMP and dCMP are the best phosphate acceptors of both CMP kinases, the specific activity with these substrates and ATP as donor are 7- to 10-fold higher in the E. coli enzyme; the relative Vm values with UMP and CMP are 0.1 for the B. subtilis CMP kinase and 0.01 for the E. coli enzyme. CMP increased the affinity of E. coli CMP kinase for ATP or for the fluorescent analog 3'-anthraniloyl dATP by one order of magnitude but had no effect on the B. subtilis enzyme. The differences in the catalytic properties of B. subtilis and E. coli CMP kinases might be reflected in the structure of the two proteins as inferred from infrared spectroscopy. Whereas the spectrum of B. subtilis CMP kinase is dominated by a band at 1633 cm-1 (representing beta type structures), the spectrum of the E. coli enzyme is dominated by two bands at 1653 and 1642 cm-1 associated with alpha-helical and unordered structures, respectively. CMP induced similar spectral changes in both proteins with a rearrangement of some of the beta-structures. ATP increases the denaturation temperature of B. subtilis CMP kinase by 9.3 degrees C, whereas in the case of the E. coli enzyme, binding of ATP has only a minor effect.

  4. Structural analysis of ARC-type inhibitor (ARC-1034) binding to protein kinase A catalytic subunit and rational design of bisubstrate analogue inhibitors of basophilic protein kinases.

    Science.gov (United States)

    Lavogina, Darja; Lust, Marje; Viil, Indrek; König, Norbert; Raidaru, Gerda; Rogozina, Jevgenia; Enkvist, Erki; Uri, Asko; Bossemeyer, Dirk

    2009-01-22

    The crystal structure of a complex of the catalytic subunit (type alpha) of cAMP-dependent protein kinase (PKA C alpha) with ARC-type inhibitor (ARC-1034), the presumed lead scaffold of previously reported adenosine-oligo-arginine conjugate-based (ARC-type) inhibitors, was solved. Structural elements important for interaction with the kinase were established with specifically modified derivatives of the lead compound. On the basis of this knowledge, a new generation of inhibitors, conjugates of adenosine-4'-dehydroxymethyl-4'-carboxylic acid moiety and oligo(D-arginine), was developed with inhibitory constants well into the subnanomolar range. The structural determinants of selectivity of the new compounds were established in assays with ROCK-II and PKBgamma.

  5. Structure of the human protein kinase CK2 catalytic subunit CK2α' and interaction thermodynamics with the regulatory subunit CK2β

    DEFF Research Database (Denmark)

    Bischoff, Nils; Olsen, Birgitte; Raaf, Jennifer;

    2011-01-01

    the limited biochemical knowledge about the second paralog (CK2α'), we developed a well-soluble catalytically active full-length mutant of human CK2α', characterized it by Michaelis-Menten kinetics and isothermal titration calorimetry, and determined its crystal structure to a resolution of 2 Å. The affinity......Protein kinase CK2 (formerly "casein kinase 2") is composed of a central dimer of noncatalytic subunits (CK2β) binding two catalytic subunits. In humans, there are two isoforms of the catalytic subunit (and an additional splicing variant), one of which (CK2α) is well characterized. To supplement...

  6. Bisubstrate analog probes for the insulin receptor protein tyrosine kinase: molecular yardsticks for analyzing catalytic mechanism and inhibitor design.

    Science.gov (United States)

    Hines, Aliya C; Parang, Keykavous; Kohanski, Ronald A; Hubbard, Stevan R; Cole, Philip A

    2005-08-01

    Bisubstrate analogs have the potential to provide enhanced specificity for protein kinase inhibition and tools to understand catalytic mechanism. Previous efforts led to the design of a peptide-ATP conjugate bisubstrate analog utilizing aminophenylalanine in place of tyrosine and a thioacetyl linker to the gamma-phosphate of ATP which was a potent inhibitor of the insulin receptor kinase (IRK). In this study, we have examined the contributions of various electrostatic and structural elements in the bisubstrate analog to IRK binding affinity. Three types of changes (seven specific analogs in all) were introduced: a Tyr isostere of the previous aminophenylalanine moiety, modifications of the spacer between the adenine and the peptide, and deletions and substitutions within the peptide moiety. These studies allowed a direct evaluation of the hydrogen bond strength between the anilino nitrogen of the bisubstrate analog and the enzyme catalytic base Asp and showed that it contributes 2.5 kcal/mol of binding energy, in good agreement with previous predictions. Modifications of the linker length resulted in weakened inhibitory affinity, consistent with the geometric requirements of an enzyme-catalyzed dissociative transition state. Alterations in the peptide motif generally led to diminished inhibitory potency, and only some of these effects could be rationalized based on prior kinetic and structural studies. Taken together, these results suggest that a combination of mechanism-based design and empirical synthetic manipulation will be necessary in producing optimized protein kinase bisubstrate analog inhibitors.

  7. Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mitosis.

    Science.gov (United States)

    Douglas, Pauline; Ye, Ruiqiong; Trinkle-Mulcahy, Laura; Neal, Jessica A; De Wever, Veerle; Morrice, Nick A; Meek, Katheryn; Lees-Miller, Susan P

    2014-06-25

    The protein kinase activity of the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and its autophosphorylation are critical for DBS (DNA double-strand break) repair via NHEJ (non-homologous end-joining). Recent studies have shown that depletion or inactivation of DNA-PKcs kinase activity also results in mitotic defects. DNA-PKcs is autophosphorylated on Ser2056, Thr2647 and Thr2609 in mitosis and phosphorylated DNA-PKcs localize to centrosomes, mitotic spindles and the midbody. DNA-PKcs also interacts with PP6 (protein phosphatase 6), and PP6 has been shown to dephosphorylate Aurora A kinase in mitosis. Here we report that DNA-PKcs is phosphorylated on Ser3205 and Thr3950 in mitosis. Phosphorylation of Thr3950 is DNA-PK-dependent, whereas phosphorylation of Ser3205 requires PLK1 (polo-like kinase 1). Moreover, PLK1 phosphorylates DNA-PKcs on Ser3205 in vitro and interacts with DNA-PKcs in mitosis. In addition, PP6 dephosphorylates DNA-PKcs at Ser3205 in mitosis and after IR (ionizing radiation). DNA-PKcs also phosphorylates Chk2 on Thr68 in mitosis and both phosphorylation of Chk2 and autophosphorylation of DNA-PKcs in mitosis occur in the apparent absence of Ku and DNA damage. Our findings provide mechanistic insight into the roles of DNA-PKcs and PP6 in mitosis and suggest that DNA-PKcs' role in mitosis may be mechanistically distinct from its well-established role in NHEJ.

  8. Gene for the catalytic subunit of mouse DNA-dependent protein kinase maps to the scid locus.

    Science.gov (United States)

    Miller, R D; Hogg, J; Ozaki, J H; Gell, D; Jackson, S P; Riblet, R

    1995-01-01

    The gene encoding the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) has been proposed recently as a candidate gene for the mouse severe combined immune deficiency (scid) locus. We have used a partial cDNA clone for human DNA-PKcs to map the mouse homologue using a large interspecific backcross panel. We found that the mouse gene for DNA-PKcs does not recombine with scid, consistent with the hypothesis that scid is a mutation in the mouse gene for DNA-PKcs. Images Fig. 3 PMID:7479885

  9. Low-density crystal packing of human protein kinase CK2 catalytic subunit in complex with resorufin or other ligands

    DEFF Research Database (Denmark)

    Klopffleisch, Karsten; Issinger, Olaf Georg; Niefind, Karsten

    2012-01-01

    adopts a closed conformation correlating to a canonically established catalytic spine as is typical for eukaryotic protein kinases. In the corresponding crystal packing the hinge/helix αD region is nearly unaffected by crystal contacts, so that largely unbiased conformational adaptions are possible....... This is documented by published human CK2α structures with the same crystal packing but with an open hinge/helix αD region, one of which has been redetermined here with a higher symmetry. An overview of all published human CK2α crystal packings serves as the basis for a discussion of the factors that determine...

  10. Cyclic AMP-dependent protein kinase I: Cyclic nucleotide binding, structural changes, and release of the catalytic subunits

    OpenAIRE

    Smith, Stephen B.; White, Hillary D.; Siegel, Jeffrey B.; Krebs, Edwin G.

    1981-01-01

    Type I cyclic AMP (cAMP)-dependent protein kinase is composed of a dimeric regulatory subunit (R2) and two catalytic subunits (C subunits). The R2 dimer binds four cAMP molecules to release the two C subunits. To characterize the cAMP binding sites and elucidate their role in the release of the C subunits, the R2 dimer has been studied by equilibrium methods. The cAMP titration of R2 was monitored by endogenous tryptophan fluorescence, and the results suggest one class of binding sites. The t...

  11. Function of Bruton's tyrosine kinase during B cell development is partially independent of its catalytic activity

    NARCIS (Netherlands)

    S. Middendorp; G.M. Dingjan (Gemma); A. Maas (Alex); K. Dahlenborg; R.W. Hendriks (Rudi)

    2003-01-01

    textabstractThe Tec family member Bruton's tyrosine kinase (Btk) is a cytoplasmic protein tyrosine kinase that transduces signals from the pre-B and B cell receptor (BCR). Btk is involved in pre-B cell maturation by regulating IL-7 responsiveness, cell surface phenotype changes, an

  12. The catalytic subunit of cAMP-dependent protein kinase induces expression of genes containing cAMP-responsive enhancer elements.

    Science.gov (United States)

    Riabowol, K T; Fink, J S; Gilman, M Z; Walsh, D A; Goodman, R H; Feramisco, J R

    1988-11-03

    Transcriptional regulation of eukaryotic genes by cyclic AMP requires a cAMP-dependent protein kinase (A kinase). Two hypotheses have been proposed to explain how the holoenzyme of the A kinase induces transcription. The regulatory subunits of the A kinase, which bind cAMP and DNA, and have amino-acid homology with the Escherichia coli catabolite activator protein could directly stimulate gene expression. Alternatively, phosphorylation by the catalytic subunits could induce transcription by activating proteins involved in gene transcription. To distinguish between these models, we microinjected purified preparations of the catalytic and regulatory subunits of A kinase into tissue culture cells and monitored expression of a stably integrated fusion gene containing a cAMP-responsive human promoter fused to a bacterial reporter gene, or of the endogenous c-fos gene. The catalytic subunit stimulated expression of these genes, whereas the regulatory subunit did not. These results indicate that the catalytic subunit of A kinase is sufficient to induce expression of two cAMP-responsive genes, without increasing levels of cAMP.

  13. Epidermal growth factor receptor levels are reduced in mice with targeted disruption of the protein kinase A catalytic subunit

    Directory of Open Access Journals (Sweden)

    Huitfeldt Henrik S

    2008-04-01

    Full Text Available Abstract Background Epidermal Growth Factor Receptor (EGFR is a key target molecule in current treatment of several neoplastic diseases. Hence, in order to develop and improve current drugs targeting EGFR signalling, an accurate understanding of how this signalling pathway is regulated is required. It has recently been demonstrated that inhibition of cAMP-dependent protein kinase (PKA induces a ligand-independent internalization of EGFR. Cyclic-AMP-dependent protein kinase consists of a regulatory dimer bound to two catalytic subunits. Results We have investigated the effect on EGFR levels after ablating the two catalytic subunits, Cα and Cβ in two different models. The first model used targeted disruption of either Cα or Cβ in mice whereas the second model used Cα and Cβ RNA interference in HeLa cells. In both models we observed a significant reduction of EGFR expression at the protein but not mRNA level. Conclusion Our results suggest that PKA may represent a target that when manipulated can maintain EGFR protein levels at the single cell level as well as in intact animals.

  14. Arginine kinase of the flagellate protozoa Trypanosoma cruzi. Regulation of its expression and catalytic activity.

    Science.gov (United States)

    Alonso, G D; Pereira, C A; Remedi, M S; Paveto, M C; Cochella, L; Ivaldi, M S; Gerez de Burgos, N M; Torres, H N; Flawiá, M M

    2001-06-01

    In epimastigotes of Trypanosoma cruzi, the etiological agent of Chagas' disease, arginine kinase activity increased continuously during the exponential phase of growth. A correlation between growth rate, enzyme-specific activity and enzyme protein was observed. Arginine kinase-specific activity, expressed as a function of enzyme protein, remains roughly constant up to 18 days of culture. In the whole range of the culture time mRNA levels showed minor changes indicating that the enzyme activity is post-transcriptionally regulated. Arginine kinase could be proposed as a modulator of energetic reserves under starvation stress condition.

  15. KSR1 is a functional protein kinase capable of serine autophosphorylation and direct phosphorylation of MEK1

    Energy Technology Data Exchange (ETDEWEB)

    Goettel, Jeremy A. [Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States); Liang, Dongchun [Department of Pediatrics, Division of Gastroenterology, Hepatology and Nutrition, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States); Hilliard, Valda C.; Edelblum, Karen L.; Broadus, Matthew R. [Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States); Gould, Kathleen L. [Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States); Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States); Hanks, Steven K. [Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States); Polk, D. Brent, E-mail: dbpolk@chla.usc.edu [Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States); Department of Pediatrics, Division of Gastroenterology, Hepatology and Nutrition, Vanderbilt University School of Medicine, Nashville, TN 37232 (United States)

    2011-02-15

    The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway is a highly conserved signaling pathway that regulates diverse cellular processes including differentiation, proliferation, and survival. Kinase suppressor of Ras-1 (KSR1) binds each of the three ERK cascade components to facilitate pathway activation. Even though KSR1 contains a C-terminal kinase domain, evidence supporting the catalytic function of KSR1 remains controversial. In this study, we produced recombinant wild-type or kinase-inactive (D683A/D700A) KSR1 proteins in Escherichia coli to test the hypothesis that KSR1 is a functional protein kinase. Recombinant wild-type KSR1, but not recombinant kinase-inactive KSR1, underwent autophosphorylation on serine residue(s), phosphorylated myelin basic protein (MBP) as a generic substrate, and phosphorylated recombinant kinase-inactive MAPK/ERK kinase-1 (MEK1). Furthermore, FLAG immunoprecipitates from KSR1{sup -/-} colon epithelial cells stably expressing FLAG-tagged wild-type KSR1 (+KSR1), but not vector (+vector) or FLAG-tagged kinase-inactive KSR1 (+D683A/D700A), were able to phosphorylate kinase-inactive MEK1. Since TNF activates the ERK pathway in colon epithelial cells, we tested the biological effects of KSR1 in the survival response downstream of TNF. We found that +vector and +D683A/D700A cells underwent apoptosis when treated with TNF, whereas +KSR1 cells were resistant. However, +KSR1 cells were sensitized to TNF-induced cell loss in the absence of MEK kinase activity. These data provide clear evidence that KSR1 is a functional protein kinase, MEK1 is an in vitro substrate of KSR1, and the catalytic activities of both proteins are required for eliciting cell survival responses downstream of TNF.

  16. Identification of DNA-dependent protein kinase catalytic subunit (DNA-PKcs as a novel target of bisphenol A.

    Directory of Open Access Journals (Sweden)

    Yuki Ito

    Full Text Available Bisphenol A (BPA forms the backbone of plastics and epoxy resins used to produce packaging for various foods and beverages. BPA is also an estrogenic disruptor, interacting with human estrogen receptors (ER and other related nuclear receptors. Nevertheless, the effects of BPA on human health remain unclear. The present study identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs as a novel BPA-binding protein. DNA-PKcs, in association with the Ku heterodimer (Ku70/80, is a critical enzyme involved in the repair of DNA double-strand breaks. Low levels of DNA-PK activity are previously reported to be associated with an increased risk of certain types of cancer. Although the Kd for the interaction between BPA and a drug-binding mutant of DNA-PKcs was comparatively low (137 nM, high doses of BPA were required before cellular effects were observed (100-300 μM. The results of an in vitro kinase assay showed that BPA inhibited DNA-PK kinase activity in a concentration-dependent manner. In M059K cells, BPA inhibited the phosphorylation of DNA-PKcs at Ser2056 and H2AX at Ser139 in response to ionizing radiation (IR-irradiation. BPA also disrupted DNA-PKcs binding to Ku70/80 and increased the radiosensitivity of M059K cells, but not M059J cells (which are DNA-PKcs-deficient. Taken together, these results provide new evidence of the effects of BPA on DNA repair in mammalian cells, which are mediated via inhibition of DNA-PK activity. This study may warrant the consideration of the possible carcinogenic effects of high doses of BPA, which are mediated through its action on DNA-PK.

  17. Conformational basis for substrate recognition and regulation of catalytic activity in Staphylococcus aureus nucleoside di-phosphate kinase.

    Science.gov (United States)

    Srivastava, Sandeep Kumar; Rajasree, Kalagiri; Gopal, B

    2011-10-01

    Nucleoside diphosphate kinases (NDK) are characterized by high catalytic turnover rates and diverse substrate specificity. These features make this enzyme an effective activator of a pro-drug-an application that has been actively pursued for a variety of therapeutic strategies. The catalytic mechanism of this enzyme is governed by a conserved histidine that coordinates a magnesium ion at the active site. Despite substantial structural and biochemical information on NDK, the mechanistic feature of the phospho-transfer that leads to auto-phosphorylation remains unclear. While the role of the histidine residue is well documented, the other active site residues, in particular the conserved serine remains poorly characterized. Studies on some homologues suggest no role for the serine residue at the active site, while others suggest a crucial role for this serine in the regulation and quaternary association of this enzyme in some species. Here we report the biochemical features of the Staphylococcus aureus NDK and the mutant enzymes. We also describe the crystal structures of the apo-NDK, as a transition state mimic with vanadate and in complex with different nucleotide substrates. These structures formed the basis for molecular dynamics simulations to understand the broad substrate specificity of this enzyme and the role of active site residues in the phospho-transfer mechanism and oligomerization. Put together, these data suggest that concerted changes in the conformation of specific residues facilitate the stabilization of nucleotide complexes thereby enabling the steps involved in the ping-pong reaction mechanism without large changes to the overall structure of this enzyme.

  18. Expression of a nonmyristylated variant of the catalytic subunit of protein kinase A during male germ-cell development.

    Science.gov (United States)

    Desseyn, J L; Burton, K A; McKnight, G S

    2000-06-06

    The catalytic subunits of protein kinase A are transcribed in all mouse tissues from two distinct genes that code for the Calpha and Cbeta isoforms. Alternative promoters exist for the Cbeta gene that are used in a tissue-specific fashion and give rise to variants that differ in their amino-terminal sequences. We have characterized an alternative promoter that is present in the first intron of the Calpha gene and is transcriptionally active in male germ cells. Transcription from this promoter is coincident with the appearance of pachytene spermatocytes and leads to a Calpha protein (Calpha2) that contains a distinctive 7 amino acid amino-terminus differing from the 14 amino acid amino-terminus of Calpha1. The Calpha2 protein does not contain the myristylation signal present on Calpha1 and migrates at a lower molecular weight on SDS/PAGE gels. By Western blotting, we estimate that most or all of the Calpha protein present in mature sperm is Calpha2. The amino-terminal sequence of Calpha2 is similar to that of ovine sperm C as previously reported [San Agustin, J. T., Leszyk, J. D., Nuwaysir, L. M. & Witman, G. B. (1998) J. Biol. Chem. 273, 24874-24883], and we show by cDNA cloning that human sperm also express a highly related Calpha2 homolog. The Calpha2 subunit forms holoenzymes with either RIIalpha or RIalpha, and both activate at the same concentration of cyclic nucleotide. Because protein kinase A is thought to play a pivotal role in sperm motility and capacitation, the distinctive biochemical properties of the unmyristylated Calpha2 may be essential for fertility in the male.

  19. A dynamically coupled allosteric network underlies binding cooperativity in Src kinase.

    Science.gov (United States)

    Foda, Zachariah H; Shan, Yibing; Kim, Eric T; Shaw, David E; Seeliger, Markus A

    2015-01-20

    Protein tyrosine kinases are attractive drug targets because many human diseases are associated with the deregulation of kinase activity. However, how the catalytic kinase domain integrates different signals and switches from an active to an inactive conformation remains incompletely understood. Here we identify an allosteric network of dynamically coupled amino acids in Src kinase that connects regulatory sites to the ATP- and substrate-binding sites. Surprisingly, reactants (ATP and peptide substrates) bind with negative cooperativity to Src kinase while products (ADP and phosphopeptide) bind with positive cooperativity. We confirm the molecular details of the signal relay through the allosteric network by biochemical studies. Experiments on two additional protein tyrosine kinases indicate that the allosteric network may be largely conserved among these enzymes. Our work provides new insights into the regulation of protein tyrosine kinases and establishes a potential conduit by which resistance mutations to ATP-competitive kinase inhibitors can affect their activity.

  20. Molecular Basis of Inactive B-RAF(WT) and B-RAF(V600E) Ligand Inhibition, Selectivity and Conformational Stability: An in Silico Study

    DEFF Research Database (Denmark)

    Fratev, Filip Filipov; Jonsdottir, Svava Osk; Mihaylova, E.

    2009-01-01

    The B-RAF kinase plays an important role both in tumor induction and maintenance in several cancers. The molecular basis of the inactive B-RAF(WT) and B-RAF(V600E) inhibition and selectivity of a series of inhibitors was examined with a combination of molecular dynamics (MD), free energy MM......-PBSA and local-binding energy (LBE) approaches. The conformational stability of the unbounded kinases and in particular the processes of the B-RAF(V600E) mutant activation were analyzed. A unique salt bridge network formed mainly by the catalytic residues was identified in the unbounded B-RAFs...

  1. Dominant Rio1 kinase/ATPase catalytic mutant induces trapping of late pre-40S biogenesis factors in 80S-like ribosomes.

    Science.gov (United States)

    Ferreira-Cerca, Sébastien; Kiburu, Irene; Thomson, Emma; LaRonde, Nicole; Hurt, Ed

    2014-07-01

    During eukaryotic ribosome biogenesis, members of the conserved atypical serine/threonine protein kinase family, the RIO kinases (Rio1, Rio2 and Rio3) function in small ribosomal subunit biogenesis. Structural analysis of Rio2 indicated a role as a conformation-sensing ATPase rather than a kinase to regulate its dynamic association with the pre-40S subunit. However, it remained elusive at which step and by which mechanism the other RIO kinase members act. Here, we have determined the crystal structure of the human Rio1-ATP-Mg(2+) complex carrying a phosphoaspartate in the active site indicative of ATPase activity. Structure-based mutations in yeast showed that Rio1's catalytic activity regulates its pre-40S association. Furthermore, we provide evidence that Rio1 associates with a very late pre-40S via its conserved C-terminal domain. Moreover, a rio1 dominant-negative mutant defective in ATP hydrolysis induced trapping of late biogenesis factors in pre-ribosomal particles, which turned out not to be pre-40S but 80S-like ribosomes. Thus, the RIO kinase fold generates a versatile ATPase enzyme, which in the case of Rio1 is activated following the Rio2 step to regulate one of the final 40S maturation events, at which time the 60S subunit is recruited for final quality control check.

  2. Elevated breast cancer risk in irradiated BALB/c mice associates with unique functional polymorphism of the Prkdc (DNA-dependent protein kinase catalytic subunit) gene

    Science.gov (United States)

    Yu, Y.; Okayasu, R.; Weil, M. M.; Silver, A.; McCarthy, M.; Zabriskie, R.; Long, S.; Cox, R.; Ullrich, R. L.

    2001-01-01

    Female BALB/c mice are unusually radiosensitive and more susceptible than C57BL/6 and other tested inbred mice to ionizing radiation (IR)-induced mammary tumors. This breast cancer susceptibility is correlated with elevated susceptibility for mammary cell transformation and genomic instability following irradiation. In this study, we report the identification of two BALB/c strain-specific polymorphisms in the coding region of Prkdc, the gene encoding the DNA-dependent protein kinase catalytic subunit, which is known to be involved in DNA double-stranded break repair and post-IR signal transduction. First, we identified an A --> G transition at base 11530 resulting in a Met --> Val conversion at codon 3844 (M3844V) in the phosphatidylinositol 3-kinase domain upstream of the scid mutation (Y4046X). Second, we identified a C --> T transition at base 6418 resulting in an Arg --> Cys conversion at codon 2140 (R2140C) downstream of the putative leucine zipper domain. This unique PrkdcBALB variant gene is shown to be associated with decreased DNA-dependent protein kinase catalytic subunit activity and with increased susceptibility to IR-induced genomic instability in primary mammary epithelial cells. The data provide the first evidence that naturally arising allelic variation in a mouse DNA damage response gene may associate with IR response and breast cancer risk.

  3. The C-terminal domain of human grp94 protects the catalytic subunit of protein kinase CK2 (CK2alpha) against thermal aggregation. Role of disulfide bonds

    DEFF Research Database (Denmark)

    Roher, N; Miró, F; Boldyreff, B

    2001-01-01

    The C-terminal domain (residues 518-803) of the 94 kDa glucose regulated protein (grp94) was expressed in Escherichia coli as a fusion protein with a His6-N-terminal tag (grp94-CT). This truncated form of grp94 formed dimers and oligomers that could be dissociated into monomers by treatment...... with dithiothreitol. Grp94-CT conferred protection against aggregation on the catalytic subunit of protein kinase CK2 (CK2alpha), although it did not protect against thermal inactivation. This anti-aggregation effect of grp94-CT was concentration dependent, with full protection achieved at grp94-CT/CK2alpha molar...

  4. Conformational plasticity of the catalytic subunit of protein kinase CK2 and its consequences for regulation and drug design

    DEFF Research Database (Denmark)

    Niefind, Karsten; Issinger, Olaf-Georg

    2010-01-01

    well to the constitutive activity of the enzyme, meaning, its independence from phosphorylation or other characteristic control factors. Most CK2alpha structures are based on the enzyme from Zea mays, supplemented by an increasing number of human CK2alpha structures. In the latter a surprising...... molecule seems to favour the partially and fully inactive states. This observation may be exploited to design effective and selective CK2 inhibitors....

  5. Identification of a nonsense mutation in the carboxyl-terminal region of DNA-dependent protein kinase catalytic subunit in the scid mouse.

    Science.gov (United States)

    Blunt, T; Gell, D; Fox, M; Taccioli, G E; Lehmann, A R; Jackson, S P; Jeggo, P A

    1996-01-01

    DNA-dependent protein kinase (DNA-PK) consists of a heterodimeric protein (Ku) and a large catalytic subunit (DNA-PKcs). The Ku protein has double-stranded DNA end-binding activity that serves to recruit the complex to DNA ends. Despite having serine/threonine protein kinase activity, DNA-PKcs falls into the phosphatidylinositol 3-kinase superfamily. DNA-PK functions in DNA double-strand break repair and V(D)J recombination, and recent evidence has shown that mouse scid cells are defective in DNA-PKcs. In this study we have cloned the cDNA for the carboxyl-terminal region of DNA-PKcs in rodent cells and identified the existence of two differently spliced products in human cells. We show that DNA-PKcs maps to the same chromosomal region as the mouse scid gene. scid cells contain approximately wild-type levels of DNA-PKcs transcripts, whereas the V-3 cell line, which is also defective in DNA-PKcs, contains very reduced transcript levels. Sequence comparison of the carboxyl-terminal region of scid and wild-type mouse cells enabled us to identify a nonsense mutation within a highly conserved region of the gene in mouse scid cells. This represents a strong candidate for the inactivating mutation in DNA-PKcs in the scid mouse. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8816792

  6. Conformational transitions of the catalytic domain of heme-regulated eukaryotic initiation factor 2α kinase, a key translational regulatory molecule.

    Science.gov (United States)

    Sreejith, R K; Suresh, C G; Bhosale, Siddharth H; Bhavnani, Varsha; Kumar, Avinash; Gaikwad, Sushama M; Pal, Jayanta K

    2012-01-01

    In mammalian cells, the heme-regulated inhibitor (HRI) plays a critical role in the regulation of protein synthesis at the initiation step through phosphorylation of α-subunit of the eukaryotic initiation factor 2 (eIF2). In this study we have cloned and performed biophysical characterization of the kinase catalytic domain (KD) of rabbit HRI. The KD described here comprises kinase 1, the kinase insertion domain (KI) and kinase 2. We report here the existence of an active and stable monomer of HRI (KD). The HRI (KD) containing three tryptophan residues was examined for its conformational transitions occurring under various denaturing conditions using steady-state and time-resolved tryptophan fluorescence, circular dichroism (CD) and hydrophobic dye binding. The parameter A and phase diagram analysis revealed multi-state unfolding and existence of three stable intermediates during guanidine hydrochloride (Gdn-HCl) induced unfolding of HRI (KD). The protein treated with 6 M Gdn-HCl showed collisional and static mechanism of acrylamide quenching and the constants (K(sv) = 3.08 M(-1) and K(s)= 5.62 M(-1)) were resolved using time resolved fluorescence titration. Based on pH, guanidine hydrochloride and temperature mediated transitions, HRI (KD) appears to exemplify a rigid molten globule-like intermediate with compact secondary structure, altered tertiary structure and exposed hydrophobic patches at pH 3.0. The results indicate the inherent structural stability of HRI (KD), a member of the class of stress response proteins.

  7. Crystal structure of the catalytic subunit of protein kinase CK2 from Zea mays at 2.1 A resolution

    DEFF Research Database (Denmark)

    Niefind, K; Guerra, B; Pinna, L A

    1998-01-01

    and probably contributes to the constitutively active nature of CK2. The active centre is occupied by a partially disordered ATP molecule with the adenine base attached to a novel binding site of low specificity. This finding explains the observation that CK2, unlike other protein kinases, can use both ATP...

  8. Crystal structure of the kinase domain of serum and glucocorticoid-regulated kinase 1 in complex with AMP–PNP

    Science.gov (United States)

    Zhao, Baoguang; Lehr, Ruth; Smallwood, Angela M.; Ho, Thau F.; Maley, Kathleen; Randall, Tanya; Head, Martha S.; Koretke, Kristin K.; Schnackenberg, Christine G.

    2007-01-01

    Serum and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine protein kinase of the AGC family which participates in the control of epithelial ion transport and is implicated in proliferation and apoptosis. We report here the 1.9 Å crystal structure of the catalytic domain of inactive human SGK1 in complex with AMP–PNP. SGK1 exists as a dimer formed by two intermolecular disulfide bonds between Cys258 in the activation loop and Cys193. Although most of the SGK1 structure closely resembles the common protein kinase fold, the structure around the active site is unique when compared to most protein kinases. The αC helix is not present in this inactive form of SGK1 crystal structure; instead, the segment corresponding to the C helix forms a β-strand that is stabilized by the N-terminal segment of the activation loop through a short antiparallel β-sheet. Since the differences from other kinases occur around the ATP binding site, this structure can provide valuable insight into the design of selective and highly potent ATP-competitive inhibitors of SGK1 kinase. PMID:17965184

  9. Crystal structure of the kinase domain of serum and glucocorticoid-regulated kinase 1 in complex with AMP-PNP

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Baoguang; Lehr, Ruth; Smallwood, Angela M; Ho, Thau F; Maley, Kathleen; Randall, Tanya; Head, Martha S; Koretke, Kristin K; Schnackenberg, Christine G [GSKPA

    2008-06-30

    Serum and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine protein kinase of the AGC family which participates in the control of epithelial ion transport and is implicated in proliferation and apoptosis. We report here the 1.9 {angstrom} crystal structure of the catalytic domain of inactive human SGK1 in complex with AMP-PNP. SGK1 exists as a dimer formed by two intermolecular disulfide bonds between Cys258 in the activation loop and Cys193. Although most of the SGK1 structure closely resembles the common protein kinase fold, the structure around the active site is unique when compared to most protein kinases. The {alpha}C helix is not present in this inactive form of SGK1 crystal structure; instead, the segment corresponding to the C helix forms a {beta}-strand that is stabilized by the N-terminal segment of the activation loop through a short antiparallel {beta}-sheet. Since the differences from other kinases occur around the ATP binding site, this structure can provide valuable insight into the design of selective and highly potent ATP-competitive inhibitors of SGK1 kinase.

  10. Protein kinase CK2: evidence for a protein kinase CK2beta subunit fraction, devoid of the catalytic CK2alpha subunit, in mouse brain and testicles

    DEFF Research Database (Denmark)

    Guerra, B; Siemer, S; Boldyreff, B;

    1999-01-01

    The highest CK2 activity was found in mouse testicles and brain, followed by spleen, liver, lung, kidney and heart. The activity values were directly correlated with the protein expression level of the CK2 subunits alpha (catalytic) and beta (regulatory). The alpha' subunit was only detected...... found for testicles and brain. The amount of CK2beta protein in brain in comparison to the other organs (except testicles) was estimated to be ca. 2-3-fold higher whereas the ratio of CK2beta between testicles and brain was estimated to be 3-4-fold. Results from the immunoprecipitation experiments...... support the notion for the existence of free CK2beta population and/or CK2beta in complex with other protein(s) present in brain and testicles. In all other mouse organs investigated, i.e. heart, lung, liver, kidney and spleen, no comparable amount of free CK2beta was observed. This is the first...

  11. Recognition of DNA Termini by the C-Terminal Region of the Ku80 and the DNA-Dependent Protein Kinase Catalytic Subunit.

    Directory of Open Access Journals (Sweden)

    Derek S Woods

    Full Text Available DNA double strand breaks (DSBs can be generated by endogenous cellular processes or exogenous agents in mammalian cells. These breaks are highly variable with respect to DNA sequence and structure and all are recognized in some context by the DNA-dependent protein kinase (DNA-PK. DNA-PK is a critical component necessary for the recognition and repair of DSBs via non-homologous end joining (NHEJ. Previously studies have shown that DNA-PK responds differentially to variations in DSB structure, but how DNA-PK senses differences in DNA substrate sequence and structure is unknown. Here we explore the enzymatic mechanisms by which DNA-PK is activated by various DNA substrates and provide evidence that the DNA-PK is differentially activated by DNA structural variations as a function of the C-terminal region of Ku80. Discrimination based on terminal DNA sequence variations, on the other hand, is independent of the Ku80 C-terminal interactions and likely results exclusively from DNA-dependent protein kinase catalytic subunit interactions with the DNA. We also show that sequence differences in DNA termini can drastically influence DNA repair through altered DNA-PK activation. These results indicate that even subtle differences in DNA substrates influence DNA-PK activation and ultimately the efficiency of DSB repair.

  12. Loss of the catalytic subunit of the DNA-dependent protein kinase in DNA double-strand-break-repair mutant mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Peterson, S.R. [Los Alamos National Lab., NM (United States)]|[Tottori Univ., Yonago (Japan); Kurimasa, Akihiro; Oshimura, Mitsuo [Tottori Univ., Yonago (Japan); Dynan, W.S. [Univ. of Colorado, Boulder, CO (United States); Bradbury, E.M. [Los Alamos National Lab., NM (United States)]|[Univ. of California, Davis, CA (United States); Chen, D.J. [Los Alamos National Lab., NM (United States)

    1995-04-11

    The DNA-dependent protein kinase (DNA-PK) consists of three polypeptide components: Ku-70, Ku-80, and an {approx}350-kDa catalytic subunit (p350). The gene encoding the Ku-80 subunit is identical to the x-ray-sensitive group 5 complementing gene XRCC5. Expression of the Ku-80 cDNA rescues both DNA double-strand break (DSB) repair and V(D)J recombination in group 5 mutant cells. The involvement of Ku-80 in these processes suggests that the underlying defect in these mutant cells may be disruption of the DNA-PK holoenzyme. In this report we show that the p350 kinase subunit is deleted in cells derived from the severe combined immunodeficiency mouse and in the Chinese hamster ovary cell line V-3, both of which are defective in DSB repair and V(D)J recombination. A centromeric fragment of human chromosome 8 that complements the scid defect also restores p350 protein expression and rescues in vitro DNA-PK activity. These data suggest the scid gene may encode the p350 protein or regulate its expression and are consistent with a model whereby DNA-PK is a critical component of the DSB-repair pathway. 38 refs., 3 figs.

  13. Crystal structures of human RIP2 kinase catalytic domain complexed with ATP-competitive inhibitors: Foundations for understanding inhibitor selectivity.

    Science.gov (United States)

    Charnley, Adam K; Convery, Máire A; Lakdawala Shah, Ami; Jones, Emma; Hardwicke, Philip; Bridges, Angela; Ouellette, Michael; Totoritis, Rachel; Schwartz, Benjamin; King, Bryan W; Wisnoski, David D; Kang, James; Eidam, Patrick M; Votta, Bartholomew J; Gough, Peter J; Marquis, Robert W; Bertin, John; Casillas, Linda

    2015-11-01

    Receptor interacting protein 2 (RIP2) is an intracellular kinase and key signaling partner for the pattern recognition receptors NOD1 and NOD2 (nucleotide-binding oligomerization domain-containing proteins 1 and 2). As such, RIP2 represents an attractive target to probe the role of these pathways in disease. In an effort to design potent and selective inhibitors of RIP2 we established a crystallographic system and determined the structure of the RIP2 kinase domain in an apo form and also in complex with multiple inhibitors including AMP-PCP (β,γ-Methyleneadenosine 5'-triphosphate, a non-hydrolysable adenosine triphosphate mimic) and structurally diverse ATP competitive chemotypes identified via a high-throughput screening campaign. These structures represent the first set of diverse RIP2-inhibitor co-crystal structures and demonstrate that the protein possesses the ability to adopt multiple DFG-in as well as DFG-out and C-helix out conformations. These structures reveal key protein-inhibitor structural insights and serve as the foundation for establishing a robust structure-based drug design effort to identify both potent and highly selective inhibitors of RIP2 kinase.

  14. Phosphatidyl-inositol-3-kinase alpha catalytic subunit mutation and response to neoadjuvant endocrine therapy for estrogen receptor positive breast cancer

    Science.gov (United States)

    Ellis, Matthew J; Lin, Li; Crowder, Robert; Tao, Yu; Hoog, Jeremy; Snider, Jacqueline; Davies, Sherri; DeSchryver, Katherine; Evans, Dean B; Steinseifer, Jutta; Bandaru, Raj; Liu, WeiHua; Gardner, Humphrey; Semiglazov, Vladimir; Watson, Mark; Hunt, Kelly; Olson, John; Baselga, José

    2010-01-01

    Background Mutations in the alpha catalytic subunit of phosphoinositol-3-kinase (PIK3CA) occur in ~30% of ER positive breast cancers. We therefore sought to determine the impact of PIK3CA mutation on response to neoadjuvant endocrine therapy. Methods Exon 9 (helical domain - HD) and Exon 20 (kinase domain- KD) mutations in PIK3CA were determined samples from four neoadjuvant endocrine therapy trials. Interactions with clinical, pathological and biomarker response parameters were examined. Results A weak negative interaction between PIK3CA mutation status and clinical response to neoadjuvant endocrine treatment was detected (N=235 P=<0.05), but not with treatment-induced changes in Ki67-based proliferation index (N=418). Despite these findings, PIK3CA KD mutation was a favorable prognostic factor for relapse-free survival (RFS log rank P=0.02) in the P024 trial (N=153). The favorable prognostic effect was maintained in a multivariable analysis (N=125) that included the preoperative prognostic index (PEPI), an approach to predicting RFS based on post neoadjuvant endocrine therapy pathological stage, ER and Ki67 levels (HR for no PIK3CA KD mutation, 14, CI 1.9–105 P=0.01). Conclusion PIK3CA mutation status did not strongly interact with neoadjuvant endocrine therapy responsiveness in estrogen receptor positive breast cancer. Nonetheless, as with other recent studies, a favorable interaction between PIK3CA kinase domain mutation and prognosis was detected. The mechanism for the favorable prognostic impact of PIK3CA mutation status therefore remains unexplained. PMID:19844788

  15. Icaritin requires Phosphatidylinositol 3 kinase (PI3K)/Akt signaling to counteract skeletal muscle atrophy following mechanical unloading

    OpenAIRE

    ZHANG, Zong-Kang; Li, Jie; Liu, Jin; Baosheng GUO; Leung, Albert; Zhang, Ge; Zhang, Bao-Ting

    2016-01-01

    Counteracting muscle atrophy induced by mechanical unloading/inactivity is of great clinical need and challenge. A therapeutic agent that could counteract muscle atrophy following mechanical unloading in safety is desired. This study showed that natural product Icaritin (ICT) could increase the phosphorylation level of Phosphatidylinositol 3 kinase (PI3K) at p110 catalytic subunit and promote PI3K/Akt signaling markers in C2C12 cells. This study further showed that the high dose ICT treatment...

  16. Protein kinase CK2α catalytic subunit ameliorates diabetic renal inflammatory fibrosis via NF-κB signaling pathway.

    Science.gov (United States)

    Huang, Junying; Chen, Zhiquan; Li, Jie; Chen, Qiuhong; Li, Jingyan; Gong, Wenyan; Huang, Jiani; Liu, Peiqing; Huang, Heqing

    2017-02-23

    Activation of casein kinase 2 (CK2) is closely linked to the body disturbance of carbohydrate metabolism and inflammatory reaction. The renal chronic inflammatory reaction in the setting of diabetes is one of the important hallmarks of diabetic renal fibrosis. However, it remains unknown whether CK2 influences the process of diabetic renal fibrosis. The current study is aimed to investigate if CK2α ameliorates renal inflammatory fibrosis in diabetes via NF-κB pathway. To explore potential regulatory mechanism of CK2α, the expression and activity of CK2α, which were studied by plasmid transfection, selective inhibitor, small-interfering RNA (siRNA) and adenovirus infection in vitro or in vivo, were analyzed by means of western blotting (WB), dual luciferase reporter assay and electrophoretic mobility shift assay (EMSA). The following findings were observed: (1) Expression of CK2α was upregulated in kidneys of db/db and KKAy diabetic mice; (2) Inhibition of CK2α kinase activity or knockdown of CK2α protein expression suppressed high glucose-induced expressions of FN and ICAM-1 in glomerular mesangial cells (GMCs); (3) Inhibition of CK2α kinase activity or knockdown of CK2α protein expression not only restrained IκB degradation, but also suppressed HG-induced nuclear accumulation, transcriptional activity and DNA binding activity of NF-κB in GMCs; (4) Treatment of TBB or CK2α RNAi adenovirus infection ameliorated renal fibrosis in diabetic animals; (5) Treatment of TBB or CK2α RNAi adenovirus infection suppressed IκB degradation and NF-κB nuclear accumulation in glomeruli of diabetic animals. This study indicates the essential role of CK2α in regulating the diabetic renal pathological process of inflammatory fibrosis via NF-κB pathway, and inhibition of CK2α may serve as a promising therapeutic strategy for diabetic nephropathy.

  17. O-GlcNAcylation of protein kinase A catalytic subunits enhances its activity: a mechanism linked to learning and memory deficits in Alzheimer's disease.

    Science.gov (United States)

    Xie, Shutao; Jin, Nana; Gu, Jianlan; Shi, Jianhua; Sun, Jianming; Chu, Dandan; Zhang, Liang; Dai, Chun-Ling; Gu, Jin-Hua; Gong, Cheng-Xin; Iqbal, Khalid; Liu, Fei

    2016-06-01

    Alzheimer's disease (AD) is characterized clinically by memory loss and cognitive decline. Protein kinase A (PKA)-CREB signaling plays a critical role in learning and memory. It is known that glucose uptake and O-GlcNAcylation are reduced in AD brain. In this study, we found that PKA catalytic subunits (PKAcs) were posttranslationally modified by O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAcylation regulated the subcellular location of PKAcα and PKAcβ and enhanced their kinase activity. Upregulation of O-GlcNAcylation in metabolically active rat brain slices by O-(2-acetamido-2-deoxy-d-glucopyranosylidenamino) N-phenylcarbamate (PUGNAc), an inhibitor of N-acetylglucosaminidase, increased the phosphorylation of tau at the PKA site, Ser214, but not at the non-PKA site, Thr205. In contrast, in rat and mouse brains, downregulation of O-GlcNAcylation caused decreases in the phosphorylation of CREB at Ser133 and of tau at Ser214, but not at Thr205. Reduction in O-GlcNAcylation through intracerebroventricular injection of 6-diazo-5-oxo-l-norleucine (DON), the inhibitor of glutamine fructose-6-phosphate amidotransferase, suppressed PKA-CREB signaling and impaired learning and memory in mice. These results indicate that in addition to cAMP and phosphorylation, O-GlcNAcylation is a novel mechanism that regulates PKA-CREB signaling. Downregulation of O-GlcNAcylation suppresses PKA-CREB signaling and consequently causes learning and memory deficits in AD.

  18. Molecular characterisation of cAMP-dependent protein kinase (PK-A) catalytic subunit isoforms in the male tick, Amblyomma hebraeum.

    Science.gov (United States)

    Tabish, Mohammad; Clegg, Roger A; Turner, Philip C; Jonczy, Jan; Rees, Huw H; Fisher, Michael J

    2006-12-01

    The cAMP-dependent protein kinase (protein kinase A, PK-A) plays a central role in the regulation of diverse aspects of cellular activity. Specifically, PK-A appears to play a key controlling role in the maturation of spermatids. Using a PCR-based approach, with degenerate primers from the highly conserved regions of the PK-A catalytic (C) subunit in combination with 5' and 3' RACE, we have cloned three cDNAs for the PK-A C-subunit of the male tick, Amblyomma hebraeum. The three cDNAs have open reading frames of 1059, 1275 and 1404bp which encode proteins of 40.6, 48.2 and 52.5kDa, respectively. These transcripts appear to arise from 5' alternative splicing of RNA derived from a single gene for the PK-A C-subunit. One isoform (AH-PK-A C1), in common with PK-A C-subunits from a range of species, contains a consensus sequence for N-myristoylation. RT-PCR and Western blot experiments suggest that the three splice variants are expressed ubiquitously; however, expression of the myristoylatable AH-PK-A C1 isoform is predominant in all investigated tissues (accessory gland, midgut, Malpighian tubules, salivary gland, testis and immature spermatids). There is no evidence for a sperm-specific PK-A C-subunit (Cs) in tick sperm; however, tyrosine protein phosphorylation, previously shown to be modulated by PK-A activity during mammalian sperm maturation, was observed in tick sperm.

  19. Photo-activated psoralen binds the ErbB2 catalytic kinase domain, blocking ErbB2 signaling and triggering tumor cell apoptosis.

    Directory of Open Access Journals (Sweden)

    Wenle Xia

    Full Text Available Photo-activation of psoralen with UVA irradiation, referred to as PUVA, is used in the treatment of proliferative skin disorders. The anti-proliferative effects of PUVA have been largely attributed to psoralen intercalation of DNA, which upon UV treatment, triggers the formation of interstrand DNA crosslinks (ICL that inhibit transcription and DNA replication. Here, we show that PUVA exerts antitumor effects in models of human breast cancer that overexpress the ErbB2 receptor tyrosine kinase oncogene, through a new mechanism. Independent of ICL formation, the antitumor effects of PUVA in ErbB2+ breast cancer models can instead be mediated through inhibition of ErbB2 activation and signaling. Using a mass spectroscopy-based approach, we show for the first time that photo-activated 8MOP (8-methoxypsoralen interacts with the ErbB2 catalytic autokinase domain. Furthermore, PUVA can reverse therapeutic resistance to lapatinib and other ErbB2 targeted therapies, including resistance mediated via expression of a phosphorylated, truncated form of ErbB2 (p85(ErbB2 that is preferentially expressed in tumor cell nuclei. Current ErbB2 targeted therapies, small molecule kinase inhibitors or antibodies, do not block the phosphorylated, activated state of p85(ErbB2. Here we show that PUVA reduced p85(ErbB2 phosphorylation leading to tumor cell apoptosis. Thus, in addition to its effects on DNA and the formation of ICL, PUVA represents a novel ErbB2 targeted therapy for the treatment of ErbB2+ breast cancers, including those that have developed resistance to other ErbB2 targeted therapies.

  20. The pandemic of physical inactivity

    DEFF Research Database (Denmark)

    Kohl, Harold W; Craig, Cora Lynn; Lambert, Estelle Victoria

    2012-01-01

    Physical inactivity is the fourth leading cause of death worldwide. We summarise present global efforts to counteract this problem and point the way forward to address the pandemic of physical inactivity. Although evidence for the benefits of physical activity for health has been available since ...

  1. Interaction of the regulatory and catalytic subunits of cAMP-dependent protein kinase. Electrostatic sites on the type Ialpha regulatory subunit.

    Science.gov (United States)

    Gibson, R M; Ji-Buechler, Y; Taylor, S S

    1997-06-27

    Since a basic surface on the catalytic (C) subunit of cAMP-dependent protein kinase is important for binding to the regulatory (R) subunit, acidic residues in R were sought that might contribute to R-C interaction. Using differential labeling by a water-soluble carbodiimide (Buechler, T. A., and Taylor, S. S. (1990) Biochemistry 29, 1937-1943), seven specific carboxylates in RIalpha were identified that were protected from chemical modification in the holoenzyme; each was then replaced with Ala. Of these, rRI(E15A/E106A/D107A)), rRI(E105A), rRI(D140A), rRI(E143A), and rRI(D258A) all were defective in holoenzyme formation and define negative electrostatic surfaces on RIalpha. An additional conserved carboxylate, Glu101 in RIalpha and the equivalent, Glu99 in RIIalpha were mutated to Ala. Replacement of Glu101 had no effect while rRII(E99A) was very defective. RIalpha and RIIalpha thus differ in the molecular details of how they recognize C. Unlike wild-type RI, two additional mutants, rRI(D170A) and rRI(K242A), inhibited C-subunit stoichiometrically in the presence of cAMP and show increases in both on- and off-rates. Asp170, which contributes directly to the hydrogen bonding network in cAMP-binding site A, thus contributes also to holoenzyme stability.

  2. miR-502 inhibits cell proliferation and tumor growth in hepatocellular carcinoma through suppressing phosphoinositide 3-kinase catalytic subunit gamma

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Suling, E-mail: suling_chen86@163.com [Department of Infectious Disease, Heping Hospital Attached to Changzhi Medical College, Changzhi 046000 (China); Li, Fang; Chai, Haiyun; Tao, Xin [Department of Infectious Disease, Heping Hospital Attached to Changzhi Medical College, Changzhi 046000 (China); Wang, Haili [Department of Hematology, Heping Hospital Attached to Changzhi Medical College, Changzhi 046000 (China); Ji, Aifang [Central Laboratory, Heping Hospital Attached to Changzhi Medical College, Changzhi 046000 (China)

    2015-08-21

    MicroRNAs (miRNAs) play a key role in carcinogenesis and tumor progression in hepatocellular carcinoma (HCC). In the present study, we demonstrated that miR-502 significantly inhibits HCC cell proliferation in vitro and tumor growth in vivo. G1/S cell cycle arrest and apoptosis of HCC cells were induced by miR-502. Phosphoinositide 3-kinase catalytic subunit gamma (PIK3CG) was identified as a direct downstream target of miR-502 in HCC cells. Notably, overexpression of PIK3CG reversed the inhibitory effects of miR-502 in HCC cells. Our findings suggest that miR-502 functions as a tumor suppressor in HCC via inhibition of PI3KCG, supporting its utility as a promising therapeutic gene target for this tumor type. - Highlights: • miR-502 suppresses HCC cell proliferation in vitro and tumorigenicity in vivo. • miR-502 regulates cell cycle and apoptosis in HCC cells. • PIK3CG is a direct target of miR-502. • miR-502 and PIK3CG expression patterns are inversely correlated in HCC tissues.

  3. Role of key residues at the flavin mononucleotide (FMN):adenylyltransferase catalytic site of the bifunctional riboflavin kinase/flavin adenine dinucleotide (FAD) Synthetase from Corynebacterium ammoniagenes.

    Science.gov (United States)

    Serrano, Ana; Frago, Susana; Velázquez-Campoy, Adrián; Medina, Milagros

    2012-11-08

    In mammals and in yeast the conversion of Riboflavin (RF) into flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) is catalysed by the sequential action of two enzymes: an ATP:riboflavin kinase (RFK) and an ATP:FMN adenylyltransferase (FMNAT). However, most prokaryotes depend on a single bifunctional enzyme, FAD synthetase (FADS), which folds into two modules: the C-terminal associated with RFK activity and the N-terminal associated with FMNAT activity. Sequence and structural analysis suggest that the 28-HxGH-31, 123-Gx(D/N)-125 and 161-xxSSTxxR-168 motifs from FADS must be involved in ATP stabilisation for the adenylylation of FMN, as well as in FAD stabilisation for FAD phyrophosphorolysis. Mutants were produced at these motifs in the Corynebacterium ammoniagenes FADS (CaFADS). Their effects on the kinetic parameters of CaFADS activities (RFK, FMNAT and FAD pyrophosphorilase), and on substrates and product binding properties indicate that H28, H31, N125 and S164 contribute to the geometry of the catalytically competent complexes at the FMNAT-module of CaFADS.

  4. Intranuclear Delivery of a Novel Antibody-Derived Radiosensitizer Targeting the DNA-Dependent Protein Kinase Catalytic Subunit

    Energy Technology Data Exchange (ETDEWEB)

    Xiong Hairong [Institute of Molecular Medicine and Genetics, Georgia Health Sciences University, Augusta, GA (Georgia); State Key Laboratory of Virology, Institute of Medical Virology, Wuhan University School of Medicine, Wuhan (China); Lee, Robert J. [Division of Pharmaceutics, College of Pharmacy, Ohio State University, Columbus, OH (United States); Haura, Eric B. [Thoracic Oncology and Experimental Therapeutics Programs, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL (United States); Edwards, John G. [Apeliotus Technologies, Inc., Atlanta, GA (United States); Dynan, William S. [Institute of Molecular Medicine and Genetics, Georgia Health Sciences University, Augusta, GA (Georgia); Li Shuyi, E-mail: sli@georgiahealth.edu [Institute of Molecular Medicine and Genetics, Georgia Health Sciences University, Augusta, GA (Georgia); Apeliotus Technologies, Inc., Atlanta, GA (United States)

    2012-07-01

    Purpose: To inhibit DNA double-strand break repair in tumor cells by delivery of a single-chain antibody variable region fragment (ScFv 18-2) to the cell nucleus. ScFv 18-2 binds to a regulatory region of the DNA-dependent protein kinase (DNA-PK), an essential enzyme in the nonhomologous end-joining pathway, and inhibits DNA end-joining in a cell-free system and when microinjected into single cells. Development as a radiosensitizer has been limited by the lack of a method for intranuclear delivery to target cells. We investigated a delivery method based on folate receptor-mediated endocytosis. Methods and Materials: A recombinant ScFv 18-2 derivative was conjugated to folate via a scissile disulfide linker. Folate-ScFv 18-2 was characterized for its ability to be internalized by tumor cells and to influence the behavior of ionizing radiation-induced repair foci. Radiosensitization was measured in a clonogenic survival assay. Survival curves were fitted to a linear-quadratic model, and between-group differences were evaluated by an F test. Sensitization ratios were determined based on mean inhibitory dose. Results: Human KB and NCI-H292 lung cancer cells treated with folate-conjugated ScFv 18-2 showed significant radiosensitization (p < 0.001). Sensitization enhancement ratios were 1.92 {+-} 0.42 for KB cells and 1.63 {+-} 0.13 for NCI-H292 cells. Studies suggest that treatment inhibits repair of radiation-induced DSBs, as evidenced by the persistence of {gamma}-H2AX-stained foci and by inhibition of staining with anti-DNA-PKcs phosphoserine 2056. Conclusions: Folate-mediated endocytosis is an effective method for intranuclear delivery of an antibody-derived DNA repair inhibitor.

  5. Protein Kinase CK2α Maintains Extracellular Signal-regulated Kinase (ERK) Activity in a CK2α Kinase-independent Manner to Promote Resistance to Inhibitors of RAF and MEK but Not ERK in BRAF Mutant Melanoma.

    Science.gov (United States)

    Zhou, Bingying; Ritt, Daniel A; Morrison, Deborah K; Der, Channing J; Cox, Adrienne D

    2016-08-19

    The protein kinase casein kinase 2 (CK2) is a pleiotropic and constitutively active kinase that plays crucial roles in cellular proliferation and survival. Overexpression of CK2, particularly the α catalytic subunit (CK2α, CSNK2A1), has been implicated in a wide variety of cancers and is associated with poorer survival and resistance to both conventional and targeted anticancer therapies. Here, we found that CK2α protein is elevated in melanoma cell lines compared with normal human melanocytes. We then tested the involvement of CK2α in drug resistance to Food and Drug Administration-approved single agent targeted therapies for melanoma. In BRAF mutant melanoma cells, ectopic CK2α decreased sensitivity to vemurafenib (BRAF inhibitor), dabrafenib (BRAF inhibitor), and trametinib (MEK inhibitor) by a mechanism distinct from that of mutant NRAS. Conversely, knockdown of CK2α sensitized cells to inhibitor treatment. CK2α-mediated RAF-MEK kinase inhibitor resistance was tightly linked to its maintenance of ERK phosphorylation. We found that CK2α post-translationally regulates the ERK-specific phosphatase dual specificity phosphatase 6 (DUSP6) in a kinase dependent-manner, decreasing its abundance. However, we unexpectedly showed, by using a kinase-inactive mutant of CK2α, that RAF-MEK inhibitor resistance did not rely on CK2α kinase catalytic function, and both wild-type and kinase-inactive CK2α maintained ERK phosphorylation upon inhibition of BRAF or MEK. That both wild-type and kinase-inactive CK2α bound equally well to the RAF-MEK-ERK scaffold kinase suppressor of Ras 1 (KSR1) suggested that CK2α increases KSR facilitation of ERK phosphorylation. Accordingly, CK2α did not cause resistance to direct inhibition of ERK by the ERK1/2-selective inhibitor SCH772984. Our findings support a kinase-independent scaffolding function of CK2α that promotes resistance to RAF- and MEK-targeted therapies.

  6. Src kinase conformational activation: thermodynamics, pathways, and mechanisms.

    Directory of Open Access Journals (Sweden)

    Sichun Yang

    2008-03-01

    Full Text Available Tyrosine kinases of the Src-family are large allosteric enzymes that play a key role in cellular signaling. Conversion of the kinase from an inactive to an active state is accompanied by substantial structural changes. Here, we construct a coarse-grained model of the catalytic domain incorporating experimental structures for the two stable states, and simulate the dynamics of conformational transitions in kinase activation. We explore the transition energy landscapes by constructing a structural network among clusters of conformations from the simulations. From the structural network, two major ensembles of pathways for the activation are identified. In the first transition pathway, we find a coordinated switching mechanism of interactions among the alphaC helix, the activation-loop, and the beta strands in the N-lobe of the catalytic domain. In a second pathway, the conformational change is coupled to a partial unfolding of the N-lobe region of the catalytic domain. We also characterize the switching mechanism for the alphaC helix and the activation-loop in detail. Finally, we test the performance of a Markov model and its ability to account for the structural kinetics in the context of Src conformational changes. Taken together, these results provide a broad framework for understanding the main features of the conformational transition taking place upon Src activation.

  7. The tyrosine kinase BMX is an essential mediator of inflammatory arthritis in a kinase-independent manner.

    Science.gov (United States)

    Gottar-Guillier, Marie; Dodeller, Francis; Huesken, Dieter; Iourgenko, Vadim; Mickanin, Craig; Labow, Mark; Gaveriaux, Samuel; Kinzel, Bernd; Mueller, Matthias; Alitalo, Kari; Littlewood-Evans, Amanda; Cenni, Bruno

    2011-05-15

    Inflammatory cytokines like TNF play a central role in autoimmune disorders such as rheumatoid arthritis. We identified the tyrosine kinase bone marrow kinase on chromosome X (BMX) as an essential component of a shared inflammatory signaling pathway. Transient depletion of BMX strongly reduced secretion of IL-8 in cell lines and primary human cells stimulated by TNF, IL-1β, or TLR agonists. BMX was required for phosphorylation of p38 MAPK and JNK, as well as activation of NF-κB. The following epistasis analysis indicated that BMX acts downstream of or at the same level as the complex TGF-β activated kinase 1 (TAK1)-TAK1 binding protein. At the cellular level, regulation of the IL-8 promoter required the pleckstrin homology domain of BMX, which could be replaced by an ectopic myristylation signal, indicating a requirement for BMX membrane association. In addition, activation of the IL-8 promoter by in vitro BMX overexpression required its catalytic activity. Genetic ablation of BMX conferred protection in the mouse arthritis model of passive K/BxN serum transfer, confirming that BMX is an essential mediator of inflammation in vivo. However, genetic replacement with a catalytically inactive BMX allele was not protective in the same arthritis animal model. We conclude that BMX is an essential component of inflammatory cytokine signaling and that catalytic, as well as noncatalytic functions of BMX are involved.

  8. Physical inactivity in Parkinson's disease

    NARCIS (Netherlands)

    Nimwegen, M.L. van; Speelman, A.D.; Hofman-van Rossum, E.J.; Overeem, S.; Deeg, D.J.G.; Borm, G.F.; Horst, M.H. van der; Bloem, B.R.; Munneke, M.

    2011-01-01

    Patients with Parkinson's disease (PD) are likely to become physically inactive, because of their motor, mental, and emotional symptoms. However, specific studies on physical activity in PD are scarce, and results are conflicting. Here, we quantified daily physical activities in a large cohort of PD

  9. Co-conserved features associated with cis regulation of ErbB tyrosine kinases.

    Directory of Open Access Journals (Sweden)

    Amar Mirza

    Full Text Available BACKGROUND: The epidermal growth factor receptor kinases, or ErbB kinases, belong to a large sub-group of receptor tyrosine kinases (RTKs, which share a conserved catalytic core. The catalytic core of ErbB kinases have functionally diverged from other RTKs in that they are activated by a unique allosteric mechanism that involves specific interactions between the kinase core and the flanking Juxtamembrane (JM and COOH-terminal tail (C-terminal tail. Although extensive studies on ErbB and related tyrosine kinases have provided important insights into the structural basis for ErbB kinase functional divergence, the sequence features that contribute to the unique regulation of ErbB kinases have not been systematically explored. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we use a Bayesian approach to identify the selective sequence constraints that most distinguish ErbB kinases from other receptor tyrosine kinases. We find that strong ErbB kinase-specific constraints are imposed on residues that tether the JM and C-terminal tail to key functional regions of the kinase core. A conserved RIxKExE motif in the JM-kinase linker region and a glutamine in the inter-lobe linker are identified as two of the most distinguishing features of the ErbB family. While the RIxKExE motif tethers the C-terminal tail to the N-lobe of the kinase domain, the glutamine tethers the C-terminal tail to hinge regions critical for inter-lobe movement. Comparison of the active and inactive crystal structures of ErbB kinases indicates that the identified residues are conformationally malleable and can potentially contribute to the cis regulation of the kinase core by the JM and C-terminal tail. ErbB3, and EGFR orthologs in sponges and parasitic worms, diverge from some of the canonical ErbB features, providing insights into sub-family and lineage-specific functional specialization. CONCLUSION/SIGNIFICANCE: Our analysis pinpoints key residues for mutational analysis, and

  10. Pyridoxal 5'-phosphate is a slow tight binding inhibitor of E. coli pyridoxal kinase.

    Directory of Open Access Journals (Sweden)

    Mohini S Ghatge

    Full Text Available Pyridoxal 5'-phosphate (PLP is a cofactor for dozens of B(6 requiring enzymes. PLP reacts with apo-B(6 enzymes by forming an aldimine linkage with the ε-amino group of an active site lysine residue, thus yielding the catalytically active holo-B(6 enzyme. During protein turnover, the PLP is salvaged by first converting it to pyridoxal by a phosphatase and then back to PLP by pyridoxal kinase. Nonetheless, PLP poses a potential toxicity problem for the cell since its reactive 4'-aldehyde moiety forms covalent adducts with other compounds and non-B(6 proteins containing thiol or amino groups. The regulation of PLP homeostasis in the cell is thus an important, yet unresolved issue. In this report, using site-directed mutagenesis, kinetic, spectroscopic and chromatographic studies we show that pyridoxal kinase from E. coli forms a complex with the product PLP to form an inactive enzyme complex. Evidence is presented that, in the inhibited complex, PLP has formed an aldimine bond with an active site lysine residue during catalytic turnover. The rate of dissociation of PLP from the complex is very slow, being only partially released after a 2-hour incubation with PLP phosphatase. Interestingly, the inactive pyridoxal kinase•PLP complex can be partially reactivated by transferring the tightly bound PLP to an apo-B(6 enzyme. These results open new perspectives on the mechanism of regulation and role of pyridoxal kinase in the Escherichia coli cell.

  11. Inactive ingredient Search for Approved Drug Products

    Data.gov (United States)

    U.S. Department of Health & Human Services — According to 21 CFR 210.3(b)(8), an inactive ingredient is any component of a drug product other than the active ingredient. Only inactive ingredients in the final...

  12. Functional intersection of ATM and DNA-dependent protein kinase catalytic subunit in coding end joining during V(D)J recombination

    DEFF Research Database (Denmark)

    Lee, Baeck-Seung; Gapud, Eric J; Zhang, Shichuan

    2013-01-01

    -PKcs deficiency leads to a nearly complete block in coding join formation, as DNA-PKcs is required to activate Artemis, the endonuclease that opens hairpin-sealed coding ends. In contrast to loss of DNA-PKcs protein, here we show that inhibition of DNA-PKcs kinase activity has no effect on coding join formation...... when ATM is present and its kinase activity is intact. The ability of ATM to compensate for DNA-PKcs kinase activity depends on the integrity of three threonines in DNA-PKcs that are phosphorylation targets of ATM, suggesting that ATM can modulate DNA-PKcs activity through direct phosphorylation of DNA...

  13. The Activation of c-Src Tyrosine Kinase: Conformational Transition Pathway and Free Energy Landscape.

    Science.gov (United States)

    Fajer, Mikolai; Meng, Yilin; Roux, Benoît

    2016-10-28

    Tyrosine kinases are important cellular signaling allosteric enzymes that regulate cell growth, proliferation, metabolism, differentiation, and migration. Their activity must be tightly controlled, and malfunction can lead to a variety of diseases, particularly cancer. The nonreceptor tyrosine kinase c-Src, a prototypical model system and a representative member of the Src-family, functions as complex multidomain allosteric molecular switches comprising SH2 and SH3 domains modulating the activity of the catalytic domain. The broad picture of self-inhibition of c-Src via the SH2 and SH3 regulatory domains is well characterized from a structural point of view, but a detailed molecular mechanism understanding is nonetheless still lacking. Here, we use advanced computational methods based on all-atom molecular dynamics simulations with explicit solvent to advance our understanding of kinase activation. To elucidate the mechanism of regulation and self-inhibition, we have computed the pathway and the free energy landscapes for the "inactive-to-active" conformational transition of c-Src for different configurations of the SH2 and SH3 domains. Using the isolated c-Src catalytic domain as a baseline for comparison, it is observed that the SH2 and SH3 domains, depending upon their bound orientation, promote either the inactive or active state of the catalytic domain. The regulatory structural information from the SH2-SH3 tandem is allosterically transmitted via the N-terminal linker of the catalytic domain. Analysis of the conformational transition pathways also illustrates the importance of the conserved tryptophan 260 in activating c-Src, and reveals a series of concerted events during the activation process.

  14. Rational design and validation of an anti-protein kinase C active-state specific antibody based on conformational changes

    Science.gov (United States)

    Pena, Darlene Aparecida; Andrade, Victor Piana de; Silva, Gabriela Ávila Fernandes; Neves, José Ivanildo; Oliveira, Paulo Sergio Lopes de; Alves, Maria Julia Manso; Devi, Lakshmi A.; Schechtman, Deborah

    2016-01-01

    Protein kinase C (PKC) plays a regulatory role in key pathways in cancer. However, since phosphorylation is a step for classical PKC (cPKC) maturation and does not correlate with activation, there is a lack of tools to detect active PKC in tissue samples. Here, a structure-based rational approach was used to select a peptide to generate an antibody that distinguishes active from inactive cPKC. A peptide conserved in all cPKCs, C2Cat, was chosen since modeling studies based on a crystal structure of PKCβ showed that it is localized at the interface between the C2 and catalytic domains of cPKCs in an inactive kinase. Anti-C2Cat recognizes active cPKCs at least two-fold better than inactive kinase in ELISA and immunoprecipitation assays, and detects the temporal dynamics of cPKC activation upon receptor or phorbol stimulation. Furthermore, the antibody is able to detect active PKC in human tissue. Higher levels of active cPKC were observed in the more aggressive triple negative breast cancer tumors as compared to the less aggressive estrogen receptor positive tumors. Thus, this antibody represents a reliable, hitherto unavailable and a valuable tool to study PKC activation in cells and tissues. Similar structure-based rational design strategies can be broadly applied to obtain active-state specific antibodies for other signal transduction molecules. PMID:26911897

  15. Rational design and validation of an anti-protein kinase C active-state specific antibody based on conformational changes.

    Science.gov (United States)

    Pena, Darlene Aparecida; Andrade, Victor Piana de; Silva, Gabriela Ávila Fernandes; Neves, José Ivanildo; Oliveira, Paulo Sergio Lopes de; Alves, Maria Julia Manso; Devi, Lakshmi A; Schechtman, Deborah

    2016-02-25

    Protein kinase C (PKC) plays a regulatory role in key pathways in cancer. However, since phosphorylation is a step for classical PKC (cPKC) maturation and does not correlate with activation, there is a lack of tools to detect active PKC in tissue samples. Here, a structure-based rational approach was used to select a peptide to generate an antibody that distinguishes active from inactive cPKC. A peptide conserved in all cPKCs, C2Cat, was chosen since modeling studies based on a crystal structure of PKCβ showed that it is localized at the interface between the C2 and catalytic domains of cPKCs in an inactive kinase. Anti-C2Cat recognizes active cPKCs at least two-fold better than inactive kinase in ELISA and immunoprecipitation assays, and detects the temporal dynamics of cPKC activation upon receptor or phorbol stimulation. Furthermore, the antibody is able to detect active PKC in human tissue. Higher levels of active cPKC were observed in the more aggressive triple negative breast cancer tumors as compared to the less aggressive estrogen receptor positive tumors. Thus, this antibody represents a reliable, hitherto unavailable and a valuable tool to study PKC activation in cells and tissues. Similar structure-based rational design strategies can be broadly applied to obtain active-state specific antibodies for other signal transduction molecules.

  16. Catalytic activity of the mouse guanine nucleotide exchanger mSOS is activated by Fyn tyrosine protein kinase and the T-cell antigen receptor in T cells.

    OpenAIRE

    1996-01-01

    mSOS, a guanine nucleotide exchange factor, is a positive regulator of Ras. Fyn tyrosine protein kinase is a potential mediator in T-cell antigen receptor signal transduction in subsets of T cells. We investigated the functional and physical interaction between mSOS and Fyn in T-cell hybridoma cells. Stimulation of the T-cell antigen receptor induced the activation of guanine nucleotide exchange activity in mSOS immunoprecipitates. Overexpression of Fyn mutants with an activated kinase mutati...

  17. Structural comparison of human mammalian ste20-like kinases.

    Directory of Open Access Journals (Sweden)

    Christopher J Record

    Full Text Available BACKGROUND: The serine/threonine mammalian Ste-20 like kinases (MSTs are key regulators of apoptosis, cellular proliferation as well as polarization. Deregulation of MSTs has been associated with disease progression in prostate and colorectal cancer. The four human MSTs are regulated differently by C-terminal regions flanking the catalytic domains. PRINCIPAL FINDINGS: We have determined the crystal structure of kinase domain of MST4 in complex with an ATP-mimetic inhibitor. This is the first structure of an inactive conformation of a member of the MST kinase family. Comparison with active structures of MST3 and MST1 revealed a dimeric association of MST4 suggesting an activation loop exchanged mechanism of MST4 auto-activation. Together with a homology model of MST2 we provide a comparative analysis of the kinase domains for all four members of the human MST family. SIGNIFICANCE: The comparative analysis identified new structural features in the MST ATP binding pocket and has also defined the mechanism for autophosphorylation. Both structural features may be further explored for inhibitors design. ENHANCED VERSION: This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

  18. The catalytic subunit of human protein kinase CK2 structurally deviates from its maize homologue in complex with the nucleotide competitive inhibitor emodin

    DEFF Research Database (Denmark)

    Raaf, Jennifer; Klopffleisch, Karsten; Issinger, Olaf-Georg;

    2008-01-01

    in the ATP-binding loop, whereas human CK2alpha shows its largest adaptations in the hinge region connecting the two main domains of the protein kinase core. These observations emphasize the importance of local plasticity for ligand binding and demonstrate that two orthologues of an enzyme can behave quite...

  19. Social background, bullying, and physical inactivity

    DEFF Research Database (Denmark)

    Henriksen, P W; Rayce, S B; Melkevik, O;

    2015-01-01

    More children from lower social backgrounds are physically inactive than those from higher ones. We studied whether bullying was a mediating factor between lower social background and physical inactivity. We also examined the combined effect of low social class and exposure to bullying on physical...... inactivity. The Danish sample of the Health Behaviour in School-aged Children (HBSC) study 2006 included 6269 schoolchildren in three age groups: 11-, 13-, and 15-year-olds from a random sample of 80 schools. The students answered the internationally standardized HBSC questionnaire. The applied definition...... leaves 4.0% in the category physically inactive. The sex and age-adjusted OR (95% CI) for physical inactivity was 2.10 (1.39-3.18) among students with low social class and unclassifiable 3.53 (2.26-5.53). Exposure to bullying was associated with physical inactivity, sex and age-adjusted OR = 2.39 (1...

  20. Inactive sites and the evolution of cooperation

    Science.gov (United States)

    da Silva Júnior, E. J.; Wardil, L. L.; da Silva, J. K. L.

    2016-10-01

    Cooperation is often conditioned on environmental factors. Behaviors may be inactive due to external factors, and yet the trait itself may not change. We study the evolution of cooperation with active and inactive sites. In inactive sites cooperators behave as defectors, receiving but not providing benefits. This unintentional mimicry provides local advantage to cooperation, but also prevents the mutual reinforcement provided by clusters of active cooperators. In general, we found that cooperation is enhanced by inactivity. In particular, if most sites are inactive, cooperation survives even if the temptation to defect is very large. Interestingly, in the square lattice with pairwise comparison rule we found that cooperation is enforced by inactive sites only up to a certain limit.

  1. Novel structural and regulatory features of rhoptry secretory kinases in Toxoplasma gondii

    Energy Technology Data Exchange (ETDEWEB)

    Qiu, Wei; Wernimont, Amy; Tang, Keliang; Taylor, Sonya; Lunin, Vladimir; Schapira, Matthieu; Fentress, Sarah; Hui, Raymond; Sibley, L. David; (Toronto); (WU-MED)

    2009-09-29

    Serine/threonine kinases secreted from rhoptry organelles constitute important virulence factors of Toxoplasma gondii. Rhoptry kinases are highly divergent and their structures and regulatory mechanism are hitherto unknown. Here, we report the X-ray crystal structures of two related pseudokinases named ROP2 and ROP8, which differ primarily in their substrate-binding site. ROP kinases contain a typical bilobate kinase fold and a novel N-terminal extension that both stabilizes the N-lobe and provides a unique means of regulation. Although ROP2 and ROP8 were catalytically inactive, they provided a template for homology modelling of the active kinase ROP18, a major virulence determinant of T. gondii. Autophosphorylation of key residues in the N-terminal extension resulted in ROP18 activation, which in turn phosphorylated ROP2 and ROP8. Mutagenesis and mass spectrometry experiments revealed that ROP18 was maximally activated when this phosphorylated N-terminus relieved autoinhibition resulting from extension of aliphatic side chains into the ATP-binding pocket. This novel means of regulation governs ROP kinases implicated in parasite virulence.

  2. Allosteric inhibition of Aurora-A kinase by a synthetic vNAR domain.

    Science.gov (United States)

    Burgess, Selena G; Oleksy, Arkadiusz; Cavazza, Tommaso; Richards, Mark W; Vernos, Isabelle; Matthews, David; Bayliss, Richard

    2016-07-01

    The vast majority of clinically approved protein kinase inhibitors target the ATP-binding pocket directly. Consequently, many inhibitors have broad selectivity profiles and most have significant off-target effects. Allosteric inhibitors are generally more selective, but are difficult to identify because allosteric binding sites are often unknown or poorly characterized. Aurora-A is activated through binding of TPX2 to an allosteric site on the kinase catalytic domain, and this knowledge could be exploited to generate an inhibitor. Here, we generated an allosteric inhibitor of Aurora-A kinase based on a synthetic, vNAR single domain scaffold, vNAR-D01. Biochemical studies and a crystal structure of the Aurora-A/vNAR-D01 complex show that the vNAR domain overlaps with the TPX2 binding site. In contrast with the binding of TPX2, which stabilizes an active conformation of the kinase, binding of the vNAR domain stabilizes an inactive conformation, in which the αC-helix is distorted, the canonical Lys-Glu salt bridge is broken and the regulatory (R-) spine is disrupted by an additional hydrophobic side chain from the activation loop. These studies illustrate how single domain antibodies can be used to characterize the regulatory mechanisms of kinases and provide a rational basis for structure-guided design of allosteric Aurora-A kinase inhibitors.

  3. Silencing expression of the catalytic subunit of DNA-dependent protein kinase by small interfering RNA sensitizes human cells for radiation-induced chromosome damage, cell killing, and mutation

    Science.gov (United States)

    Peng, Yuanlin; Zhang, Qinming; Nagasawa, Hatsumi; Okayasu, Ryuichi; Liber, Howard L.; Bedford, Joel S.

    2002-01-01

    Targeted gene silencing in mammalian cells by RNA interference (RNAi) using small interfering RNAs (siRNAs) was recently described by Elbashir et al. (S. M. Elbashir et al., Nature (Lond.), 411: 494-498, 2001). We have used this methodology in several human cell strains to reduce expression of the Prkdc (DNA-PKcs) gene coding for the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) that is involved in the nonhomologous end joining of DNA double-strand breaks. We have also demonstrated a radiosensitization for several phenotypic endpoints of radiation damage. In low-passage normal human fibroblasts, siRNA knock-down of DNA-PKcs resulted in a reduced capacity for restitution of radiation-induced interphase chromosome breaks as measured by premature chromosome condensation, an increased yield of acentric chromosome fragments at the first postirradiation mitosis, and an increased radiosensitivity for cell killing. For three strains of related human lymphoblasts, DNA-PKcs-targeted siRNA transfection resulted in little or no increase in radiosensitivity with respect to cell killing, a 1.5-fold decrease in induced mutant yield in TK6- and p53-null NH32 cells, but about a 2-fold increase in induced mutant yield in p53-mutant WTK1 cells at both the hypoxanthine quanine phosphoribosyl transferase (hprt) and the thymidine kinase loci.

  4. Inactive DNMT3B Splice Variants Modulate De Novo DNA Methylation

    OpenAIRE

    Gordon, Catherine A.; Hartono, Stella R; Frédéric Chédin

    2013-01-01

    Inactive DNA methyltransferase (DNMT) 3B splice isoforms are associated with changes in DNA methylation, yet the mechanisms by which they act remain largely unknown. Using biochemical and cell culture assays, we show here that the inactive DNMT3B3 and DNMT3B4 isoforms bind to and regulate the activity of catalytically competent DNMT3A or DNMT3B molecules. DNMT3B3 modestly stimulated the de novo methylation activity of DNMT3A and also counteracted the stimulatory effects of DNMT3L, therefore l...

  5. Catalytic roles of lysines (K9, K27, K31) in the N-terminal domain in human adenylate kinase by random site-directed mutagenesis.

    Science.gov (United States)

    Ayabe, T; Park, S K; Takenaka, H; Sumida, M; Uesugi, S; Takenaka, O; Hamada, M

    1996-11-01

    To elucidate lysine residues in the N-terminal domain of human cytosolic adenylate kinase (hAK1, EC 2.7.4.3), random site-directed mutagenesis of K9, K27, and K31 residues was performed, and six mutants were analyzed by steady-state kinetics. K9 residue may play an important role in catalysis by interacting with AMP2-. K27 and K31 residues appear to play a functional role in catalysis by interacting with MgATP2-. In human AK, the epsilon-amino group in the side chain of these lysine residues would be essential for phosphoryl transfer between MgATP2- and AMP2- during transition state.

  6. The structure of arabidopsis thaliana OST1 provides insights into the kinase regulation mechanism in response to osmotic stress

    KAUST Repository

    Yunta, Cristina

    2011-11-01

    SnRK [SNF1 (sucrose non-fermenting-1)-related protein kinase] 2.6 [open stomata 1 (OST1)] is well characterized at molecular and physiological levels to control stomata closure in response to water-deficit stress. OST1 is a member of a family of 10 protein kinases from Arabidopsis thaliana (SnRK2) that integrates abscisic acid (ABA)-dependent and ABA-independent signals to coordinate the cell response to osmotic stress. A subgroup of protein phosphatases type 2C binds OST1 and keeps the kinase dephosphorylated and inactive. Activation of OST1 relies on the ABA-dependent inhibition of the protein phosphatases type 2C and the subsequent self-phosphorylation of the kinase. The OST1 ABA-independent activation depends on a short sequence motif that is conserved among all the members of the SnRK2 family. However, little is known about the molecular mechanism underlying this regulation. The crystallographic structure of OST1 shows that ABA-independent regulation motif stabilizes the conformation of the kinase catalytically essential α C helix, and it provides the basis of the ABA-independent regulation mechanism for the SnRK2 family of protein kinases. © 2011 Elsevier Ltd. All rights reserved.

  7. The inaction approach to gauge theories

    CERN Document Server

    Pivovarov, Grigorii

    2012-01-01

    The inaction approach introduced previously for phi^4 is generalized to gauge theories. It combines the advantages of the effective field theory and causal approaches to quantum fields. Also, it suggests ways to generalizing gauge theories.

  8. Amplified detection of DNA ligase and polynucleotide kinase/phosphatase on the basis of enrichment of catalytic G-quadruplex DNAzyme by rolling circle amplification.

    Science.gov (United States)

    Jiang, Hong-Xin; Kong, De-Ming; Shen, Han-Xi

    2014-05-15

    As two commonly used tool enzymes, DNA ligase and polynucleotide kinase/phosphatase (PNKP) play important roles in DNA metabolism. More and more studies show that regulation of their activity represents promising means for cancer therapy. To detect the activity of DNA ligase with high sensitivity and specificity, a G-quadruplex DNAzyme-based DNA ligase sensor was developed. In this sensor, the use of G-quadruplex DNAzyme eliminated the needs for any labeled oligonucleotide probes, thus making label-free detection possible. The introduction of rolling circle amplification (RCA) reaction could lead to the formation of multimeric G-quadruplexes containing thousands of G-quadruplex units, which can provide highly active hemin-binding sites, thus significantly improving the sensitivity of the sensor. The proposed sensor allowed specific detection of T4 DNA ligase with a detection limit of 0.0019 U/mL. By adding a PNKP-triggered 5'-phosphroylation step of the template DNA, the above sensing strategy could be easily extended to the design of PNKP sensor. The established sensor allowed specific detection of T4 PNKP with a detection limit of 0.0018 U/mL. In addition, these two sensors could also be used for the studies on inhibitors of these two enzymes.

  9. Realtime (31)P NMR Investigation on the Catalytic Behavior of the Enzyme Adenylate kinase in the Matrix of a Switchable Ionic Liquid.

    Science.gov (United States)

    Rogne, Per; Sparrman, Tobias; Anugwom, Ikenna; Mikkola, Jyri-Pekka; Wolf-Watz, Magnus

    2015-11-01

    The integration of highly efficient enzymatic catalysis with the solvation properties of ionic liquids for an environmentally friendly and efficient use of raw materials such as wood requires fundamental knowledge about the influence of relevant ionic liquids on enzymes. Switchable ionic liquids (SIL) are promising candidates for implementation of enzymatic treatments of raw materials. One industrially interesting SIL is constituted by monoethanol amine (MEA) and 1,8-diazabicyclo-[5.4.0]-undec-7-ene (DBU) formed with sulfur dioxide (SO2) as the coupling media (DBU-SO2-MEASIL). It has the ability to solubilize the matrix of lignocellulosic biomass while leaving the cellulose backbone intact. Using a novel (31)P NMR-based real-time assay we show that this SIL is compatible with enzymatic catalysis because a model enzyme, adenylate kinase, retains its activity in up to at least 25 wt % of DBU-SO2-MEASIL. Thus this SIL appears suitable for, for example, enzymatic degradation of hemicellulose.

  10. Overexpression of nuclear protein kinase CK2 α catalytic subunit (CK2α as a poor prognosticator in human colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Kai-Yuan Lin

    Full Text Available BACKGROUND: Colorectal cancer (CRC is one of the most common malignancies but the current therapeutic approaches for advanced CRC are less efficient. Thus, novel therapeutic approaches are badly needed. The purpose of this study is to investigate the involvement of nuclear protein kinase CK2 α subunit (CK2α in tumor progression, and in the prognosis of human CRC. METHODOLOGY/PRINCIPAL FINDINGS: Expression levels of nuclear CK2α were analyzed in 245 colorectal tissues from patients with CRC by immunohistochemistry, quantitative real-time PCR and Western blot. We correlated the expression levels with clinicopathologic parameters and prognosis in human CRC patients. Overexpression of nuclear CK2α was significantly correlated with depth of invasion, nodal status, American Joint Committee on Cancer (AJCC staging, degree of differentiation, and perineural invasion. Patients with high expression levels of nuclear CK2α had a significantly poorer overall survival rate compared with patients with low expression levels of nuclear CK2α. In multi-variate Cox regression analysis, overexpression of nuclear CK2α was proven to be an independent prognostic marker for CRC. In addition, DLD-1 human colon cancer cells were employed as a cellular model to study the role of CK2α on cell growth, and the expression of CK2α in DLD-1 cells was inhibited by using siRNA technology. The data indicated that CK2α-specific siRNA treatment resulted in growth inhibition. CONCLUSIONS/SIGNIFICANCE: Taken together, overexpression of nuclear CK2α can be a useful marker for predicting the outcome of patients with CRC.

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

    Directory of Open Access Journals (Sweden)

    Mutsuki Amano

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

  12. A dynamic model of interactions of Ca2+, calmodulin, and catalytic subunits of Ca2+/calmodulin-dependent protein kinase II.

    Directory of Open Access Journals (Sweden)

    Shirley Pepke

    2010-02-01

    Full Text Available During the acquisition of memories, influx of Ca2+ into the postsynaptic spine through the pores of activated N-methyl-D-aspartate-type glutamate receptors triggers processes that change the strength of excitatory synapses. The pattern of Ca2+influx during the first few seconds of activity is interpreted within the Ca2+-dependent signaling network such that synaptic strength is eventually either potentiated or depressed. Many of the critical signaling enzymes that control synaptic plasticity,including Ca2+/calmodulin-dependent protein kinase II (CaMKII, are regulated by calmodulin, a small protein that can bindup to 4 Ca2+ ions. As a first step toward clarifying how the Ca2+-signaling network decides between potentiation or depression, we have created a kinetic model of the interactions of Ca2+, calmodulin, and CaMKII that represents our best understanding of the dynamics of these interactions under conditions that resemble those in a postsynaptic spine. We constrained parameters of the model from data in the literature, or from our own measurements, and then predicted time courses of activation and autophosphorylation of CaMKII under a variety of conditions. Simulations showed that species of calmodulin with fewer than four bound Ca2+ play a significant role in activation of CaMKII in the physiological regime,supporting the notion that processing of Ca2+ signals in a spine involves competition among target enzymes for binding to unsaturated species of CaM in an environment in which the concentration of Ca2+ is fluctuating rapidly. Indeed, we showed that dependence of activation on the frequency of Ca2+ transients arises from the kinetics of interaction of fluctuating Ca2+with calmodulin/CaMKII complexes. We used parameter sensitivity analysis to identify which parameters will be most beneficial to measure more carefully to improve the accuracy of predictions. This model provides a quantitative base from which to build more complex dynamic

  13. SGX523 is an exquisitely selective, ATP-competitive inhibitor of the MET receptor tyrosine kinase with antitumor activity in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Buchanan, Sean G.; Hendle, Jorg; Lee, Patrick S.; Smith, Christopher R.; Bounaud, Pierre-Yves; Jessen, Katti A.; Tang, Crystal M.; Huser, Nanni H.; Felce, Jeremy D.; Froning, Karen J.; Peterman, Marshall C.; Aubol, Brandon E.; Gessert, Steve F.; Sauder, J. Michael; Schwinn, Kenneth D.; Russell, Marijane; Rooney, Isabelle A.; Adams, Jason; Leon, Barbara C.; Do, Tuan H.; Blaney, Jeff M.; Sprengeler, Paul A.; Thompson, Devon A.; Smyth, Lydia; Pelletier, Laura A.; Atwell, Shane; Holme, Kevin; Wasserman, Stephen R.; Emtage, Spencer; Burley, Stephen K.; Reich, Siegfried H.; (Ventana); (SGX); (Genentech); (Amira)

    2010-01-12

    The MET receptor tyrosine kinase has emerged as an important target for the development of novel cancer therapeutics. Activation of MET by mutation or gene amplification has been linked to kidney, gastric, and lung cancers. In other cancers, such as glioblastoma, autocrine activation of MET has been demonstrated. Several classes of ATP-competitive inhibitor have been described, which inhibit MET but also other kinases. Here, we describe SGX523, a novel, ATP-competitive kinase inhibitor remarkable for its exquisite selectivity for MET. SGX523 potently inhibited MET with an IC{sub 50} of 4 nmol/L and is >1,000-fold selective versus the >200-fold selectivity of other protein kinases tested in biochemical assays. Crystallographic study revealed that SGX523 stabilizes MET in a unique inactive conformation that is inaccessible to other protein kinases, suggesting an explanation for the selectivity. SGX523 inhibited MET-mediated signaling, cell proliferation, and cell migration at nanomolar concentrations but had no effect on signaling dependent on other protein kinases, including the closely related RON, even at micromolar concentrations. SGX523 inhibition of MET in vivo was associated with the dose-dependent inhibition of growth of tumor xenografts derived from human glioblastoma and lung and gastric cancers, confirming the dependence of these tumors on MET catalytic activity. Our results show that SGX523 is the most selective inhibitor of MET catalytic activity described to date and is thus a useful tool to investigate the role of MET kinase in cancer without the confounding effects of promiscuous protein kinase inhibition.

  14. Extracellular-regulated kinase 2 is activated by the enhancement of hinge flexibility.

    Science.gov (United States)

    Sours, Kevin M; Xiao, Yao; Ahn, Natalie G

    2014-05-01

    Protein motions underlie conformational and entropic contributions to enzyme catalysis; however, relatively little is known about the ways in which this occurs. Studies of the mitogen-activated protein kinase ERK2 (extracellular-regulated protein kinase 2) by hydrogen-exchange mass spectrometry suggest that activation enhances backbone flexibility at the linker between N- and C-terminal domains while altering nucleotide binding mode. Here, we address the hypothesis that enhanced backbone flexibility within the hinge region facilitates kinase activation. We show that hinge mutations enhancing flexibility promote changes in the nucleotide binding mode consistent with domain movement, without requiring phosphorylation. They also lead to the activation of monophosphorylated ERK2, a form that is normally inactive. The hinge mutations bypass the need for pTyr but not pThr, suggesting that Tyr phosphorylation controls hinge motions. In agreement, monophosphorylation of pTyr enhances both hinge flexibility and nucleotide binding mode, measured by hydrogen-exchange mass spectrometry. Our findings demonstrate that regulated protein motions underlie kinase activation. Our working model is that constraints to domain movement in ERK2 are overcome by phosphorylation at pTyr, which increases hinge dynamics to promote the active conformation of the catalytic site.

  15. Structural basis for basal activity and autoactivation of abscisic acid (ABA) signaling SnRK2 kinases

    Energy Technology Data Exchange (ETDEWEB)

    Ng, Ley-Moy; Soon, Fen-Fen; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Suino-Powell, Kelly M.; Chalmers, Michael J.; Li, Jun; Yong, Eu-Leong; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric (Van Andel); (Scripps); (Purdue); (NU Singapore)

    2014-10-02

    Abscisic acid (ABA) is an essential hormone that controls plant growth, development, and responses to abiotic stresses. Central for ABA signaling is the ABA-mediated autoactivation of three monomeric Snf1-related kinases (SnRK2.2, -2.3, and -2.6). In the absence of ABA, SnRK2s are kept in an inactive state by forming physical complexes with type 2C protein phosphatases (PP2Cs). Upon relief of this inhibition, SnRK2 kinases can autoactivate through unknown mechanisms. Here, we report the crystal structures of full-length Arabidopsis thaliana SnRK2.3 and SnRK2.6 at 1.9- and 2.3-{angstrom} resolution, respectively. The structures, in combination with biochemical studies, reveal a two-step mechanism of intramolecular kinase activation that resembles the intermolecular activation of cyclin-dependent kinases. First, release of inhibition by PP2C allows the SnRK2s to become partially active because of an intramolecular stabilization of the catalytic domain by a conserved helix in the kinase regulatory domain. This stabilization enables SnRK2s to gain full activity by activation loop autophosphorylation. Autophosphorylation is more efficient in SnRK2.6, which has higher stability than SnRK2.3 and has well-structured activation loop phosphate acceptor sites that are positioned next to the catalytic site. Together, these data provide a structural framework that links ABA-mediated release of PP2C inhibition to activation of SnRK2 kinases.

  16. Short-term regulation of tyrosine hydroxylase in tonically-active and in tonically-inactive dopamine neurons: effects of haloperidol and protein phosphorylation.

    Science.gov (United States)

    Iuvone, P M

    1983-09-26

    Dopamine (DA)-containing neurons of retina were employed as an experimental model for studying the short-term regulation of tyrosine hydroxylase (TH) in tonically-active and tonically-inactive neurons. These DA-containing neurons are trans-synaptically activated by light. Two mechanisms have been observed in this system for regulation of TH activity. A short-term activation of TH that is characterized by a decreased apparent Km for pteridine cofactors occurs in response to rapid increases of neuronal activity. A second mechanism occurs in response to prolonged, tonic changes of neuronal activity and is characterized by changes of Vmax. Both the Km changes and Vmax changes represent changes of specific activity of TH rather than enzyme induction. To determine the effects of short-term increases of neuronal activity on TH in tonically-active and tonically-inactive neurons, the effects of acute administration of haloperidol were examined in rats that were continuously light-exposed or light-deprived for 4 days. Haloperidol increased TH activity in both light-exposed and light-deprived retinas. The drug elicited the same percent stimulation in both experimental conditions. However, because the basal activity of TH was higher in the light-exposed than the light-deprived retinas, the absolute increase of TH specific activity was greater in the light-exposed samples. The effect of protein phosphorylation on TH activity in extracts of chronically light-exposed or light-deprived retinas was also examined to determine if the differences in the response to haloperidol might be due to a difference in the amount of TH available for short-term activation. Phosphorylation by endogenous cyclic AMP-dependent protein kinase (APK) or by purified catalytic subunit of APK resulted in larger increases of TH specific activity in extracts of light-exposed retinas than in those of light-deprived retinas. As was observed for haloperidol-induced activation, the percent stimulation elicited

  17. Properties of inactive Photosystem II centers.

    Science.gov (United States)

    Lavergne, J; Leci, E

    1993-03-01

    A fraction (usually in the range of 10-25%) of PS II centers is unable to transfer electrons from the primary quinone acceptor QA to the secondary acceptor QB. These centers are inactive with respect to O2 evolution since their reopening after photochemical charge separation to the S2OA (-) state involves predominantly a back reaction to S1QA in the few seconds time range (slower phases are also occurring). Several properties of these centers are analyzed by fluorescence and absorption change experiments. The initial rise phase Fo-Fpl of fluorescence induction under weak illumination reflects both the closure of inactive centers and the modulation of the fluorescence yield by the S-states of the oxygen-evolving system: We estimate typical relative amplitudes of these contributions as, respectively, 65 and 35% of the Fo-Fpl amplitude. The half-rise time of this phase is significantly shorter than for the fluorescence induction in the presence of DCMU (in which all centers are involved). This finding is shown to be consistent with inactive centers sharing the same light-harvesting antenna as normal centers, a view which is also supported by comparing the dependence of the fluorescence yield on the amount of closed active or inactive centers estimated through absorption changes. It is argued that the exponential kinetics of the Fo-Fpl phase does not indicate absence of excitation energy transfer between the antennas of inactive and active centers. We show that the acceptor dichlorobenzoquinone does not restore electron transfer in inactive centers, in disagreement with previous suggestions. We confirm, however, the enhancement of steady-state electron flow caused by this quinone and suggest that it acts by relieving a blocking step involved in the reoxidation of a fraction of the plastoquinone pool. Part of the discrepancies between the present results and those from previous literature may arise from the confusion of inactive centers characterized on a single

  18. Detrimental effects of physical inactivity on neurogenesis

    Directory of Open Access Journals (Sweden)

    Trenton Lippert

    2016-01-01

    Full Text Available Patients diagnosed with neurological disorders exhibit a variety of physical and psychiatric symptoms, including muscle atrophy, general immobility, and depression. Patients who participate in physical rehabilitation at times show unexpected clinical improvement, which includes diminished depression and other stress-related behaviors. Regenerative medicine has advanced two major stem cell-based therapies for central nervous system (CNS disorders, transplantation of exogenous stem cells, and enhancing the endogenous neurogenesis. The latter therapy utilizes a natural method of re-innervating the injured brain, which may mend neurological impairments. In this study, we examine how inactivity-induced atrophy, using the hindlimb suspension model, alters neurogenesis in rats. The hypothesis is that inactivity inhibits neurogenesis by decreasing circulation growth or trophic factors, such as vascular endothelial growth or neurotrophic factors. The restriction modifies neurogenesis and stem cell differentiation in the CNS, the stem cell microenvironment is examined by the trophic and growth factors, including stress-related proteins. Despite growing evidence revealing the benefits of "increased" exercise on neurogenesis, the opposing theory involving "physical inactivity," which simulates pathological states, continues to be neglected. This novel theory will allow us to explore the effects on neurogenesis by an intransigent stem cell microenvironment likely generated by inactivity. 5-bromo-2-deoxyuridine labeling of proliferative cells, biochemical assays of serum, cerebrospinal fluid, and brain levels of trophic factors, growth factors, and stress-related proteins are suggested identifiers of neurogenesis, while evaluation of spontaneous movements will give insight into the psychomotor effects of inactivity. Investigations devised to show how in vivo stimulation, or lack thereof, affects the stem cell microenvironment are necessary to establish

  19. A Novel Mode of Protein Kinase Inhibition Exploiting Hydrophobic Motifs of Autoinhibited Kinases

    Energy Technology Data Exchange (ETDEWEB)

    S Eathiraj; R Palma; M Hirschi; E Volckova; E Nakuci; J Castro; C Chen; T Chan; D France; M Ashwell

    2011-12-31

    Protein kinase inhibitors with enhanced selectivity can be designed by optimizing binding interactions with less conserved inactive conformations because such inhibitors will be less likely to compete with ATP for binding and therefore may be less impacted by high intracellular concentrations of ATP. Analysis of the ATP-binding cleft in a number of inactive protein kinases, particularly in the autoinhibited conformation, led to the identification of a previously undisclosed non-polar region in this cleft. This ATP-incompatible hydrophobic region is distinct from the previously characterized hydrophobic allosteric back pocket, as well as the main pocket. Generalized hypothetical models of inactive kinases were constructed and, for the work described here, we selected the fibroblast growth factor receptor (FGFR) tyrosine kinase family as a case study. Initial optimization of a FGFR2 inhibitor identified from a library of commercial compounds was guided using structural information from the model. We describe the inhibitory characteristics of this compound in biophysical, biochemical, and cell-based assays, and have characterized the binding mode using x-ray crystallographic studies. The results demonstrate, as expected, that these inhibitors prevent activation of the autoinhibited conformation, retain full inhibitory potency in the presence of physiological concentrations of ATP, and have favorable inhibitory activity in cancer cells. Given the widespread regulation of kinases by autoinhibitory mechanisms, the approach described herein provides a new paradigm for the discovery of inhibitors by targeting inactive conformations of protein kinases.

  20. Inactive DNMT3B splice variants modulate de novo DNA methylation.

    Science.gov (United States)

    Gordon, Catherine A; Hartono, Stella R; Chédin, Frédéric

    2013-01-01

    Inactive DNA methyltransferase (DNMT) 3B splice isoforms are associated with changes in DNA methylation, yet the mechanisms by which they act remain largely unknown. Using biochemical and cell culture assays, we show here that the inactive DNMT3B3 and DNMT3B4 isoforms bind to and regulate the activity of catalytically competent DNMT3A or DNMT3B molecules. DNMT3B3 modestly stimulated the de novo methylation activity of DNMT3A and also counteracted the stimulatory effects of DNMT3L, therefore leading to subtle and contrasting effects on activity. DNMT3B4, by contrast, significantly inhibited de novo DNA methylation by active DNMT3 molecules, most likely due to its ability to reduce the DNA binding affinity of co-complexes, thereby sequestering them away from their substrate. Immunocytochemistry experiments revealed that in addition to their effects on the intrinsic catalytic function of active DNMT3 enzymes, DNMT3B3 and DNMT34 drive distinct types of chromatin compaction and patterns of histone 3 lysine 9 tri-methylation (H3K9me3) deposition. Our findings suggest that regulation of active DNMT3 members through the formation of co-complexes with inactive DNMT3 variants is a general mechanism by which DNMT3 variants function. This may account for some of the changes in DNA methylation patterns observed during development and disease.

  1. Inactive DNMT3B splice variants modulate de novo DNA methylation.

    Directory of Open Access Journals (Sweden)

    Catherine A Gordon

    Full Text Available Inactive DNA methyltransferase (DNMT 3B splice isoforms are associated with changes in DNA methylation, yet the mechanisms by which they act remain largely unknown. Using biochemical and cell culture assays, we show here that the inactive DNMT3B3 and DNMT3B4 isoforms bind to and regulate the activity of catalytically competent DNMT3A or DNMT3B molecules. DNMT3B3 modestly stimulated the de novo methylation activity of DNMT3A and also counteracted the stimulatory effects of DNMT3L, therefore leading to subtle and contrasting effects on activity. DNMT3B4, by contrast, significantly inhibited de novo DNA methylation by active DNMT3 molecules, most likely due to its ability to reduce the DNA binding affinity of co-complexes, thereby sequestering them away from their substrate. Immunocytochemistry experiments revealed that in addition to their effects on the intrinsic catalytic function of active DNMT3 enzymes, DNMT3B3 and DNMT34 drive distinct types of chromatin compaction and patterns of histone 3 lysine 9 tri-methylation (H3K9me3 deposition. Our findings suggest that regulation of active DNMT3 members through the formation of co-complexes with inactive DNMT3 variants is a general mechanism by which DNMT3 variants function. This may account for some of the changes in DNA methylation patterns observed during development and disease.

  2. TLR and TNF-R1 activation of the MKK3/MKK6-p38α axis in macrophages is mediated by TPL-2 kinase.

    Science.gov (United States)

    Pattison, Michael J; Mitchell, Olivia; Flynn, Helen R; Chen, Chao-Sheng; Yang, Huei-Ting; Ben-Addi, Hakem; Boeing, Stefan; Snijders, Ambrosius P; Ley, Steven C

    2016-09-15

    Previous studies suggested that Toll-like receptor (TLR) stimulation of the p38α MAP kinase (MAPK) is mediated by transforming growth factor-β-activated kinase 1 (TAK1) activation of MAPK kinases, MKK3, MKK4 and MKK6. We used quantitative mass spectrometry to monitor tumour progression locus 2 (TPL-2)-dependent protein phosphorylation following TLR4 stimulation with lipopolysaccharide, comparing macrophages from wild-type mice and Map3k8(D270A/D270A) mice expressing catalytically inactive TPL-2 (MAP3K8). In addition to the established TPL-2 substrates MKK1/2, TPL-2 kinase activity was required to phosphorylate the activation loops of MKK3/6, but not of MKK4. MKK3/6 activation required IκB kinase (IKK) phosphorylation of the TPL-2 binding partner nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB1) p105, similar to MKK1/2 activation. Tumour necrosis factor (TNF) stimulation of MKK3/6 phosphorylation was similarly dependent on TPL-2 catalytic activity and IKK phosphorylation of NF-κB1 p105. Owing to redundancy of MKK3/6 with MKK4, Map3k8(D270A) mutation only fractionally decreased lipopolysaccharide activation of p38α. TNF activation of p38α, which is mediated predominantly via MKK3/6, was substantially reduced. TPL-2 catalytic activity was also required for MKK3/6 and p38α activation following macrophage stimulation with Mycobacterium tuberculosis and Listeria monocytogenes Our experiments demonstrate that the IKK/NF-κB1 p105/TPL-2 signalling pathway, downstream of TAK1, regulates MKK3/6 and p38α activation in macrophages in inflammation.

  3. Exploring human inactivity in computer power consumption

    Science.gov (United States)

    Candrawati, Ria; Hashim, Nor Laily Binti

    2016-08-01

    Managing computer power consumption has become an important challenge in computer society and this is consistent with a trend where a computer system is more important to modern life together with a request for increased computing power and functions continuously. Unfortunately, previous approaches are still inadequately designed to handle the power consumption problem due to unpredictable workload of a system caused by unpredictable human behaviors. This is happens due to lack of knowledge in a software system and the software self-adaptation is one approach in dealing with this source of uncertainty. Human inactivity is handled by adapting the behavioral changes of the users. This paper observes human inactivity in the computer usage and finds that computer power usage can be reduced if the idle period can be intelligently sensed from the user activities. This study introduces Control, Learn and Knowledge model that adapts the Monitor, Analyze, Planning, Execute control loop integrates with Q Learning algorithm to learn human inactivity period to minimize the computer power consumption. An experiment to evaluate this model was conducted using three case studies with same activities. The result show that the proposed model obtained those 5 out of 12 activities shows the power decreasing compared to others.

  4. 40 CFR 256.25 - Recommendation for inactive facilities.

    Science.gov (United States)

    2010-07-01

    ... 40 Protection of Environment 24 2010-07-01 2010-07-01 false Recommendation for inactive facilities. 256.25 Section 256.25 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) SOLID... Disposal Programs § 256.25 Recommendation for inactive facilities. Inactive facilities that continue...

  5. Redox potential tuning by redox-inactive cations in nature's water oxidizing catalyst and synthetic analogues.

    Science.gov (United States)

    Krewald, Vera; Neese, Frank; Pantazis, Dimitrios A

    2016-04-28

    The redox potential of synthetic oligonuclear transition metal complexes has been shown to correlate with the Lewis acidity of a redox-inactive cation connected to the redox-active transition metals of the cluster via oxo or hydroxo bridges. Such heterometallic clusters are important cofactors in many metalloenzymes, where it is speculated that the redox-inactive constituent ion of the cluster serves to optimize its redox potential for electron transfer or catalysis. A principal example is the oxygen-evolving complex in photosystem II of natural photosynthesis, a Mn4CaO5 cofactor that oxidizes water into dioxygen, protons and electrons. Calcium is critical for catalytic function, but its precise role is not yet established. In analogy to synthetic complexes it has been suggested that Ca(2+) fine-tunes the redox potential of the manganese cluster. Here we evaluate this hypothesis by computing the relative redox potentials of substituted derivatives of the oxygen-evolving complex with the cations Sr(2+), Gd(3+), Cd(2+), Zn(2+), Mg(2+), Sc(3+), Na(+) and Y(3+) for two sequential transitions of its catalytic cycle. The theoretical approach is validated with a series of experimentally well-characterized Mn3AO4 cubane complexes that are structural mimics of the enzymatic cluster. Our results reproduce perfectly the experimentally observed correlation between the redox potential and the Lewis acidities of redox-inactive cations for the synthetic complexes. However, it is conclusively demonstrated that this correlation does not hold for the oxygen evolving complex. In the enzyme the redox potential of the cluster only responds to the charge of the redox-inactive cations and remains otherwise insensitive to their precise identity, precluding redox-tuning of the metal cluster as a primary role for Ca(2+) in biological water oxidation.

  6. A multiscale modeling approach to investigate molecular mechanisms of pseudokinase activation and drug resistance in the HER3/ErbB3 receptor tyrosine kinase signaling network.

    Science.gov (United States)

    Telesco, Shannon E; Shih, Andrew J; Jia, Fei; Radhakrishnan, Ravi

    2011-06-01

    Multiscale modeling provides a powerful and quantitative platform for investigating the complexity inherent in intracellular signaling pathways and rationalizing the effects of molecular perturbations on downstream signaling events and ultimately, on the cell phenotype. Here we describe the application of a multiscale modeling scheme to the HER3/ErbB3 receptor tyrosine kinase (RTK) signaling network, which regulates critical cellular processes including proliferation, migration and differentiation. The HER3 kinase is a topic of current interest and investigation, as it has been implicated in mechanisms of resistance to tyrosine kinase inhibition (TKI) of EGFR and HER2 in the treatment of many human malignancies. Moreover, the commonly regarded status of HER3 as a catalytically inactive 'pseudokinase' has recently been challenged by our previous study, which demonstrated robust residual kinase activity for HER3. Through our multiscale model, we investigate the most significant molecular interactions that contribute to potential mechanisms of HER3 activity and the physiological relevance of this activity to mechanisms of drug resistance in an ErbB-driven tumor cell in silico. The results of our molecular-scale simulations support the characterization of HER3 as a weakly active kinase that, in contrast to its fully-active ErbB family members, depends upon a unique hydrophobic interface to coordinate the alignment of specific catalytic residues required for its activity. Translating our molecular simulation results of the uniquely active behavior of the HER3 kinase into a physiologically relevant environment, our HER3 signaling model demonstrates that even a weak level of HER3 activity may be sufficient to induce AKT signaling and TKI resistance in the context of an ErbB signaling-dependent tumor cell, and therefore therapeutic targeting of HER3 may represent a superior treatment strategy for specific ErbB-driven cancers.

  7. Identification of inactivity behavior in smart home.

    Science.gov (United States)

    Poujaud, J; Noury, N; Lundy, J-E

    2008-01-01

    To help elderly people live independently at home, the TIMC-IMAG laboratory developed Health Smart Homes called 'HIS'. These smart Homes are composed of several sensors to monitor the activities of daily living of the patients. Volunteers have accepted to be monitored during 2 years in their own flats. During one year, we carried out our survey on one elderly patient. Thanks to this experimentation, we will access to relevant information like physiological, environmental and activity. This paper focuses on daily living activity. We will introduce an original data splitting method based on the relationship between the frame of time and the location in the flat. Moreover we will present two different methods to determine a threshold of critical inactivity and eventually we will discuss their possible utilities.

  8. The secret life of kinases: functions beyond catalysis.

    LENUS (Irish Health Repository)

    Rauch, Jens

    2011-10-28

    Abstract Protein phosphorylation participates in the regulation of all fundamental biological processes, and protein kinases have been intensively studied. However, while the focus was on catalytic activities, accumulating evidence suggests that non-catalytic properties of protein kinases are essential, and in some cases even sufficient for their functions. These non-catalytic functions include the scaffolding of protein complexes, the competition for protein interactions, allosteric effects on other enzymes, subcellular targeting, and DNA binding. This rich repertoire often is used to coordinate phosphorylation events and enhance the specificity of substrate phosphorylation, but also can adopt functions that do not rely on kinase activity. Here, we discuss such kinase independent functions of protein and lipid kinases focussing on kinases that play a role in the regulation of cell proliferation, differentiation, apoptosis, and motility.

  9. The secret life of kinases: functions beyond catalysis

    Directory of Open Access Journals (Sweden)

    Romano David

    2011-10-01

    Full Text Available Abstract Protein phosphorylation participates in the regulation of all fundamental biological processes, and protein kinases have been intensively studied. However, while the focus was on catalytic activities, accumulating evidence suggests that non-catalytic properties of protein kinases are essential, and in some cases even sufficient for their functions. These non-catalytic functions include the scaffolding of protein complexes, the competition for protein interactions, allosteric effects on other enzymes, subcellular targeting, and DNA binding. This rich repertoire often is used to coordinate phosphorylation events and enhance the specificity of substrate phosphorylation, but also can adopt functions that do not rely on kinase activity. Here, we discuss such kinase independent functions of protein and lipid kinases focussing on kinases that play a role in the regulation of cell proliferation, differentiation, apoptosis, and motility.

  10. ERK kinases modulate the activation of PI3 kinase related kinases (PIKKs) in DNA damage response.

    Science.gov (United States)

    Lin, Xiaozeng; Yan, Judy; Tang, Damu

    2013-12-01

    DNA damage response (DDR) is the critical surveillance mechanism in maintaining genome integrity. The mechanism activates checkpoints to prevent cell cycle progression in the presence of DNA lesions, and mediates lesion repair. DDR is coordinated by three apical PI3 kinase related kinases (PIKKs), including ataxia-telangiectasia mutated (ATM), ATM- and Rad3-related (ATR), and DNA-PKcs (the catalytic subunit of the DNA dependent protein kinase). These kinases are activated in response to specific DNA damage or lesions, resulting in checkpoint activation and DNA lesion repair. While it is clear that the pathways of ATM, ATR, and DNA-PK are the core components of DDR, there is accumulating evidence revealing the involvement of other cellular pathways in regulating DDR; this is in line with the concept that in addition to being a nuclear event DDR is also a cellular process. One of these pathways is the extracellular signal-regulated kinase (ERK) MAPK (mitogen-activated protein kinase) pathway. ERK is a converging point of multiple signal transduction pathways involved in cell proliferation, differentiation, and apoptosis. Adding to this list of pathways is the recent development of ERK in DDR. The ERK kinases (ERK1 and ERK2) contribute to the proper execution of DDR in terms of checkpoint activation and the repair of DNA lesions. This review summarizes the contributions of ERK to DDR with emphasis on the relationship of ERK kinases with the activation of ATM, ATR, and DNA-PKcs.

  11. Gene targeting of CK2 catalytic subunits

    Science.gov (United States)

    Lou, David Y.; Toselli, Paul; Landesman-Bollag, Esther; Dominguez, Isabel

    2013-01-01

    Protein kinase CK2 is a highly conserved and ubiquitous serine–threonine kinase. It is a tetrameric enzyme that is made up of two regulatory CK2β subunits and two catalytic subunits, either CK2α/CK2α, CK2α/ CK2α′, or CK2α′/CK2α′. Although the two catalytic subunits diverge in their C termini, their enzymatic activities are similar. To identify the specific function of the two catalytic subunits in development, we have deleted them individually from the mouse genome by homologous recombination. We have previously reported that CK2α′is essential for male germ cell development, and we now demonstrate that CK2α has an essential role in embryogenesis, as mice lacking CK2α die in mid-embryogenesis, with cardiac and neural tube defects. PMID:18594950

  12. Gene targeting of CK2 catalytic subunits.

    Science.gov (United States)

    Seldin, David C; Lou, David Y; Toselli, Paul; Landesman-Bollag, Esther; Dominguez, Isabel

    2008-09-01

    Protein kinase CK2 is a highly conserved and ubiquitous serine-threonine kinase. It is a tetrameric enzyme that is made up of two regulatory CK2beta subunits and two catalytic subunits, either CK2alpha/CK2alpha, CK2alpha/CK2alpha', or CK2alpha'/CK2alpha'. Although the two catalytic subunits diverge in their C termini, their enzymatic activities are similar. To identify the specific function of the two catalytic subunits in development, we have deleted them individually from the mouse genome by homologous recombination. We have previously reported that CK2alpha' is essential for male germ cell development, and we now demonstrate that CK2alpha has an essential role in embryogenesis, as mice lacking CK2alpha die in mid-embryogenesis, with cardiac and neural tube defects.

  13. Molecular Determinants Underlying Binding Specificities of the ABL Kinase Inhibitors: Combining Alanine Scanning of Binding Hot Spots with Network Analysis of Residue Interactions and Coevolution.

    Directory of Open Access Journals (Sweden)

    Amanda Tse

    Full Text Available Quantifying binding specificity and drug resistance of protein kinase inhibitors is of fundamental importance and remains highly challenging due to complex interplay of structural and thermodynamic factors. In this work, molecular simulations and computational alanine scanning are combined with the network-based approaches to characterize molecular determinants underlying binding specificities of the ABL kinase inhibitors. The proposed theoretical framework unveiled a relationship between ligand binding and inhibitor-mediated changes in the residue interaction networks. By using topological parameters, we have described the organization of the residue interaction networks and networks of coevolving residues in the ABL kinase structures. This analysis has shown that functionally critical regulatory residues can simultaneously embody strong coevolutionary signal and high network centrality with a propensity to be energetic hot spots for drug binding. We have found that selective (Nilotinib and promiscuous (Bosutinib, Dasatinib kinase inhibitors can use their energetic hot spots to differentially modulate stability of the residue interaction networks, thus inhibiting or promoting conformational equilibrium between inactive and active states. According to our results, Nilotinib binding may induce a significant network-bridging effect and enhance centrality of the hot spot residues that stabilize structural environment favored by the specific kinase form. In contrast, Bosutinib and Dasatinib can incur modest changes in the residue interaction network in which ligand binding is primarily coupled only with the identity of the gate-keeper residue. These factors may promote structural adaptability of the active kinase states in binding with these promiscuous inhibitors. Our results have related ligand-induced changes in the residue interaction networks with drug resistance effects, showing that network robustness may be compromised by targeted mutations

  14. Characterization of inactive renin from human kidney and plasma. Evidence of a renal source of circulating inactive renin.

    OpenAIRE

    Hsueh, W A; Carlson, E J; Dzau, V J

    1983-01-01

    An inactive form of renin has been isolated from human plasma. It has been suggested that this may represent renin precursor secreted from the kidney. However, early studies failed to isolate inactive renin from human renal tissue. In this investigation, rapid processing of human kidney cortex at temperatures below 4 degrees C in the presence of protease inhibitors followed by cibacron-blue affinity chromatography allowed us to extract a totally inactive form of renal renin. Furthermore, we f...

  15. Effects of Physical (Inactivity on Platelet Function

    Directory of Open Access Journals (Sweden)

    Stefan Heber

    2015-01-01

    Full Text Available As platelet activation is closely related to the liberation of growth factors and inflammatory mediators, platelets play a central role in the development of CVD. Virtually all cardiovascular risk factors favor platelet hyperreactivity and, accordingly, also physical (inactivity affects platelet function. Within this paper, we will summarize and discuss the current knowledge on the impact of acute and habitual exercise on platelet function. Although there are apparent discrepancies regarding the reported effects of acute, strenuous exercise on platelet activation, a deeper analysis of the available literature reveals that the applied exercise intensity and the subjects’ cardiorespiratory fitness represent critical determinants for the observed effects. Consideration of these factors leads to the summary that (i acute, strenuous exercise can lead to platelet activation, (ii regular physical activity and/or physical fitness diminish or prevent platelet activation in response to acute exercise, and (iii habitual physical activity and/or physical fitness also favorably modulate platelet function at physical rest. Notably, these effects of exercise on platelet function show obvious similarities to the well-recognized relation between exercise and the risk for cardiovascular events where vigorous exercise transiently increases the risk for myocardial infarction and a physically active lifestyle dramatically reduces cardiovascular mortality.

  16. Physical inactivity, depression, and risk of cardiovascular mortality

    NARCIS (Netherlands)

    Kamphuis, M.H.; Geerlings, M.I.; Tijhuis, M.A.R.; Giampaoli, S.; Nissinen, A.; Grobbee, D.E.; Kromhout, D.

    2007-01-01

    Purpose: Studies indicate that depression may increase risk of cardiovascular disease (CVD) in addition to classical risk factors. One of the hypotheses to explain this relation is that depressed subjects become physically inactive. We set out to determine the role of physical inactivity in the rela

  17. Physical inactivity and arterial stiffness in COPD

    Directory of Open Access Journals (Sweden)

    Sievi NA

    2015-09-01

    Full Text Available Noriane A Sievi,1 Daniel Franzen,1 Malcolm Kohler,1,2 Christian F Clarenbach1 1Division of Pulmonology, University Hospital of Zurich, 2Zurich Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland Background: Arterial stiffness is an important predictor of cardiovascular risk besides classic cardiovascular risk factors. Previous studies showed that arterial stiffness is increased in patients with COPD compared to healthy controls and exercise training may reduce arterial stiffness. Since physical inactivity is frequently observed in patients with COPD and exercise training may improve arterial stiffness, we hypothesized that low daily physical activity may be associated with increased arterial stiffness. Methods: In 123 patients with COPD (72% men; mean [standard deviation] age: 62 [7.5] years; median [quartile] forced expiratory volume in 1 second 35 [27/65] %predicted, arterial stiffness was assessed by augmentation index (AI. Daily physical activity level (PAL was measured by an activity monitor (SenseWear Pro™ >1 week. The association between AI and PAL was investigated by univariate and multivariate regression analysis, taking into account disease-specific characteristics and comorbidities. Results: Patients suffered from moderate (35%, severe (32%, and very severe (33% COPD, and 22% were active smokers. Median (quartile PAL was 1.4 (1.3/1.5 and mean (standard deviation AI 26% (9.2%. PAL showed a negative association with AI (B=-9.32, P=0.017 independent of age, sex, blood pressure, and airflow limitation. Conclusion: In COPD patients, a higher PAL seems to favorably influence arterial stiffness and therefore may reduce cardiovascular risk. Clinical Trial Registration: www.ClinicalTrials.gov, NCT01527773 Keywords: activity monitor, airflow limitation, COPD, physical activity level

  18. Structure of protein kinase CK2: dimerization of the human beta-subunit

    DEFF Research Database (Denmark)

    Boldyreff, B; Mietens, U; Issinger, O G

    1996-01-01

    Protein kinase CK2 has been shown to be elevated in all so far investigated solid tumors and its catalytic subunit has been shown to serve as an oncogene product. CK2 is a heterotetrameric serine-threonine kinase composed of two catalytic (alpha and/or alpha') and two regulatory beta...

  19. Recombinant, catalytically inactive juvenile hormone esterase enhances efficacy of baculovirus insecticides

    NARCIS (Netherlands)

    Meer, van M.M.M.; Bonning, B.C.; Ward, V.K.; Vlak, J.M.; Hammock, B.D.

    2000-01-01

    The insecticidal efficacy of baculoviruses can be enhanced by engineering the viral genome to express proteins that disrupt the physiology of the host insect. Here we describe the development of a genetically engineered Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) which expresses

  20. Identification of Novel Small Molecule Inhibitors of Oncogenic RET Kinase

    OpenAIRE

    Marialuisa Moccia; Qingsong Liu; Teresa Guida; Giorgia Federico; Annalisa Brescia; Zheng Zhao; Hwan Geun Choi; Xianming Deng; Li Tan; Jinhua Wang; Marc Billaud; Gray, Nathanael S.; Francesca Carlomagno; Massimo Santoro

    2015-01-01

    Oncogenic mutation of the RET receptor tyrosine kinase is observed in several human malignancies. Here, we describe three novel type II RET tyrosine kinase inhibitors (TKI), ALW-II-41-27, XMD15-44 and HG-6-63-01, that inhibit the cellular activity of oncogenic RET mutants at two digit nanomolar concentration. These three compounds shared a 3-trifluoromethyl-4-methylpiperazinephenyl pharmacophore that stabilizes the 'DFG-out' inactive conformation of RET activation loop. They blocked RET-media...

  1. Anaplastic lymphoma kinase (ALK) inhibitors in the treatment of ALK-driven lung cancers.

    Science.gov (United States)

    Roskoski, Robert

    2017-03-01

    Anaplastic lymphoma kinase is expressed in two-thirds of the anaplastic large-cell lymphomas as an NPM-ALK fusion protein. Physiological ALK is a receptor protein-tyrosine kinase within the insulin receptor superfamily of proteins that participates in nervous system development. The EML4-ALK fusion protein and four other ALK-fusion proteins play a fundamental role in the development in about 5% of non-small cell lung cancers. The amino-terminal portions of the ALK fusion proteins result in dimerization and subsequent activation of the ALK protein kinase domain that plays a key role in the pathogenesis of various tumors. Downstream signaling from the ALK fusion protein leads to the activation of the Ras/Raf/MEK/ERK1/2 cell proliferation module and the JAK/STAT cell survival pathways. Moreover, nearly two dozen ALK activating mutations are involved in the pathogenesis of childhood neuroblastomas. The occurrence of oncogenic ALK-fusion proteins, particularly in non-small cell lung cancer, has fostered considerable interest in the development of ALK inhibitors. Crizotinib was the first such inhibitor approved by the US Food and Drug Administration for the treatment of ALK-positive non-small cell lung cancer in 2011. The median time for the emergence of crizotinib drug resistance is 10.5 months after the initiation of therapy. Such resistance prompted the development of second-generation drugs including ceritinib and alectinib, which are approved for the treatment of non-small cell lung cancer. Unlike the single gatekeeper mutation that occurs in drug-resistant epidermal growth factor receptor in lung cancer, nearly a dozen different mutations in the catalytic domain of ALK fusion proteins have been discovered that result in crizotinib resistance. Crizotinib, ceritinib, and alectinib form a complex within the front cleft between the small and large lobes of an inactive ALK protein-kinase domain with a compact activation segment. These drugs are classified as type I½ B

  2. Expression, purification and functional characterization of IkappaB kinase-2 (IKK-2) mutants.

    Science.gov (United States)

    Mathialagan, Sumathy; Poda, Gennadiy I; Kurumbail, Ravi G; Selness, Shaun R; Hall, Troii; Reitz, Beverly A; Weinberg, Robin A; Kishore, Nandini; Mbalaviele, Gabriel

    2010-08-01

    NF-kappaB signaling plays a pivotal role in a variety of pathological conditions. Because of its central role in the overall NF-kappaB regulation, IKK-2 is a viable target for drug discovery. In order to enable structure-based design of IKK-2 inhibitors, we carried out a rational generation of IKK-2 mutants based on induced-fit docking of a selective IKK-2 inhibitor, PHA-408, into the homology model of IKK-2. One mutant we have characterized is a catalytically inactive form of IKK-2, D145A IKK-2, wherein the catalytic aspartic acid, D145 was replaced with alanine. Unlike the WT enzyme, D145A IKK-2 is devoid of kinase activity despite its ability to bind ATP with high affinity and is not phosphorylated at the T loop. In addition, this mutant binds a diverse collection of inhibitors with comparable binding affinities to WT IKK-2. Another interesting mutant we have characterized is F26A IKK-2 (F26 is an aromatic residue located at the very tip of the Gly-rich loop). Pre-incubation of F26A IKK-2 with PHA-408 revealed the role of F26 in the time-dependent binding of this inhibitor. Thus, functional characterization of these mutants provides the first evidence showing the role of a Gly-rich loop residue of a kinase in binding kinetics. These two mutants along with others that we have identified could be used to validate homology models and probe the interactions of IKK-2 with a variety of inhibitors.

  3. Reversible dissociation of active octamer of cyanase to inactive dimer promoted by alteration of the sulfhydryl group.

    Science.gov (United States)

    Anderson, P M; Johnson, W V; Korte, J J; Xiong, X F; Sung, Y C; Fuchs, J A

    1988-04-25

    Cyanase is an inducible enzyme in Escherichia coli that catalyzes the reaction of cyanate with bicarbonate resulting in the decomposition of cyanate to ammonia and bicarbonate. In this study, the role of the single sulfhydryl group in each of the eight identical subunits of cyanase was investigated. Tetranitromethane, methyl methanethiosulfonate, N-ethylmaleimide, and Hg2+ all reacted with the sulfhydryl group to give derivatives which had reduced activities and which dissociated reversibly to inactive dimer. Association of inactive dimer to active octamer was facilitated by the presence of azide (cyanate analog) and bicarbonate, increased temperature and enzyme concentration, and presence of phosphate. Nitration of tyrosine residues by tetranitromethane occurred only in the absence of azide and bicarbonate, suggesting that at least some of the tyrosine residues become exposed when octamer dissociates to dimer. Site-directed mutagenesis was used to prepare a mutant enzyme in which serine was substituted for cysteine. The mutant enzyme was catalytically active and had properties very similar to native enzyme, except that it was less stable to treatment with urea and to high temperatures. These results establish that in native cyanase the sulfhydryl group per se is not required for catalytic activity, but it may play a role in stabilizing octameric structure, and that octameric structure is required for catalytic activity.

  4. Chronic Recreational Physical Inactivity and Epithelial Ovarian Cancer Risk

    DEFF Research Database (Denmark)

    Cannioto, Rikki; LaMonte, Michael J; Risch, Harvey A

    2016-01-01

    BACKGROUND: Despite a large body of literature evaluating the association between recreational physical activity and epithelial ovarian cancer (EOC) risk, the extant evidence is inconclusive, and little is known about the independent association between recreational physical inactivity and EOC ri...

  5. MEK kinases are regulated by EGF and selectively interact with Rac/Cdc42.

    Science.gov (United States)

    Fanger, G R; Johnson, N L; Johnson, G L

    1997-08-15

    MEK kinases (MEKKs) 1, 2, 3 and 4 are members of sequential kinase pathways that regulate MAP kinases including c-Jun NH2-terminal kinases (JNKs) and extracellular regulated kinases (ERKs). Confocal immunofluorescence microscopy of COS cells demonstrated differential MEKK subcellular localization: MEKK1 was nuclear and in post-Golgi vesicular-like structures; MEKK2 and 4 were localized to distinct Golgi-associated vesicles that were dispersed by brefeldin A. MEKK1 and 2 were activated by EGF, and kinase-inactive mutants of each MEKK partially inhibited EGF-stimulated JNK activity. Kinase-inactive MEKK1, but not MEKK2, 3 or 4, strongly inhibited EGF-stimulated ERK activity. In contrast to MEKK2 and 3, MEKK1 and 4 specifically associated with Rac and Cdc42 and kinase-inactive mutants blocked Rac/Cdc42 stimulation of JNK activity. Inhibitory mutants of MEKK1-4 did not affect p21-activated kinase (PAK) activation of JNK, indicating that the PAK-regulated JNK pathway is independent of MEKKs. Thus, in different cellular locations, specific MEKKs are required for the regulation of MAPK family members, and MEKK1 and 4 are involved in the regulation of JNK activation by Rac/Cdc42 independent of PAK. Differential MEKK subcellular distribution and interaction with small GTP-binding proteins provides a mechanism to regulate MAP kinase responses in localized regions of the cell and to different upstream stimuli.

  6. Prevalence of physical inactivity in Iran: a systematic review

    Science.gov (United States)

    Fakhrzadeh, Hossein; Djalalinia, Shirin; Mirarefin, Mojdeh; Arefirad, Tahereh; Asayesh, Hamid; Safiri, Saeid; Samami, Elham; Mansourian, Morteza; Shamsizadeh, Morteza; Qorbani, Mostafa

    2016-01-01

    Introduction: Physical inactivity is one of the most important risk factors for chronic diseases, including cardiovascular disease, cancer, and stroke. We aim to conduct a systematic review of the prevalence of physical inactivity in Iran. Methods: We searched international databases; ISI, PubMed/Medline, Scopus, and national databases Irandoc, Barakat knowledge network system, and Scientific Information Database (SID). We collected data for outcome measures of prevalence of physical inactivity by sex, age, province, and year. Quality assessment and data extraction has been conducted independently by two independent research experts. There were no limitations for time and language. Results: We analyzed data for prevalence of physical inactivity in Iranian population. According to our search strategy we found 254 records; of them 185 were from international databases and the remaining 69 were obtained from national databases after refining the data, 34 articles that met eligible criteria remained for data extraction. From them respectively; 9, 20, 2 and 3 studies were at national, provincial, regional and local levels. The estimates for inactivity ranged from approximately 30% to almost 70% and had considerable variation between sexes and studied sub-groups. Conclusion: In Iran, most of studies reported high prevalence of physical inactivity. Our findings reveal a heterogeneity of reported values, often from differences in study design, measurement tools and methods, different target groups and sub-population sampling. These data do not provide the possibility of aggregation of data for a comprehensive inference. PMID:27777692

  7. Association of protein kinase Cmu with type II phosphatidylinositol 4-kinase and type I phosphatidylinositol-4-phosphate 5-kinase.

    Science.gov (United States)

    Nishikawa, K; Toker, A; Wong, K; Marignani, P A; Johannes, F J; Cantley, L C

    1998-09-04

    Protein kinase Cmu (PKCmu), also named protein kinase D, is an unusual member of the PKC family that has a putative transmembrane domain and pleckstrin homology domain. This enzyme has a substrate specificity distinct from other PKC isoforms (Nishikawa, K., Toker, A., Johannes, F. J., Songyang, Z., and Cantley, L. C. (1997) J. Biol. Chem. 272, 952-960), and its mechanism of regulation is not yet clear. Here we show that PKCmu forms a complex in vivo with a phosphatidylinositol 4-kinase and a phosphatidylinositol-4-phosphate 5-kinase. A region of PKCmu between the amino-terminal transmembrane domain and the pleckstrin homology domain is shown to be involved in the association with the lipid kinases. Interestingly, a kinase-dead point mutant of PKCmu failed to associate with either lipid kinase activity, indicating that autophosphorylation may be required to expose the lipid kinase interaction domain. Furthermore, the subcellular distribution of the PKCmu-associated lipid kinases to the particulate fraction depends on the presence of the amino-terminal region of PKCmu including the predicted transmembrane region. These results suggest a novel model in which the non-catalytic region of PKCmu acts as a scaffold for assembly of enzymes involved in phosphoinositide synthesis at specific membrane locations.

  8. Active cAMP-dependent protein kinase incorporated within highly purified HIV-1 particles is required for viral infectivity and interacts with viral capsid protein.

    Science.gov (United States)

    Cartier, Christine; Hemonnot, Bénédicte; Gay, Bernard; Bardy, Martine; Sanchiz, Céline; Devaux, Christian; Briant, Laurence

    2003-09-12

    Host cell components, including protein kinases such as ERK-2/mitogen-activated protein kinase, incorporated within human immunodeficiency virus type 1 (HIV-1) virions play a pivotal role in the ability of HIV to infect and replicate in permissive cells. The present work provides evidence that the catalytic subunit of cAMP-dependent protein kinase (C-PKA) is packaged within HIV-1 virions as demonstrated using purified subtilisin-digested viral particles. Virus-associated C-PKA was shown to be enzymatically active and able to phosphorylate synthetic substrate in vitro. Suppression of virion-associated C-PKA activity by specific synthetic inhibitor had no apparent effect on viral precursor maturation and virus assembly. However, virus-associated C-PKA activity was demonstrated to regulate HIV-1 infectivity as assessed by single round infection assays performed by using viruses produced from cells expressing an inactive form of C-PKA. In addition, virus-associated C-PKA was found to co-precipitate with and to phosphorylate the CAp24gag protein. Altogether our results indicate that virus-associated C-PKA regulates HIV-1 infectivity, possibly by catalyzing phosphorylation of the viral CAp24gag protein.

  9. Prevalence and factors associated with physical inactivity among Malaysian adults.

    Science.gov (United States)

    Ying, Chanying; Kuay, Lim Kuang; Huey, Teh Chien; Hock, Lim Kuang; Hamid, Hamizatul Akmal Abd; Omar, Mohd Azahadi; Ahmad, Noor Ani; Cheong, Kee Chee

    2014-03-01

    Using data from the Third National Health and Morbidity Survey (NHMS III) in 2006, this study examined the association between socio-demographic factors and physical inactivity in a sample of 33,949 adults aged 18 years and above by gender. Physical activity levels were measured using the Global Physical Activity Questionnaire (GPAQ vers 1). Physical inactivity was defined as having a total physical activity level of less than 600 metabolic equivalents-minutes per week (METs-minutes/week) contributed by all three different life domains.Logistic regression analyses were conducted.The prevalence of overall physical inactivity was 43.7% (95% CI: 42.9-44.5). The mean total physical activity level was 894.2 METs-minutes/ week. The means METs-minutes/week for the domain of work, travelling, and leisure time were 518.4, 288.1, and 134.8, respectively. Multivariable logistic regression analyses indicated that females were more likely to be physically inactive than males were (aOR=1.62; 95% CI: 1.53-1.72). Among women, being a housewife (aOR = 1.78; 95% CI: 1.56-2.03), widow/divorcee (aOR = 1.23; 95% CI: 1.05-1.43), and those with no formal education (aOR = 1.20; 95% CI: 1.01-1.43) were found to be significantly associated with physical inactivity.Urban residents, older adults aged 65 years and above, private employees, nonworking group, and those with a monthly household income level of MYR5,000 and above appeared to be consistently associated with physical inactivity across men, women, and combined group (both). Specific health intervention strategies to promote physical activity should be targeted on population subgroups who are inactive.

  10. ERK1 and ERK2 mitogen-activated protein kinases affect Ras-dependent cell signaling differentially

    Directory of Open Access Journals (Sweden)

    Bonini Chiara

    2006-06-01

    Full Text Available Abstract Background The mitogen-activated protein (MAP kinases p44ERK1 and p42ERK2 are crucial components of the regulatory machinery underlying normal and malignant cell proliferation. A currently accepted model maintains that ERK1 and ERK2 are regulated similarly and contribute to intracellular signaling by phosphorylating a largely common subset of substrates, both in the cytosol and in the nucleus. Results Here, we show that ablation of ERK1 in mouse embryo fibroblasts and NIH 3T3 cells by gene targeting and RNA interference results in an enhancement of ERK2-dependent signaling and in a significant growth advantage. By contrast, knockdown of ERK2 almost completely abolishes normal and Ras-dependent cell proliferation. Ectopic expression of ERK1 but not of ERK2 in NIH 3T3 cells inhibits oncogenic Ras-mediated proliferation and colony formation. These phenotypes are independent of the kinase activity of ERK1, as expression of a catalytically inactive form of ERK1 is equally effective. Finally, ectopic expression of ERK1 but not ERK2 is sufficient to attenuate Ras-dependent tumor formation in nude mice. Conclusion These results reveal an unexpected interplay between ERK1 and ERK2 in transducing Ras-dependent cell signaling and proliferation. Whereas ERK2 seems to have a positive role in controlling normal and Ras-dependent cell proliferation, ERK1 probably affects the overall signaling output of the cell by antagonizing ERK2 activity.

  11. Casein kinases

    DEFF Research Database (Denmark)

    Issinger, O G

    1993-01-01

    subunits are highly conserved during evolution. The relationship between CK-2 alpha from humans and plants is still 73%. Similar relationships are reported for the beta-subunit. Chromosomal assignment of CK-2 alpha shows two gene loci, one of which is a pseudogene. They are located on different chromosomes......, no genetic changes are necessarily involved; the observed changes may be entirely due to a signal transduction pathway where CK-2 could be phosphorylated by another kinase(s). CK-2 cDNAs from various organisms have been isolated and characterized. From the deduced amino acid sequence it turns out that CK-2......-subunit affecting: (i) stability, (ii) enzyme specificity and (iii) enzyme activity. The question where CK-2 and its subunits are located throughout the cell cycle has also been addressed, mainly because of the large discrepancies that still exist between results obtained by different investigators. Tissue...

  12. Assessing compliance: Active versus inactive trainees in a memory intervention

    Directory of Open Access Journals (Sweden)

    Dana K Bagwell

    2008-06-01

    Full Text Available Dana K Bagwell, Robin L WestDepartment of Psychology, University of Florida, Gainesville, FL, USAAbstract: Extensive research on memory interventions has confirmed their success with older adults, but the individual difference factors that predict successful training outcomes remain relatively unexplored. In the current intervention, trainees were identified as active (compliant with training regimens or inactive using trainer ratings based on attendance, homework completion, and class participation. The active group showed significantly greater training-related gains than the inactive group and the control group on most measures. Compliance was predicted by health, education, and self-efficacy. Specifically, active trainees were more likely to have advanced degrees and somewhat higher self-efficacy, and to have higher vitality and fewer functional limitations than the inactive trainees. This research may assist future investigators to target interventions to those who will show the most benefit.Keywords: compliance, memory training, aging, intervention

  13. Comparative Analysis of Two Gene-Targeting Approaches Challenges the Tumor-Suppressive Role of the Protein Kinase MK5/PRAK.

    Directory of Open Access Journals (Sweden)

    Natalia Ronkina

    Full Text Available MK5 (MAPK-activated protein kinase 5 or PRAK (p38-regulated and -activated kinase are alternative names for a serine/threonine protein kinase downstream to ERK3/4 and p38 MAPK. A previous gene targeting approach for MK5/PRAK (termed here MK5/PRAK-Δex8 revealed a seemingly tumor-suppressive role of MK5/PRAK in DMBA-induced one step skin carcinogenesis and Ras-induced transformation. Here we demonstrate that an alternative targeting strategy of MK5/PRAK (termed MK5/PRAK-Δex6 increased neither tumor incidence in the one step skin carcinogenesis model, nor Ras-induced transformation in primary cells. Interestingly, due to the targeting strategies and exon skipping both knockouts do not completely abolish the generation of MK5/PRAK protein, but express MK5/PRAK deletion mutants with different biochemical properties depending on the exon targeted: Targeting of exon 6 leads to expression of an unstable cytoplasmic catalytically inactive MK5/PRAK-Δex6 mutant while targeting of exon 8 results in a more stable nuclear MK5/PRAK-Δex8 mutant with residual catalytic activity. The different properties of the MK5/PRAK deletion mutants could be responsible for the observed discrepancy between the knockout strains and challenge the role of MK5/PRAK in p53-dependent tumor suppression. Further MK5/PRAK knockout and knock-in mouse strains will be necessary to assign a physiological function to MK5/PRAK in this model organism.

  14. The economic benefits of reducing physical inactivity: an Australian example

    Directory of Open Access Journals (Sweden)

    Cumming Toby B

    2011-09-01

    Full Text Available Abstract Background Physical inactivity has major impacts on health and productivity. Our aim was to estimate the health and economic benefits of reducing the prevalence of physical inactivity in the 2008 Australian adult population. The economic benefits were estimated as 'opportunity cost savings', which represent resources utilized in the treatment of preventable disease that are potentially available for re-direction to another purpose from fewer incident cases of disease occurring in communities. Methods Simulation models were developed to show the effect of a 10% feasible, reduction target for physical inactivity from current Australian levels (70%. Lifetime cohort health benefits were estimated as fewer incident cases of inactivity-related diseases; deaths; and Disability Adjusted Life Years (DALYs by age and sex. Opportunity costs were estimated as health sector cost impacts, as well as paid and unpaid production gains and leisure impacts from fewer disease events associated with reduced physical inactivity. Workforce production gains were estimated by comparing surveyed participation and absenteeism rates of physically active and inactive adults, and valued using the friction cost approach. The impact of an improvement in health status on unpaid household production and leisure time were modeled from time use survey data, as applied to the exposed and non-exposed population subgroups and valued by suitable proxy. Potential costs associated with interventions to increase physical activity were not included. Multivariable uncertainty analyses and univariate sensitivity analyses were undertaken to provide information on the strength of the conclusions. Results A 10% reduction in physical inactivity would result in 6,000 fewer incident cases of disease, 2,000 fewer deaths, 25,000 fewer DALYs and provide gains in working days (114,000, days of home-based production (180,000 while conferring a AUD96 million reduction in health sector costs

  15. The Crystal Structure of Toxoplasma gondii Pyruvate Kinase 1

    Energy Technology Data Exchange (ETDEWEB)

    Bakszt, R.; Wernimont, A; Allali-Hassani, A; Mok, M; Hills, T; Hui, R; Pizarro, J

    2010-01-01

    Pyruvate kinase (PK), which catalyzes the final step in glycolysis converting phosphoenolpyruvate to pyruvate, is a central metabolic regulator in most organisms. Consequently PK represents an attractive therapeutic target in cancer and human pathogens, like Apicomplexans. The phylum Aplicomplexa, a group of exclusively parasitic organisms, includes the genera Plasmodium, Cryptosporidium and Toxoplasma, the etiological agents of malaria, cryptosporidiosis and toxoplasmosis respectively. Toxoplasma gondii infection causes a mild illness and is a very common infection affecting nearly one third of the world's population. We have determined the crystal structure of the PK1 enzyme from T. gondii, with the B domain in the open and closed conformations. We have also characterized its enzymatic activity and confirmed glucose-6-phosphate as its allosteric activator. This is the first description of a PK enzyme in a closed inactive conformation without any bound substrate. Comparison of the two tetrameric TgPK1 structures indicates a reorientation of the monomers with a concomitant change in the buried surface among adjacent monomers. The change in the buried surface was associated with significant B domain movements in one of the interacting monomers. We hypothesize that a loop in the interface between the A and B domains plays an important role linking the position of the B domain to the buried surface among monomers through two {alpha}-helices. The proposed model links the catalytic cycle of the enzyme with its domain movements and highlights the contribution of the interface between adjacent subunits. In addition, an unusual ordered conformation was observed in one of the allosteric binding domains and it is related to a specific apicomplexan insertion. The sequence and structural particularity would explain the atypical activation by a mono-phosphorylated sugar. The sum of peculiarities raises this enzyme as an emerging target for drug discovery.

  16. Moonlighting kinases with guanylate cyclase activity can tune regulatory signal networks

    KAUST Repository

    Irving, Helen R.

    2012-02-01

    Guanylate cyclase (GC) catalyzes the formation of cGMP and it is only recently that such enzymes have been characterized in plants. One family of plant GCs contains the GC catalytic center encapsulated within the intracellular kinase domain of leucine rich repeat receptor like kinases such as the phytosulfokine and brassinosteroid receptors. In vitro studies show that both the kinase and GC domain have catalytic activity indicating that these kinase-GCs are examples of moonlighting proteins with dual catalytic function. The natural ligands for both receptors increase intracellular cGMP levels in isolated mesophyll protoplast assays suggesting that the GC activity is functionally relevant. cGMP production may have an autoregulatory role on receptor kinase activity and/or contribute to downstream cell expansion responses. We postulate that the receptors are members of a novel class of receptor kinases that contain functional moonlighting GC domains essential for complex signaling roles.

  17. Novel Library of Selenocompounds as Kinase Modulators

    Directory of Open Access Journals (Sweden)

    Carmen Sanmartín

    2011-07-01

    Full Text Available Although the causes of cancer lie in mutations or epigenic changes at the genetic level, their molecular manifestation is the dysfunction of biochemical pathways at the protein level. The 518 protein kinases encoded by the human genome play a central role in various diseases, a fact that has encouraged extensive investigations on their biological function and three dimensional structures. Selenium (Se is an important nutritional trace element involved in different physiological functions with antioxidative, antitumoral and chemopreventive properties. The mechanisms of action for selenocompounds as anticancer agents are not fully understood, but kinase modulation seems to be a possible pathway. Various organosulfur compounds have shown antitumoral and kinase inhibition effects but, in many cases, the replacement of sulfur by selenium improves the antitumoral effect of compounds. Although Se atom possesses a larger atomic volume and nucleophilic character than sulfur, Se can also formed interactions with aminoacids of the catalytic centers of proteins. So, we propose a novel chemical library that includes organoselenium compounds as kinase modulators. In this study thirteen selenocompounds have been evaluated at a concentration of 3 or 10 µM in a 24 kinase panel using a Caliper LabChip 3000 Drug Discover Platform. Several receptor (EGFR, IGFR1, FGFR1… and non-receptor (Abl kinases have been selected, as well as serine/threonine/lipid kinases (AurA, Akt, CDKs, MAPKs… implicated in main cancer pathways: cell cycle regulation, signal transduction, angiogenesis regulation among them. The obtained results showed that two compounds presented inhibition values higher than 50% in at least four kinases and seven derivatives selectively inhibited one or two kinases. Furthermore, three compounds selectively activated IGF-1R kinase with values ranging from −98% to −211%. In conclusion, we propose that the replacement of sulfur by selenium seems to be

  18. Autoinhibition of Bruton's tyrosine kinase (Btk) and activation by soluble inositol hexakisphosphate.

    Science.gov (United States)

    Wang, Qi; Vogan, Erik M; Nocka, Laura M; Rosen, Connor E; Zorn, Julie A; Harrison, Stephen C; Kuriyan, John

    2015-02-20

    Bruton's tyrosine kinase (Btk), a Tec-family tyrosine kinase, is essential for B-cell function. We present crystallographic and biochemical analyses of Btk, which together reveal molecular details of its autoinhibition and activation. Autoinhibited Btk adopts a compact conformation like that of inactive c-Src and c-Abl. A lipid-binding PH-TH module, unique to Tec kinases, acts in conjunction with the SH2 and SH3 domains to stabilize the inactive conformation. In addition to the expected activation of Btk by membranes containing phosphatidylinositol triphosphate (PIP3), we found that inositol hexakisphosphate (IP6), a soluble signaling molecule found in both animal and plant cells, also activates Btk. This activation is a consequence of a transient PH-TH dimerization induced by IP6, which promotes transphosphorylation of the kinase domains. Sequence comparisons with other Tec-family kinases suggest that activation by IP6 is unique to Btk.

  19. Inaction inertia, regret, and valuation : A closer look

    NARCIS (Netherlands)

    Zeelenberg, Marcel; Nijstad, Bernard A.; van Putten, Marijke; van Dijk, Eric

    2006-01-01

    Inaction inertia is the phenomenon that one is not likely to act on an attractive opportunity after having bypassed an even more attractive opportunity. So far, all published work has assumed a causal role for the emotion regret in this effect. In a series of 5 experiments we found no support for th

  20. Home Delivery Medicament Program: access, inactivity and cardiovascular risk

    Directory of Open Access Journals (Sweden)

    Roque da Silva Araújo

    Full Text Available ABSTRACT Objective: to verify causes of inactivity in the Home Delivery Medicament Program, as referred by users from a Primary Health Care Service in São Paulo, comparing them to the causes registered in the program and analyzing them in the theoretical model Concept of Access to Health. Methods: cross-sectional study, interviewing 111 inactive users; and documentary study in the program records. Results: half of the users did not know the condition of inactivity. Discrepancies were found between the user's and the program's information, observing different levels of agreement: Absence of physician and administrative staff member 0%; Transfer to other service 25%; Death 50%; Option to quit 50%; Address change 57% and Change in therapeutic schedule 80%. The users' feeling of accepting the program was observed. In the health access concept, inactivity can be explained in the information dimension, in the degree of asymmetry between the patient's and the health professional's knowledge, identified through the indicators: education, knowledge and information sources. Conclusions: due to the low education level, the user does not assimilate the information on the steps of the program flowchart, does not return for the assessment that guarantees its continuity. Consequently, (she stops receiving the medication and spends a long time without treatment, increasing the cardiovascular risk of hypertensive (92% of the sample, diabetic (44% and dyslipidemic patients (31%.

  1. Automatic Detection of Inactive Solar Cell Cracks in Electroluminescence Images

    DEFF Research Database (Denmark)

    Spataru, Sergiu; Hacke, Peter; Sera, Dezso

    2017-01-01

    We propose an algorithm for automatic determination of the electroluminescence (EL) signal threshold level corresponding to inactive solar cell cracks, resulting from their disconnection from the electrical circuit of the cell. The method enables automatic quantification of the cell crack size...

  2. Physical inactivity and muscle oxidative capacity in humans

    DEFF Research Database (Denmark)

    Gram, Martin; Dahl, Rannvá; Dela, Flemming

    2014-01-01

    of proteins related to oxidative phosphorylation. With such a substantial down-regulation, it is likely that a range of adenosine triphosphate (ATP)-dependent pathways such as calcium signalling, respiratory capacity and apoptosis are affected by physical inactivity. However, this has not been investigated...

  3. Muscle activity and inactivity periods during normal daily life.

    Science.gov (United States)

    Tikkanen, Olli; Haakana, Piia; Pesola, Arto J; Häkkinen, Keijo; Rantalainen, Timo; Havu, Marko; Pullinen, Teemu; Finni, Taija

    2013-01-01

    Recent findings suggest that not only the lack of physical activity, but also prolonged times of sedentary behaviour where major locomotor muscles are inactive, significantly increase the risk of chronic diseases. The purpose of this study was to provide details of quadriceps and hamstring muscle inactivity and activity during normal daily life of ordinary people. Eighty-four volunteers (44 females, 40 males, 44.1±17.3 years, 172.3±6.1 cm, 70.1±10.2 kg) were measured during normal daily life using shorts measuring muscle electromyographic (EMG) activity (recording time 11.3±2.0 hours). EMG was normalized to isometric MVC (EMG(MVC)) during knee flexion and extension, and inactivity threshold of each muscle group was defined as 90% of EMG activity during standing (2.5±1.7% of EMG(MVC)). During normal daily life the average EMG amplitude was 4.0±2.6% and average activity burst amplitude was 5.8±3.4% of EMG(MVC) (mean duration of 1.4±1.4 s) which is below the EMG level required for walking (5 km/h corresponding to EMG level of about 10% of EMG(MVC)). Using the proposed individual inactivity threshold, thigh muscles were inactive 67.5±11.9% of the total recording time and the longest inactivity periods lasted for 13.9±7.3 min (2.5-38.3 min). Women had more activity bursts and spent more time at intensities above 40% EMG(MVC) than men (p<0.05). In conclusion, during normal daily life the locomotor muscles are inactive about 7.5 hours, and only a small fraction of muscle's maximal voluntary activation capacity is used averaging only 4% of the maximal recruitment of the thigh muscles. Some daily non-exercise activities such as stair climbing produce much higher muscle activity levels than brisk walking, and replacing sitting by standing can considerably increase cumulative daily muscle activity.

  4. Muscle activity and inactivity periods during normal daily life.

    Directory of Open Access Journals (Sweden)

    Olli Tikkanen

    Full Text Available Recent findings suggest that not only the lack of physical activity, but also prolonged times of sedentary behaviour where major locomotor muscles are inactive, significantly increase the risk of chronic diseases. The purpose of this study was to provide details of quadriceps and hamstring muscle inactivity and activity during normal daily life of ordinary people. Eighty-four volunteers (44 females, 40 males, 44.1±17.3 years, 172.3±6.1 cm, 70.1±10.2 kg were measured during normal daily life using shorts measuring muscle electromyographic (EMG activity (recording time 11.3±2.0 hours. EMG was normalized to isometric MVC (EMG(MVC during knee flexion and extension, and inactivity threshold of each muscle group was defined as 90% of EMG activity during standing (2.5±1.7% of EMG(MVC. During normal daily life the average EMG amplitude was 4.0±2.6% and average activity burst amplitude was 5.8±3.4% of EMG(MVC (mean duration of 1.4±1.4 s which is below the EMG level required for walking (5 km/h corresponding to EMG level of about 10% of EMG(MVC. Using the proposed individual inactivity threshold, thigh muscles were inactive 67.5±11.9% of the total recording time and the longest inactivity periods lasted for 13.9±7.3 min (2.5-38.3 min. Women had more activity bursts and spent more time at intensities above 40% EMG(MVC than men (p<0.05. In conclusion, during normal daily life the locomotor muscles are inactive about 7.5 hours, and only a small fraction of muscle's maximal voluntary activation capacity is used averaging only 4% of the maximal recruitment of the thigh muscles. Some daily non-exercise activities such as stair climbing produce much higher muscle activity levels than brisk walking, and replacing sitting by standing can considerably increase cumulative daily muscle activity.

  5. Halogen Chemistry on Catalytic Surfaces.

    Science.gov (United States)

    Moser, Maximilian; Pérez-Ramírez, Javier

    2016-01-01

    Halogens are key building blocks for the manufacture of high-value products such as chemicals, plastics, and pharmaceuticals. The catalytic oxidation of HCl and HBr is an attractive route to recover chlorine and bromine in order to ensure the sustainability of the production processes. Very few materials withstand the high corrosiveness and the strong exothermicity of the reactions and among them RuO2 and CeO2-based catalysts have been successfully applied in HCl oxidation. The search for efficient systems for HBr oxidation was initiated by extrapolating the results of HCl oxidation based on the chemical similarity of these reactions. Interestingly, despite its inactivity in HCl oxidation, TiO2 was found to be an outstanding HBr oxidation catalyst, which highlighted that the latter reaction is more complex than previously assumed. Herein, we discuss the results of recent comparative studies of HCl and HBr oxidation on both rutile-type (RuO2, IrO2, and TiO2) and ceria-based catalysts using a combination of advanced experimental and theoretical methods to provide deeper molecular-level understanding of the reactions. This knowledge aids the design of the next-generation catalysts for halogen recycling.

  6. An allosteric signaling pathway of human 3-phosphoglycerate kinase from force distribution analysis.

    Directory of Open Access Journals (Sweden)

    Zoltan Palmai

    2014-01-01

    Full Text Available 3-Phosphogycerate kinase (PGK is a two domain enzyme, which transfers a phosphate group between its two substrates, 1,3-bisphosphoglycerate bound to the N-domain and ADP bound to the C-domain. Indispensable for the phosphoryl transfer reaction is a large conformational change from an inactive open to an active closed conformation via a hinge motion that should bring substrates into close proximity. The allosteric pathway resulting in the active closed conformation has only been partially uncovered. Using Molecular Dynamics simulations combined with Force Distribution Analysis (FDA, we describe an allosteric pathway, which connects the substrate binding sites to the interdomain hinge region. Glu192 of alpha-helix 7 and Gly394 of loop L14 act as hinge points, at which these two secondary structure elements straighten, thereby moving the substrate-binding domains towards each other. The long-range allosteric pathway regulating hPGK catalytic activity, which is partially validated and can be further tested by mutagenesis, highlights the virtue of monitoring internal forces to reveal signal propagation, even if only minor conformational distortions, such as helix bending, initiate the large functional rearrangement of the macromolecule.

  7. An allosteric signaling pathway of human 3-phosphoglycerate kinase from force distribution analysis.

    Science.gov (United States)

    Palmai, Zoltan; Seifert, Christian; Gräter, Frauke; Balog, Erika

    2014-01-01

    3-Phosphogycerate kinase (PGK) is a two domain enzyme, which transfers a phosphate group between its two substrates, 1,3-bisphosphoglycerate bound to the N-domain and ADP bound to the C-domain. Indispensable for the phosphoryl transfer reaction is a large conformational change from an inactive open to an active closed conformation via a hinge motion that should bring substrates into close proximity. The allosteric pathway resulting in the active closed conformation has only been partially uncovered. Using Molecular Dynamics simulations combined with Force Distribution Analysis (FDA), we describe an allosteric pathway, which connects the substrate binding sites to the interdomain hinge region. Glu192 of alpha-helix 7 and Gly394 of loop L14 act as hinge points, at which these two secondary structure elements straighten, thereby moving the substrate-binding domains towards each other. The long-range allosteric pathway regulating hPGK catalytic activity, which is partially validated and can be further tested by mutagenesis, highlights the virtue of monitoring internal forces to reveal signal propagation, even if only minor conformational distortions, such as helix bending, initiate the large functional rearrangement of the macromolecule.

  8. Purification and kinase assay of PKN.

    Science.gov (United States)

    Mukai, Hideyuki; Ono, Yoshitaka

    2006-01-01

    PKN is a serine/threonine protein kinase, which has a catalytic domain highly homologous to that of protein kinase C (PKC) in the carboxyl-terminal region and three repeats of the antiparallel coiled coil (ACC) domain in the amino-terminal region. Mammalian PKN has three isoforms each derived from different genes, PKN1 (PKNalpha/PRK1/PAK1), PKN2 (PRK2/PAK2/PKNgamma), and PKN3 (PKNbeta). PKN isoforms show different enzymatic properties and tissue distributions and have been implicated in various distinct cellular processes (reviewed in Mukai [2003]). This chapter discusses methods to prepare purified enzymes and to assay substrate phosphorylation activities.

  9. Do Running and Strength Exercises Reduce Daily Muscle Inactivity Time?

    Directory of Open Access Journals (Sweden)

    Taija Finni

    2016-09-01

    Full Text Available Understanding how a specific exercise changes daily activity patterns is important when designing physical activity interventions. We examined the effects of strength and interval running exercise sessions on daily activity patterns using recordings of quadriceps and hamstring muscle electromyographic (EMG activity and inactivity. Five male and five female subjects taking part in a 10-week training programme containing both strength and interval running training sessions were measured for daily muscle EMG activities during three days: on a strength day, an interval running day, and a day without exercise. EMG was measured using textile electrodes embedded into sport shorts that were worn 9.1 ± 1.4 hours/day and results are given as % of recording time. During the total measurement time the muscles were inactive 55 ± 26%, 53 ± 30% and 71 ± 12% during strength training day, interval running day, and day without exercise (n.s.. When compared to the day without exercise, the change in muscle inactivity correlated negatively with change in light muscle activity in strength (r = -0.971,p< 0.001 and interval running days (r = -0.965,p< 0.001. While interval running exercise bout induced a more systematic decrease in muscle inactivity time (from 62 ± 15% to 6 ± 6%,p< 0.001, reductions in muscle inactivity in response to strength exercise were highly individual (range 5–70 pp despite the same training programme. Strength, but not running exercise bout, increased muscle activity levels occurring above 50% MVC (p< 0.05 when compared to a similar period without exercise. The effect of strength exercise bout on totaldaily recording time increased the EMG amplitudes across the entire intensity spectrum. While strength and interval running exercise are effective in increasing muscle moderate-to-vigorous activity when compared to a similar period without exercise, it comprises only a small part of the day and does not seem to have a systematic effect

  10. Inhibition of c-Abl kinase activity renders cancer cells highly sensitive to mitoxantrone.

    Directory of Open Access Journals (Sweden)

    Kemal Alpay

    Full Text Available Although c-Abl has increasingly emerged as a key player in the DNA damage response, its role in this context is far from clear. We studied the effect of inhibition of c-Abl kinase activity by imatinib with chemotherapy drugs and found a striking difference in cell survival after combined mitoxantrone (MX and imatinib treatment compared to a panel of other chemotherapy drugs. The combinatory treatment induced apoptosis in HeLa cells and other cancer cell lines but not in primary fibroblasts. The difference in MX and doxorubicin was related to significant augmentation of DNA damage. Transcriptionally active p53 accumulated in cells in which human papillomavirus E6 normally degrades p53. The combination treatment resulted in caspase activation and apoptosis, but this effect did not depend on either p53 or p73 activity. Despite increased p53 activity, the cells arrested in G2 phase became defective in this checkpoint, allowing cell cycle progression. The effect after MX treatment depended partially on c-Abl: Short interfering RNA knockdown of c-Abl rendered HeLa cells less sensitive to MX. The effect of imatinib was decreased by c-Abl siRNA suggesting a role for catalytically inactive c-Abl in the death cascade. These findings indicate that MX has a unique cytotoxic effect when the kinase activity of c-Abl is inhibited. The treatment results in increased DNA damage and c-Abl-dependent apoptosis, which may offer new possibilities for potentiation of cancer chemotherapy.

  11. Protein kinase clk/STY is differentially regulated during erythroleukemia cell differentiation: a bias toward the skipped splice variant characterizes postcommitment stages

    Institute of Scientific and Technical Information of China (English)

    Ana GARC(I)A-SACRIST(A)N; María J.FERN(A)NDEZ-NESTOSA; Pablo HERN(A)NDEZ; Jorge B.SCHVARTZMAN; Dora B.KRIMER

    2005-01-01

    Clk/STY is a LAMMER protein kinase capable to phosphorylate serine/arginine-rich (SR) proteins that modulate premRNA splicing.Clk/STY alternative splicing generates transcripts encoding a full-length kinase and a truncated catalytically inactive protein.Here we showed that clk/STY,as well as other members of the family (e.g.clk2,clk3 and clk4),are up-regulated during HMBA-induced erythroleukemia cell differentiation.mRNAs coding for the full-length and the truncated forms were responsible for the overall increased expression.In clk/STY,however,a switch was observed for the ratio of the two alternative spliced products.In undifferentiated cells the full-length transcript was more abundant whereas the transcript encoding for the truncated form predominated at latter stages of differentiation.Surprisingly,overexpression of clk/STY did not alter the splicing switch upon differentiation in MEL cells.These results suggest that clk/STY might contribute to control erythroid differentiation by a mechanism that implicates a balance between these two isoforms.

  12. Catalytic combustor for hydrogen

    Energy Technology Data Exchange (ETDEWEB)

    Mercea, J.; Grecu, E.; Fodor, T.; Kreibik, S.

    1982-01-01

    The performance of catalytic combustors for hydrogen using platinum-supported catalysts is described. Catalytic plates of different sizes were constructed using fibrous and ceramic supports. The temperature distribution as well as the reaction efficiency as a function of the fuel input rate was determined, and a comparison between the performances of different plates is discussed.

  13. Phosphorylation of the regulatory beta-subunit of protein kinase CK2 by checkpoint kinase Chk1: identification of the in vitro CK2beta phosphorylation site

    DEFF Research Database (Denmark)

    Kristensen, Lars P; Larsen, Martin Røssel; Højrup, Peter;

    2004-01-01

    The regulatory beta-subunit of protein kinase CK2 mediates the formation of the CK2 tetrameric form and it has functions independent of CK2 catalytic subunit through interaction with several intracellular proteins. Recently, we have shown that CK2beta associates with the human checkpoint kinase Chk...... by the modification of Thr213 but it does require the presence of an active Chk1 kinase....

  14. KLIFS: a knowledge-based structural database to navigate kinase-ligand interaction space.

    Science.gov (United States)

    van Linden, Oscar P J; Kooistra, Albert J; Leurs, Rob; de Esch, Iwan J P; de Graaf, Chris

    2014-01-23

    Protein kinases regulate the majority of signal transduction pathways in cells and have become important targets for the development of designer drugs. We present a systematic analysis of kinase-ligand interactions in all regions of the catalytic cleft of all 1252 human kinase-ligand cocrystal structures present in the Protein Data Bank (PDB). The kinase-ligand interaction fingerprints and structure database (KLIFS) contains a consistent alignment of 85 kinase ligand binding site residues that enables the identification of family specific interaction features and classification of ligands according to their binding modes. We illustrate how systematic mining of kinase-ligand interaction space gives new insights into how conserved and selective kinase interaction hot spots can accommodate the large diversity of chemical scaffolds in kinase ligands. These analyses lead to an improved understanding of the structural requirements of kinase binding that will be useful in ligand discovery and design studies.

  15. Comparative analysis of human and bovine protein kinases reveals unique relationship and functional diversity

    Directory of Open Access Journals (Sweden)

    Nuzhat N. Kabir

    2011-01-01

    Full Text Available Reversible protein phosphorylation by protein kinases and phosphatases is a common event in various cellular processes. The eukaryotic protein kinase superfamily, which is one of the largest superfamilies of eukaryotic proteins, plays several roles in cell signaling and diseases. We identified 482 eukaryotic protein kinases and 39 atypical protein kinases in the bovine genome, by searching publicly accessible genetic-sequence databases. Bovines have 512 putative protein kinases, each orthologous to a human kinase. Whereas orthologous kinase pairs are, on an average, 90.6% identical, orthologous kinase catalytic domain pairs are, on an average, 95.9% identical at the amino acid level. This bioinformatic study of bovine protein kinases provides a suitable framework for further characterization of their functional and structural properties.

  16. ERICA: leisure-time physical inactivity in Brazilian adolescents

    Science.gov (United States)

    Cureau, Felipe Vogt; da Silva, Thiago Luiz Nogueira; Bloch, Katia Vergetti; Fujimori, Elizabeth; Belfort, Dilson Rodrigues; de Carvalho, Kênia Mara Baiocchi; de Leon, Elisa Brosina; de Vasconcellos, Mauricio Teixeira Leite; Ekelund, Ulf; Schaan, Beatriz D

    2016-01-01

    ABSTRACT OBJECTIVE To evaluate the prevalence of leisure-time physical inactivity in Brazilian adolescents and their association with geographical and sociodemographic variables. METHODS The sample was composed by 74,589 adolescents participating in the Study of Cardiovascular Risks in Adolescents (ERICA). This cross-sectional study of school basis with national scope involved adolescents aged from 12 to 17 years in Brazilian cities with more than 100 thousand inhabitants. The prevalence of leisure-time physical inactivity was categorized according to the volume of weekly practice (< 300; 0 min). The prevalences were estimated for the total sample and by sex. Poisson regression models were used to assess associated factors. RESULTS The prevalence of leisure-time physical inactivity was 54.3% (95%CI 53.4-55.2), and higher for the female sex (70.7%, 95%CI 69.5-71.9) compared to the male (38.0%, 95%CI 36.7-39.4). More than a quarter of adolescents (26.5%, 95%CI 25.8-27.3) reported not practicing physical activity in the leisure time, a condition more prevalent for girls (39.8%, 95%CI 38.8-40.9) than boys (13.4%, 95%CI 12.4-14.4). For girls, the variables that were associated with physical inactivity were: reside in the Northeast (RP = 1.13, 95%CI 1.08-1.19), Southeast (RP = 1.16, 95%CI 1.11-1.22) and South (RP = 1.12, 95%CI 1.06-1.18); have 16-17 years (RP = 1.06, 95%CI 1.12-1.15); and belong to the lower economic class (RP = 1.33, 95%CI 1.20-1.48). The same factors, except reside in the Southeast and South, were also associated with not practicing physical activity in the leisure time for the same group. In males, as well as the region, being older (p < 0.001) and declaring to be indigenous (RP = 0.37, 95%CI 0.19-0.73) were also associated with not practicing physical activities in the leisure time. CONCLUSIONS The prevalence of leisure-time physical inactivity in Brazilian adolescents is high. It presents regional variations and is associated with age and low

  17. Crystal structure of human nicotinamide riboside kinase.

    Science.gov (United States)

    Khan, Javed A; Xiang, Song; Tong, Liang

    2007-08-01

    Nicotinamide riboside kinase (NRK) has an important role in the biosynthesis of NAD(+) as well as the activation of tiazofurin and other NR analogs for anticancer therapy. NRK belongs to the deoxynucleoside kinase and nucleoside monophosphate (NMP) kinase superfamily, although the degree of sequence conservation is very low. We report here the crystal structures of human NRK1 in a binary complex with the reaction product nicotinamide mononucleotide (NMN) at 1.5 A resolution and in a ternary complex with ADP and tiazofurin at 2.7 A resolution. The active site is located in a groove between the central parallel beta sheet core and the LID and NMP-binding domains. The hydroxyl groups on the ribose of NR are recognized by Asp56 and Arg129, and Asp36 is the general base of the enzyme. Mutation of residues in the active site can abolish the catalytic activity of the enzyme, confirming the structural observations.

  18. Crystal Structure of Human Nicotinamide Riboside Kinase

    Energy Technology Data Exchange (ETDEWEB)

    Khan,J.; Xiang, S.; Tong, L.

    2007-01-01

    Nicotinamide riboside kinase (NRK) has an important role in the biosynthesis of NAD{sup +} as well as the activation of tiazofurin and other NR analogs for anticancer therapy. NRK belongs to the deoxynucleoside kinase and nucleoside monophosphate (NMP) kinase superfamily, although the degree of sequence conservation is very low. We report here the crystal structures of human NRK1 in a binary complex with the reaction product nicotinamide mononucleotide (NMN) at 1.5 {angstrom} resolution and in a ternary complex with ADP and tiazofurin at 2.7 {angstrom} resolution. The active site is located in a groove between the central parallel {beta} sheet core and the LID and NMP-binding domains. The hydroxyl groups on the ribose of NR are recognized by Asp56 and Arg129, and Asp36 is the general base of the enzyme. Mutation of residues in the active site can abolish the catalytic activity of the enzyme, confirming the structural observations.

  19. CMP kinase from Escherichia coli is structurally related to other nucleoside monophosphate kinases.

    Science.gov (United States)

    Bucurenci, N; Sakamoto, H; Briozzo, P; Palibroda, N; Serina, L; Sarfati, R S; Labesse, G; Briand, G; Danchin, A; Bărzu, O; Gilles, A M

    1996-02-02

    CMP kinase from Escherichia coli is a monomeric protein of 225 amino acid residues. The protein exhibits little overall sequence similarities with other known NMP kinases. However, residues involved in binding of substrates and/or in catalysis were found conserved, and sequence comparison suggested conservation of the global fold found in adenylate kinases or in several CMP/UMP kinases. The enzyme was purified to homogeneity, crystallized, and analyzed for its structural and catalytic properties. The crystals belong to the hexagonal space group P6(3), have unit cell parameters a = b = 82.3 A and c = 60.7 A, and diffract x-rays to a 1.9 A resolution. The bacterial enzyme exhibits a fluorescence emission spectrum with maximum at 328 nm upon excitation at 295 nm, which suggests that the single tryptophan residue (Trp30) is located in a hydrophobic environment. Substrate specificity studies showed that CMP kinase from E. coli is active with ATP, dATP, or GTP as donors and with CMP, dCMP, and arabinofuranosyl-CMP as acceptors. This is in contrast with CMP/UMP kinase from Dictyostelium discoideum, an enzyme active on CMP or UMP but much less active on the corresponding deoxynucleotides. Binding of CMP enhanced the affinity of E. coli CMP kinase for ATP or ADP, a particularity never described in this family of proteins that might explain inhibition of enzyme activity by excess of nucleoside monophosphate.

  20. Tumor necrosis factor-alpha induced expression of matrix metalloproteinase-9 through p21-activated Kinase-1

    Directory of Open Access Journals (Sweden)

    Garner Warren

    2009-03-01

    Full Text Available Abstract Background Expressed in embryonic development, matrix metalloprotein-9 (MMP-9 is absent in most of developed adult tissues, but recurs in inflammation during tissue injury, wound healing, tumor formation and metastasis. Expression of MMP-9 is tightly controlled by extracellular cues including pro-inflammatory cytokines and extracellular matrix (ECM. While the pathologic functions of MMP-9 are evident, the intracellular signaling pathways to control its expression are not fully understood. In this study we investigated mechanism of cytokine induced MMP-9 with particular emphasis on the role of p21-activated-kinase-1 (PAK1 and the down stream signaling. Results In response to TNF-alpha or IL-1alpha, PAK1 was promptly activated, as characterized by a sequential phosphorylation, initiated at threonine-212 followed by at threonine-423 in the activation loop of the kinase, in human skin keratinocytes, dermal fibroblasts, and rat hepatic stellate cells. Ectopic expression of PAK1 variants, but not p38 MAP kinase, impaired the TNF-alpha-induced MMP-9 expression, while other MMPs such as MMP-2, -3 and -14 were not affected. Activation of Jun N-terminal kinase (JNK and NF-kappaB has been demonstrated to be essential for MMP-9 expression. Expression of inactive PAK1 variants impaired JNK but not NF-kappaB activation, which consequently suppressed the 5'-promoter activities of the MMP-9 gene. After the cytokine-induced phosphorylation, both ectopically expressed and endogenous PAK1 proteins were promptly accumulated even in the condition of suppressing protein synthesis, suggesting the PAK1 protein is stabilized upon TNF-alpha stimulation. Stabilization of PAK1 protein by TNF-alpha treatment is independent of the kinase catalytic activity and p21 GTPase binding capacities. In contrast to epithelial cells, mesenchymal cells require 3-dimensional type-I collagen in response to TNF-alpha to massively express MMP-9. The collagen effect is mediated, in

  1. The role of DNA dependent protein kinase in synapsis of DNA ends

    NARCIS (Netherlands)

    E.P.W.C. Weterings (Eric); N.S. Verkaik (Nicole); H.T. Brüggenwirth (Hennie); J.H.J. Hoeijmakers (Jan); D.C. van Gent (Dik)

    2003-01-01

    textabstractDNA dependent protein kinase (DNA-PK) plays a central role in the non-homologous end-joining pathway of DNA double strand break repair. Its catalytic subunit (DNA-PK(CS)) functions as a serine/threonine protein kinase. We show that DNA-PK forms a stable complex at DNA termini that blocks

  2. Inhibition of ErbB3 by a monoclonal antibody that locks the extracellular domain in an inactive configuration.

    Science.gov (United States)

    Lee, Sangwon; Greenlee, Etienne B; Amick, Joseph R; Ligon, Gwenda F; Lillquist, Jay S; Natoli, Edward J; Hadari, Yaron; Alvarado, Diego; Schlessinger, Joseph

    2015-10-27

    ErbB3 (HER3) is a member of the EGF receptor (EGFR) family of receptor tyrosine kinases, which, unlike the other three family members, contains a pseudo kinase in place of a tyrosine kinase domain. In cancer, ErbB3 activation is driven by a ligand-dependent mechanism through the formation of heterodimers with EGFR, ErbB2, or ErbB4 or via a ligand-independent process through heterodimerization with ErbB2 overexpressed in breast tumors or other cancers. Here we describe the crystal structure of the Fab fragment of an antagonistic monoclonal antibody KTN3379, currently in clinical development in human cancer patients, in complex with the ErbB3 extracellular domain. The structure reveals a unique allosteric mechanism for inhibition of ligand-dependent or ligand-independent ErbB3-driven cancers by binding to an epitope that locks ErbB3 in an inactive conformation. Given the similarities in the mechanism of ErbB receptor family activation, these findings could facilitate structure-based design of antibodies that inhibit EGFR and ErbB4 by an allosteric mechanism.

  3. Catalytic Functions of Standards

    NARCIS (Netherlands)

    K. Blind (Knut)

    2009-01-01

    textabstractThe three different areas and the examples have illustrated several catalytic functions of standards for innovation. First, the standardisation process reduces the time to market of inventions, research results and innovative technologies. Second, standards themselves promote the diffusi

  4. Catalytic Synthesis Lactobionic Acid

    Directory of Open Access Journals (Sweden)

    V.G. Borodina

    2014-07-01

    Full Text Available Gold nanoparticles are obtained, characterized and deposited on the carrier. Conducted catalytic synthesis of lactobionic acid from lactose. Received lactobionic acid identify on the IR spectrum.

  5. Catalytic distillation process

    Science.gov (United States)

    Smith, Jr., Lawrence A.

    1982-01-01

    A method for conducting chemical reactions and fractionation of the reaction mixture comprising feeding reactants to a distillation column reactor into a feed zone and concurrently contacting the reactants with a fixed bed catalytic packing to concurrently carry out the reaction and fractionate the reaction mixture. For example, a method for preparing methyl tertiary butyl ether in high purity from a mixed feed stream of isobutene and normal butene comprising feeding the mixed feed stream to a distillation column reactor into a feed zone at the lower end of a distillation reaction zone, and methanol into the upper end of said distillation reaction zone, which is packed with a properly supported cationic ion exchange resin, contacting the C.sub.4 feed and methanol with the catalytic distillation packing to react methanol and isobutene, and concurrently fractionating the ether from the column below the catalytic zone and removing normal butene overhead above the catalytic zone.

  6. Catalytic distillation process

    Science.gov (United States)

    Smith, L.A. Jr.

    1982-06-22

    A method is described for conducting chemical reactions and fractionation of the reaction mixture comprising feeding reactants to a distillation column reactor into a feed zone and concurrently contacting the reactants with a fixed bed catalytic packing to concurrently carry out the reaction and fractionate the reaction mixture. For example, a method for preparing methyl tertiary butyl ether in high purity from a mixed feed stream of isobutene and normal butene comprising feeding the mixed feed stream to a distillation column reactor into a feed zone at the lower end of a distillation reaction zone, and methanol into the upper end of said distillation reaction zone, which is packed with a properly supported cationic ion exchange resin, contacting the C[sub 4] feed and methanol with the catalytic distillation packing to react methanol and isobutene, and concurrently fractionating the ether from the column below the catalytic zone and removing normal butene overhead above the catalytic zone.

  7. Catalytic distillation structure

    Science.gov (United States)

    Smith, L.A. Jr.

    1984-04-17

    Catalytic distillation structure is described for use in reaction distillation columns, and provides reaction sites and distillation structure consisting of a catalyst component and a resilient component intimately associated therewith. The resilient component has at least about 70 volume % open space and is present with the catalyst component in an amount such that the catalytic distillation structure consists of at least 10 volume % open space. 10 figs.

  8. Inhibition of ERBB2-overexpressing Tumors by Recombinant Human Prolidase and Its Enzymatically Inactive Mutant

    Directory of Open Access Journals (Sweden)

    Lu Yang

    2015-05-01

    Full Text Available ERBB2 is an oncogenic receptor tyrosine kinase overexpressed in a subset of human breast cancer and other cancers. We recently found that human prolidase (PEPD, a dipeptidase, is a high affinity ERBB2 ligand and cross-links two ERBB2 monomers. Here, we show that recombinant human PEPD (rhPEPD strongly inhibits ERBB2-overexpressing tumors in mice, whereas it does not impact tumors without ERBB2 overexpression. rhPEPD causes ERBB2 depletion, disrupts oncogenic signaling orchestrated by ERBB2 homodimers and heterodimers, and induces apoptosis. The impact of enzymatically-inactive mutant rhPEPDG278D on ERBB2 is indistinguishable from that of rhPEPD, but rhPEPDG278D is superior to rhPEPD for tumor inhibition. The enzymatic function of rhPEPD stimulates HIF-1α and other pro-survival factors in tumors, which likely attenuates its antitumor activity. rhPEPDG278D is also attractive in that it may not interfere with the physiologic function of endogenous PEPD in normal cells. Collectively, we have identified a human protein as an inhibitory ERBB2 ligand that inhibits ERBB2-overexpressing tumors in vivo. Several anti-ERBB2 agents are on the market but are hampered by drug resistance and high drug cost. rhPEPDG278D may synergize with these agents and may also be highly cost-effective, since it targets ERBB2 with a different mechanism and can be produced in bacteria.

  9. Evaluating the predictivity of virtual screening for ABL kinase inhibitors to hinder drug resistance

    DEFF Research Database (Denmark)

    Gani, Osman A B S M; Narayanan, Dilip; Engh, Richard A

    2013-01-01

    Virtual screening methods are now widely used in early stages of drug discovery, aiming to rank potential inhibitors. However, any practical ligand set (of active or inactive compounds) chosen for deriving new virtual screening approaches cannot fully represent all relevant chemical space for pot...... inactive ligands, even without assuming knowledge of the binding mode. We believe that this study can be extended to other therapeutically important kinases in prospective virtual screening studies....... for potential new compounds. In this study, we have taken a retrospective approach to evaluate virtual screening methods for the leukemia target kinase ABL1 and its drug-resistant mutant ABL1-T315I. 'Dual active' inhibitors against both targets were grouped together with inactive ligands chosen from different...

  10. Inositol hexakisphosphate kinase 1 (IP6K1) activity is required for cytoplasmic dynein-driven transport

    Science.gov (United States)

    Chanduri, Manasa; Rai, Ashim; Malla, Aushaq Bashir; Wu, Mingxuan; Fiedler, Dorothea; Mallik, Roop; Bhandari, Rashna

    2016-01-01

    Inositol pyrophosphates, such as diphosphoinositol pentakisphosphate (IP7), are conserved eukaryotic signaling molecules that possess pyrophosphate and monophosphate moieties. Generated predominantly by inositol hexakisphosphate kinases (IP6Ks), inositol pyrophosphates can modulate protein function by posttranslational serine pyrophosphorylation. Here, we report inositol pyrophosphates as novel regulators of cytoplasmic dynein-driven vesicle transport. Mammalian cells lacking IP6K1 display defects in dynein-dependent trafficking pathways, including endosomal sorting, vesicle movement, and Golgi maintenance. Expression of catalytically active but not inactive IP6K1 reverses these defects, suggesting a role for inositol pyrophosphates in these processes. Endosomes derived from slime mold lacking inositol pyrophosphates also display reduced dynein-directed microtubule transport. We demonstrate that Ser51 in the dynein intermediate chain (IC) is a target for pyrophosphorylation by IP7, and this modification promotes the interaction of the IC N-terminus with the p150Glued subunit of dynactin. IC–p150Glued interaction is decreased, and IC recruitment to membranes is reduced in cells lacking IP6K1. Our study provides the first evidence for the involvement of IP6Ks in dynein function and proposes that inositol pyrophosphate-mediated pyrophosphorylation may act as a regulatory signal to enhance dynein-driven transport. PMID:27474409

  11. Structure of the kinase domain of Gilgamesh from Drosophila melanogaster.

    Science.gov (United States)

    Han, Ni; Chen, CuiCui; Shi, Zhubing; Cheng, Dianlin

    2014-04-01

    The CK1 family kinases regulate multiple cellular aspects and play important roles in Wnt/Wingless and Hedgehog signalling. The kinase domain of Drosophila Gilgamesh isoform I (Gilgamesh-I), a homologue of human CK1-γ, was purified and crystallized. Crystals of methylated Gilgamesh-I kinase domain with a D210A mutation diffracted to 2.85 Å resolution and belonged to space group P43212, with unit-cell parameters a = b = 52.025, c = 291.727 Å. The structure of Gilgamesh-I kinase domain, which was determined by molecular replacement, has conserved catalytic elements and an active conformation. Structural comparison indicates that an extended loop between the α1 helix and the β4 strand exists in the Gilgamesh-I kinase domain. This extended loop may regulate the activity and function of Gilgamesh-I.

  12. A KIND OF INACTIVE TITANIUM SPECIES IN TITANIUM SILICALITE%钛硅沸石中的一种非活性钛物种

    Institute of Scientific and Technical Information of China (English)

    李钢; 郭新闻; 王祥生; 周卓华; 赵琦; 包信和; 韩秀文; 林励吾

    1999-01-01

    @@ Synthesis of the titanium silicalite TS-1 was first reported by Taramasso et al[1]in 1983. TS-1 has received considerable interest during the last decade because of its unique catalytic properties in oxidation reactions involving H2O2 as the oxidant. It is accepted that the extraframework Ti species in TS-1 favor the decomposition of H2O2and should be avoided. But in the present study, it was observed that there was a kind of Ti species inactive in both the oxidation reaction and the decomposition of H2O2.

  13. Feasibility study of home telerehabilitation for physically inactive veterans

    Directory of Open Access Journals (Sweden)

    Nancy D. Harada, PhD

    2010-07-01

    Full Text Available The aim of this study was to develop a system for and determine the feasibility of monitoring home exercise for physically inactive older adults using a Health Buddy (HB text messaging device (Robert Bosch Healthcare; Palo Alto, California. Questions and messages related to exercise adherence are displayed on the HB screen and participants choose a response by pressing the corresponding button on the device. Responses are transmitted through a landline connection and high-risk responses are highlighted by the system for follow-up. We developed the questions and messages based on input from patient and clinician focus groups. We evaluated feasibility by administering the intervention to inpatient and outpatient adults aged 60 or older. We gave participants a choice of exercise monitoring by HB (n = 20 or telephone (n = 18. The results showed that home exercise monitoring by HB and telephone is safe, as evidenced by low adverse event rates. We saw a decline in exercise adherence rates to both the HB and telephone after 8 weeks, although adherence was better for HB than telephone. Taken together, the results demonstrate the feasibility of using text messaging to monitor home exercise adherence in physically inactive older adults.

  14. Kepler Flares I. Active and Inactive M dwarfs

    CERN Document Server

    Hawley, Suzanne L; Kowalski, Adam F; Wisniewski, John P; Hebb, Leslie; Deitrick, Russell; Hilton, Eric J

    2014-01-01

    We analyzed Kepler short-cadence M dwarf observations. Spectra from the ARC 3.5m telescope identify magnetically active (H$\\alpha$ in emission) stars. The active stars are of mid-M spectral type, have numerous flares, and well-defined rotational modulation due to starspots. The inactive stars are of early-M type, exhibit less starspot signature, and have fewer flares. A Kepler to U-band energy scaling allows comparison of the Kepler flare frequency distributions with previous ground-based data. M dwarfs span a large range of flare frequency and energy, blurring the distinction between active and inactive stars designated solely by the presence of H$\\alpha$. We analyzed classical and complex (multiple peak) flares on GJ 1243, finding strong correlations between flare energy, amplitude, duration and decay time, with only a weak dependence on rise time. Complex flares last longer and have higher energy at the same amplitude, and higher energy flares are more likely to be complex. A power law fits the energy dist...

  15. Inactivity amplifies the catabolic response of skeletal muscle to cortisol

    Science.gov (United States)

    Ferrando, A. A.; Stuart, C. A.; Sheffield-Moore, M.; Wolfe, R. R.

    1999-01-01

    Severe injury or trauma is accompanied by both hypercortisolemia and prolonged inactivity or bed rest (BR). Trauma and BR alone each result in a loss of muscle nitrogen, albeit through different metabolic alterations. Although BR alone can result in a 2-3% loss of lean body mass, the effects of severe trauma can be 2- to 3-fold greater. We investigated the combined effects of hypercortisolemia and prolonged inactivity on muscle protein metabolism in healthy volunteers. Six males were studied before and after 14 days of strict BR using a model based on arteriovenous sampling and muscle biopsy. Fractional synthesis and breakdown rates of skeletal muscle protein were also directly calculated. Each assessment of protein metabolism was conducted during a 12-h infusion of hydrocortisone sodium succinate (120 microg/kg x h), resulting in blood cortisol concentrations that mimic severe injury (approximately 31 microg/dL). After 14 days of strict BR, hypercortisolemia increased phenylalanine efflux from muscle by 3-fold (P catabolic effects of hypercortisolemia. Furthermore, these effects on healthy volunteers are analogous to those seen after severe injury.

  16. Structure of the intact ATM/Tel1 kinase.

    Science.gov (United States)

    Wang, Xuejuan; Chu, Huanyu; Lv, Mengjuan; Zhang, Zhihui; Qiu, Shuwan; Liu, Haiyan; Shen, Xuetong; Wang, Weiwu; Cai, Gang

    2016-05-27

    The ataxia-telangiectasia mutated (ATM) protein is an apical kinase that orchestrates the multifaceted DNA-damage response. Normally, ATM kinase is in an inactive, homodimer form and is transformed into monomers upon activation. Besides a conserved kinase domain at the C terminus, ATM contains three other structural modules, referred to as FAT, FATC and N-terminal helical solenoid. Here we report the first cryo-EM structure of ATM kinase, which is an intact homodimeric ATM/Tel1 from Schizosaccharomyces pombe. We show that two monomers directly contact head-to-head through the FAT and kinase domains. The tandem N-terminal helical solenoid tightly packs against the FAT and kinase domains. The structure suggests that ATM/Tel1 dimer interface and the consecutive HEAT repeats inhibit the binding of kinase substrates and regulators by steric hindrance. Our study provides a structural framework for understanding the mechanisms of ATM/Tel1 regulation as well as the development of new therapeutic agents.

  17. Purification and characterization of a casein kinase 2-type protein kinase from pea nuclei

    Science.gov (United States)

    Li, H.; Roux, S. J.

    1992-01-01

    Almost all the polyamine-stimulated protein kinase activity associated with the chromatin fraction of nuclei purified from etiolated pea (Pisum sativum L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.35 molar NaCl. This protein kinase can be further purified over 2000-fold by salt fractionation and anion-exchange and casein-agarose column chromatography, after which it is more than 90% pure. The purified kinase has a specific activity of about 650 nanomoles per minute per milligram protein in the absence of polyamines, with either ATP or GTP as phosphoryl donor. Spermidine can stimulate its activity fourfold, with half-maximal activation at about 2 millimolar. Spermine and putrescine also stimulate activity, although somewhat less effectively. This kinase has a tetrameric alpha 2 beta 2 structure with a native molecular weight of 130,000, and subunit molecular weights of 36,000 for the catalytic subunit (alpha) and 29,000 for the regulatory subunit (beta). In western blot analyses, only the alpha subunit reacts strongly with polyclonal antibodies to a Drosophila casein kinase II. The pea kinase can use casein and phosvitin as artificial substrates, phosphorylating both the serine and threonine residues of casein. It has a pH optimum near 8.0, a Vmax of 1.5 micromoles per minute per milligram protein, and a Km for ATP of approximately 75 micromolar. Its activity can be almost completely inhibited by heparin at 5 micrograms per milliliter, but is relatively insensitive to concentrations of staurosporine, K252a, and chlorpromazine that strongly antagonize Ca(2+) -regulated protein kinases. These results are discussed in relation to recent findings that casein kinase 2-type kinases may phosphorylate trans-acting factors that bind to light-regulated promoters in plants.

  18. Crystal structures of human pyridoxal kinase in complex with the neurotoxins, ginkgotoxin and theophylline: insights into pyridoxal kinase inhibition.

    Directory of Open Access Journals (Sweden)

    Amit K Gandhi

    Full Text Available Several drugs and natural compounds are known to be highly neurotoxic, triggering epileptic convulsions or seizures, and causing headaches, agitations, as well as other neuronal symptoms. The neurotoxic effects of some of these compounds, including theophylline and ginkgotoxin, have been traced to their inhibitory activity against human pyridoxal kinase (hPL kinase, resulting in deficiency of the active cofactor form of vitamin B₆, pyridoxal 5'-phosphate (PLP. Pyridoxal (PL, an inactive form of vitamin B₆ is converted to PLP by PL kinase. PLP is the B₆ vitamer required as a cofactor for over 160 enzymatic activities essential in primary and secondary metabolism. We have performed structural and kinetic studies on hPL kinase with several potential inhibitors, including ginkgotoxin and theophylline. The structural studies show ginkgotoxin and theophylline bound at the substrate site, and are involved in similar protein interactions as the natural substrate, PL. Interestingly, the phosphorylated product of ginkgotoxin is also observed bound at the active site. This work provides insights into the molecular basis of hPL kinase inhibition and may provide a working hypothesis to quickly screen or identify neurotoxic drugs as potential hPL kinase inhibitors. Such adverse effects may be prevented by administration of an appropriate form of vitamin B₆, or provide clues of how to modify these drugs to help reduce their hPL kinase inhibitory effects.

  19. Crystal structure of human protein kinase CK2: insights into basic properties of the CK2 holoenzyme

    DEFF Research Database (Denmark)

    Niefind, K; Guerra, B; Ermakowa, I;

    2001-01-01

    subunits, which make no direct contact with one another. Each catalytic subunit interacts with both regulatory chains, predominantly via an extended C-terminal tail of the regulatory subunit. The CK2 structure is consistent with its constitutive activity and with a flexible role of the regulatory subunit......The crystal structure of a fully active form of human protein kinase CK2 (casein kinase 2) consisting of two C-terminally truncated catalytic and two regulatory subunits has been determined at 3.1 A resolution. In the CK2 complex the regulatory subunits form a stable dimer linking the two catalytic...... as a docking partner for various protein kinases. Furthermore it shows an inter-domain mobility in the catalytic subunit known to be functionally important in protein kinases and detected here for the first time directly within one crystal structure....

  20. Characterization of G-protein coupled receptor kinase interaction with the neurokinin-1 receptor using bioluminescence resonance energy transfer

    DEFF Research Database (Denmark)

    Jorgensen, Rasmus; Holliday, Nicholas D; Hansen, Jakob L

    2007-01-01

    To analyze the interaction between the neurokinin-1 (NK-1) receptor and G-protein coupled receptor kinases (GRKs), we performed bioluminescence resonance energy transfer(2) (BRET(2)) measurements between the family A NK-1 receptor and GRK2 and GRK5 as well as their respective kinase-inactive muta......To analyze the interaction between the neurokinin-1 (NK-1) receptor and G-protein coupled receptor kinases (GRKs), we performed bioluminescence resonance energy transfer(2) (BRET(2)) measurements between the family A NK-1 receptor and GRK2 and GRK5 as well as their respective kinase...

  1. The N-terminal domain of the tomato immune protein Prf contains multiple homotypic and Pto kinase interaction sites.

    Science.gov (United States)

    Saur, Isabel Marie-Luise; Conlan, Brendon Francis; Rathjen, John Paul

    2015-05-01

    Resistance to Pseudomonas syringae bacteria in tomato (Solanum lycopersicum) is conferred by the Prf recognition complex, composed of the nucleotide-binding leucine-rich repeats protein Prf and the protein kinase Pto. The complex is activated by recognition of the P. syringae effectors AvrPto and AvrPtoB. The N-terminal domain is responsible for Prf homodimerization, which brings two Pto kinases into close proximity and holds them in inactive conformation in the absence of either effector. Negative regulation is lost by effector binding to the catalytic cleft of Pto, leading to disruption of its P+1 loop within the activation segment. This change is translated through Prf to a second Pto molecule in the complex. Here we describe a schematic model of the unique Prf N-terminal domain dimer and its interaction with the effector binding determinant Pto. Using heterologous expression in Nicotiana benthamiana, we define multiple sites of N domain homotypic interaction and infer that it forms a parallel dimer folded centrally to enable contact between the N and C termini. Furthermore, we found independent binding sites for Pto at either end of the N-terminal domain. Using the constitutively active mutant ptoL205D, we identify a potential repression site for Pto in the first ∼100 amino acids of Prf. Finally, we find that the Prf leucine-rich repeats domain also binds the N-terminal region, highlighting a possible mechanism for transfer of the effector binding signal to the NB-LRR regulatory unit (consisting of a central nucleotide binding and C-terminal leucine-rich repeats).

  2. Social background, bullying, and physical inactivity: National study of 11- to 15-year-olds.

    Science.gov (United States)

    Henriksen, P W; Rayce, S B; Melkevik, O; Due, P; Holstein, B E

    2016-10-01

    More children from lower social backgrounds are physically inactive than those from higher ones. We studied whether bullying was a mediating factor between lower social background and physical inactivity. We also examined the combined effect of low social class and exposure to bullying on physical inactivity. The Danish sample of the Health Behaviour in School-aged Children (HBSC) study 2006 included 6269 schoolchildren in three age groups: 11-, 13-, and 15-year-olds from a random sample of 80 schools. The students answered the internationally standardized HBSC questionnaire. The applied definition leaves 4.0% in the category physically inactive. The sex and age-adjusted OR (95% CI) for physical inactivity was 2.10 (1.39-3.18) among students with low social class and unclassifiable 3.53 (2.26-5.53). Exposure to bullying was associated with physical inactivity, sex and age-adjusted OR = 2.39 (1.67-3.41). Exposure to bullying did not explain the association between social class and physical inactivity. The association between social class and physical inactivity was more pronounced among participants also exposed to bullying. In conclusion, there was a significantly increased odds ratio for physical inactivity among students from lower social classes and for students exposed to bullying. There was a combined effect of low social class and bullying on physical inactivity.

  3. Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET

    Science.gov (United States)

    Perdios, Louis; Lowe, Alan R.; Saladino, Giorgio; Bunney, Tom D.; Thiyagarajan, Nethaji; Alexandrov, Yuriy; Dunsby, Christopher; French, Paul M. W.; Chin, Jason W.; Gervasio, Francesco Luigi; Tate, Edward W.; Katan, Matilda

    2017-01-01

    Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo.

  4. Conformational transition of FGFR kinase activation revealed by site-specific unnatural amino acid reporter and single molecule FRET

    Science.gov (United States)

    Perdios, Louis; Lowe, Alan R.; Saladino, Giorgio; Bunney, Tom D.; Thiyagarajan, Nethaji; Alexandrov, Yuriy; Dunsby, Christopher; French, Paul M. W.; Chin, Jason W.; Gervasio, Francesco Luigi; Tate, Edward W.; Katan, Matilda

    2017-01-01

    Protein kinases share significant structural similarity; however, structural features alone are insufficient to explain their diverse functions. Thus, bridging the gap between static structure and function requires a more detailed understanding of their dynamic properties. For example, kinase activation may occur via a switch-like mechanism or by shifting a dynamic equilibrium between inactive and active states. Here, we utilize a combination of FRET and molecular dynamics (MD) simulations to probe the activation mechanism of the kinase domain of Fibroblast Growth Factor Receptor (FGFR). Using genetically-encoded, site-specific incorporation of unnatural amino acids in regions essential for activation, followed by specific labeling with fluorescent moieties, we generated a novel class of FRET-based reporter to monitor conformational differences corresponding to states sampled by non phosphorylated/inactive and phosphorylated/active forms of the kinase. Single molecule FRET analysis in vitro, combined with MD simulations, shows that for FGFR kinase, there are populations of inactive and active states separated by a high free energy barrier resulting in switch-like activation. Compared to recent studies, these findings support diversity in features of kinases that impact on their activation mechanisms. The properties of these FRET-based constructs will also allow further studies of kinase dynamics as well as applications in vivo. PMID:28045057

  5. Modeling the Relationship Between Social Network Activity, Inactivity, and Growth

    CERN Document Server

    Ribeiro, Bruno

    2013-01-01

    Online Social Networks (OSNs) are multi-billion dollar enterprises. Surprisingly, little is known about the mechanisms that drive them to growth, stability, or death. This study sheds light on these mechanisms. We are particularly interested in OSNs where current subscribers can invite new users to join the network (e.g., Facebook, LinkedIn). Measuring the relationship between subscriber activity and network growth of a large OSN over five years, we formulate three hypotheses that together describe the observed OSN subscriber behavior. We then provide a model (and extensions) that simultaneously satisfies all three hypotheses. Our model provides deep insights into the dynamics of subscriber activity, inactivity, and network growth rates, even predicting four types of OSNs with respect to subscriber activity evolution. Finally, we present activity data of nearly thirty OSN websites, measured over five years, and show that the observed activity is well described by one of the four activity time series predicted...

  6. TALE proteins bind to both active and inactive chromatin.

    Science.gov (United States)

    Scott, James N F; Kupinski, Adam P; Kirkham, Christopher M; Tuma, Roman; Boyes, Joan

    2014-02-15

    TALE (transcription activator-like effector) proteins can be tailored to bind to any DNA sequence of choice and thus are of immense utility for genome editing and the specific delivery of transcription activators. However, to perform these functions, they need to occupy their sites in chromatin. In the present study, we have systematically assessed TALE binding to chromatin substrates and find that in vitro TALEs bind to their target site on nucleosomes at the more accessible entry/exit sites, but not at the nucleosome dyad. We show further that in vivo TALEs bind to transcriptionally repressed chromatin and that transcription increases binding by only 2-fold. These data therefore imply that TALEs are likely to bind to their target in vivo even at inactive loci.

  7. Helicobacter pylori CagA Inhibits PAR1-MARK Family Kinases by Mimicking Host Substrates

    Energy Technology Data Exchange (ETDEWEB)

    Nesic, D.; Miller, M; Quinkert, Z; Stein, M; Chait, B; Stebbins, C

    2010-01-01

    The CagA protein of Helicobacter pylori interacts with numerous cellular factors and is associated with increased virulence and risk of gastric carcinoma. We present here the cocrystal structure of a subdomain of CagA with the human kinase PAR1b/MARK2, revealing that a CagA peptide mimics substrates of this kinase family, resembling eukaryotic protein kinase inhibitors. Mutagenesis of conserved residues central to this interaction renders CagA inactive as an inhibitor of MARK2.

  8. Catalytic hydrotreating process

    Science.gov (United States)

    Karr, Jr., Clarence; McCaskill, Kenneth B.

    1978-01-01

    Carbonaceous liquids boiling above about 300.degree. C such as tars, petroleum residuals, shale oils and coal-derived liquids are catalytically hydrotreated by introducing the carbonaceous liquid into a reaction zone at a temperature in the range of 300.degree. to 450.degree. C and a pressure in the range of 300 to 4000 psig for effecting contact between the carbonaceous liquid and a catalytic transition metal sulfide in the reaction zone as a layer on a hydrogen permeable transition metal substrate and then introducing hydrogen into the reaction zone by diffusing the hydrogen through the substrate to effect the hydrogenation of the carbonaceous liquid in the presence of the catalytic sulfide layer.

  9. Protein kinase CK2 in health and disease: Protein kinase CK2: from structures to insights

    DEFF Research Database (Denmark)

    Niefind, K; Raaf, J; Issinger, Olaf-Georg

    2009-01-01

    Within the last decade, 40 crystal structures corresponding to protein kinase CK2 (former name 'casein kinase 2'), to its catalytic subunit CK2alpha and to its regulatory subunit CK2beta were published. Together they provide a valuable, yet by far not complete basis to rationalize the biochemical...... features of the enzyme, such as its constitutive activity, acidophilic substrate specificity, dual-cosubstrate specificity and its heterotetrameric quarternary structure. Comprehensive sets of structural superimpositions reveal that both CK2alpha and CK2beta are relatively rigid molecules. In CK2beta...... the critical region of CK2alpha recruitment is pre-formed in the unbound state. In CK2alpha the activation segment - a key element of protein kinase regulation - adapts invariably the typical conformation of the active enzymes. Recent structures of human CK2alpha revealed a surprising plasticity in the ATP...

  10. Differential membrane fluidization by active and inactive cannabinoid analogues.

    Science.gov (United States)

    Mavromoustakos, T; Papahatjis, D; Laggner, P

    2001-06-06

    The effects of the two cannabinomimetic drugs (-)-2-(6a,7,10,10a-tetrahydro-6,6,9-trimethyl-1-hydroxy-6H-dibenzo[b,d]pyranyl-2-(hexyl)-1,3-dithiolane (AMG-3) and its pharmacologically less active 1-methoxy analogue (AMG-18) on the thermotropic and structural properties of dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC) liposomes have been studied by X-ray diffraction and differential scanning calorimetry (DSC). DSC data revealed that the incorporation of the drugs affect differently the thermotropic properties of DPPC. The presence of the more active drug distinctly broadened and attenuated both the pretransition and main phase transition of DPPC bilayers, while the inactive analogue had only minor effects. Small and wide angle X-ray diffraction data showed that the two cannabinoids have different effects on the lipid phase structures and on the hydrocarbon chain packing. The pharmacologically active analogue, AMG-3, was found to efficiently fluidize domains of the lipids in the L(beta)' gel phase, and to perturb the regular multibilayer lattice. In the liquid crystalline L(alpha) phase, AMG-3 was also found to cause irregularities in packing, suggesting that the drug induces local curvature. At the same concentration, the inactive AMG-18 had only minor structural effects on the lipids. At about 10-fold or higher concentrations, AMG-18 was found to produce similar but still less pronounced effects in comparison to those observed by AMG-3. The dose-dependent, different thermotropic and structural effects by the two cannabinoid analogues suggest that these may be related to their biological activity.

  11. Abl family kinases regulate FcγR-mediated phagocytosis in murine macrophages.

    Science.gov (United States)

    Greuber, Emileigh K; Pendergast, Ann Marie

    2012-12-01

    Phagocytosis of Ab-coated pathogens is mediated through FcγRs, which activate intracellular signaling pathways to drive actin cytoskeletal rearrangements. Abl and Arg define a family of nonreceptor tyrosine kinases that regulate actin-dependent processes in a variety of cell types, including those important in the adaptive immune response. Using pharmacological inhibition as well as dominant negative and knockout approaches, we demonstrate a role for the Abl family kinases in phagocytosis by macrophages and define a mechanism whereby Abl kinases regulate this process. Bone marrow-derived macrophages from mice lacking Abl and Arg kinases exhibit inefficient phagocytosis of sheep erythrocytes and zymosan particles. Treatment with the Abl kinase inhibitors imatinib and GNF-2 or overexpression of kinase-inactive forms of the Abl family kinases also impairs particle internalization in murine macrophages, indicating Abl kinase activity is required for efficient phagocytosis. Further, Arg kinase is present at the phagocytic cup, and Abl family kinases are activated by FcγR engagement. The regulation of phagocytosis by Abl family kinases is mediated in part by the spleen tyrosine kinase (Syk). Loss of Abl and Arg expression or treatment with Abl inhibitors reduced Syk phosphorylation in response to FcγR ligation. The link between Abl family kinases and Syk may be direct, as purified Arg kinase phosphorylates Syk in vitro. Further, overexpression of membrane-targeted Syk in cells treated with Abl kinase inhibitors partially rescues the impairment in phagocytosis. Together, these findings reveal that Abl family kinases control the efficiency of phagocytosis in part through the regulation of Syk function.

  12. Heterologous expression and catalytic properties of the C-terminal domain of starfish cdc25 dual-specificity phosphatase, a cell cycle regulator.

    Science.gov (United States)

    Deshimaru, Shungo; Miyake, Yasuo; Ohmiya, Tadamasa; Tatsu, Yoshiro; Endo, Yasuko; Yumoto, Noboru; Toraya, Tetsuo

    2002-05-01

    The 3'-terminal region of starfish Asterina pectinifera cdc25 cDNA encoding the C-terminal catalytic domain was overexpressed in Escherichia coli. The C-terminal domain consisted of 226 amino acid residues containing the signature motif HCxxxxxR, a motif highly conserved among protein tyrosine and dual-specificity phosphatases, and showed phosphatase activity toward p-nitrophenyl phosphate. The enzyme activity was strongly inhibited by SH inhibitors. Mutational studies indicated that the cysteine and arginine residues in the conserved motif are essential for activity, but the histidine residue is not. These results suggest that the enzyme catalyzes the reaction through a two-step mechanism involving a phosphocysteine intermediate like in the cases of other protein tyrosine and dual-specificity phosphatases. The C-terminal domain of Cdc25 activated the histone H1 kinase activity of the purified, inactive form of Cdc2.cyclin B complex (preMPF) from extracts of immature starfish oocytes. Synthetic diphosphorylated di- to nonadecapeptides mimicking amino acid sequences around the dephosphorylation site of Cdc2 still retained substrate activity. Phosphotyrosine and phosphothreonine underwent dephosphorylation in this order. This is the reverse order to that reported for the in vivo and in vitro dephosphorylation of preMPF. Monophosphopeptides having the same sequence served as much poorer substrates. As judged from the results with synthetic phosphopeptides, the presence of two phosphorylated residues was important for specific recognition of substrates by the Cdc25 phosphatase.

  13. Giardia lamblia Nek1 and Nek2 kinases affect mitosis and excystation.

    Science.gov (United States)

    Smith, Alias J; Lauwaet, Tineke; Davids, Barbara J; Gillin, Frances D

    2012-04-01

    The NIMA-related serine/threonine kinases (Neks) function in the cell cycle and regulate ciliary and flagellar length. The Giardia lamblia genome encodes 198 Neks, of which 56 are predicted to be active. Here we believe that we report the first functional analysis of two G. lamblia Neks. The GlNek1 and GlNek2 kinase domains share 57% and 43% identity to the kinase domains of human Nek1 and Nek2, respectively. Both GlNeks are active in vitro, have dynamic relocalisation during the cell cycle, and are expressed throughout the life cycle, with GlNek1 being upregulated in cysts. Over-expression of inactive GlNek1 delays disassembly of the parental attachment disc and cytokinesis, whilst over-expression of either wild type GlNek1 or inactive mutant GlNek2 inhibits excystation.

  14. Autophosphorylation properties of inactive and active JNK2.

    Science.gov (United States)

    Pimienta, Genaro; Ficarro, Scott B; Gutierrez, Gustavo J; Bhoumik, Anindita; Peters, Eric C; Ronai, Ze'ev; Pascual, Jaime

    2007-07-15

    The c-Jun N-terminal kinases (JNKs) are ubiquitous proteins that phosphorylate their substrates, such as transcription factors, in response to physical stress, cytokines or UV radiation. This leads to changes in gene expression, ensuing either cell cycle progression or apoptosis. Active phospho JNK1 is the main in vivo kinase component of the JNK cascade, whereas JNK2 is presumed not to participate as a kinase during JNK signalling. However, there is evidence that JNK isoforms interact functionally in vivo. Also, a recent chemical genetics investigation has confirmed that JNK transient activation leads to cellular proliferation, whereas a sustained one is pro-apoptotic. Here we investigate the phosphorylation pattern of JNK2, with protein biochemistry tools and tandem mass spectrometry. We choose to focus on JNK2 because of its reported constitutive activity in glioma cells. Our results indicate that purified JNK2 from transfected nonstressed 293T cells is a mixture of the mono-sites pThr183 and pTyr185 of its activation loop and of pThr386 along its unique C-terminal region. Upon UV stimulation, its phosphorylation stoichiometry is upregulated on the activation loop, generating a mixture of mono-pTyr185 and the expected dual-pThr183/pTyr185 species, with the pThr386 specie present but unaltered respect to the basal conditions.

  15. Dynamic Allostery Mediated by a Conserved Tryptophan in the Tec Family Kinases.

    Directory of Open Access Journals (Sweden)

    Nikita Chopra

    2016-03-01

    Full Text Available Bruton's tyrosine kinase (Btk is a Tec family non-receptor tyrosine kinase that plays a critical role in immune signaling and is associated with the immunological disorder X-linked agammaglobulinemia (XLA. Our previous findings showed that the Tec kinases are allosterically activated by the adjacent N-terminal linker. A single tryptophan residue in the N-terminal 17-residue linker mediates allosteric activation, and its mutation to alanine leads to the complete loss of activity. Guided by hydrogen/deuterium exchange mass spectrometry results, we have employed Molecular Dynamics simulations, Principal Component Analysis, Community Analysis and measures of node centrality to understand the details of how a single tryptophan mediates allostery in Btk. A specific tryptophan side chain rotamer promotes the functional dynamic allostery by inducing coordinated motions that spread across the kinase domain. Either a shift in the rotamer population, or a loss of the tryptophan side chain by mutation, drastically changes the coordinated motions and dynamically isolates catalytically important regions of the kinase domain. This work also identifies a new set of residues in the Btk kinase domain with high node centrality values indicating their importance in transmission of dynamics essential for kinase activation. Structurally, these node residues appear in both lobes of the kinase domain. In the N-lobe, high centrality residues wrap around the ATP binding pocket connecting previously described Catalytic-spine residues. In the C-lobe, two high centrality node residues connect the base of the R- and C-spines on the αF-helix. We suggest that the bridging residues that connect the catalytic and regulatory architecture within the kinase domain may be a crucial element in transmitting information about regulatory spine assembly to the catalytic machinery of the catalytic spine and active site.

  16. Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases

    Energy Technology Data Exchange (ETDEWEB)

    Soon, Fen-Fen; Ng, Ley-Moy; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Tan, M.H. Eileen; Suino-Powell, Kelly M.; He, Yuanzheng; Xu, Yong; Chalmers, Michael J.; Brunzelle, Joseph S.; Zhang, Huiming; Yang, Huaiyu; Jiang, Hualiang; Li, Jun; Yong, Eu-Leong; Cutler, Sean; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric (Van Andel); (Scripps); (NWU); (Purdue); (UCR); (Chinese Aca. Sci.); (NU Singapore)

    2014-10-02

    Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, while the conserved ABA-sensing tryptophan of PP2C inserts into the kinase catalytic cleft, thus mimicking receptor-PP2C interactions. These structural results provide a simple mechanism that directly couples ABA binding to SnRK2 kinase activation and highlight a new paradigm of kinase-phosphatase regulation through mutual packing of their catalytic sites.

  17. Catalytic efficiency of designed catalytic proteins.

    Science.gov (United States)

    Korendovych, Ivan V; DeGrado, William F

    2014-08-01

    The de novo design of catalysts that mimic the affinity and specificity of natural enzymes remains one of the Holy Grails of chemistry. Despite decades of concerted effort we are still unable to design catalysts as efficient as enzymes. Here we critically evaluate approaches to (re)design of novel catalytic function in proteins using two test cases: Kemp elimination and ester hydrolysis. We show that the degree of success thus far has been modest when the rate enhancements seen for the designed proteins are compared with the rate enhancements by small molecule catalysts in solvents with properties similar to the active site. Nevertheless, there are reasons for optimism: the design methods are ever improving and the resulting catalyst can be efficiently improved using directed evolution.

  18. Catalytic Phosphination and Arsination

    Institute of Scientific and Technical Information of China (English)

    Kwong Fuk Yee; Chan Kin Shing

    2004-01-01

    The catalytic, user-friendly phosphination and arsination of aryl halides and triflates by triphenylphosphine and triphenylarsine using palladium catalysts have provided a facile synthesis of functionalized aryl phosphines and arsines in neutral media. Modification of the cynaoarisne yielded optically active N, As ligands which will be screened in various asymmetric catalysis.

  19. Functional diversity of human protein kinase splice variants marks significant expansion of human kinome

    Directory of Open Access Journals (Sweden)

    Anamika Krishanpal

    2009-12-01

    Full Text Available Abstract Background Protein kinases are involved in diverse spectrum of cellular processes. Availability of draft version of the human genomic data in the year 2001 enabled recognition of repertoire of protein kinases. However, over the years the human genomic data is being refined and the current release of human genomic data has helped us to recognize a larger repertoire of over 900 human protein kinases represented mainly by splice variants. Results Many of these identified protein kinases are alternatively spliced products. Interestingly, some of the human kinase splice variants appear to be significantly diverged in terms of their functional properties as represented by incorporation or absence of one or more domains. Many sets of protein kinase splice variants have substantially different domain organization and in a few sets of splice variants kinase domains belong to different subfamilies of kinases suggesting potential participation in different signal transduction pathways. Conclusions Addition or deletion of a domain between splice variants of multi-domain kinases appears to be a means of generating differences in the functional features of otherwise similar kinases. It is intriguing that marked sequence diversity within the catalytic regions of some of the splice variant kinases result in kinases belonging to different subfamilies. These human kinase splice variants with different functions might contribute to diversity of eukaryotic cellular signaling.

  20. Catalytic and non-catalytic roles for the mono-ADP-ribosyltransferase Arr in the mycobacterial DNA damage response.

    Science.gov (United States)

    Stallings, Christina L; Chu, Linda; Li, Lucy X; Glickman, Michael S

    2011-01-01

    Recent evidence indicates that the mycobacterial response to DNA double strand breaks (DSBs) differs substantially from previously characterized bacteria. These differences include the use of three DSB repair pathways (HR, NHEJ, SSA), and the CarD pathway, which integrates DNA damage with transcription. Here we identify a role for the mono-ADP-ribosyltransferase Arr in the mycobacterial DNA damage response. Arr is transcriptionally induced following DNA damage and cellular stress. Although Arr is not required for induction of a core set of DNA repair genes, Arr is necessary for suppression of a set of ribosomal protein genes and rRNA during DNA damage, placing Arr in a similar pathway as CarD. Surprisingly, the catalytic activity of Arr is not required for this function, as catalytically inactive Arr was still able to suppress ribosomal protein and rRNA expression during DNA damage. In contrast, Arr substrate binding and catalytic activities were required for regulation of a small subset of other DNA damage responsive genes, indicating that Arr has both catalytic and noncatalytic roles in the DNA damage response. Our findings establish an endogenous cellular function for a mono-ADP-ribosyltransferase apart from its role in mediating Rifampin resistance.

  1. Catalytic and non-catalytic roles for the mono-ADP-ribosyltransferase Arr in the mycobacterial DNA damage response.

    Directory of Open Access Journals (Sweden)

    Christina L Stallings

    Full Text Available Recent evidence indicates that the mycobacterial response to DNA double strand breaks (DSBs differs substantially from previously characterized bacteria. These differences include the use of three DSB repair pathways (HR, NHEJ, SSA, and the CarD pathway, which integrates DNA damage with transcription. Here we identify a role for the mono-ADP-ribosyltransferase Arr in the mycobacterial DNA damage response. Arr is transcriptionally induced following DNA damage and cellular stress. Although Arr is not required for induction of a core set of DNA repair genes, Arr is necessary for suppression of a set of ribosomal protein genes and rRNA during DNA damage, placing Arr in a similar pathway as CarD. Surprisingly, the catalytic activity of Arr is not required for this function, as catalytically inactive Arr was still able to suppress ribosomal protein and rRNA expression during DNA damage. In contrast, Arr substrate binding and catalytic activities were required for regulation of a small subset of other DNA damage responsive genes, indicating that Arr has both catalytic and noncatalytic roles in the DNA damage response. Our findings establish an endogenous cellular function for a mono-ADP-ribosyltransferase apart from its role in mediating Rifampin resistance.

  2. Effect of physical inactivity on major non-communicable diseases worldwide

    DEFF Research Database (Denmark)

    Lee, I-Min; Shiroma, Eric J; Lobelo, Felipe;

    2012-01-01

    Strong evidence shows that physical inactivity increases the risk of many adverse health conditions, including major non-communicable diseases such as coronary heart disease, type 2 diabetes, and breast and colon cancers, and shortens life expectancy. Because much of the world's population...... is inactive, this link presents a major public health issue. We aimed to quantify the eff ect of physical inactivity on these major non-communicable diseases by estimating how much disease could be averted if inactive people were to become active and to estimate gain in life expectancy at the population level....

  3. Structural organization of the inactive X chromosome in the mouse.

    Science.gov (United States)

    Giorgetti, Luca; Lajoie, Bryan R; Carter, Ava C; Attia, Mikael; Zhan, Ye; Xu, Jin; Chen, Chong Jian; Kaplan, Noam; Chang, Howard Y; Heard, Edith; Dekker, Job

    2016-07-28

    X-chromosome inactivation (XCI) involves major reorganization of the X chromosome as it becomes silent and heterochromatic. During female mammalian development, XCI is triggered by upregulation of the non-coding Xist RNA from one of the two X chromosomes. Xist coats the chromosome in cis and induces silencing of almost all genes via its A-repeat region, although some genes (constitutive escapees) avoid silencing in most cell types, and others (facultative escapees) escape XCI only in specific contexts. A role for Xist in organizing the inactive X (Xi) chromosome has been proposed. Recent chromosome conformation capture approaches have revealed global loss of local structure on the Xi chromosome and formation of large mega-domains, separated by a region containing the DXZ4 macrosatellite. However, the molecular architecture of the Xi chromosome, in both the silent and expressed regions,remains unclear. Here we investigate the structure, chromatin accessibility and expression status of the mouse Xi chromosome in highly polymorphic clonal neural progenitors (NPCs) and embryonic stem cells. We demonstrate a crucial role for Xist and the DXZ4-containing boundary in shaping Xi chromosome structure using allele-specific genome-wide chromosome conformation capture (Hi-C) analysis, an assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) and RNA sequencing. Deletion of the boundary disrupts mega-domain formation, and induction of Xist RNA initiates formation of the boundary and the loss of DNA accessibility. We also show that in NPCs, the Xi chromosome lacks active/inactive compartments and topologically associating domains (TADs), except around genes that escape XCI. Escapee gene clusters display TAD-like structures and retain DNA accessibility at promoter-proximal and CTCF-binding sites. Furthermore, altered patterns of facultative escape genes indifferent neural progenitor clones are associated with the presence of different TAD

  4. Mitsunobu Reactions Catalytic in Phosphine and a Fully Catalytic System.

    Science.gov (United States)

    Buonomo, Joseph A; Aldrich, Courtney C

    2015-10-26

    The Mitsunobu reaction is renowned for its mild reaction conditions and broad substrate tolerance, but has limited utility in process chemistry and industrial applications due to poor atom economy and the generation of stoichiometric phosphine oxide and hydrazine by-products that complicate purification. A catalytic Mitsunobu reaction using innocuous reagents to recycle these by-products would overcome both of these shortcomings. Herein we report a protocol that is catalytic in phosphine (1-phenylphospholane) employing phenylsilane to recycle the catalyst. Integration of this phosphine catalytic cycle with Taniguchi's azocarboxylate catalytic system provided the first fully catalytic Mitsunobu reaction.

  5. Engineering and Functional Analysis of Mitotic Kinases Through Chemical Genetics.

    Science.gov (United States)

    Jones, Mathew J K; Jallepalli, Prasad V

    2016-01-01

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

  6. Molecular mechanism of regulation of the atypical protein kinase C by N-terminal domains and an allosteric small compound

    DEFF Research Database (Denmark)

    Zhang, Hua; Neimanis, Sonja; Lopez-Garcia, Laura A;

    2014-01-01

    Protein kinases play important regulatory roles in cells and organisms. Therefore, they are subject to specific and tight mechanisms of regulation that ultimately converge on the catalytic domain and allow the kinases to be activated or inhibited only upon the appropriate stimuli. AGC protein kin...

  7. Network social capital, social participation, and physical inactivity in an urban adult population.

    Science.gov (United States)

    Legh-Jones, Hannah; Moore, Spencer

    2012-05-01

    Research on individual social capital and physical activity has tended to focus on the association among physical activity, generalized trust, and social participation. Less is known about the association between network social capital, i.e., the resources accessed through one's social connections, and physical inactivity. Using formal network measures of social capital, this study examined which specific dimension of network capital (i.e. diversity, reach and range) was associated with physical inactivity, and whether network social capital mediated the association between physical inactivity and social participation. Data came from the 2008 Montreal (Canada) Neighbourhood Networks and Healthy Aging survey, in which 2707 adults 25 years and older in 300 Montreal neighbourhoods were surveyed. Physical activity was self-reported using the International Physical Activity Questionnaire (IPAQ). IPAQ guidelines provided the basis for the physical inactivity cutoff. Network social capital was measured with a position generator instrument. Multilevel logistic methods were used to examine the association between physical inactivity and individual social capital dimensions, while adjusting for socio-demographic and -economic factors. Higher network diversity was associated with a decreased likelihood of physical inactivity. Consistent with previous findings, individuals who did not participate in any formal associations were more likely to be physically inactive compared to those with high levels of participation. Network diversity mediated the association between physical inactivity and participation. Generalized trust and the network components of reach and range were not shown associated with physical inactivity. Findings highlight the importance of social participation and network social capital and the added value of network measures in the study of social capital and physical inactivity. Population-based programs targeting physical inactivity among adults might

  8. Structural basis for morpheein-type allosteric regulation of Escherichia coli glucosamine-6-phosphate synthase: equilibrium between inactive hexamer and active dimer.

    Science.gov (United States)

    Mouilleron, Stéphane; Badet-Denisot, Marie-Ange; Pecqueur, Ludovic; Madiona, Karine; Assrir, Nadine; Badet, Bernard; Golinelli-Pimpaneau, Béatrice

    2012-10-01

    The amino-terminal cysteine of glucosamine-6-phosphate synthase (GlmS) acts as a nucleophile to release and transfer ammonia from glutamine to fructose 6-phosphate through a channel. The crystal structure of the C1A mutant of Escherichia coli GlmS, solved at 2.5 Å resolution, is organized as a hexamer, where the glutaminase domains adopt an inactive conformation. Although the wild-type enzyme is active as a dimer, size exclusion chromatography, dynamic and quasi-elastic light scattering, native polyacrylamide gel electrophoresis, and ultracentrifugation data show that the dimer is in equilibrium with a hexameric state, in vitro and in cellulo. The previously determined structures of the wild-type enzyme, alone or in complex with glucosamine 6-phosphate, are also consistent with a hexameric assembly that is catalytically inactive because the ammonia channel is not formed. The shift of the equilibrium toward the hexameric form in the presence of cyclic glucosamine 6-phosphate, together with the decrease of the specific activity with increasing enzyme concentration, strongly supports product inhibition through hexamer stabilization. Altogether, our data allow us to propose a morpheein model, in which the active dimer can rearrange into a transiently stable form, which has the propensity to form an inactive hexamer. This would account for a physiologically relevant allosteric regulation of E. coli GlmS. Finally, in addition to cyclic glucose 6-phosphate bound at the active site, the hexameric organization of E. coli GlmS enables the binding of another linear sugar molecule. Targeting this sugar-binding site to stabilize the inactive hexameric state is therefore suggested for the development of specific antibacterial inhibitors.

  9. Ras CAAX peptidomimetic FTI-277 selectively blocks oncogenic Ras signaling by inducing cytoplasmic accumulation of inactive Ras-Raf complexes.

    Science.gov (United States)

    Lerner, E C; Qian, Y; Blaskovich, M A; Fossum, R D; Vogt, A; Sun, J; Cox, A D; Der, C J; Hamilton, A D; Sebti, S M

    1995-11-10

    Ras-induced malignant transformation requires Ras farnesylation, a lipid posttranslational modification catalyzed by farnesyltransferase (FTase). Inhibitors of this enzyme have been shown to block Ras-dependent transformation, but the mechanism by which this occurs remains largely unknown. We have designed FTI-276, a peptide mimetic of the COOH-terminal Cys-Val-Ile-Met of K-Ras4B that inhibited potently FTase in vitro (IC50 = 500 pM) and was highly selective for FTase over geranylgeranyltransferase I (GGTase I) (IC50 = 50 nM). FTI-277, the methyl ester derivative of FTI-276, was extremely potent (IC50 = 100 nM) at inhibiting H-Ras, but not the geranylgeranylated Rap1A processing in whole cells. Treatment of H-Ras oncogene-transformed NIH 3T3 cells with FTI-277 blocked recruitment to the plasma membrane and subsequent activation of the serine/threonine kinase c-Raf-1 in cells transformed by farnesylated Ras (H-RasF), but not geranylgeranylated, Ras (H-RasGG). FTI-277 induced accumulation of cytoplasmic non-farnesylated H-Ras that was able to bind Raf and form cytoplasmic Ras/Raf complexes in which Raf kinase was not activated. Furthermore, FTI-277 blocked constitutive activation of mitogen-activated protein kinase (MAPK) in H-RasF, but not H-RasGG, or Raf-transformed cells. FTI-277 also inhibited oncogenic K-Ras4B processing and constitutive activation of MAPK, but the concentrations required were 100-fold higher than those needed for H-Ras inhibition. The results demonstrate that FTI-277 blocks Ras oncogenic signaling by accumulating inactive Ras/Raf complexes in the cytoplasm, hence preventing constitutive activation of the MAPK cascade.

  10. Thymidine kinases in archaea

    DEFF Research Database (Denmark)

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

    2006-01-01

    Twenty-six fully sequenced archaeal genomes were searched for genes coding for putative deoxyribonucleoside kinases (dNKs). We identified only 5 human-like thymidine kinase 1 genes (TK1s) and none for non-TK1 kinases. Four TK1s were identified in the Euryarchaea and one was found in the Crenarchaea...... that a functional deoxyribonucleoside salvage pathway is not crucial for the archaeal cell....

  11. Associations of unhealthy lifestyle factors with sexual inactivity and sexual dysfunctions in Denmark

    DEFF Research Database (Denmark)

    Christensen, Birgitte S; Grønbaek, Morten; Pedersen, Bo V;

    2011-01-01

    Studies have linked obesity, a sedentary lifestyle, and tobacco smoking to erectile dysfunction, but the evidence linking unhealthy lifestyle factors to other sexual dysfunctions or to sexual inactivity is conflicting.......Studies have linked obesity, a sedentary lifestyle, and tobacco smoking to erectile dysfunction, but the evidence linking unhealthy lifestyle factors to other sexual dysfunctions or to sexual inactivity is conflicting....

  12. 38 CFR 3.372 - Initial grant following inactivity of tuberculosis.

    Science.gov (United States)

    2010-07-01

    ... inactivity of tuberculosis. 3.372 Section 3.372 Pensions, Bonuses, and Veterans' Relief DEPARTMENT OF... Considerations Relative to Specific Diseases § 3.372 Initial grant following inactivity of tuberculosis. When... tuberculosis and there is satisfactory evidence that the condition was active previously but is now...

  13. Murine Extracellular Superoxide Dismutase Is Converted into the Inactive Fold by the Ser195Cys Mutation

    DEFF Research Database (Denmark)

    Scavenius, Carsten; Petersen, Jane Savskov; Thomsen, Line Rold

    2013-01-01

    We have previously shown that human extracellular superoxide dismutase (EC-SOD) exists as two variants with differences in their disulfide bridge patterns: one form is the active enzyme (aEC-SOD), and the other is inactive (iEC-SOD). The availability of both active and inactive folding variants...

  14. Interleukin 7 receptor functions by recruiting the tyrosine kinase p59fyn through a segment of its cytoplasmic tail.

    OpenAIRE

    1992-01-01

    Engagement of the cell surface receptor for interleukin 7 (IL-7R) provokes protein tyrosine phosphorylation, although the receptor lacks a kinase catalytic domain in its cytoplasmic tail. The molecular basis of this response is not known. Here we report that the IL-7R functions by recruiting p59fyn, an intracellular tyrosine kinase of the src family. Treatment of pre-B cells with IL-7 causes an enhancement of the catalytic activity of p59fyn, but not of the related kinase p62yes. IL-7-depende...

  15. Redox regulation of Janus kinase: The elephant in the room.

    Science.gov (United States)

    Duhé, Roy J

    2013-10-01

    The redox regulation of Janus kinases (JAKs) is a complex subject. Due to other redox-sensitive kinases in the kinome, redox-sensitive phosphatases, and cellular antioxidant systems and reactive oxygen species (ROS) production systems, the net biological outcomes of oxidative stress on JAK-dependent signal transduction vary according to the specific biological system examined. This review begins with a discussion of the biochemical evidence for a cysteine-based redox switch in the catalytic domain of JAKs, proceeds to consider direct and indirect regulatory mechanisms involved in biological experiments, and ends with a discussion of the role(s) of redox regulation of JAKs in various diseases.

  16. Structural diversity of the active N-terminal kinase domain of p90 ribosomal S6 kinase 2.

    Directory of Open Access Journals (Sweden)

    Margarita Malakhova

    Full Text Available The p90 ribosomal protein kinase 2 (RSK2 is a highly expressed Ser/Thr kinase activated by growth factors and is involved in cancer cell proliferation and tumor promoter-induced cell transformation. RSK2 possesses two non-identical kinase domains, and the structure of its N-terminal domain (NTD, which is responsible for phosphorylation of a variety of substrates, is unknown. The crystal structure of the NTD RSK2 was determined at 1.8 A resolution in complex with AMP-PNP. The N-terminal kinase domain adopted a unique active conformation showing a significant structural diversity of the kinase domain compared to other kinases. The NTD RSK2 possesses a three-stranded betaB-sheet inserted in the N-terminal lobe, resulting in displacement of the alphaC-helix and disruption of the Lys-Glu interaction, classifying the kinase conformation as inactive. The purified protein was phosphorylated at Ser227 in the T-activation loop and exhibited in vitro kinase activity. A key characteristic is the appearance of a new contact between Lys216 (betaB-sheet and the beta-phosphate of AMP-PNP. Mutation of this lysine to alanine impaired both NTDs in vitro and full length RSK2 ex vivo activity, emphasizing the importance of this interaction. Even though the N-terminal lobe undergoes structural re-arrangement, it possesses an intact hydrophobic groove formed between the alphaC-helix, the beta4-strand, and the betaB-sheet junction, which is occupied by the N-terminal tail. The presence of a unique betaB-sheet insert in the N-lobe suggests a different type of activation mechanism for RSK2.

  17. Muscle phosphorylase kinase deficiency

    DEFF Research Database (Denmark)

    Preisler, N; Orngreen, M C; Echaniz-Laguna, A;

    2012-01-01

    To examine metabolism during exercise in 2 patients with muscle phosphorylase kinase (PHK) deficiency and to further define the phenotype of this rare glycogen storage disease (GSD).......To examine metabolism during exercise in 2 patients with muscle phosphorylase kinase (PHK) deficiency and to further define the phenotype of this rare glycogen storage disease (GSD)....

  18. Kinetic Analysis of a Globin-Coupled Histidine Kinase, AfGcHK: Effects of the Heme Iron Complex, Response Regulator, and Metal Cations on Autophosphorylation Activity.

    Science.gov (United States)

    Fojtikova, Veronika; Stranava, Martin; Vos, Marten H; Liebl, Ursula; Hranicek, Jakub; Kitanishi, Kenichi; Shimizu, Toru; Martinkova, Marketa

    2015-08-18

    The globin-coupled histidine kinase, AfGcHK, is a part of the two-component signal transduction system from the soil bacterium Anaeromyxobacter sp. Fw109-5. Activation of its sensor domain significantly increases its autophosphorylation activity, which targets the His183 residue of its functional domain. The phosphate group of phosphorylated AfGcHK is then transferred to the cognate response regulator. We investigated the effects of selected variables on the autophosphorylation reaction's kinetics. The kcat values of the heme Fe(III)-OH(-), Fe(III)-cyanide, Fe(III)-imidazole, and Fe(II)-O2 bound active AfGcHK forms were 1.1-1.2 min(-1), and their Km(ATP) values were 18.9-35.4 μM. However, the active form bearing a CO-bound Fe(II) heme had a kcat of 1.0 min(-1) but a very high Km(ATP) value of 357 μM, suggesting that its active site structure differs strongly from the other active forms. The Fe(II) heme-bound inactive form had kcat and Km(ATP) values of 0.4 min(-1) and 78 μM, respectively, suggesting that its low activity reflects a low affinity for ATP relative to that of the Fe(III) form. The heme-free form exhibited low activity, with kcat and Km(ATP) values of 0.3 min(-1) and 33.6 μM, respectively, suggesting that the heme iron complex is essential for high catalytic activity. Overall, our results indicate that the coordination and oxidation state of the sensor domain heme iron profoundly affect the enzyme's catalytic activity because they modulate its ATP binding affinity and thus change its kcat/Km(ATP) value. The effects of the response regulator and different divalent metal cations on the autophosphorylation reaction are also discussed.

  19. Detailed search for protein kinase(s) involved in plasma membrane H+-ATPase activity regulation of yeast cells.

    Science.gov (United States)

    Pereira, Renata R; Castanheira, Diogo; Teixeira, Janaina A; Bouillet, Leoneide E M; Ribeiro, Erica M C; Trópia, Maria M J; Alvarez, Florencia; Correa, Lygia F M; Mota, Bruno E F; Conceição, Luis Eduardo F R; Castro, Ieso M; Brandão, Rogelio L

    2015-03-01

    This study displays a screening using yeast strains deficient in protein kinases known to exist in Saccharomyces cerevisiae. From 95 viable single mutants, 20 mutants appear to be affected in the glucose-induced extracellular acidification. The mutants that are unaffected in calcium signaling were tested for their sensitivity to hygromycin B. Furthermore, we verified whether the remaining mutants produced enzymes that are appropriately incorporated at plasma membrane. Finally, we measure the kinetic properties of the enzyme in purified plasma membranes from glucose-starved as well as glucose-fermenting cells. We confirmed the kinase Ptk2 involvement in H(+)-ATPase regulation (increase of affinity for ATP). However, the identification of the kinase(s) responsible for phosphorylation that leads to an increase in Vmax appears to be more complex. Complementary experiments were performed to check how those protein kinases could be related to the control of the plasma membrane H(+)-ATPase and/or the potential membrane. In summary, our results did not permit us to identify the protein kinase(s) involved in regulating the catalytic efficiency of the plasma membrane H(+)-ATPase. Therefore, our results indicate that the current regulatory model based on the phosphorylation of two different sites located in the C-terminus tail of the enzyme could be inappropriate.

  20. Ternary structure reveals mechanism of a membrane diacylglycerol kinase

    Science.gov (United States)

    Li, Dianfan; Stansfeld, Phillip J.; Sansom, Mark S. P.; Keogh, Aaron; Vogeley, Lutz; Howe, Nicole; Lyons, Joseph A.; Aragao, David; Fromme, Petra; Fromme, Raimund; Basu, Shibom; Grotjohann, Ingo; Kupitz, Christopher; Rendek, Kimberley; Weierstall, Uwe; Zatsepin, Nadia A.; Cherezov, Vadim; Liu, Wei; Bandaru, Sateesh; English, Niall J.; Gati, Cornelius; Barty, Anton; Yefanov, Oleksandr; Chapman, Henry N.; Diederichs, Kay; Messerschmidt, Marc; Boutet, Sébastien; Williams, Garth J.; Marvin Seibert, M.; Caffrey, Martin

    2015-12-01

    Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution.

  1. A protein phosphatase methylesterase (PME-1) is one of several novel proteins stably associating with two inactive mutants of protein phosphatase 2A.

    Science.gov (United States)

    Ogris, E; Du, X; Nelson, K C; Mak, E K; Yu, X X; Lane, W S; Pallas, D C

    1999-05-14

    Carboxymethylation of proteins is a highly conserved means of regulation in eukaryotic cells. The protein phosphatase 2A (PP2A) catalytic (C) subunit is reversibly methylated at its carboxyl terminus by specific methyltransferase and methylesterase enzymes which have been purified, but not cloned. Carboxymethylation affects PP2A activity and varies during the cell cycle. Here, we report that substitution of glutamine for either of two putative active site histidines in the PP2A C subunit results in inactivation of PP2A and formation of stable complexes between PP2A and several cellular proteins. One of these cellular proteins, herein named protein phosphatase methylesterase-1 (PME-1), was purified and microsequenced, and its cDNA was cloned. PME-1 is conserved from yeast to human and contains a motif found in lipases having a catalytic triad-activated serine as their active site nucleophile. Bacterially expressed PME-1 demethylated PP2A C subunit in vitro, and okadaic acid, a known inhibitor of the PP2A methylesterase, inhibited this reaction. To our knowledge, PME-1 represents the first mammalian protein methylesterase to be cloned. Several lines of evidence indicate that, although there appears to be a role for C subunit carboxyl-terminal amino acids in PME-1 binding, amino acids other than those at the extreme carboxyl terminus of the C subunit also play an important role in PME-1 binding to a catalytically inactive mutant.

  2. Genome-wide analysis of the phosphoinositide kinome from two ciliates reveals novel evolutionary links for phosphoinositide kinases in eukaryotic cells.

    Directory of Open Access Journals (Sweden)

    George Leondaritis

    Full Text Available BACKGROUND: The complexity of phosphoinositide signaling in higher eukaryotes is partly due to expansion of specific families and types of phosphoinositide kinases (PIKs that can generate all phosphoinositides via multiple routes. This is particularly evident in the PI3Ks and PIPKs, and it is considered an evolutionary trait associated with metazoan diversification. Yet, there are limited comprehensive studies on the PIK repertoire of free living unicellular organisms. METHODOLOGY/PRINCIPAL FINDINGS: We undertook a genome-wide analysis of putative PIK genes in two free living ciliated cells, Tetrahymena and Paramecium. The Tetrahymena thermophila and Paramecium tetraurelia genomes were probed with representative kinases from all families and types. Putative homologs were verified by EST, microarray and deep RNA sequencing database searches and further characterized for domain structure, catalytic efficiency, expression patterns and phylogenetic relationships. In total, we identified and characterized 22 genes in the Tetrahymena thermophila genome and 62 highly homologues genes in Paramecium tetraurelia suggesting a tight evolutionary conservation in the ciliate lineage. Comparison to the kinome of fungi reveals a significant expansion of PIK genes in ciliates. CONCLUSIONS/SIGNIFICANCE: Our study highlights four important aspects concerning ciliate and other unicellular PIKs. First, ciliate-specific expansion of PI4KIII-like genes. Second, presence of class I PI3Ks which, at least in Tetrahymena, are associated with a metazoan-type machinery for PIP3 signaling. Third, expansion of divergent PIPK enzymes such as the recently described type IV transmembrane PIPKs. Fourth, presence of possible type II PIPKs and presumably inactive PIKs (hence, pseudo-PIKs not previously described. Taken together, our results provide a solid framework for future investigation of the roles of PIKs in ciliates and indicate that novel functions and novel regulatory

  3. Physical inactivity and obesity: relation to asthma and chronic obstructive pulmonary disease?

    Science.gov (United States)

    ten Hacken, Nick H T

    2009-12-01

    Physical inactivity and obesity are modifiable risk factors for many chronic diseases, including cardiovascular disease, diabetes mellitus, osteoporosis, osteoarthritis, and depression. Both physical inactivity and obesity are associated with low-grade systemic inflammation that may contribute to the inflammatory processes present in many chronic diseases. In asthma, almost no studies are available in which physical inactivity has been studied using performance-based instruments. In contrast, the association between obesity and a higher prevalence of asthma has often been suggested in a large number of studies. In chronic obstructive pulmonary disease (COPD) physical inactivity has been demonstrated in a few studies that used performance-based instruments; this was associated with the higher COPD Global Initiative on Obstructive Lung Disease (GOLD) stages and a higher degree of systemic inflammation, independent of body mass index. In contrast to physical inactivity, obesity in COPD is associated with the lower GOLD stages. Additionally, obesity is associated with the chronic obstructive phenotype and features of the metabolic syndrome. To elucidate the independent relation of physical inactivity and obesity with systemic inflammation, performance-based studies of physical inactivity in asthma and COPD are highly needed.

  4. Prevalence of Irritable Bowel Syndrome–like Symptoms in Japanese Patients with Inactive Inflammatory Bowel Disease

    Science.gov (United States)

    Tomita, Toshihiko; Kato, Yu; Takimoto, Mayu; Yamasaki, Takahisa; Kondo, Takashi; Kono, Tomoaki; Tozawa, Katsuyuki; Yokoyama, Yoko; Ikehara, Hisatomo; Ohda, Yoshio; Oshima, Tadayuki; Fukui, Hirokazu; Tanaka, Shigemi; Shima, Masayuki; Watari, Jiro; Miwa, Hiroto

    2016-01-01

    Background/Aims Few studies are available that have investigated the risk factors for overlapping irritable bowel syndrome (IBS)-like symptoms in patients with inactive inflammatory bowel disease (IBD). The present study has 3 objectives: (1) to assess the prevalence of IBS-like symptoms in Japanese patients with inactive IBD using Rome III criteria, (2) to examine the relationship of IBS-like symptoms to health related quality of life (HR-QOL), and (3) to investigate associations for developing IBS-like symptoms in patients with inactive IBD. Methods IBS-like symptoms were evaluated using the Rome III questionnaire for functional gastrointestinal disorders. HR-QOL and hospital anxiety and depression scale were evaluated. Results IBS-like symptoms were found in 17.5% (7/40) of patients with inactive ulcerative colitis, 27.1% (29/107) of patients with inactive Crohn’s disease (CD), and 5.3% (23/438) of healthy control subjects. The QOL level was significantly lower and anxiety score was significantly higher in inactive CD patients with IBS-like symptoms than in those without such symptoms (P = 0.003, P = 0.009). Use of anti-anxiety drugs was associated with the presence of IBS symptoms (P = 0.045). HR-QOL score was lower and anxiety score was higher in patients with inactive ulcerative colitis, but the difference was not statistically significant. Conclusions The prevalence of IBS-like symptoms in inactive IBD patients was significantly higher than in healthy controls. Inactive CD patients with IBS-like symptoms has low QOL and anxiety; suggesting that anxiety may be associated with symptom development in such patients. PMID:27193973

  5. Novel Catalytic Membrane Reactors

    Energy Technology Data Exchange (ETDEWEB)

    Stuart Nemser, PhD

    2010-10-01

    There are many industrial catalytic organic reversible reactions with amines or alcohols that have water as one of the products. Many of these reactions are homogeneously catalyzed. In all cases removal of water facilitates the reaction and produces more of the desired chemical product. By shifting the reaction to right we produce more chemical product with little or no additional capital investment. Many of these reactions can also relate to bioprocesses. Given the large number of water-organic compound separations achievable and the ability of the Compact Membrane Systems, Inc. (CMS) perfluoro membranes to withstand these harsh operating conditions, this is an ideal demonstration system for the water-of-reaction removal using a membrane reactor. Enhanced reaction synthesis is consistent with the DOE objective to lower the energy intensity of U.S. industry 25% by 2017 in accord with the Energy Policy Act of 2005 and to improve the United States manufacturing competitiveness. The objective of this program is to develop the platform technology for enhancing homogeneous catalytic chemical syntheses.

  6. Complement receptor-3 negatively regulates the phagocytosis of degenerated myelin through tyrosine kinase Syk and cofilin

    Directory of Open Access Journals (Sweden)

    Hadas Smadar

    2012-07-01

    Full Text Available Abstract Background Intact myelin, which normally surrounds axons, breaks down in Wallerian degeneration following axonal injury and during neurodegenerative diseases such as multiple sclerosis. Clearance of degenerated myelin by phagocytosis is essential since myelin impedes repair and exacerbates damage. CR3 (complement receptor-3 is a principal phagocytic receptor in myelin phagocytosis. We studied how tyrosine kinase Syk (spleen tyrosine kinase and cofilin control phagocytosis of degenerated myelin by CR3 in microglia and macrophages. Syk is a non-receptor tyrosine kinase that CR3 recruits to convey cellular functions. Cofilin is an actin-depolymerizing protein that controls F-actin (filamentous actin remodeling (i.e., disassembly and reassembly by shifting between active unphosphorylated and inactive phosphorylated states. Results Syk was continuously activated during prolonged phagocytosis. Phagocytosis increased when Syk activity and expression were reduced, suggesting that normally Syk down regulates CR3-mediated myelin phagocytosis. Levels of inactive p-cofilin (phosphorylated cofilin decreased transiently during prolonged phagocytosis. In contrast, p-cofilin levels decreased continuously when Syk activity and expression were continuously reduced, suggesting that normally Syk advances the inactive state of cofilin. Observations also revealed inverse relationships between levels of phagocytosis and levels of inactive p-cofilin, suggesting that active unphosphorylated cofilin advances phagocytosis. Active cofilin could advance phagocytosis by promoting F-actin remodeling, which supports the production of membrane protrusions (e.g., filopodia, which, as we also revealed, are instrumental in myelin phagocytosis. Conclusions CR3 both activates and downregulates myelin phagocytosis at the same time. Activation was previously documented. We presently demonstrate that downregulation is mediated through Syk, which advances the inactive

  7. wKinMut-2: Identification and Interpretation of Pathogenic Variants in Human Protein Kinases

    DEFF Research Database (Denmark)

    Vazquez, Miguel; Pons, Tirso; Brunak, Søren;

    2016-01-01

    is often scattered across different sources, which makes the integrative analysis complex and laborious. wKinMut-2 constitutes a solution to facilitate the interpretation of the consequences of human protein kinase variation. Nine methods predict their pathogenicity, including a kinase-specific random...... forest approach. To understand the biological mechanisms causative of human diseases and cancer, information from pertinent reference knowledgebases and the literature is automatically mined, digested and homogenized. Variants are visualized in their structural contexts and residues affecting catalytic...

  8. Receptor Tyrosine Kinase and Tyrosine Kinase Inhibitors: New Hope for Success in Multiple Sclerosis Therapy.

    Science.gov (United States)

    Mirshafiey, Abbas; Ghalamfarsa, Ghasem; Asghari, Babak; Azizi, Gholamreza

    2014-07-01

    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.

  9. Structural analysis of Staphylococcus aureus serine/threonine kinase PknB.

    Directory of Open Access Journals (Sweden)

    Sonja Rakette

    Full Text Available Effective treatment of infections caused by the bacterium Staphylococcus aureus remains a worldwide challenge, in part due to the constant emergence of new strains that are resistant to antibiotics. The serine/threonine kinase PknB is of particular relevance to the life cycle of S. aureus as it is involved in the regulation of purine biosynthesis, autolysis, and other central metabolic processes of the bacterium. We have determined the crystal structure of the kinase domain of PknB in complex with a non-hydrolyzable analog of the substrate ATP at 3.0 Å resolution. Although the purified PknB kinase is active in solution, it crystallized in an inactive, autoinhibited state. Comparison with other bacterial kinases provides insights into the determinants of catalysis, interactions of PknB with ligands, and the pathway of activation.

  10. Structural analysis of Staphylococcus aureus serine/threonine kinase PknB.

    Science.gov (United States)

    Rakette, Sonja; Donat, Stefanie; Ohlsen, Knut; Stehle, Thilo

    2012-01-01

    Effective treatment of infections caused by the bacterium Staphylococcus aureus remains a worldwide challenge, in part due to the constant emergence of new strains that are resistant to antibiotics. The serine/threonine kinase PknB is of particular relevance to the life cycle of S. aureus as it is involved in the regulation of purine biosynthesis, autolysis, and other central metabolic processes of the bacterium. We have determined the crystal structure of the kinase domain of PknB in complex with a non-hydrolyzable analog of the substrate ATP at 3.0 Å resolution. Although the purified PknB kinase is active in solution, it crystallized in an inactive, autoinhibited state. Comparison with other bacterial kinases provides insights into the determinants of catalysis, interactions of PknB with ligands, and the pathway of activation.

  11. Identification of potent Yes1 kinase inhibitors using a library screening approach.

    Science.gov (United States)

    Patel, Paresma R; Sun, Hongmao; Li, Samuel Q; Shen, Min; Khan, Javed; Thomas, Craig J; Davis, Mindy I

    2013-08-01

    Yes1 kinase has been implicated as a potential therapeutic target in a number of cancers including melanomas, breast cancers, and rhabdomyosarcomas. Described here is the development of a robust and miniaturized biochemical assay for Yes1 kinase that was applied in a high throughput screen (HTS) of kinase-focused small molecule libraries. The HTS provided 144 (17% hit rate) small molecule compounds with IC₅₀ values in the sub-micromolar range. Three of the most potent Yes1 inhibitors were then examined in a cell-based assay for inhibition of cell survival in rhabdomyosarcoma cell lines. Homology models of Yes1 were generated in active and inactive conformations, and docking of inhibitors supports binding to the active conformation (DFG-in) of Yes1. This is the first report of a large high throughput enzymatic activity screen for identification of Yes1 kinase inhibitors, thereby elucidating the polypharmacology of a variety of small molecules and clinical candidates.

  12. A new light on DNA replication from the inactive X chromosome.

    Science.gov (United States)

    Aladjem, Mirit I; Fu, Haiqing

    2014-06-01

    While large portions of the mammalian genome are known to replicate sequentially in a distinct, tissue-specific order, recent studies suggest that the inactive X chromosome is duplicated rapidly via random, synchronous DNA synthesis at numerous adjacent regions. The rapid duplication of the inactive X chromosome was observed in high-resolution studies visualizing DNA replication patterns in the nucleus, and by allele-specific DNA sequencing studies measuring the extent of DNA synthesis. These studies conclude that inactive X chromosomes complete replication earlier than previously thought and suggest that the strict order of DNA replication detected in the majority of genomic regions is not preserved in non-transcribed, "silent" chromatin. These observations alter current concepts about the regulation of DNA replication in non-transcribed portions of the genome in general and in the inactive X-chromosome in particular.

  13. 2014 consensus statement from the first Economics of Physical Inactivity Consensus (EPIC) conference (Vancouver).

    Science.gov (United States)

    Davis, Jennifer C; Verhagen, Evert; Bryan, Stirling; Liu-Ambrose, Teresa; Borland, Jeff; Buchner, David; Hendriks, Marike R C; Weiler, Richard; Morrow, James R; van Mechelen, Willem; Blair, Steven N; Pratt, Mike; Windt, Johann; al-Tunaiji, Hashel; Macri, Erin; Khan, Karim M

    2014-06-01

    This article describes major topics discussed from the 'Economics of Physical Inactivity Consensus Workshop' (EPIC), held in Vancouver, Canada, in April 2011. Specifically, we (1) detail existing evidence on effective physical inactivity prevention strategies; (2) introduce economic evaluation and its role in health policy decisions; (3) discuss key challenges in establishing and building health economic evaluation evidence (including accurate and reliable costs and clinical outcome measurement) and (4) provide insight into interpretation of economic evaluations in this critically important field. We found that most methodological challenges are related to (1) accurately and objectively valuing outcomes; (2) determining meaningful clinically important differences in objective measures of physical inactivity; (3) estimating investment and disinvestment costs and (4) addressing barriers to implementation. We propose that guidelines specific for economic evaluations of physical inactivity intervention studies are developed to ensure that related costs and effects are robustly, consistently and accurately measured. This will also facilitate comparisons among future economic evidence.

  14. Studying Kinetochore Kinases

    NARCIS (Netherlands)

    Saurin, Adrian T; Kops, Geert J P L

    2016-01-01

    Mitotic kinetochores are signaling network hubs that regulate chromosome movements, attachment error-correction, and the spindle assembly checkpoint. Key switches in these networks are kinases and phosphatases that enable rapid responses to changing conditions. Describing the mechanisms and dynamics

  15. HYDROGEN TRANSFER IN CATALYTIC CRACKING

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Hydrogen transfer is an important secondary reaction of catalytic cracking reactions, which affects product yield distribution and product quality. It is an exothermic reaction with low activation energy around 43.3 kJ/mol. Catalyst properties and operation parameters in catalytic cracking greatly influence the hydrogen transfer reaction. Satisfactory results are expected through careful selection of proper catalysts and operation conditions.

  16. Update on Aurora Kinase Targeted Therapeutics in Oncology

    Science.gov (United States)

    Green, Myke R.; Woolery, Joseph E.

    2011-01-01

    Introduction Mammalian cells contain three distinct serine/threonine protein kinases with highly conserved catalytic domains, including aurora A and B kinases that are essential regulators of mitotic entry and progression. Overexpression of aurora A and/or B kinase is associated with high proliferation rates and poor prognosis, making them ideal targets for anti-cancer therapy. Disruption of mitotic machinery is a proven anti-cancer strategy employed by multiple chemotherapeutic agents. Numerous small molecule inhibitors of the aurora kinases have been discovered and tested in vivo and in vitro, with a few currently in phase II testing. Areas covered This review provides the reader with updated results from both preclinical and human studies for each of the aurora kinase inhibitors (AKI) that are currently being investigated. The paper also covers in detail the late breaking and phase I data presented for AKIs thereby allowing the reader to compare and contrast individual and classrelated effects of AKIs. Expert opinion While the successful development and approval of an AKI for anti-cancer therapy remains unresolved, pre-clinical identification of resistant mechanisms would help design better early phase clinical trials where relevant combinations may be evaluated prior to phase II testing. The authors believe that aurora kinases are important anti-cancer targets that operate in collaboration with other oncogenes intimately involved in uncontrolled tumor proliferation and by providing a unique, targeted and complimentary anti-cancer mechanism, expand the available armamentarium against cancer. PMID:21556291

  17. Proteolytic activation of ETK/Bmx tyrosine kinase by caspases.

    Science.gov (United States)

    Wu, Y M; Huang, C L; Kung, H J; Huang, C Y

    2001-05-25

    Etk/Bmx is a member of the Btk/Tec family of kinases, which are characterized by having a pleckstrin homology domain at the N terminus, in addition to the Src homology 3 (SH3), SH2, and the catalytic domains, shared with the Src family kinases. Etk, or Btk kinases in general, has been implicated in the regulation of apoptosis. To test whether Etk is the substrate for caspases during apoptosis, in vitro translated [(35)S]methionine-labeled Etk was incubated with different apoptotic extracts and recombinant caspases, respectively. Results showed that Etk was proteolyzed in all conditions tested with identical cleavage patterns. Caspase-mediated cleavage of Etk generated a C-terminal fragment, containing the complete SH2 and tyrosine kinase domains, but without intact pleckstrin homology and SH3 domains. This fragment has 4-fold higher kinase activity than that of the full-length Etk. Ectopic expression of the C-terminal fragment of Etk sensitized the PC3 prostate cancer cells to apoptosis in response to apoptosis-inducing stimuli. The finding, together with an earlier report that Etk is potentially antiapoptotic, suggests that Etk may serve as an apoptotic switch, depending on the forms of Etk existing inside the cells. To our knowledge, this is the first case where the activity of a tyrosine kinase is induced by caspase cleavage.

  18. Metabolic rates and biochemical compositions of Apostichopus japonicus (Selenka) tissue during periods of inactivity

    Institute of Scientific and Technical Information of China (English)

    包杰; 董双林; 田相利; 王芳; 高勤峰; 董云伟

    2010-01-01

    Estivation, hibernation, and starvation are indispensable inactive states of sea cucumbers Apostichopus japonicus in nature and in culture ponds. Generally, temperature is the principal factor that induces estivation or hibernation in the sea cucumber. The present study provided insight into the physiological adaptations of A. japonicus during the three types of inactivity (hibernation, estivation, and starvation) by measuring the oxygen consumption rates (Vo2) and biochemical compositions under laboratory ...

  19. Light saturation response of inactive photosystem II reaction centers in spinach.

    Science.gov (United States)

    Chylla, R A; Whitmarsh, J

    1990-07-01

    The effective absorption cross section of inactive photosystem II (PS II) centers, which is the product of the effective antenna size and the quantum yield for photochemistry, was investigated by comparing the light saturation curves of inactive PS II and active reaction centers in intact chloroplasts and thylakoid membranes of spinach (Spinacia oleracea). Inactive PS II centers are defined as the impaired PS II reaction centers that require greater than 50 ms for the reoxidation of QA (-) subsequent to a single turnover flash. Active reaction centers are defined as the rapidly turning over PS II centers (recovery time less than 50 ms) and all of the PS I centers. The electrochromic shift, measured by the flash-induced absorbance increase at 518 nm, was used to probe the activity of the reaction centers. Light saturation curves were generated for inactive PS II centers and active reaction centers by measuring the extent of the absorbance increase at 518 nm induced by red actinic flashes of variable energy. The light saturation curves show that inactive PS II centers required over twice as many photons as active reaction centers to achieve the same yield. The ratio of the flash energy required for 50% saturation for active reaction centers (PS II active + PS I) compared to inactive PS II centers was 0.45±0.04 in intact chloroplasts, and 0.54±0.11 in thylakoid membranes. Analysis of the light saturation curves using a Poisson statistical model in which the ratio of the antenna size of active PS II centers to that of PS I is considered to range from 1 to 1.5, indicates that the effective absorption cross section of inactive PS II centers was 0.54-0.37 times that of active PS II centers. If the quantum yield for photochemistry is assumed to be one, we estimate that the antenna system serving the inactive PS II centers contains approx. 110 chlorophyll molecules.

  20. Elucidating the role of the TRPM7 alpha-kinase: TRPM7 kinase inactivation leads to magnesium deprivation resistance phenotype in mice.

    Science.gov (United States)

    Ryazanova, Lillia V; Hu, Zhixian; Suzuki, Sayuri; Chubanov, Vladimir; Fleig, Andrea; Ryazanov, Alexey G

    2014-12-23

    TRPM7 is an unusual bi-functional protein containing an ion channel covalently linked to a protein kinase domain. TRPM7 is implicated in regulating cellular and systemic magnesium homeostasis. While the biophysical properties of TRPM7 ion channel and its function are relatively well characterized, the function of the TRPM7 enzymatically active kinase domain is not understood yet. To investigate the physiological role of TRPM7 kinase activity, we constructed mice carrying an inactive TRPM7 kinase. We found that these mice were resistant to dietary magnesium deprivation, surviving three times longer than wild type mice; also they displayed decreased chemically induced allergic reaction. Interestingly, mutant mice have lower magnesium bone content compared to wild type mice when fed regular diet; unlike wild type mice, mutant mice placed on magnesium-depleted diet did not alter their bone magnesium content. Furthermore, mouse embryonic fibroblasts isolated from TRPM7 kinase-dead animals exhibited increased resistance to magnesium deprivation and oxidative stress. Finally, electrophysiological data revealed that the activity of the kinase-dead TRPM7 channel was not significantly altered. Together, our results suggest that TRPM7 kinase is a sensor of magnesium status and provides coordination of cellular and systemic responses to magnesium deprivation.

  1. Catalytic quantum error correction

    CERN Document Server

    Brun, T; Hsieh, M H; Brun, Todd; Devetak, Igor; Hsieh, Min-Hsiu

    2006-01-01

    We develop the theory of entanglement-assisted quantum error correcting (EAQEC) codes, a generalization of the stabilizer formalism to the setting in which the sender and receiver have access to pre-shared entanglement. Conventional stabilizer codes are equivalent to dual-containing symplectic codes. In contrast, EAQEC codes do not require the dual-containing condition, which greatly simplifies their construction. We show how any quaternary classical code can be made into a EAQEC code. In particular, efficient modern codes, like LDPC codes, which attain the Shannon capacity, can be made into EAQEC codes attaining the hashing bound. In a quantum computation setting, EAQEC codes give rise to catalytic quantum codes which maintain a region of inherited noiseless qubits. We also give an alternative construction of EAQEC codes by making classical entanglement assisted codes coherent.

  2. TYK2 kinase activity is required for functional type I interferon responses in vivo.

    Directory of Open Access Journals (Sweden)

    Michaela Prchal-Murphy

    Full Text Available Tyrosine kinase 2 (TYK2 is a member of the Janus kinase (JAK family and is involved in cytokine signalling. In vitro analyses suggest that TYK2 also has kinase-independent, i.e., non-canonical, functions. We have generated gene-targeted mice harbouring a mutation in the ATP-binding pocket of the kinase domain. The Tyk2 kinase-inactive (Tyk2(K923E mice are viable and show no gross abnormalities. We show that kinase-active TYK2 is required for full-fledged type I interferon- (IFN induced activation of the transcription factors STAT1-4 and for the in vivo antiviral defence against viruses primarily controlled through type I IFN actions. In addition, TYK2 kinase activity was found to be required for the protein's stability. An inhibitory function was only observed upon over-expression of TYK2(K923Ein vitro. Tyk2(K923E mice represent the first model for studying the kinase-independent function of a JAK in vivo and for assessing the consequences of side effects of JAK inhibitors.

  3. Effects of selective inhibition of protein kinase C, cyclic AMP-dependent protein kinase, and Ca(2+)-calmodulin-dependent protein kinase on neurite development in cultured rat hippocampal neurons.

    Science.gov (United States)

    Cabell, L; Audesirk, G

    1993-06-01

    A variety of experimental evidence suggests that calmodulin and protein kinases, especially protein kinase C, may participate in regulating neurite development in cultured neurons, particularly neurite initiation. However, the results are somewhat contradictory. Further, the roles of calmodulin and protein kinases on many aspects of neurite development, such as branching or elongation of axons vs dendrites, have not been extensively studied. Cultured embryonic rat hippocampal pyramidal neurons develop readily identifiable axons and dendrites. We used this culture system and the new generation of highly specific protein kinase inhibitors to investigate the roles of protein kinases and calmodulin in neurite development. Neurons were cultured for 2 days in the continuous presence of calphostin C (a specific inhibitor of protein kinase C), KT5720 (inhibitor of cyclic AMP-dependent protein kinase), KN62 (inhibitor of Ca(2+)-calmodulin-dependent protein kinase II), or calmidazolium (inhibitor of calmodulin), each at concentrations from approximately 1 to 10 times the concentration reported in the literature to inhibit each kinase by 50%. The effects of phorbol 12-myristate 13-acetate (an activator of protein kinase C) and 4 alpha-phorbol 12,13-didecanoate (an inactive phorbol ester) were also tested. At concentrations that had no effect on neuronal viability, calphostin C reduced neurite initiation and axon branching without significantly affecting the number of dendrites per neuron, dendrite branching, dendrite length, or axon length. Phorbol 12-myristate 13-acetate increased axon branching and the number of dendrites per cell, compared to the inactive 4 alpha-phorbol 12,13-didecanoate. KT5720 inhibited only axon branching. KN62 reduced axon length, the number of dendrites per neuron, and both axon and dendrite branching. At low concentrations, calmidazolium had no effect on any aspect of neurite development, but at high concentrations, calmidazolium inhibited every

  4. Method of empirical dependences in estimation and prediction of activity of creatine kinase isoenzymes in cerebral ischemia

    Science.gov (United States)

    Sergeeva, Tatiana F.; Moshkova, Albina N.; Erlykina, Elena I.; Khvatova, Elena M.

    2016-04-01

    Creatine kinase is a key enzyme of energy metabolism in the brain. There are known cytoplasmic and mitochondrial creatine kinase isoenzymes. Mitochondrial creatine kinase exists as a mixture of two oligomeric forms - dimer and octamer. The aim of investigation was to study catalytic properties of cytoplasmic and mitochondrial creatine kinase and using of the method of empirical dependences for the possible prediction of the activity of these enzymes in cerebral ischemia. Ischemia was revealed to be accompanied with the changes of the activity of creatine kinase isoenzymes and oligomeric state of mitochondrial isoform. There were made the models of multiple regression that permit to study the activity of creatine kinase system in cerebral ischemia using a calculating method. Therefore, the mathematical method of empirical dependences can be applied for estimation and prediction of the functional state of the brain by the activity of creatine kinase isoenzymes in cerebral ischemia.

  5. Catalytic Combustion of Gasified Waste

    Energy Technology Data Exchange (ETDEWEB)

    Kusar, Henrik

    2003-09-01

    This thesis concerns catalytic combustion for gas turbine application using a low heating-value (LHV) gas, derived from gasified waste. The main research in catalytic combustion focuses on methane as fuel, but an increasing interest is directed towards catalytic combustion of LHV fuels. This thesis shows that it is possible to catalytically combust a LHV gas and to oxidize fuel-bound nitrogen (NH{sub 3}) directly into N{sub 2} without forming NO{sub x} The first part of the thesis gives a background to the system. It defines waste, shortly describes gasification and more thoroughly catalytic combustion. The second part of the present thesis, paper I, concerns the development and testing of potential catalysts for catalytic combustion of LHV gases. The objective of this work was to investigate the possibility to use a stable metal oxide instead of noble metals as ignition catalyst and at the same time reduce the formation of NO{sub x} In paper II pilot-scale tests were carried out to prove the potential of catalytic combustion using real gasified waste and to compare with the results obtained in laboratory scale using a synthetic gas simulating gasified waste. In paper III, selective catalytic oxidation for decreasing the NO{sub x} formation from fuel-bound nitrogen was examined using two different approaches: fuel-lean and fuel-rich conditions. Finally, the last part of the thesis deals with deactivation of catalysts. The various deactivation processes which may affect high-temperature catalytic combustion are reviewed in paper IV. In paper V the poisoning effect of low amounts of sulfur was studied; various metal oxides as well as supported palladium and platinum catalysts were used as catalysts for combustion of a synthetic gas. In conclusion, with the results obtained in this thesis it would be possible to compose a working catalytic system for gas turbine application using a LHV gas.

  6. Mechanism of Ribonuclease III Catalytic Regulation by Serine Phosphorylation

    Science.gov (United States)

    Gone, Swapna; Alfonso-Prieto, Mercedes; Paudyal, Samridhdi; Nicholson, Allen W.

    2016-05-01

    Ribonuclease III (RNase III) is a conserved, gene-regulatory bacterial endonuclease that cleaves double-helical structures in diverse coding and noncoding RNAs. RNase III is subject to multiple levels of control, reflective of its global regulatory functions. Escherichia coli (Ec) RNase III catalytic activity is known to increase during bacteriophage T7 infection, reflecting the expression of the phage-encoded protein kinase, T7PK. However, the mechanism of catalytic enhancement is unknown. This study shows that Ec-RNase III is phosphorylated on serine in vitro by purified T7PK, and identifies the targets as Ser33 and Ser34 in the N-terminal catalytic domain. Kinetic experiments reveal a 5-fold increase in kcat and a 1.4-fold decrease in Km following phosphorylation, providing a 7.4–fold increase in catalytic efficiency. Phosphorylation does not change the rate of substrate cleavage under single-turnover conditions, indicating that phosphorylation enhances product release, which also is the rate-limiting step in the steady-state. Molecular dynamics simulations provide a mechanism for facilitated product release, in which the Ser33 phosphomonoester forms a salt bridge with the Arg95 guanidinium group, thereby weakening RNase III engagement of product. The simulations also show why glutamic acid substitution at either serine does not confer enhancement, thus underscoring the specific requirement for a phosphomonoester.

  7. Unsteady catalytic processes and sorption-catalytic technologies

    Energy Technology Data Exchange (ETDEWEB)

    Zagoruiko, A N [G.K. Boreskov Institute of Catalysis, Siberian Branch of the Russian Academy of Sciences, Novosibirsk (Russian Federation)

    2007-07-31

    Catalytic processes that occur under conditions of the targeted unsteady state of the catalyst are considered. The highest efficiency of catalytic processes was found to be ensured by a controlled combination of thermal non-stationarity and unsteady composition of the catalyst surface. The processes based on this principle are analysed, in particular, catalytic selective reduction of nitrogen oxides, deep oxidation of volatile organic impurities, production of sulfur by the Claus process and by hydrogen sulfide decomposition, oxidation of sulfur dioxide, methane steam reforming and anaerobic combustion, selective oxidation of hydrocarbons, etc.

  8. Structural basis for catalytically restrictive dynamics of a high-energy enzyme state

    Science.gov (United States)

    Kovermann, Michael; Ådén, Jörgen; Grundström, Christin; Elisabeth Sauer-Eriksson, A.; Sauer, Uwe H.; Wolf-Watz, Magnus

    2015-07-01

    An emerging paradigm in enzymology is that transient high-energy structural states play crucial roles in enzymatic reaction cycles. Generally, these high-energy or `invisible' states cannot be studied directly at atomic resolution using existing structural and spectroscopic techniques owing to their low populations or short residence times. Here we report the direct NMR-based detection of the molecular topology and conformational dynamics of a catalytically indispensable high-energy state of an adenylate kinase variant. On the basis of matching energy barriers for conformational dynamics and catalytic turnover, it was found that the enzyme's catalytic activity is governed by its dynamic interconversion between the high-energy state and a ground state structure that was determined by X-ray crystallography. Our results show that it is possible to rationally tune enzymes' conformational dynamics and hence their catalytic power--a key aspect in rational design of enzymes catalysing novel reactions.

  9. Mass spectrometry and site-directed mutagenesis identify several autophosphorylated residues required for the activity of PrkC, a Ser/Thr kinase from Bacillus subtilis

    DEFF Research Database (Denmark)

    Madec, Edwige; Stensballe, Allan; Kjellström, Sven;

    2003-01-01

    structure of PrkC modelled on the structure of known Hanks kinases, the first cluster of phospho-threonine residues falls precisely in the activation loop, controlling the access of substrate and ATP to the catalytic site of many eukaryotic receptor kinases, whereas the second cluster is located...

  10. Role of a novel PH-kinase domain interface in PKB/Akt regulation: structural mechanism for allosteric inhibition.

    Directory of Open Access Journals (Sweden)

    Véronique Calleja

    2009-01-01

    Full Text Available Protein kinase B (PKB/Akt belongs to the AGC superfamily of related serine/threonine protein kinases. It is a key regulator downstream of various growth factors and hormones and is involved in malignant transformation and chemo-resistance. Full-length PKB protein has not been crystallised, thus studying the molecular mechanisms that are involved in its regulation in relation to its structure have not been simple. Recently, the dynamics between the inactive and active conformer at the molecular level have been described. The maintenance of PKB's inactive state via the interaction of the PH and kinase domains prevents its activation loop to be phosphorylated by its upstream activator, phosphoinositide-dependent protein kinase-1 (PDK1. By using a multidisciplinary approach including molecular modelling, classical biochemical assays, and Förster resonance energy transfer (FRET/two-photon fluorescence lifetime imaging microscopy (FLIM, a detailed model depicting the interaction between the different domains of PKB in its inactive conformation was demonstrated. These findings in turn clarified the molecular mechanism of PKB inhibition by AKT inhibitor VIII (a specific allosteric inhibitor and illustrated at the molecular level its selectivity towards different PKB isoforms. Furthermore, these findings allude to the possible function of the C-terminus in sustaining the inactive conformer of PKB. This study presents essential insights into the quaternary structure of PKB in its inactive conformation. An understanding of PKB structure in relation to its function is critical for elucidating its mode of activation and discovering how to modulate its activity. The molecular mechanism of inhibition of PKB activation by the specific drug AKT inhibitor VIII has critical implications for determining the mechanism of inhibition of other allosteric inhibitors and for opening up opportunities for the design of new generations of modulator drugs.

  11. The dsRBP and Inactive Editor ADR-1 Utilizes dsRNA Binding to Regulate A-to-I RNA Editing across the C. elegans Transcriptome

    OpenAIRE

    Michael C. Washburn; Boyko Kakaradov; Balaji Sundararaman; Emily Wheeler; Shawn Hoon; Gene W. Yeo; Heather A. Hundley

    2014-01-01

    Inadequate adenosine-to-inosine editing of noncoding regions occurs in disease but is often uncorrelated with ADAR levels, underscoring the need to study deaminase-independent control of editing. C. elegans have two ADAR proteins, ADR-2 and the theoretically catalytically inactive ADR-1. Using high-throughput RNA sequencing of wild-type and adr mutant worms, we expand the repertoire of C. elegans edited transcripts over 5-fold and confirm that ADR-2 is the only active deaminase in vivo. Despi...

  12. Catalytic production of biodiesel

    Energy Technology Data Exchange (ETDEWEB)

    Theilgaard Madsen, A.

    2011-07-01

    The focus of this thesis is the catalytic production of diesel from biomass, especially emphasising catalytic conversion of waste vegetable oils and fats. In chapter 1 an introduction to biofuels and a review on different catalytic methods for diesel production from biomass is given. Two of these methods have been used industrially for a number of years already, namely the transesterification (and esterification) of oils and fats with methanol to form fatty acid methyl esters (FAME), and the hydrodeoxygenation (HDO) of fats and oils to form straight-chain alkanes. Other possible routes to diesel include upgrading and deoxygenation of pyrolysis oils or aqueous sludge wastes, condensations and reductions of sugars in aqueous phase (aqueous-phase reforming, APR) for monofunctional hydrocarbons, and gasification of any type of biomass followed by Fischer-Tropsch-synthesis for alkane biofuels. These methods have not yet been industrialised, but may be more promising due to the larger abundance of their potential feedstocks, especially waste feedstocks. Chapter 2 deals with formation of FAME from waste fats and oils. A range of acidic catalysts were tested in a model fat mixture of methanol, lauric acid and trioctanoin. Sulphonic acid-functionalised ionic liquids showed extremely fast convertion of lauric acid to methyl laurate, and trioctanoate was converted to methyl octanoate within 24 h. A catalyst based on a sulphonated carbon-matrix made by pyrolysing (or carbonising) carbohydrates, so-called sulphonated pyrolysed sucrose (SPS), was optimised further. No systematic dependency on pyrolysis and sulphonation conditions could be obtained, however, with respect to esterification activity, but high activity was obtained in the model fat mixture. SPS impregnated on opel-cell Al{sub 2}O{sub 3} and microporous SiO{sub 2} (ISPS) was much less active in the esterification than the original SPS powder due to low loading and thereby low number of strongly acidic sites on the

  13. Exploring the Linkage between Activity-Friendly Zoning, Inactivity, and Cancer Incidence in the United States.

    Science.gov (United States)

    Nicholson, Lisa M; Leider, Julien; Chriqui, Jamie F

    2017-03-07

    Background: Physical activity (PA) protects against cancer and enhances cancer survivorship. Given high inactivity rates nationwide, population-level physical activity facilitators are needed. Several authoritative bodies have recognized that zoning and planning helps create activity-friendly environments. This study examined the association between activity-friendly zoning, inactivity, and cancer in 478 of the most populous U.S. counties.Methods: County geocodes linked county-level data: cancer incidence and smoking (State Cancer Profiles), inactivity (Behavioral Risk Factor Surveillance System), 11 zoning measures (compiled by the study team), and covariates (from the American Community Survey and NAVTEQ). For each zoning measure, single mediation regression models and Sobel tests examined whether activity-friendly zoning was associated with reduced cancer incidence, and whether inactivity mediated those associations. All models were clustered on state with robust SEs and significance at the P Zoning for crosswalks, bike-pedestrian connectivity, and bike-pedestrian trails/paths were associated with reduced cancer incidence (β between -0.71 and -1.27, P zoning. Except for crosswalks, each association was mediated by inactivity. However, county smoking attenuated these results, with only crosswalks remaining significant. Results were similar for males (with zoning for bike-pedestrian connectivity, street connectivity, and bike-pedestrian trails/paths), but not females, alone.Conclusions: Zoning can help to create activity-friendly environments that support decreased inactivity, and possibly reduced cancer incidence.Impact: Given low physical activity levels nationwide, cross-sectoral collaborations with urban planning can inform cancer prevention and public health efforts to decrease inactivity and cancer. Cancer Epidemiol Biomarkers Prev; 1-9. ©2017 AACR.See all the articles in this CEBP Focus section, "Geospatial Approaches to Cancer Control and Population

  14. High prevalence of inactivity among young patients with type 1 diabetes in south Spain

    Directory of Open Access Journals (Sweden)

    José Vicente Gutiérrez Manzanedo

    2014-04-01

    Full Text Available Objectives: To describe usual physical activity level and analyze its association with metabolic control and presence of microvascular complications in a cohort of patients with type 1 diabetes (DM-1 in south Spain. Methods: Observational, cross-sectional study that included one hundred thirty patients, aged 33.9 ± 11.5 years-old with disease duration of 16.5 ± 9.5 years that consecutively were recruited among patients attending the Endocrinology Service of Puerta del Mar University Hospital (Cádiz, Spain. Usual physical activity level was assessed using the "General Practice Physical Activity Questionnaire" (GPPAQ together with clinical, anthropometric, metabolic parameters and microvascular complications. Results: DM-1 patients were grouped in four categories of physical activity level: inactive (n = 33; 25.3%, moderately inactive (n = 31; 23.8%, moderately active (n = 26; 20.0% and active (n = 40; 30.9%. We observed no significant differences in glycated haemoglobin (HbA1c level between the different physical activity groups analyzed. Patients classified as moderately active and active were more often men, significantly younger and presented lower plasmatic levels of triglycerides than patients classified as inactive or moderately inactive, with no differences in other clinical or anthropometric variables. In addition, active and moderately active patients had a lower prevalence of diabetic retinopathy and microvascular complications in general compared to inactive or moderately inactive patients. Conclusions: Half of patients with type 1 diabetes evaluated were classified as inactive and these patients had a higher prevalence of diabetic retinopathy than active patients. No difference in HbA1c levels was documented among different groups of physical activity.

  15. Catalytic Membrane Sensors

    Energy Technology Data Exchange (ETDEWEB)

    Boyle, T.J.; Brinker, C.J.; Gardner, T.J.; Hughes, R.C.; Sault, A.G.

    1998-12-01

    The proposed "catalytic membrane sensor" (CMS) was developed to generate a device which would selectively identify a specific reagent in a complex mixture of gases. This was to be accomplished by modifying an existing Hz sensor with a series of thin films. Through selectively sieving the desired component from a complex mixture and identifying it by decomposing it into Hz (and other by-products), a Hz sensor could then be used to detect the presence of the select component. The proposed "sandwich-type" modifications involved the deposition of a catalyst layered between two size selective sol-gel layers on a Pd/Ni resistive Hz sensor. The role of the catalyst was to convert organic materials to Hz and organic by-products. The role of the membraneo was to impart both chemical specificity by molecukir sieving of the analyte and converted product streams, as well as controlling access to the underlying Pd/Ni sensor. Ultimately, an array of these CMS elements encompassing different catalysts and membranes were to be developed which would enable improved selectivity and specificity from a compiex mixture of organic gases via pattern recognition methodologies. We have successfully generated a CMS device by a series of spin-coat deposited methods; however, it was determined that the high temperature required to activate the catalyst, destroys the sensor.

  16. Catalytic gasification of biomass

    Science.gov (United States)

    Robertus, R. J.; Mudge, L. K.; Sealock, L. J., Jr.; Mitchell, D. H.; Weber, S. L.

    1981-12-01

    Methane and methanol synthesis gas can be produced by steam gasification of biomass in the presence of appropriate catalysts. This concept is to use catalysts in a fluidized bed reactor which is heated indirectly. The objective is to determine the technical and economic feasibility of the concept. Technically the concept has been demonstrated on a 50 lb per hr scale. Potential advantages over conventional processes include: no oxygen plant is needed, little tar is produced so gas and water treatment are simplified, and yields and efficiencies are greater than obtained by conventional gasification. Economic studies for a plant processing 2000 T/per day dry wood show that the cost of methanol from wood by catalytic gasification is competitive with the current price of methanol. Similar studies show the cost of methane from wood is competitive with projected future costs of synthetic natural gas. When the plant capacity is decreased to 200 T per day dry wood, neither product is very attractive in today's market.

  17. Catalytic cracking of lignites

    Energy Technology Data Exchange (ETDEWEB)

    Seitz, M.; Nowak, S.; Naegler, T.; Zimmermann, J. [Hochschule Merseburg (Germany); Welscher, J.; Schwieger, W. [Erlangen-Nuernberg Univ. (Germany); Hahn, T. [Halle-Wittenberg Univ., Halle (Germany)

    2013-11-01

    A most important factor for the chemical industry is the availability of cheap raw materials. As the oil price of crude oil is rising alternative feedstocks like coal are coming into focus. This work, the catalytic cracking of lignite is part of the alliance ibi (innovative Braunkohlenintegration) to use lignite as a raw material to produce chemicals. With this new one step process without an input of external hydrogen, mostly propylene, butenes and aromatics and char are formed. The product yield depends on manifold process parameters. The use of acid catalysts (zeolites like MFI) shows the highest amount of the desired products. Hydrogen rich lignites with a molar H/C ratio of > 1 are to be favoured. Due to primary cracking and secondary reactions the ratio between catalyst and lignite, temperature and residence time are the most important parameter to control the product distribution. Experiments at 500 C in a discontinuous rotary kiln reactor show yields up to 32 wt-% of hydrocarbons per lignite (maf - moisture and ash free) and 43 wt-% char, which can be gasified. Particularly, the yields of propylene and butenes as main products can be enhanced four times to about 8 wt-% by the use of catalysts while the tar yield decreases. In order to develop this innovative process catalyst systems fixed on beads were developed for an easy separation and regeneration of the used catalyst from the formed char. (orig.)

  18. Chromosomal mapping and mutational analysis of the coding region of the glycogen synthase kinase-3alpha and beta isoforms in patients with NIDDM

    DEFF Research Database (Denmark)

    Hansen, L; Arden, K C; Rasmussen, S B

    1997-01-01

    Activation of glycogen synthesis in skeletal muscle in response to insulin results from the combined inactivation of glycogen synthase kinase-3 (GSK-3) and activation of the protein phosphatase-1, changing the ratio between the inactive phosphorylated state of the glycogen synthase to the active ...

  19. Enterococcus faecalis phosphomevalonate kinase.

    Science.gov (United States)

    Doun, Stephanie S; Burgner, John W; Briggs, Scott D; Rodwell, Victor W

    2005-05-01

    The six enzymes of the mevalonate pathway of isopentenyl diphosphate biosynthesis represent potential for addressing a pressing human health concern, the development of antibiotics against resistant strains of the Gram-positive streptococci. We previously characterized the first four of the mevalonate pathway enzymes of Enterococcus faecalis, and here characterize the fifth, phosphomevalonate kinase (E.C. 2.7.4.2). E. faecalis genomic DNA and the polymerase chain reaction were used to clone DNA thought to encode phosphomevalonate kinase into pET28b(+). Double-stranded DNA sequencing verified the sequence of the recombinant gene. The encoded N-terminal hexahistidine-tagged protein was expressed in Escherichia coli with induction by isopropylthiogalactoside and purified by Ni(++) affinity chromatography, yield 20 mg protein per liter. Analysis of the purified protein by MALDI-TOF mass spectrometry established it as E. faecalis phosphomevalonate kinase. Analytical ultracentrifugation revealed that the kinase exists in solution primarily as a dimer. Assay for phosphomevalonate kinase activity used pyruvate kinase and lactate dehydrogenase to couple the formation of ADP to the oxidation of NADH. Optimal activity occurred at pH 8.0 and at 37 degrees C. The activation energy was approximately 5.6 kcal/mol. Activity with Mn(++), the preferred cation, was optimal at about 4 mM. Relative rates using different phosphoryl donors were 100 (ATP), 3.6 (GTP), 1.6 (TTP), and 0.4 (CTP). K(m) values were 0.17 mM for ATP and 0.19 mM for (R,S)-5-phosphomevalonate. The specific activity of the purified enzyme was 3.9 micromol substrate converted per minute per milligram protein. Applications to an immobilized enzyme bioreactor and to drug screening and design are discussed.

  20. Immigration process in catalytic medium

    Institute of Scientific and Technical Information of China (English)

    洪文明; 王梓坤

    2000-01-01

    The longtime behavior of the immigration process associated with a catalytic super-Brown-ian motion is studied. A large number law is proved in dimension d≤3 and a central limit theorem is proved for dimension d = 3.

  1. Immigration process in catalytic medium

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The longtime behavior of the immigration process associated with a catalytic super-Brownian motion is studied. A large number law is proved in dimension d≤3 and a central limit theorem is proved for dimension d=3.

  2. Association between Natural Resources for Outdoor Activities and Physical Inactivity: Results from the Contiguous United States

    Directory of Open Access Journals (Sweden)

    Yan Jiang

    2016-08-01

    Full Text Available Protected areas including national/state parks and recreational waters are excellent natural resources that promote physical activity and interaction with Nature, which can relieve stress and reduce disease risk. Despite their importance, however, their contribution to human health has not been properly quantified. This paper seeks to evaluate quantitatively how national/state parks and recreational waters are associated with human health and well-being, taking into account of the spatial dependence of environmental variables for the contiguous U.S., at the county level. First, we describe available natural resources for outdoor activities (ANROA, using national databases that include features from the Protected Areas Database, NAVSTREETS, and ATTAINSGEO 305(b Waters. We then use spatial regression techniques to explore the association of ANROA and socioeconomic status factors on physical inactivity rates. Finally, we use variance analysis to analyze ANROA’s influence on income-related health inequality. We found a significantly negative association between ANROA and the rate of physical inactivity: ANROA and the spatial effect explained 69%, nationwide, of the variation in physical inactivity. Physical inactivity rate showed a strong spatial dependence—influenced not only by its own in-county ANROA, but also by that of its neighbors ANROA. Furthermore, community groups at the same income level and with the highest ANROA, always had the lowest physical inactivity rate. This finding may help to guide future land use planning and community development that will benefit human health and well-being.

  3. Association between Natural Resources for Outdoor Activities and Physical Inactivity: Results from the Contiguous United States.

    Science.gov (United States)

    Jiang, Yan; Yuan, Yongping; Neale, Anne; Jackson, Laura; Mehaffey, Megan

    2016-08-17

    Protected areas including national/state parks and recreational waters are excellent natural resources that promote physical activity and interaction with Nature, which can relieve stress and reduce disease risk. Despite their importance, however, their contribution to human health has not been properly quantified. This paper seeks to evaluate quantitatively how national/state parks and recreational waters are associated with human health and well-being, taking into account of the spatial dependence of environmental variables for the contiguous U.S., at the county level. First, we describe available natural resources for outdoor activities (ANROA), using national databases that include features from the Protected Areas Database, NAVSTREETS, and ATTAINSGEO 305(b) Waters. We then use spatial regression techniques to explore the association of ANROA and socioeconomic status factors on physical inactivity rates. Finally, we use variance analysis to analyze ANROA's influence on income-related health inequality. We found a significantly negative association between ANROA and the rate of physical inactivity: ANROA and the spatial effect explained 69%, nationwide, of the variation in physical inactivity. Physical inactivity rate showed a strong spatial dependence-influenced not only by its own in-county ANROA, but also by that of its neighbors ANROA. Furthermore, community groups at the same income level and with the highest ANROA, always had the lowest physical inactivity rate. This finding may help to guide future land use planning and community development that will benefit human health and well-being.

  4. Comparative stability and catalytic and chemical properties of the sulfate-activating enzymes from Penicillium chrysogenum (mesophile) and Penicillium duponti (thermophile).

    Science.gov (United States)

    Renosto, F; Schultz, T; Re, E; Mazer, J; Chandler, C J; Barron, A; Segel, I H

    1985-11-01

    ATP sulfurylases from Penicillium chrysogenum (a mesophile) and from Penicillium duponti (a thermophile) had a native molecular weight of about 440,000 and a subunit molecular weight of about 69,000. (The P. duponti subunit appeared to be a little smaller than the P. chrysogenum subunit.) The P. duponti enzyme was about 100 times more heat stable than the P. chrysogenum enzyme; k inact (the first-order rate constant for inactivation) at 65 degrees C = 3.3 X 10(-4) s-1 for P. duponti and 3.0 X 10(-2) s-1 for P. chrysogenum. The P. duponti enzyme was also more stable to low pH and urea at 30 degrees C. Rabbit serum antibodies to each enzyme showed heterologous cross-reaction. Amino acid analyses disclosed no major compositional differences between the two enzymes. The analogous Km and Ki values of the forward and reverse reactions were also essentially identical at 30 degrees C. At 30 degrees C, the physiologically important adenosine 5'-phosphosulfate (APS) synthesis activity of the P. duponti enzyme was 4 U mg of protein-1, which is about half that of the P. chrysogenum enzyme. The molybdolysis and ATP synthesis activities of the P. duponti enzyme at 30 degrees C were similar to those of the P. chrysogenum enzyme. At 50 degrees C, the APS synthesis activity of the P. duponti enzyme was 12 to 19 U mg of protein-1, which was higher than that of the P. chrysogenum enzyme at 30 degrees C (8 +/- 1 U mg of protein-1). Treatment of the P. chrysogenum enzyme with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) at 30 degrees C under nondenaturing conditions modified one free sulfhydryl group per subunit. Vmax was not significantly altered, but the catalytic activity at low magnesium-ATP or SO4(2-) (or MoO4(2-)) was markedly reduced. Chemical modification with tetranitromethane had the same results on the kinetics. The native P. duponti enzyme was relatively unreactive toward DTNB or tetranitromethane at 30 degrees C and pH 8.0 or pH 9.0, but at 50 degrees C and pH 8.0, DTNB rapidly

  5. Hsp90/p50cdc37 is required for mixed-lineage kinase (MLK) 3 signaling.

    Science.gov (United States)

    Zhang, Hua; Wu, Wei; Du, Yan; Santos, Sarah J; Conrad, Susan E; Watson, Jack T; Grammatikakis, Nicholas; Gallo, Kathleen A

    2004-05-07

    Mixed-lineage kinase 3 (MLK3) is a mitogen-activated protein kinase (MAPK) kinase kinase that activates MAPK pathways, including the c-Jun NH(2)-terminal kinase (JNK) and p38 pathways. MLK3 and its family members have been implicated in JNK-mediated apoptosis. A survey of human cell lines revealed high levels of MLK3 in breast cancer cells. To learn more about MLK3 regulation and its signaling pathways in breast cancer cells, we engineered the estrogen-responsive human breast cancer cell line, MCF-7, to stably, inducibly express FLAG epitope-tagged MLK3. FLAG.MLK3 complexes were isolated by affinity purification, and associated proteins were identified by in-gel trypsin digestion followed by liquid chromatography/tandem mass spectrometry. Among the proteins identified were heat shock protein 90alpha,beta (Hsp90) and its kinase-specific co-chaperone p50(cdc37). We show that endogenous MLK3 complexes with Hsp90 and p50(cdc37). Further experiments demonstrate that MLK3 associates with Hsp90/p50(cdc37) through its catalytic domain in an activity-independent manner. Upon treatment of MCF-7 cells with geldanamycin, an ansamycin antibiotic that inhibits Hsp90 function, MLK3 levels decrease dramatically. Furthermore, tumor necrosis factor alpha-induced activation of MLK3 and JNK in MCF-7 cells is blocked by geldanamycin treatment. Our finding that geldanamycin treatment does not affect the cellular levels of the downstream signaling components, MAPK kinase 4, MAPK kinase 7, and JNK, suggests that Hsp90/p50(cdc37) regulates JNK signaling at the MAPK kinase kinase level. Previously identified Hsp90/p50(cdc37) clients include oncoprotein kinases and protein kinases that promote cellular proliferation and survival. Our findings reveal that Hsp90/p50(cdc37) also regulates protein kinases involved in apoptotic signaling.

  6. Establishment of X chromosome inactivation and epigenomic features of the inactive X depend on cellular contexts.

    Science.gov (United States)

    Vallot, Céline; Ouimette, Jean-François; Rougeulle, Claire

    2016-09-01

    X chromosome inactivation (XCI) is an essential epigenetic process that ensures X-linked gene dosage equilibrium between sexes in mammals. XCI is dynamically regulated during development in a manner that is intimately linked to differentiation. Numerous studies, which we review here, have explored the dynamics of X inactivation and reactivation in the context of development, differentiation and diseases, and the phenotypic and molecular link between the inactive status, and the cellular context. Here, we also assess whether XCI is a uniform mechanism in mammals by analyzing epigenetic signatures of the inactive X (Xi) in different species and cellular contexts. It appears that the timing of XCI and the epigenetic signature of the inactive X greatly vary between species. Surprisingly, even within a given species, various Xi configurations are found across cellular states. We discuss possible mechanisms underlying these variations, and how they might influence the fate of the Xi.

  7. A novel PWM scheme to eliminate the diode freewheeling In the Inactive phase in BLDC motor

    Institute of Scientific and Technical Information of China (English)

    WEI Kun; HU Chang-sheng; ZHANG Zhong-chao

    2006-01-01

    The brushless DC motor (BLDCM) with trapezoidal electromotive force (back-EMF) waveform is used widely.In principle,when the motor runs in the 120°conduction mode,two of the three phases are active while the other phase is inactive at all times.However,a ripple current occurs in the inactive phase due to the diode freewheeling during the non-commutation period in the traditional pulse width modulation (PWM) methods,which aggravates the torque ripples.A new PWM method is proposed in this paper to eliminate the diode freewheeling during the non-commutation period in the inactive phase.As a result,the torque ripple is suppressed using the proposed method.The simulation and experimental results are demonstrated to verify the validity of the proposed PWM method.

  8. Aerobic exercise and cold pressor test induce hypoalgesia in active and inactive men and women

    DEFF Research Database (Denmark)

    Vaegter, Henrik Bjarke; Handberg, Gitte; Jørgensen, Maria N.;

    2015-01-01

    and after exercise, PPTs increased to the same degree in active and inactive subjects, and the CPM and EIH responses were correlated (P aerobic exercise caused......). Conditioned pain modulation (CPM) was assessed by cold pressor testing. Exercise-induced hypoalgesia (EIH) was assessed after 15 minutes bicycling at a heart rate corresponding to 75% VO2max. A control session of 15 minutes quiet rest was also included. Pressure pain thresholds (PPTs) were recorded...... at the dominant arm and leg before, immediately after, and 15 minutes after conditioning and exercise as well as before and after rest. PPTs were also recorded during conditioning. RESULTS: At baseline, PPTs in inactive men were increased compared with inactive women (P 

  9. Dual-wavelength polymer laser based on an active/inactive/active sandwich-like structure

    Science.gov (United States)

    Zhai, Tianrui; Wu, Xiaofeng; Wang, Meng; Tong, Fei; Li, Songtao; Ma, Yanbin; Deng, Jinxiang; Zhang, Xinping

    2016-09-01

    Dual-wavelength laser emission is achieved by using an active/inactive/active sandwich-like structure, which can be conveniently fabricated using spin coating technique. Poly [(9, 9-dioctylfluorenyl-2, 7-diyl)-alt-co-(1, 4-benzo-(2, 1', 3) -thiadiazole)] and polyvinyl alcohol are employed as the active and the inactive materials, respectively. Two laser wavelengths are simultaneously observed, which are attributed to the difference of the surrounding refractive index of two active waveguides in the sandwich-like structure. Each wavelength is controlled by the respective waveguide structure, meaning that multi-wavelength laser can be designed by stacking the active/inactive layer pair. These results provide more flexibility to design compact laser sources.

  10. Remarks Regarding the Activity of Taxpayers Declared Inactive and Subsequent Economical Consequences

    Directory of Open Access Journals (Sweden)

    Despina-Martha ILUCA

    2013-12-01

    Full Text Available Declaring a taxpayer as inactive is a procedure founded on expressly stipulated law conditions and it has both individual effects and effects on the relation established between an inactive taxpayer and an active one. The primary effect, which is the cause for all of the others, is the ex officio cancellation of the VAT registration. This marks the loss of the taxpayer’s right to deduct the VAT using the “downstream VAT – upstream VAT” mechanism, becoming just a final consumer. The active taxpayer engaging in commercial transactions with an inactive taxpayer cannot deduct expenses, nor the VAT, as these operations are based on a document that is not issued in accordance with the legal provisions.

  11. Plant phosphatidylinositol 3-kinase

    NARCIS (Netherlands)

    Lee, Y.; Munnik, T.; Munnik, T.

    2010-01-01

    Phosphatidylinositol 3-kinase (PI3K) phosphorylates the D-3 position of phosphoinositides. In Arabidopsis, only one PI3K exists, which belongs to the class-III PI3K subfamily which makes phosphatidylinositol 3-phosphate (PtdIns3P). The single AtPI3K gene is essential for survival, since loss of its

  12. Changes in diagnosed diabetes, obesity, and physical inactivity prevalence in US counties, 2004-2012

    Science.gov (United States)

    Kirtland, Karen; Lin, Ji; Shrestha, Sundar; Thompson, Ted; Albright, Ann; Gregg, Edward W.

    2017-01-01

    Recent studies suggest that prevalence of diagnosed diabetes in the United States reached a plateau or slowed around 2008, and that this change coincided with obesity plateaus and increases in physical activity. However, national estimates can obscure important variations in geographic subgroups. We examine whether a slowing or leveling off in diagnosed diabetes, obesity, and leisure time physical inactivity prevalence is also evident across the 3143 counties of the United States. We used publicly available county estimates of the age-adjusted prevalence of diagnosed diabetes, obesity, and leisure-time physical inactivity, which were generated by the Centers for Disease Control and Prevention (CDC). Using a Bayesian multilevel regression that included random effects by county and year and applied cubic splines to smooth these estimates over time, we estimated the average annual percentage point change (APPC) from 2004 to 2008 and from 2008 to 2012 for diabetes, obesity, and physical inactivity prevalence in each county. Compared to 2004–2008, the median APPCs for diabetes, obesity, and physical inactivity were lower in 2008–2012 (diabetes APPC difference = 0.16, 95%CI 0.14, 0.18; obesity APPC difference = 0.65, 95%CI 0.59, 0.70; physical inactivity APPC difference = 0.43, 95%CI 0.37, 0.48). APPCs and APPC differences between time periods varied among counties and U.S. regions. Despite improvements, levels of these risk factors remained high with most counties merely slowing rather than reversing, which suggests that all counties would likely benefit from reductions in these risk factors. The diversity of trajectories in the prevalence of these risk factors across counties underscores the continued need to identify high risk areas and populations for preventive interventions. Awareness of how these factors are changing might assist local policy makers in targeting and tracking the impact of efforts to reduce diabetes, obesity and physical inactivity. PMID

  13. Light saturation curves show competence of the water splitting complex in inactive Photosystem II reaction centers.

    Science.gov (United States)

    Nedbal, L; Gibas, C; Whitmarsh, J

    1991-12-01

    Photosystem II complexes of higher plants are structurally and functionally heterogeneous. While the only clearly defined structural difference is that Photosystem II reaction centers are served by two distinct antenna sizes, several types of functional heterogeneity have been demonstrated. Among these is the observation that in dark-adapted leaves of spinach and pea, over 30% of the Photosystem II reaction centers are unable to reduce plastoquinone to plastoquinol at physiologically meaningful rates. Several lines of evidence show that the impaired reaction centers are effectively inactive, because the rate of oxidation of the primary quinone acceptor, QA, is 1000 times slower than in normally active reaction centers. However, there are conflicting opinions and data over whether inactive Photosystem II complexes are capable of oxidizing water in the presence of certain artificial electron acceptors. In the present study we investigated whether inactive Photosystem II complexes have a functional water oxidizing system in spinach thylakoid membranes by measuring the flash yield of water oxidation products as a function of flash intensity. At low flash energies (less that 10% saturation), selected to minimize double turnovers of reaction centers, we found that in the presence of the artificial quinone acceptor, dichlorobenzoquinone (DCBQ), the yield of proton release was enhanced 20±2% over that observed in the presence of dimethylbenzoquinone (DMBQ). We argue that the extra proton release is from the normally inactive Photosystem II reaction centers that have been activated in the presence of DCBQ, demonstrating their capacity to oxidize water in repetitive flashes, as concluded by Graan and Ort (Biochim Biophys Acta (1986) 852: 320-330). The light saturation curves indicate that the effective antenna size of inactive reaction centers is 55±12% the size of active Photosystem II centers. Comparison of the light saturation dependence of steady state oxygen evolution

  14. The association of ambient air pollution and physical inactivity in the United States.

    Directory of Open Access Journals (Sweden)

    Jennifer D Roberts

    Full Text Available BACKGROUND: Physical inactivity, ambient air pollution and obesity are modifiable risk factors for non-communicable diseases, with the first accounting for 10% of premature deaths worldwide. Although community level interventions may target each simultaneously, research on the relationship between these risk factors is lacking. OBJECTIVES: After comparing spatial interpolation methods to determine the best predictor for particulate matter (PM2.5; PM10 and ozone (O3 exposures throughout the U.S., we evaluated the cross-sectional association of ambient air pollution with leisure-time physical inactivity among adults. METHODS: In this cross-sectional study, we assessed leisure-time physical inactivity using individual self-reported survey data from the Centers for Disease Control and Prevention's 2011 Behavioral Risk Factor Surveillance System. These data were combined with county-level U.S. Environmental Protection Agency air pollution exposure estimates using two interpolation methods (Inverse Distance Weighting and Empirical Bayesian Kriging. Finally, we evaluated whether those exposed to higher levels of air pollution were less active by performing logistic regression, adjusting for demographic and behavioral risk factors, and after stratifying by body weight category. RESULTS: With Empirical Bayesian Kriging air pollution values, we estimated a statistically significant 16-35% relative increase in the odds of leisure-time physical inactivity per exposure class increase of PM2.5 in the fully adjusted model across the normal weight respondents (p-value<0.0001. Evidence suggested a relationship between the increasing dose of PM2.5 exposure and the increasing odds of physical inactivity. CONCLUSIONS: In a nationally representative, cross-sectional sample, increased community level air pollution is associated with reduced leisure-time physical activity particularly among the normal weight. Although our design precludes a causal inference, these

  15. Linking geology and microbiology: inactive pockmarks affect sediment microbial community structure.

    Directory of Open Access Journals (Sweden)

    Thomas H A Haverkamp

    Full Text Available Pockmarks are geological features that are found on the bottom of lakes and oceans all over the globe. Some are active, seeping oil or methane, while others are inactive. Active pockmarks are well studied since they harbor specialized microbial communities that proliferate on the seeping compounds. Such communities are not found in inactive pockmarks. Interestingly, inactive pockmarks are known to have different macrofaunal communities compared to the surrounding sediments. It is undetermined what the microbial composition of inactive pockmarks is and if it shows a similar pattern as the macrofauna. The Norwegian Oslofjord contains many inactive pockmarks and they are well suited to study the influence of these geological features on the microbial community in the sediment. Here we present a detailed analysis of the microbial communities found in three inactive pockmarks and two control samples at two core depth intervals. The communities were analyzed using high-throughput amplicon sequencing of the 16S rRNA V3 region. Microbial communities of surface pockmark sediments were indistinguishable from communities found in the surrounding seabed. In contrast, pockmark communities at 40 cm sediment depth had a significantly different community structure from normal sediments at the same depth. Statistical analysis of chemical variables indicated significant differences in the concentrations of total carbon and non-particulate organic carbon between 40 cm pockmarks and reference sample sediments. We discuss these results in comparison with the taxonomic classification of the OTUs identified in our samples. Our results indicate that microbial communities at the sediment surface are affected by the water column, while the deeper (40 cm sediment communities are affected by local conditions within the sediment.

  16. Linking geology and microbiology: inactive pockmarks affect sediment microbial community structure.

    Science.gov (United States)

    Haverkamp, Thomas H A; Hammer, Øyvind; Jakobsen, Kjetill S

    2014-01-01

    Pockmarks are geological features that are found on the bottom of lakes and oceans all over the globe. Some are active, seeping oil or methane, while others are inactive. Active pockmarks are well studied since they harbor specialized microbial communities that proliferate on the seeping compounds. Such communities are not found in inactive pockmarks. Interestingly, inactive pockmarks are known to have different macrofaunal communities compared to the surrounding sediments. It is undetermined what the microbial composition of inactive pockmarks is and if it shows a similar pattern as the macrofauna. The Norwegian Oslofjord contains many inactive pockmarks and they are well suited to study the influence of these geological features on the microbial community in the sediment. Here we present a detailed analysis of the microbial communities found in three inactive pockmarks and two control samples at two core depth intervals. The communities were analyzed using high-throughput amplicon sequencing of the 16S rRNA V3 region. Microbial communities of surface pockmark sediments were indistinguishable from communities found in the surrounding seabed. In contrast, pockmark communities at 40 cm sediment depth had a significantly different community structure from normal sediments at the same depth. Statistical analysis of chemical variables indicated significant differences in the concentrations of total carbon and non-particulate organic carbon between 40 cm pockmarks and reference sample sediments. We discuss these results in comparison with the taxonomic classification of the OTUs identified in our samples. Our results indicate that microbial communities at the sediment surface are affected by the water column, while the deeper (40 cm) sediment communities are affected by local conditions within the sediment.

  17. Unemployment, Employment and Inactivity in Denmark: An Analysis of Event History Data

    DEFF Research Database (Denmark)

    Lauzadyté, Agné

    and inactivity. The less educated and unskilled workers are found to be another risk group to face the marginalisation from the labour market. Being previously employed reduces the risk of OLF, and increases the re-entry to employment probability, while living in the biggest Danish cities makes persons......In this paper I estimate a discrete time hazard model for the exits from the different labour market states - unemployment, employment and inactivity (or OLF) - in the Danish labour market. I find that women and individuals over fifty are more likely to experience the long-term unemployment...

  18. The role of Src kinase in the biology and pathogenesis of Acanthamoeba castellanii

    Directory of Open Access Journals (Sweden)

    Siddiqui Ruqaiyyah

    2012-06-01

    Full Text Available Abstract Background Acanthamoeba species are the causative agents of fatal granulomatous encephalitis in humans. Haematogenous spread is thought to be a primary step, followed by blood–brain barrier penetration, in the transmission of Acanthmaoeba into the central nervous system, but the associated molecular mechanisms remain unclear. Here, we evaluated the role of Src, a non-receptor protein tyrosine kinase in the biology and pathogenesis of Acanthamoeba. Methods Amoebistatic and amoebicidal assays were performed by incubating amoeba in the presence of Src kinase-selective inhibitor, PP2 (4-amino-5-(4-chlorophenyl-7-(t-butylpyrazolo[3,4-d]pyrimidine and its inactive analog, PP3 (4-amino-7-phenylpyrazolo[3,4-d]pyrimidine. Using this inhibitor, the role of Src kinase in A. castellanii interactions with Escherichia coli was determined. Zymographic assays were performed to study effects of Src kinase on extracellular proteolytic activities of A. castellanii. The human brain microvascular endothelial cells were used to determine the effects of Src kinase on A. castellanii adhesion to and cytotoxicity of host cells. Results Inhibition of Src kinase using a specific inhibitor, PP2 (4-amino-5-(4 chlorophenyl-7-(t-butylpyrazolo [3,4-d] pyrimidine but not its inactive analog, PP3 (4-amino-7-phenylpyrazolo[3,4-d] pyrimidine, had detrimental effects on the growth of A. castellanii (keratitis isolate, belonging to the T4 genotype. Interestingly, inhibition of Src kinase hampered the phagocytic ability of A. castellanii, as measured by the uptake of non-invasive bacteria, but, on the contrary, invasion by pathogenic bacteria was enhanced. Zymographic assays revealed that inhibition of Src kinases reduced extracellular protease activities of A. castellanii. Src kinase inhibition had no significant effect on A. castellanii binding to and cytotoxicity of primary human brain microvascular endothelial cells, which constitute the blood–brain barrier

  19. Casein kinase II protein kinase is bound to lamina-matrix and phosphorylates lamin-like protein in isolated pea nuclei

    Science.gov (United States)

    Li, H.; Roux, S. J.

    1992-01-01

    A casein kinase II (CK II)-like protein kinase was identified and partially isolated from a purified envelope-matrix fraction of pea (Pisum sativum L.) nuclei. When [gamma-32P]ATP was directly added to the envelope-matrix preparation, the three most heavily labeled protein bands had molecular masses near 71, 48, and 46 kDa. Protein kinases were removed from the preparation by sequential extraction with Triton X-100, EGTA, 0.3 M NaCl, and a pH 10.5 buffer, but an active kinase still remained bound to the remaining lamina-matrix fraction after these treatments. This kinase had properties resembling CK II kinases previously characterized from animal and plant sources: it preferred casein as an artificial substrate, could use GTP as efficiently as ATP as the phosphoryl donor, was stimulated by spermine, was calcium independent, and had a catalytic subunit of 36 kDa. Some animal and plant CK II kinases have regulatory subunits near 29 kDa, and a lamina-matrix-bound protein of this molecular mass was recognized on immunoblot by anti-Drosophila CK II polyclonal antibodies. Also found associated with the envelope-matrix fraction of pea nuclei were p34cdc2-like and Ca(2+)-dependent protein kinases, but their properties could not account for the protein kinase activity bound to the lamina. The 71-kDa substrate of the CK II-like kinase was lamin A-like, both in its molecular mass and in its cross-reactivity with anti-intermediate filament antibodies. Lamin phosphorylation is considered a crucial early step in the entry of cells into mitosis, so lamina-bound CK II kinases may be important control points for cellular proliferation.

  20. Structural Mechanisms of Allostery and Autoinhibition in JNK Family Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Laughlin, J. D.; Nwachukwu, J. C.; Figuera-Losada, M.; Cherry, L.; Nettles, K. W.; LoGrasso, P. V.

    2012-12-05

    c-Jun N-terminal (JNK) family kinases have a common peptide-docking site used by upstream activating kinases, substrates, scaffold proteins, and phosphatases, where the ensemble of bound proteins determines signaling output. Although there are many JNK structures, little is known about mechanisms of allosteric regulation between the catalytic and peptide-binding sites, and the activation loop, whose phosphorylation is required for catalytic activity. Here, we compare three structures of unliganded JNK3 bound to different peptides. These were compared as a class to structures that differ in binding of peptide, small molecule ligand, or conformation of the kinase activation loop. Peptide binding induced an inhibitory interlobe conformer that was reversed by alterations in the activation loop. Structure class analysis revealed the subtle structural mechanisms for allosteric signaling between the peptide-binding site and activation loop. Biochemical data from isothermal calorimetry, fluorescence energy transfer, and enzyme inhibition demonstrated affinity differences among the three peptides that were consistent with structural observations.

  1. Activation of protein kinase C inhibits potassium currents in cultured endothelial cells.

    Science.gov (United States)

    Zhang, H; Weir, B; Daniel, E E

    1995-04-01

    The effect of protein kinase C on potassium channels in cultured endothelial cells was investigated by using whole-cell patch-clamp techniques. Activation of protein kinase C by phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu), but not phorbol 12-monomyristate (PMM), an inactive analogue of phorbol esters, depressed an outward calcium-dependent potassium current. The inhibitory actions of PMA and PDBu could be reversed by the kinase inhibitor H-7. Cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum calcium pump, and LP-805, a novel vasodilator which also releases endothelium-derived relaxing factors, activated the outward calcium-dependent potassium conductance. PMA and PDBu, but not PMM, reduced the outward conductance induced by cyclopiazonic acid and LP-805. These effects of PMA and PDBu on potassium currents may be mediated either by phosphorylation of ion channels, or by decreasing intracellular calcium concentration.

  2. Calcium is the switch in the moonlighting dual function of the ligand-activated receptor kinase phytosulfokine receptor 1

    KAUST Repository

    Muleya, Victor

    2014-09-23

    Background: A number of receptor kinases contain guanylate cyclase (GC) catalytic centres encapsulated in the cytosolic kinase domain. A prototypical example is the phytosulfokine receptor 1 (PSKR1) that is involved in regulating growth responses in plants. PSKR1 contains both kinase and GC activities however the underlying mechanisms regulating the dual functions have remained elusive. Findings: Here, we confirm the dual activity of the cytoplasmic domain of the PSKR1 receptor. We show that mutations within the guanylate cyclase centre modulate the GC activity while not affecting the kinase catalytic activity. Using physiologically relevant Ca2+ levels, we demonstrate that its GC activity is enhanced over two-fold by Ca2+ in a concentration-dependent manner. Conversely, increasing Ca2+ levels inhibits kinase activity up to 500-fold at 100 nM Ca2+. Conclusions: Changes in calcium at physiological levels can regulate the kinase and GC activities of PSKR1. We therefore propose a functional model of how calcium acts as a bimodal switch between kinase and GC activity in PSKR1 that could be relevant to other members of this novel class of ligand-activated receptor kinases.

  3. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C. International Federation of Clinical Chemistry and Laboratory Medicine. Part 4. Reference procedure for the measurement of catalytic concentration of alanine aminotransferase.

    Science.gov (United States)

    Schumann, Gerhard; Bonora, Roberto; Ceriotti, Ferruccio; Férard, Georges; Ferrero, Carlo A; Franck, Paul F H; Gella, F Javier; Hoelzel, Wieland; Jørgensen, Poul Jørgen; Kanno, Takashi; Kessner, Art; Klauke, Rainer; Kristiansen, Nina; Lessinger, Jean-Marc; Linsinger, Thomas P J; Misaki, Hideo; Panteghini, Mauro; Pauwels, Jean; Schiele, Françoise; Schimmel, Heinz G; Weidemann, Gerhard; Siekmann, Lothar

    2002-07-01

    This paper is the fourth in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C and the certification of reference preparations. Other parts deal with: Part 1. The Concept of Reference Procedures for the Measurement of Catalytic Activity Concentrations of Enzymes; Part 2. Reference Procedure for the Measurement of Catalytic Concentration of Creatine Kinase; Part 3. Reference Procedure for the Measurement of Catalytic Concentration of Lactate Dehydrogenase; Part 5. Reference Procedure for the Measurement of Catalytic Concentration of Aspartate Aminotransferase; Part 6. Reference Procedure for the Measurement of Catalytic Concentration of Gamma-Glutamyltransferase; Part 7. Certification of Four Reference Materials for the Determination of Enzymatic Activity of Gamma-Glutamyltransferase, Lactate Dehydrogenase, Alanine Aminotransferase and Creatine Kinase at 37 degrees C. A document describing the determination of preliminary upper reference limits is also in preparation. The procedure described here is deduced from the previously described 30 degrees C IFCC reference method. Differences are tabulated and commented on in Appendix 2.

  4. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C. International Federation of Clinical Chemistry and Laboratory Medicine. Part 6. Reference procedure for the measurement of catalytic concentration of gamma-glutamyltransferase.

    Science.gov (United States)

    Schumann, Gerhard; Bonora, Roberto; Ceriotti, Ferruccio; Férard, Georges; Ferrero, Carlo A; Franck, Paul F H; Gella, F Javier; Hoelzel, Wieland; Jørgensen, Poul Jørgen; Kanno, Takashi; Kessner, Art; Klauke, Rainer; Kristiansen, Nina; Lessinger, Jean-Marc; Linsinger, Thomas P J; Misaki, Hideo; Panteghini, Mauro; Pauwels, Jean; Schiele, Françoise; Schimmel, Heinz G; Weidemann, Gerhard; Siekmann, Lothar

    2002-07-01

    This paper is the sixth in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 degrees C and the certification of reference preparations. Other parts deal with: Part 1. The Concept of Reference Procedures for the Measurement of Catalytic Activity Concentrations of Enzymes; Part 2. Reference Procedure for the Measurement of Catalytic Concentration of Creatine Kinase; Part 3. Reference Procedure for the Measurement of Catalytic Concentration of Lactate Dehydrogenase; Part 4. Reference Procedure for the Measurement of Catalytic Concentration of Alanine Aminotransferase; Part 5. Reference Procedure for the Measurement of Catalytic Concentration of Aspartate Aminotransferase; Part 7. Certification of Four Reference Materials for the Determination of Enzymatic Activity of Gamma-Glutamyltransferase, Lactate Dehydrogenase, Alanine Aminotransferase and Creatine Kinase at 37 degrees C A document describing the determination of preliminary upper reference limits is also in preparation. The procedure described here is deduced from the previously described 30 degrees C IFCC reference method. Differences are tabulated and commented on in Appendix 1.

  5. Engineering reactors for catalytic reactions

    Indian Academy of Sciences (India)

    Vivek V Ranade

    2014-03-01

    Catalytic reactions are ubiquitous in chemical and allied industries. A homogeneous or heterogeneous catalyst which provides an alternative route of reaction with lower activation energy and better control on selectivity can make substantial impact on process viability and economics. Extensive studies have been conducted to establish sound basis for design and engineering of reactors for practising such catalytic reactions and for realizing improvements in reactor performance. In this article, application of recent (and not so recent) developments in engineering reactors for catalytic reactions is discussed. Some examples where performance enhancement was realized by catalyst design, appropriate choice of reactor, better injection and dispersion strategies and recent advances in process intensification/ multifunctional reactors are discussed to illustrate the approach.

  6. Catalytic activity of Au nanoparticles

    DEFF Research Database (Denmark)

    Larsen, Britt Hvolbæk; Janssens, Ton V.W.; Clausen, Bjerne;

    2007-01-01

    Au is usually viewed as an inert metal, but surprisingly it has been found that Au nanoparticles less than 3–5 nm in diameter are catalytically active for several chemical reactions. We discuss the origin of this effect, focusing on the way in which the chemical activity of Au may change with par......Au is usually viewed as an inert metal, but surprisingly it has been found that Au nanoparticles less than 3–5 nm in diameter are catalytically active for several chemical reactions. We discuss the origin of this effect, focusing on the way in which the chemical activity of Au may change...... with particle size. We find that the fraction of low-coordinated Au atoms scales approximately with the catalytic activity, suggesting that atoms on the corners and edges of Au nanoparticles are the active sites. This effect is explained using density functional calculations....

  7. Tyrosine kinases in rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Kobayashi Akiko

    2011-08-01

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

  8. Teacher Educators' In-Action Mental Models in Different Teaching Situations

    Science.gov (United States)

    Mevorach, Miriam; Strauss, Sidney

    2012-01-01

    In previous studies on teachers' cognition, we discovered that teachers' teaching can be described via a general in-action mental model (IAMM) concerning the structure of the mind and the roles of teaching in fostering children's learning. The purpose of our study was to examine teacher educators' IAMM regarding student teachers' minds and…

  9. Physical inactivity and obesity: Using a novel environmental quality measure to control confounding

    Science.gov (United States)

    Physical inactivity is well-established as a contributor to obesity prevalence in the US. Many aspects of the ambient environment (e.g., air pollution, food deserts, neighborhood socioeconomics) have also been associated with obesity. Yet, controlling for the overall ambient envi...

  10. A conceptualisation of help-avoidance as motivated inaction: implications for theory, research, and society

    NARCIS (Netherlands)

    Täuber, Susanne; Zagefka, Hanna; van Leeuwen, Esther

    2017-01-01

    This chapter zooms in on the strategic motives of help-avoidance, an intriguing yet under-researched phenomenon. Conceptualising this phenomenon as a particular form of inaction, I propose that help-avoidance is a strategic response to disadvantage that is motivated by identity concerns. I provide t

  11. Job strain as a risk factor for leisure-time physical inactivity

    DEFF Research Database (Denmark)

    Fransson, Eleonor I; Heikkilä, Katriina; Nyberg, Solja T

    2012-01-01

    Unfavorable work characteristics, such as low job control and too high or too low job demands, have been suggested to increase the likelihood of physical inactivity during leisure time, but this has not been verified in large-scale studies. The authors combined individual-level data from 14 Europ...

  12. Comparison of fractions of inactive modules between Run1 and Run2

    CERN Document Server

    Motohashi, Kazuki; The ATLAS collaboration

    2015-01-01

    Fraction of inactive modules for each component of the ATLAS pixel detector at the end of Run 1 and the beginning of Run 2. A similar plot which uses a result of functionality tests during LS1 can be found in ATL-INDET-SLIDE-2014-388.

  13. Coronary Heart Disease Risk between Active and Inactive Women with Multiple Sclerosis.

    Science.gov (United States)

    Slawta, Jennifer N.; McCubbin, Jeffrey A.; Wilcox, Anthony R.; Fox, Susan D.; Nalle, Darek J.; Anderson, Gail

    2002-01-01

    Investigated whether abdominal fat accumulation and levels of triglyceride, high-density lipoprotein cholesterol, and glucose differed between 123 active and inactive women with multiple sclerosis (MS). Results indicated that low-to-moderate leisure time physical activity significantly related to less abdominal fat accumulation, lower triglyceride…

  14. Social Cognitive Correlates of Physical Activity in Inactive Adults with Multiple Sclerosis

    Science.gov (United States)

    Dlugonski, Deirdre; Wojcicki, Thomas R.; McAuley, Edward; Motl, Robert W.

    2011-01-01

    Persons with multiple sclerosis (MS) are often physically inactive. This observation has prompted the search for modifiable constructs derived from established theories that act as correlates of physical activity. This study investigated self efficacy, outcome expectations, impediments, and goal setting as correlates of physical activity in…

  15. Peripheral aneurysm rupture in a patient with inactive systemic lupus erythematosus

    Energy Technology Data Exchange (ETDEWEB)

    Engelke, Christoph; Sabharwal, Tarun; Reidy, John F. [Department of Radiology, Guy' s and St. Thomas' Hospital Trust, St. Thomas' Street, London SE1 9RT (United Kingdom); Mohan, Aarthi R. [Department of Chest Medicine, Guy' s and St. Thomas' Hospital Trust, St. Thomas' Street, London SE1 9RT (United Kingdom)

    2002-12-01

    We describe a patient with inactive systemic lupus erythematosus (SLE) presenting with sudden haemothorax, due to a ruptured internal mammary artery (IMA) aneurysm 7 years after the corticosteroid treatment was terminated. The unusual imaging findings and the treatment with embolization are discussed with a view to the role of a regular vascular screening in this patient group. (orig.)

  16. Sauna-induced body mass loss in physically inactive young women and men

    Directory of Open Access Journals (Sweden)

    Podstawski Robert

    2016-01-01

    Full Text Available Study aim: The aim of this study was to evaluate the relationship between basic somatic features (body mass and height and body mass loss in physically inactive young women and men exposed to thermal stress in a dry sauna.

  17. 38 CFR 3.375 - Determination of inactivity (complete arrest) in tuberculosis.

    Science.gov (United States)

    2010-07-01

    ... inactivity (complete arrest) in tuberculosis. 3.375 Section 3.375 Pensions, Bonuses, and Veterans' Relief...) in tuberculosis. (a) Pulmonary tuberculosis. A veteran shown to have had pulmonary tuberculosis will...) Nonpulmonary disease. Determination of complete arrest of nonpulmonary tuberculosis requires absence...

  18. Reactivation of inactive X chromosome in buccal smear of carcinoma of breast

    Directory of Open Access Journals (Sweden)

    Natekar Prashant

    2008-01-01

    Full Text Available Buccal mucosal smears of 100 female patients of carcinoma of breast were compared with 100 controls matched accordingly. The frequency of Barr bodies was significantly lower in carcinoma of breast patients (menstruating and menopausal women P < 0.001 when compared with controls indicating reactivation of the inactive X chromosome.

  19. Obesity, smoking, and physical inactivity as risk factors for CKD: Are men more vulnerable?

    NARCIS (Netherlands)

    S. Hallan; R. de Mutsert; S. Carlsen; F.W. Dekker; K. Aasarod; J. Holmen

    2006-01-01

    Background: The incidence of end-stage renal disease is especially high in men, and some studies indicated that smoking is a risk factor for men only. We investigated associations between obesity, smoking, and physical inactivity and chronic kidney disease (CKD) in the general population and whether

  20. Cosmopolitan Utilitarianism and the Problem of Local Inaction in a Globalized World

    Directory of Open Access Journals (Sweden)

    Fausto Corvino

    2015-10-01

    Full Text Available This article explores the problem of the public acceptability of political inaction as an extreme consequence of cosmopolitan utilitarianism. The case of political inaction as the utility-maximizing public policy option emerges more clearly in the globalized world, because of a misalignment between the electoral body and the persons that the government ought to consider while evaluating the consequences of a given policy. In this context, a situation can easily occur in which the only way to maximize utility in a global context is by renouncing action at the national or local level. However, the problem of inaction should not be interpreted simply as a by-product of globalization. Its origins can be traced to the basic structure of utilitarianism as a normative consequentialist theory. This drawback can even present itself at the local level in a less visible form. One example is that in which the performance of a supererogatory act in the exercise of public office leads to a reduction in overall utility. The aim of the article is to demonstrate that cosmopolitan utilitarianism can bind the decision maker to a series of inactions at the global and local levels that contradict his own mandate, generating a dangerous moral confusion in the implementation of public policies. This can seriously threaten the universal applicability of cosmopolitan utilitarianism as a normative political theory, especially in the age of globalization.

  1. Structure of the two-domain hexameric APS kinase from Thiobacillus denitrificans: structural basis for the absence of ATP sulfurylase activity

    Energy Technology Data Exchange (ETDEWEB)

    Gay, Sean C. [Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616 (United States); Segel, Irwin H. [Section of Molecular and Cellular Biology, University of California, One Shields Avenue, Davis, CA 95616 (United States); Fisher, Andrew J., E-mail: fisher@chem.ucdavis.edu [Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616 (United States); Section of Molecular and Cellular Biology, University of California, One Shields Avenue, Davis, CA 95616 (United States)

    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.

  2. Enterococcus faecalis phosphomevalonate kinase

    OpenAIRE

    Doun, Stephanie S.; Burgner, John W.; Briggs, Scott D.; Rodwell, Victor W.

    2005-01-01

    The six enzymes of the mevalonate pathway of isopentenyl diphosphate biosynthesis represent potential for addressing a pressing human health concern, the development of antibiotics against resistant strains of the Gram-positive streptococci. We previously characterized the first four of the mevalonate pathway enzymes of Enterococcus faecalis, and here characterize the fifth, phosphomevalonate kinase (E.C. 2.7.4.2). E. faecalis genomic DNA and the polymerase chain reaction were used to clone D...

  3. Oncoprotein protein kinase

    Energy Technology Data Exchange (ETDEWEB)

    Karin, Michael (San Diego, CA); Hibi, Masahiko (San Diego, CA); Lin, Anning (La Jolla, CA); Davis, Roger (Princeton, MA); Derijard, Benoit (Shrewsbury, MA)

    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.

  4. Catalytic Decoupling of Quantum Information

    DEFF Research Database (Denmark)

    Majenz, Christian; Berta, Mario; Dupuis, Frédéric

    2017-01-01

    The decoupling technique is a fundamental tool in quantum information theory with applications ranging from quantum thermodynamics to quantum many body physics to the study of black hole radiation. In this work we introduce the notion of catalytic decoupling, that is, decoupling in the presence...... of an uncorrelated ancilla system. This removes a restriction on the standard notion of decoupling, which becomes important for structureless resources, and yields a tight characterization in terms of the max-mutual information. Catalytic decoupling naturally unifies various tasks like the erasure of correlations...... and quantum state merging, and leads to a resource theory of decoupling....

  5. Comparing Levels of Depression in Healthy Active and Inactive Elders versus Those with Knee Osteoarthritis Disease

    Directory of Open Access Journals (Sweden)

    Amir Shams

    2016-04-01

    Full Text Available Knee osteoarthritis can have negative effects on the physical and mental conditions, social and family relations, general health and positive feelings of elders. For example, severe limitation of motion and increased depression, which are results of this disease, can have negative impacts on elders. The purpose of this study was to compare the levels of depression in healthy active and inactive elders versus those with knee osteoarthritis disease. Two hundred and twenty (220 elders with knee osteoarthritis disease (110 active and 110 inactive patients and 220 healthy elders (110 active and 110 inactive were voluntarily selected, and they filled the questionnaire about elders’ personal characteristics, physical activity and depression levels. Four groups had differences with respect to education, age and gender. The results of chi-square test revealed that women with knee osteoarthritis disease had increased levels of depression compared to men with the same condition (p<0.05. Older patients had increased levels of depression and educated elders reported lower levels of depression. The results of the one way analysis of variance (ANOVA revealed that there exist a significant difference in depression scores of healthy active and healthy inactive elders and those with knee osteoarthritis disease. The results of Tukey’s test revealed that healthy active elder had lower depression scores compared to inactive elders with knee osteoarthritis disease. Carrying out exercises and physical activities can help improve the health state of patients with knee osteoarthritis problem and can also improve the elders’ mental condition and thus decrease their pain and depression.

  6. A national survey of 'inactive' physicians in the United States of America: enticements to reentry

    Directory of Open Access Journals (Sweden)

    Brotherton Sarah E

    2011-02-01

    Full Text Available Abstract Background Physicians leaving and reentering clinical practice can have significant medical workforce implications. We surveyed inactive physicians younger than typical retirement age to determine their reasons for clinical inactivity and what barriers, real or perceived, there were to reentry into the medical workforce. Methods A random sample of 4975 inactive physicians aged under 65 years was drawn from the Physician Masterfile of the American Medical Association in 2008. Physicians were mailed a survey about activity in medicine and perceived barriers to reentry. Chi-square statistics were used for significance tests of the association between categorical variables and t-tests were used to test differences between means. Results Our adjusted response rate was 36.1%. Respondents were fully retired (37.5%, not currently active in medicine (43.0% or now active (reentered, 19.4%. Nearly half (49.5% were in or had practiced primary care. Personal health was the top reason for leaving for fully retired physicians (37.8% or those not currently active in medicine (37.8% and the second highest reason for physicians who had reentered (28.8%. For reentered (47.8% and inactive (51.5% physicians, the primary reason for returning or considering returning to practice was the availability of part-time work or flexible scheduling. Retired and currently inactive physicians used similar strategies to explore reentry, and 83% of both groups thought it would be difficult; among those who had reentered practice, 35.9% reported it was difficult to reenter. Retraining was uncommon for this group (37.5%. Conclusion Availability of part-time work and flexible scheduling have a strong influence on decisions to leave or reenter clinical practice. Lack of retraining before reentry raises questions about patient safety and the clinical competence of reentered physicians.

  7. Germinal-center kinase-like kinase co-crystal structure reveals a swapped activation loop and C-terminal extension.

    Science.gov (United States)

    Marcotte, Douglas; Rushe, Mia; M Arduini, Robert; Lukacs, Christine; Atkins, Kateri; Sun, Xin; Little, Kevin; Cullivan, Michael; Paramasivam, Murugan; Patterson, Thomas A; Hesson, Thomas; D McKee, Timothy; May-Dracka, Tricia L; Xin, Zhili; Bertolotti-Ciarlet, Andrea; Bhisetti, Govinda R; Lyssikatos, Joseph P; Silvian, Laura F

    2017-02-01

    Germinal-center kinase-like kinase (GLK, Map4k3), a GCK-I family kinase, plays multiple roles in regulating apoptosis, amino acid sensing, and immune signaling. We describe here the crystal structure of an activation loop mutant of GLK kinase domain bound to an inhibitor. The structure reveals a weakly associated, activation-loop swapped dimer with more than 20 amino acids of ordered density at the carboxy-terminus. This C-terminal PEST region binds intermolecularly to the hydrophobic groove of the N-terminal domain of a neighboring molecule. Although the GLK activation loop mutant crystallized demonstrates reduced kinase activity, its structure demonstrates all the hallmarks of an "active" kinase, including the salt bridge between the C-helix glutamate and the catalytic lysine. Our compound displacement data suggests that the effect of the Ser170Ala mutation in reducing kinase activity is likely due to its effect in reducing substrate peptide binding affinity rather than reducing ATP binding or ATP turnover. This report details the first structure of GLK; comparison of its activation loop sequence and P-loop structure to that of Map4k4 suggests ideas for designing inhibitors that can distinguish between these family members to achieve selective pharmacological inhibitors.

  8. Mechanism of Activation and Inhibition of the HER4/ErbB4 Kinase

    Energy Technology Data Exchange (ETDEWEB)

    Qiu,C.; Tarrant, M.; Choi, S.; Sathyamurthy, A.; Bose, R.; Banjade, S.; Pal, A.; Bornmann, W.; Lemmon, M.; et al

    2008-01-01

    HER4/ErbB4 is a ubiquitously expressed member of the EGF/ErbB family of receptor tyrosine kinases that is essential for normal development of the heart, nervous system, and mammary gland. We report here crystal structures of the ErbB4 kinase domain in active and lapatinib-inhibited forms. Active ErbB4 kinase adopts an asymmetric dimer conformation essentially identical to that observed to be important for activation of the EGF receptor/ErbB1 kinase. Mutagenesis studies of intact ErbB4 in Ba/F3 cells confirm the importance of this asymmetric dimer for activation of intact ErbB4. Lapatinib binds to an inactive form of the ErbB4 kinase in a mode equivalent to its interaction with the EGF receptor. All ErbB4 residues contacted by lapatinib are conserved in the EGF receptor and HER2/ErbB2, which lapatinib also targets. These results demonstrate that key elements of kinase activation and inhibition are conserved among ErbB family members.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  10. Regulation of Autophagy by Kinases

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-06-09

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

  11. NITRIC OXIDE BINDS TO AND MODULATES THE ACTIVITY OF A POLLEN SPECIFIC ARABIDOPSIS DIACYLGLYCEROL KINASE

    KAUST Repository

    Wong, Aloysius Tze

    2014-06-01

    Nitric oxide (NO) is an important signaling molecule in plants. In the pollen of Arabidopsis thaliana, NO causes re-orientation of the growing tube and this response is mediated by 3′,5′-cyclic guanosine monophosphate (cGMP). However, in plants, NO-sensors have remained somewhat elusive. Here, the findings of an NO-binding candidate, Arabidopsis thaliana DIACYLGLYCEROL KINASE 4 (ATDGK4; AT5G57690) is presented. In addition to the annotated diacylglycerol kinase domain, this molecule also harbors a predicted heme-NO/oxygen (H-NOX) binding site and a guanylyl cyclase (GC) catalytic domain which have been identified based on the alignment of functionally conserved amino acid residues across species. A 3D model of the molecule was constructed, and from which the locations of the kinase catalytic center, the ATP-binding site, the GC and H-NOX domains were estimated. Docking of ATP to the kinase catalytic center was also modeled. The recombinant ATDGK4 demonstrated kinase activity in vitro, catalyzing the ATP-dependent conversion of sn-1,2-diacylglycerol (DAG) to phosphatidic acid (PA). This activity was inhibited by the mammalian DAG kinase inhibitor R59949 and importantly also by the NO donors diethylamine NONOate (DEA NONOate) and sodium nitroprusside (SNP). Recombinant ATDGK4 also has GC activity in vitro, catalyzing the conversion of guanosine-5\\'-triphosphate (GTP) to cGMP. The catalytic domains of ATDGK4 kinase and GC may be independently regulated since the kinase but not the GC, was inhibited by NO while Ca2+ only stimulates the GC. It is likely that the DAG kinase product, PA, causes the release of Ca2+ from the intracellular stores and Ca2+ in turn activates the GC domain of ATDGK4 through a feedback mechanism. Analysis of publicly available microarray data has revealed that ATDGK4 is highly expressed in the pollen. Here, the pollen tubes of mis-expressing atdgk4 recorded slower growth rates than the wild-type (Col-0) and importantly, they showed altered

  12. Catalytic performance and molecular dynamic simulation of immobilized CC bond hydrolase based on carbon nanotube matrix.

    Science.gov (United States)

    Zhou, Hao; Qu, Yuanyuan; Kong, Chunlei; Li, Duanxing; Shen, E; Ma, Qiao; Zhang, Xuwang; Wang, Jingwei; Zhou, Jiti

    2014-04-01

    Carbon nanotube (CNT) has been proved to be a kind of novel support for enzyme immobilization. In this study, we tried to find the relationship between conformation and catalytic performance of immobilized enzyme. Two CC bond hydrolases BphD and MfphA were immobilized on CNTs (SWCNT and MWCNT) via physical adsorption and covalent attachment. Among the conjugates, the immobilized BphD on chemically functionalized SWCNT (BphD-CSWCNT) retained the highest catalytic efficiency (kcat/Km value) compared to free BphD (92.9%). On the other hand, when MfphA bound to pristine SWCNT (MfphA-SWCNT), it was completely inactive. Time-resolved fluorescence spectrum indicated the formation of static ground complexes during the immobilization processes. Circular dichroism (CD) showed that the secondary structures of immobilized enzymes changed in varying degrees. In order to investigate the inhibition mechanism of MfphA by SWCNT, molecular dynamics simulation was employed to analyze the adsorption process, binding sites and time evolution of substrate tunnels. The results showed that the preferred binding sites (Trp201 and Met81) of MfphA for SWCNT blocked the main substrate access tunnel, thus making the enzyme inactive. The "tunnel-block" should be a novel possible inhibition mechanism for enzyme-nanotube conjugate.

  13. Relief of autoinhibition by conformational switch explains enzyme activation by a catalytically dead paralog

    Energy Technology Data Exchange (ETDEWEB)

    Volkov, Oleg A.; Kinch, Lisa; Ariagno, Carson; Deng, Xiaoyi; Zhong, Shihua; Grishin, Nick; Tomchick, Diana R.; Chen, Zhe; Phillips, Margaret A.

    2016-12-15

    Catalytically inactive enzyme paralogs occur in many genomes. Some regulate their active counterparts but the structural principles of this regulation remain largely unknown. We report X-ray structures ofTrypanosoma brucei S-adenosylmethionine decarboxylase alone and in functional complex with its catalytically dead paralogous partner, prozyme. We show monomericTbAdoMetDC is inactive because of autoinhibition by its N-terminal sequence. Heterodimerization with prozyme displaces this sequence from the active site through a complex mechanism involving acis-to-transproline isomerization, reorganization of a β-sheet, and insertion of the N-terminal α-helix into the heterodimer interface, leading to enzyme activation. We propose that the evolution of this intricate regulatory mechanism was facilitated by the acquisition of the dimerization domain, a single step that can in principle account for the divergence of regulatory schemes in the AdoMetDC enzyme family. These studies elucidate an allosteric mechanism in an enzyme and a plausible scheme by which such complex cooperativity evolved.

  14. Simple, chemoselective, catalytic olefin isomerization.

    Science.gov (United States)

    Crossley, Steven W M; Barabé, Francis; Shenvi, Ryan A

    2014-12-01

    Catalytic amounts of Co(Sal(tBu,tBu))Cl and organosilane irreversibly isomerize terminal alkenes by one position. The same catalysts effect cycloisomerization of dienes and retrocycloisomerization of strained rings. Strong Lewis bases like amines and imidazoles, and labile functionalities like epoxides, are tolerated.

  15. Catalytic Asymmetric Bromocyclization of Polyenes.

    Science.gov (United States)

    Samanta, Ramesh C; Yamamoto, Hisashi

    2017-02-01

    The first catalytic asymmetric bromonium ion-induced polyene cyclization has been achieved by using a chiral BINOL-derived thiophosphoramide catalyst and 1,3-dibromo-5,5-dimethylhydantoin as an electrophilic bromine source. Bromocyclization products are obtained in high yields, with good enantiomeric ratios and high diastereoselectivity, and are abundantly found as scaffolds in natural products.

  16. A High-Affinity Adenosine Kinase from Anopheles Gambiae

    Energy Technology Data Exchange (ETDEWEB)

    M Cassera; M Ho; E Merino; E Burgos; A Rinaldo-Matthis; S Almo; V Schramm

    2011-12-31

    Genome analysis revealed a mosquito orthologue of adenosine kinase in Anopheles gambiae (AgAK; the most important vector for the transmission of Plasmodium falciparum in Africa). P. falciparum are purine auxotrophs and do not express an adenosine kinase but rely on their hosts for purines. AgAK was kinetically characterized and found to have the highest affinity for adenosine (K{sub m} = 8.1 nM) of any known adenosine kinase. AgAK is specific for adenosine at the nucleoside site, but several nucleotide triphosphate phosphoryl donors are tolerated. The AgAK crystal structure with a bound bisubstrate analogue Ap{sub 4}A (2.0 {angstrom} resolution) reveals interactions for adenosine and ATP and the geometry for phosphoryl transfer. The polyphosphate charge is partly neutralized by a bound Mg{sup 2+} ion and an ion pair to a catalytic site Arg. The AgAK structure consists of a large catalytic core in a three-layer {alpha}/{beta}/{alpha} sandwich, and a small cap domain in contact with adenosine. The specificity and tight binding for adenosine arise from hydrogen bond interactions of Asn14, Leu16, Leu40, Leu133, Leu168, Phe168, and Thr171 and the backbone of Ile39 and Phe168 with the adenine ring as well as through hydrogen bond interactions between Asp18, Gly64, and Asn68 and the ribosyl 2'- and 3'-hydroxyl groups. The structure is more similar to that of human adenosine kinase (48% identical) than to that of AK from Toxoplasma gondii (31% identical). With this extraordinary affinity for AgAK, adenosine is efficiently captured and converted to AMP at near the diffusion limit, suggesting an important role for this enzyme in the maintenance of the adenine nucleotide pool. mRNA analysis verifies that AgAK transcripts are produced in the adult insects.

  17. Distinct expression patterns of ICK/MAK/MOK protein kinases in the intestine implicate functional diversity.

    Directory of Open Access Journals (Sweden)

    Tufeng Chen

    Full Text Available ICK/MRK (intestinal cell kinase/MAK-related kinase, MAK (male germ cell-associated kinase, and MOK (MAPK/MAK/MRK-overlapping kinase are closely related serine/threonine protein kinases in the protein kinome. The biological functions and regulatory mechanisms of the ICK/MAK/MOK family are still largely elusive. Despite significant similarities in their catalytic domains, they diverge markedly in the sequence and structural organization of their C-terminal non-catalytic domains, raising the question as to whether they have distinct, overlapping, or redundant biological functions. In order to gain insights into their biological activities and lay a fundamental groundwork for functional studies, we investigated the spatio-temporal distribution patterns and the expression dynamics of ICK/MAK/MOK protein kinases in the intestine. We found that ICK/MAK/MOK proteins display divergent expression patterns along the duodenum-to-colon axis and during postnatal murine development. Furthermore, they are differentially partitioned between intestinal epithelium and mesenchyme. A significant increase in the protein level of ICK, but not MAK, was induced in human primary colon cancer specimens. ICK protein level was up-regulated whereas MOK protein level was down-regulated in mouse intestinal adenomas as compared with their adjacent normal intestinal mucosa. These data suggest distinct roles for ICK/MAK/MOK protein kinases in the regulation of intestinal neoplasia. Taken together, our findings demonstrate that the expressions of ICK/MAK/MOK proteins in the intestinal tract can be differentially and dynamically regulated, implicating a significant functional diversity within this group of protein kinases.

  18. Mutations in the catalytic loop HRD motif alter the activity and function of Drosophila Src64.

    Directory of Open Access Journals (Sweden)

    Taylor C Strong

    Full Text Available The catalytic loop HRD motif is found in most protein kinases and these amino acids are predicted to perform functions in catalysis, transition to, and stabilization of the active conformation of the kinase domain. We have identified mutations in a Drosophila src gene, src64, that alter the three HRD amino acids. We have analyzed the mutants for both biochemical activity and biological function during development. Mutation of the aspartate to asparagine eliminates biological function in cytoskeletal processes and severely reduces fertility, supporting the amino acid's critical role in enzymatic activity. The arginine to cysteine mutation has little to no effect on kinase activity or cytoskeletal reorganization, suggesting that the HRD arginine may not be critical for coordinating phosphotyrosine in the active conformation. The histidine to leucine mutant retains some kinase activity and biological function, suggesting that this amino acid may have a biochemical function in the active kinase that is independent of its side chain hydrogen bonding interactions in the active site. We also describe the phenotypic effects of other mutations in the SH2 and tyrosine kinase domains of src64, and we compare them to the phenotypic effects of the src64 null allele.

  19. Enzyme kinetics and distinct modulation of the protein kinase N family of kinases by lipid activators and small molecule inhibitors

    Science.gov (United States)

    Falk, Matthew D.; Liu, Wei; Bolaños, Ben; Unsal-Kacmaz, Keziban; Klippel, Anke; Grant, Stephan; Brooun, Alexei; Timofeevski, Sergei

    2014-01-01

    The PKN (protein kinase N) family of Ser/Thr protein kinases regulates a diverse set of cellular functions, such as cell migration and cytoskeletal organization. Inhibition of tumour PKN activity has been explored as an oncology therapeutic approach, with a PKN3-targeted RNAi (RNA interference)-derived therapeutic agent in Phase I clinical trials. To better understand this important family of kinases, we performed detailed enzymatic characterization, determining the kinetic mechanism and lipid sensitivity of each PKN isoform using full-length enzymes and synthetic peptide substrate. Steady-state kinetic analysis revealed that PKN1–3 follows a sequential ordered Bi–Bi kinetic mechanism, where peptide substrate binding is preceded by ATP binding. This kinetic mechanism was confirmed by additional kinetic studies for product inhibition and affinity of small molecule inhibitors. The known lipid effector, arachidonic acid, increased the catalytic efficiency of each isoform, mainly through an increase in kcat for PKN1 and PKN2, and a decrease in peptide KM for PKN3. In addition, a number of PKN inhibitors with various degrees of isoform selectivity, including potent (Ki<10 nM) and selective PKN3 inhibitors, were identified by testing commercial libraries of small molecule kinase inhibitors. This study provides a kinetic framework and useful chemical probes for understanding PKN biology and the discovery of isoform-selective PKN-targeted inhibitors. PMID:27919031

  20. Phosphatidylinositol 3-kinase in myogenesis.

    Science.gov (United States)

    Kaliman, P; Zorzano, A

    1997-08-01

    Phosphatidylinositol 3-kinase (PI 3-kinase) has been cloned and characterized in a wide range of organisms. PI 3-kinases are activated by a diversity of extracellular stimuli and are involved in multiple cell processes such as cell proliferation, protein trafficking, cell motility, differentiation, regulation of cytoskeletal structure, and apoptosis. It has recently been shown that PI 3-kinase is a crucial second messenger in the signaling of myogenesis. Two structurally unrelated highly specific inhibitors of PI 3-kinase-wortmannin and LY294002-block the morphological and biochemical differentiation program of different skeletal-muscle cell models. Moreover, L6E9 myoblasts overexpressing a dominant-negative mutant of PI 3-kinase p85 regulatory subunit (Δp85) are unable to differentiate. Furthermore, PI 3-kinase is specifically involved in the insulinlike growth factor (IGF)-dependent myogenic pathway. Indeed, the ability of IGF-I, des-1,3-IGF-I, and IGF-II to promote cell fusion and muscle-specific protein expression is impaired after treatment with PI 3-kinase inhibitors or in cells overexpressing Δp85. The identification of additional key downstream elements of the IGF/PI 3-kinase myogenic cascade is crucial to a detailed understanding of the process of muscle differentiation and may generate new tools for skeletal and cardiac muscle regeneration therapies. (Trends Cardiovasc Med 1997;7:198-202). © 1997, Elsevier Science Inc.

  1. Ethanol Metabolism Activates Cell Cycle Checkpoint Kinase, Chk2

    Science.gov (United States)

    Clemens, Dahn L.; Mahan Schneider, Katrina J.; Nuss, Robert F.

    2011-01-01

    Chronic ethanol abuse results in hepatocyte injury and impairs hepatocyte replication. We have previously shown that ethanol metabolism results in cell cycle arrest at the G2/M transition, which is partially mediated by inhibitory phosphorylation of the cyclin-dependent kinase, Cdc2. To further delineate the mechanisms by which ethanol metabolism mediates this G2/M arrest, we investigated the involvement of upstream regulators of Cdc2 activity. Cdc2 is activated by the phosphatase Cdc25C. The activity of Cdc25C can, in turn, be regulated by the checkpoint kinase, Chk2, which is regulated by the kinase ataxia telangiectasia mutated (ATM). To investigate the involvement of these regulators of Cdc2 activity, VA-13 cells, which are Hep G2 cells modified to efficiently express alcohol dehydrogenase, were cultured in the presence or absence of 25 mM ethanol. Immunoblots were performed to determine the effects of ethanol metabolism on the activation of Cdc25C, Chk2, and ATM. Ethanol metabolism increased the active forms of ATM, and Chk2, as well as the phosphorylated form of Cdc25C. Additionally, inhibition of ATM resulted in approximately 50% of the cells being rescued from the G2/M cell cycle arrest, and ameliorated the inhibitory phosphorylation of Cdc2. Our findings demonstrate that ethanol metabolism activates ATM. ATM can activate the checkpoint kinase Chk2, resulting in phosphorylation of Cdc25C, and ultimately in the accumulation of inactive Cdc2. This may, in part, explain the ethanol metabolism-mediated impairment in hepatocyte replication, which may be important in the initiation and progression of alcoholic liver injury. PMID:21924579

  2. Arenavirus nucleoprotein targets interferon regulatory factor-activating kinase IKKε.

    Science.gov (United States)

    Pythoud, Christelle; Rodrigo, W W Shanaka I; Pasqual, Giulia; Rothenberger, Sylvia; Martínez-Sobrido, Luis; de la Torre, Juan Carlos; Kunz, Stefan

    2012-08-01

    Arenaviruses perturb innate antiviral defense by blocking induction of type I interferon (IFN) production. Accordingly, the arenavirus nucleoprotein (NP) was shown to block activation and nuclear translocation of interferon regulatory factor 3 (IRF3) in response to virus infection. Here, we sought to identify cellular factors involved in innate antiviral signaling targeted by arenavirus NP. Consistent with previous studies, infection with the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) prevented phosphorylation of IRF3 in response to infection with Sendai virus, a strong inducer of the retinoic acid-inducible gene I (RIG-I)/mitochondrial antiviral signaling (MAVS) pathway of innate antiviral signaling. Using a combination of coimmunoprecipitation and confocal microscopy, we found that LCMV NP associates with the IκB kinase (IKK)-related kinase IKKε but that, rather unexpectedly, LCMV NP did not bind to the closely related TANK-binding kinase 1 (TBK-1). The NP-IKKε interaction was highly conserved among arenaviruses from different clades. In LCMV-infected cells, IKKε colocalized with NP but not with MAVS located on the outer membrane of mitochondria. LCMV NP bound the kinase domain (KD) of IKKε (IKBKE) and blocked its autocatalytic activity and its ability to phosphorylate IRF3, without undergoing phosphorylation. Together, our data identify IKKε as a novel target of arenavirus NP. Engagement of NP seems to sequester IKKε in an inactive complex. Considering the important functions of IKKε in innate antiviral immunity and other cellular processes, the NP-IKKε interaction likely plays a crucial role in arenavirus-host interaction.

  3. 3D Studies of Neutral and Ionised Gas and Stars in Seyfert and Inactive Galaxies

    CERN Document Server

    Mundell, C G; Schinnerer, E; Nagar, N; Haan, S; Wilcots, E; Wilson, A S; Emsellem, E; Ferruit, P; Peletier, R F; De Zeeuw, P T

    2006-01-01

    We are conducting the first systematic 3D spectroscopic imaging survey to quantify the properties of the atomic gas (HI) in a distance-limited sample of 28 Seyfert galaxies and a sample of 28 inactive control galaxies with well-matched optical properties (the VHIKINGS survey). This study aims to address the role of the host galaxy in nuclear activity and confront outstanding controversies in optical/IR imaging surveys. Early results show possible relationships between Seyfert activity and HI extent, content and the prevalence of small, nearby gas-rich dwarf galaxies (M(HI)~10^7 Msun); results will be tested via rigorous comparison with control galaxies. Initial results from our optical followup study of 15 of our galaxies using the SAURON integral field unit on the WHT suggest a possible difference between Seyfert and inactive stellar and gaseous kinematics that support the conclusion that internal kinematics of galaxies are the key to nuclear activity.

  4. Revegetation and rock cover for stabilization of inactive uranium mill tailings disposal sites. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Beedlow, P.A.

    1984-05-01

    Guidelines for using vegetation and rock to protect inactive uranium mill tailings from erosion were developed by Pacific Northwest Laboratory as part of the Department of Energy's Uranium Mill Tailings Remedial Action Project (UMTRAP) Technology Development program. Information on soils, climate, and vegetation were collected for 20 inactive tailings sites in the western United States. Sites were grouped according to similarities in climate and vegetation. Soil loss for those sites was characterized using the Universal Soil Loss Equation. Test plots were used to evaluate (1) the interaction between vegetation and sealant barrier systems and (2) the effects of surface rock on soil water and vegetation. Lysimeter and simulation studies were used to direct and support field experiments. 49 references, 17 figures, 16 tables.

  5. Transport dynamics of molecular motors that switch between an active and inactive state.

    Science.gov (United States)

    Pinkoviezky, I; Gov, N S

    2013-08-01

    Molecular motors are involved in key transport processes in the cell. Many of these motors can switch from an active to a nonactive state, either spontaneously or depending on their interaction with other molecules. When active, the motors move processively along the filaments, while when inactive they are stationary. We treat here the simple case of spontaneously switching motors, between the active and inactive states, along an open linear track. We use our recent analogy with vehicular traffic, where we go beyond the mean-field description. We map the phase diagram of this system, and find that it clearly breaks the symmetry between the different phases, as compared to the standard total asymmetric exclusion process. We make several predictions that may be testable using molecular motors in vitro and in living cells.

  6. Heterogeneous reallocation of presynaptic efficacy in recurrent excitatory circuits adapting to inactivity.

    Science.gov (United States)

    Mitra, Ananya; Mitra, Siddhartha S; Tsien, Richard W

    2011-12-18

    Recurrent excitatory circuits face extreme challenges in balancing efficacy and stability. We recorded from CA3 pyramidal neuron pairs in rat hippocampal slice cultures to characterize synaptic and circuit-level changes in recurrent synapses resulting from long-term inactivity. Chronic tetrodotoxin treatment greatly reduced the percentage of connected CA3-CA3 neurons, but enhanced the strength of the remaining connections; presynaptic release probability sharply increased, whereas quantal size was unaltered. Connectivity was decreased in activity-deprived circuits by functional silencing of synapses, whereas three-dimensional anatomical analysis revealed no change in spine or bouton density or aggregate dendrite length. The silencing arose from enhanced Cdk5 activity and could be reverted by acute Cdk5 inhibition with roscovitine. Our results suggest that recurrent circuits adapt to chronic inactivity by reallocating presynaptic weights heterogeneously, strengthening certain connections while silencing others. This restricts synaptic output and input, preserving signaling efficacy among a subset of neuronal ensembles while protecting network stability.

  7. A new algorithm for inactive orbital optimization in valence bond theory

    Institute of Scientific and Technical Information of China (English)

    CHEN ZhenHua; ZHANG QianEr; WU Wei

    2009-01-01

    This paper presents an efficient algorithm for energy gradients in valence bond self-consistent field (VBSCF) method with non-orthogonal orbitals.The frozen core approximation method is extended to the case of non-orthogonal orbitals.The expressions for the total energy and its gradients are presented by introducing auxiliary orbitals,where inactive orbitals are orthogonal,while active orbitals are non-orthogonal themselves but orthogonal to inactive orbitsls.It is shown that our new algorithm has a low scaling of (N_a+ 1)m~4,where N_a and m are the numbers of the active orbitals and basis functions,respectively,and is more efficient than the existing VBSCF algorithms.

  8. A new algorithm for inactive orbital optimization in valence bond theory

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    This paper presents an efficient algorithm for energy gradients in valence bond self-consistent field(VBSCF) method with non-orthogonal orbitals.The frozen core approximation method is extended to the case of non-orthogonal orbitals.The expressions for the total energy and its gradients are presented by introducing auxiliary orbitals,where inactive orbitals are orthogonal,while active orbitals are non-orthogonal themselves but orthogonal to inactive orbitals.It is shown that our new algorithm has a low scaling of(Na+1)m4,where Na and m are the numbers of the active orbitals and basis functions,respectively,and is more efficient than the existing VBSCF algorithms.

  9. The effects of exergaming on physical activity among inactive children in a physical education classroom.

    Science.gov (United States)

    Fogel, Victoria A; Miltenberger, Raymond G; Graves, Rachel; Koehler, Shannon

    2010-01-01

    Childhood obesity, which is due in part to lack of physical activity, is a serious concern that requires the attention of the behavioral community. Although excessive video game play has been noted in the literature as a contributor to childhood obesity, newer video gaming technology, called exergaming, has been designed to capitalize on the reinforcing effects of video games to increase physical activity in children. This study evaluated the effects of exergaming on physical activity among 4 inactive children in a physical education (PE) classroom. Results showed that exergaming produced substantially more minutes of physical activity and more minutes of opportunity to engage in physical activity than did the standard PE program. In addition, exergaming was socially acceptable to both the students and the PE teacher. Exergaming appears to hold promise as a method for increasing physical activity among inactive children and might be a possible intervention for childhood obesity.

  10. Recreational physical inactivity and mortality in women with invasive epithelial ovarian cancer

    DEFF Research Database (Denmark)

    Cannioto, Rikki A; LaMonte, Michael J; Kelemen, Linda E

    2016-01-01

    and mortality. METHODS: Participants included 6806 women with a primary diagnosis of invasive EOC. In accordance with the Physical Activity Guidelines for Americans, women reporting no regular, weekly recreational physical activity were classified as inactive. We utilised Cox proportional hazard models......BACKGROUND: Little is known about modifiable behaviours that may be associated with epithelial ovarian cancer (EOC) survival. We conducted a pooled analysis of 12 studies from the Ovarian Cancer Association Consortium to investigate the association between pre-diagnostic physical inactivity.......12-1.33) further adjustment for residual disease, respectively. CONCLUSION: In this large pooled analysis, lack of recreational physical activity was associated with increased mortality among women with invasive EOC....

  11. Influence of radiative processes on the ignition of deuterium-tritium plasma containing inactive impurities

    Science.gov (United States)

    Gus'kov, S. Yu.; Sherman, V. E.

    2016-08-01

    The degree of influence of radiative processes on the ignition of deuterium-tritium (DT) plasma has been theoretically studied as dependent on the content of inactive impurities in plasma. The analytic criterion of plasma ignition in inertial confinement fusion (ICF) targets is modified taking into account the absorption of intrinsic radiation from plasma in the ignition region. The influence of radiative processes on the DT plasma ignition has been analytically and numerically studied for plasma that contains a significant fraction of inactive impurities either as a result of DT fuel mixing with ICF target ablator material or as a result of using light metal DT-hydrides as solid noncryogenic fuel. It has been shown that the effect of the absorption of intrinsic radiation leads to lower impurity-induced increase in the ignition energy as compared to that calculated in the approximation of optically transparent ignition region.

  12. The burden of abdominal obesity with physical inactivity on health expenditure in Brazil

    Directory of Open Access Journals (Sweden)

    Jamile S. Codogno

    2015-03-01

    Full Text Available The purpose of this study was to analyze the association between the clustering of physical inactivity with abdominal obesity and public health care expenditure in Brazilian adults. The sample was composed of 963 patients of both genders, randomly selected in the Brazilian Public Health care System during 2010. Entire health care expenditures during the last year were computed and stratified into: medical consultations, medication dispensing, laboratory tests and overall expenditure. Waist circumference was used to diagnose abdominal obesity and physical activity was assessed by previously validated questionnaire. Sedentary and abdominally obese patients (OR= 3.01 [OR95%CI= 1.81-4.99] had higher likelihood be inserted in the group of higher expenditures than only abdominally obese patients (OR= 1.66 [OR95%CI= 1.07-2.59]. There is a synergic effect between abdominal obesity and physical inactivity on overall health care expenditures.

  13. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 °C. Part 9: reference procedure for the measurement of catalytic concentration of alkaline phosphatase International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Scientific Division, Committee on Reference Systems of Enzymes (C-RSE) (1)).

    Science.gov (United States)

    Schumann, Gerhard; Klauke, Rainer; Canalias, Francesca; Bossert-Reuther, Steffen; Franck, Paul F H; Gella, F-Javier; Jørgensen, Poul J; Kang, Dongchon; Lessinger, Jean-Marc; Panteghini, Mauro; Ceriotti, Ferruccio

    2011-09-01

    Abstract This paper is the ninth in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 °C and the certification of reference preparations. Other parts deal with: Part 1. The concept of reference procedures for the measurement of catalytic activity concentrations of enzymes; Part 2. Reference procedure for the measurement of catalytic concentration of creatine kinase; Part 3. Reference procedure for the measurement of catalytic concentration of lactate dehydrogenase; Part 4. Reference procedure for the measurement of catalytic concentration of alanine aminotransferase; Part 5. Reference procedure for the measurement of catalytic concentration of aspartate aminotransferase; Part 6. Reference procedure for the measurement of catalytic concentration of γ-glutamyltransferase; Part 7. Certification of four reference materials for the determination of enzymatic activity of γ-glutamyltransferase, lactate dehydrogenase, alanine aminotransferase and creatine kinase at 37 °C; Part 8. Reference procedure for the measurement of catalytic concentration of α-amylase. The procedure described here is derived from the previously described 30 °C IFCC reference method. Differences are tabulated and commented on in Appendix 1.

  14. Food reward in active compared to inactive men: Roles for gastric emptying and body fat.

    Science.gov (United States)

    Horner, Katy M; Finlayson, Graham; Byrne, Nuala M; King, Neil A

    2016-06-01

    Habitual exercise could contribute to weight management by altering processes of food reward via the gut-brain axis. We investigated hedonic processes of food reward in active and inactive men and characterised relationships with gastric emptying and body fat. Forty-four men (active: n=22; inactive: n=22, BMI range 21-36kg/m(2); percent fat mass range 9-42%) were studied. Participants were provided with a standardised fixed breakfast and an ad libitum lunch meal 5h later. Explicit liking, implicit wanting and preference among high-fat, low-fat, sweet and savoury food items were assessed immediately post-breakfast (fed state) and again pre-lunch (hungry state) using the Leeds Food Preference Questionnaire. Gastric emptying was assessed by (13)C-octanoic acid breath test. Active individuals exhibited a lower liking for foods overall and a greater implicit wanting for low-fat savoury foods in the fed state, compared to inactive men. Differences in the fed state remained significant after adjusting for percent fat mass. Active men also had a greater increase in liking for savoury foods in the interval between breakfast and lunch. Faster gastric emptying was associated with liking for savoury foods and with an increase in liking for savoury foods in the postprandial interval. In contrast, greater implicit wanting for high-fat foods was associated with slower gastric emptying. These associations were independent of each other, activity status and body fat. In conclusion, active and inactive men differ in processes of food reward. The rate of gastric emptying may play a role in the association between physical activity status and food reward, via the gut-brain axis.

  15. Differences in some physical characteristics and motor abilities at dance active and dance inactive children

    OpenAIRE

    Petrič, Anja

    2012-01-01

    In Degree dissertation titled “Differences in some physical characteristics and motor abilities at dance active and dance inactive children” we compared some physical characteristics and motor abilities in the two groups of children: the ones that are taking the interests in dance and the ones that are not interested in dance. In theoretical part of dissertation we presented the offer of dance schools in Slovenia, projects School dance festival, recognition of dance in elementary schools, in...

  16. Pyridinediimine Iron Complexes with Pendant Redox-Inactive Metals Located in the Secondary Coordination Sphere.

    Science.gov (United States)

    Delgado, Mayra; Ziegler, Joshua M; Seda, Takele; Zakharov, Lev N; Gilbertson, John D

    2016-01-19

    A series of pyridinediimine (PDI) iron complexes that contain a pendant 15-crown-5 located in the secondary coordination sphere were synthesized and characterized. The complex Fe((15c5)PDI)(CO)2 (2) was shown in both the solid state and solution to encapsulate redox-inactive metal ions. Modest shifts in the reduction potential of the metal-ligand scaffold were observed upon encapsulation of either Na(+) or Li(+).

  17. Discrimination between acid and alkali-labile phosphorylated residues on Immobilon: phosphorylation studies of nucleoside diphosphate kinase

    DEFF Research Database (Denmark)

    Biondi, R M; Walz, K; Issinger, O G

    1996-01-01

    to deplete phosphate from membranes incubated successively under acid and basic conditions. The technique was applied to the study of nucleoside diphosphate kinase (NDP kinase) phosphorylation. In this enzyme, autophosphorylation of active site histidine is an accepted intermediate step in the catalytic...... phosphate transfer activity of nucleoside diphosphate kinase (NDP kinase). Nonetheless, a significant degree of autophosphorylation on other residues has been reported by several laboratories, and the hypothesis has been advanced that this nonhistidine phosphorylation may play an important role in NDP...... of phosphoserine after strong acid hydrolysis of the histidine autophosphorylated enzyme is in fact a nonenzymatic transphosphorylation from phosphohistidine due to the harsh acid treatment. This methodology was also applied to in vivo phosphorylation studies of C. albicans NDP kinase. We believe...

  18. Inactive vaccine derived from velogenic strain of local Newcastle disease virus .

    Directory of Open Access Journals (Sweden)

    Darminto

    1996-03-01

    Full Text Available The objective of this research is to evaluate an application of an inactive Newcastle disease (ND vaccine derived from velogenic strain of local Newcastle disease virus (NDV. In this research . the Ira strain of velogenic ND virus was grown in specific pathogen free (SPF eggs and then was inactivated by formalin at a final concentration of 1 :1,000 at 4°C. The inactive antigen was then emulsified with an oil adjuvant or aluminium hydroxide gel before being administered for vaccination in layers and compared to a commercial inactive ND vaccine . Results indicated that application of these inactivated ND vaccines for booster vaccination following vaccination with an active lentogenic ND virus in pullets nearly producing eggs, resulted in high antibody titre which persisted for considerable long period of time and capable of protecting layers from sick of ND and from reducing egg production . Hence, it could be concluded that the inactivated vaccine emulsified in either oil-adjuvant (lanolin-paraffin or aluminium hydroxide gel were considered to be highly immunogenic and capable of protecting layers from sick of ND and from reducing egg production

  19. Active and Inactive Enhancers Cooperate to Exert Localized and Long-Range Control of Gene Regulation

    Directory of Open Access Journals (Sweden)

    Charlotte Proudhon

    2016-06-01

    Full Text Available V(DJ recombination relies on the presence of proximal enhancers that activate the antigen receptor (AgR loci in a lineage- and stage-specific manner. Unexpectedly, we find that both active and inactive AgR enhancers cooperate to disseminate their effects in a localized and long-range manner. Here, we demonstrate the importance of short-range contacts between active enhancers that constitute an Igk super-enhancer in B cells. Deletion of one element reduces the interaction frequency between other enhancers in the hub, which compromises the transcriptional output of each component. Furthermore, we establish that, in T cells, long-range contact and cooperation between the inactive Igk enhancer MiEκ and the active Tcrb enhancer Eβ alters enrichment of CBFβ binding in a manner that impacts Tcrb recombination. These findings underline the complexities of enhancer regulation and point to a role for localized and long-range enhancer-sharing between active and inactive elements in lineage- and stage-specific control.

  20. GTP plus water mimic ATP in the active site of protein kinase CK2

    DEFF Research Database (Denmark)

    Niefind, K; Pütter, M; Guerra, B;

    1999-01-01

    The structures of the catalytic subunit of protein kinase CK2 from Zea mays complexed with Mg2+ and with analogs of ATP or GTP were determined to 2.2 A resolution. Unlike most other protein kinases, CK2 from various sources shows 'dual-cosubstrate specificity', that is, the ability to efficiently...... use either ATP or GTP as a cosubstrate. The structures of these complexes demonstrate that water molecules are critical to switch the active site of CK2 from an ATP- to a GTP-compatible state. An understanding of the structural basis of dual-cosubstrate specificity may help in the design of drugs...

  1. Template electrodeposition of catalytic nanomotors.

    Science.gov (United States)

    Wang, Joseph

    2013-01-01

    The combination of nanomaterials with electrode materials has opened new horizons in electroanalytical chemistry, and in electrochemistry in general. Over the past two decades we have witnessed an enormous activity aimed at designing new electrochemical devices based on nanoparticles, nanotubes or nanowires, and towards the use of electrochemical routes--particularly template-assisted electrodeposition--for preparing nanostructured materials. The power of template-assisted electrochemical synthesis is demonstrated in this article towards the preparation and the realization of self-propelled catalytic nanomotors, ranging from Pt-Au nanowire motors to polymer/Pt microtube engines. Design considerations affecting the propulsion behavior of such catalytic nanomotors are discussed along with recent bioanalytical and environmental applications. Despite recent major advances, artificial nanomotors have a low efficiency compared to their natural counterparts. Hopefully, the present Faraday Discussion will stimulate other electrochemistry teams to contribute to the fascinating area of artificial nanomachines.

  2. Role of Intrinsic and Extrinsic Factors in the Regulation of the Mitotic Checkpoint Kinase Bub1.

    Directory of Open Access Journals (Sweden)

    Claudia Breit

    Full Text Available The spindle assembly checkpoint (SAC monitors microtubule attachment to kinetochores to ensure accurate sister chromatid segregation during mitosis. The SAC members Bub1 and BubR1 are paralogs that underwent significant functional specializations during evolution. We report an in-depth characterization of the kinase domains of Bub1 and BubR1. BubR1 kinase domain binds nucleotides but is unable to deliver catalytic activity in vitro. Conversely, Bub1 is an active kinase regulated by intra-molecular phosphorylation at the P+1 loop. The crystal structure of the phosphorylated Bub1 kinase domain illustrates a hitherto unknown conformation of the P+1 loop docked into the active site of the Bub1 kinase. Both Bub1 and BubR1 bind Bub3 constitutively. A hydrodynamic characterization of Bub1:Bub3 and BubR1:Bub3 demonstrates both complexes to have 1:1 stoichiometry, with no additional oligomerization. Conversely, Bub1:Bub3 and BubR1:Bub3 combine to form a heterotetramer. Neither BubR1:Bub3 nor Knl1, the kinetochore receptor of Bub1:Bub3, modulate the kinase activity of Bub1 in vitro, suggesting autonomous regulation of the Bub1 kinase domain. We complement our study with an analysis of the Bub1 substrates. Our results contribute to the mechanistic characterization of a crucial cell cycle checkpoint.

  3. Targeting the SH2-Kinase Interface in Bcr-Abl Inhibits Leukemogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Grebien, Florian; Hantschel, Oliver; Wojcik, John; Kaupe, Ines; Kovacic, Boris; Wyrzucki, Arkadiusz M.; Gish, Gerald D.; Cerny-Reiterer, Sabine; Koide, Akiko; Beug, Hartmut; Pawson, Tony; Valent, Peter; Koide, Shohei; Superti-Furga, Giulio (AAS); (Mount Sinai Hospital); (Med U. Vienna); (UC); (IMP-CNRS)

    2012-10-25

    Chronic myelogenous leukemia (CML) is caused by the constitutively active tyrosine kinase Bcr-Abl and treated with the tyrosine kinase inhibitor (TKI) imatinib. However, emerging TKI resistance prevents complete cure. Therefore, alternative strategies targeting regulatory modules of Bcr-Abl in addition to the kinase active site are strongly desirable. Here, we show that an intramolecular interaction between the SH2 and kinase domains in Bcr-Abl is both necessary and sufficient for high catalytic activity of the enzyme. Disruption of this interface led to inhibition of downstream events critical for CML signaling and, importantly, completely abolished leukemia formation in mice. Furthermore, disruption of the SH2-kinase interface increased sensitivity of imatinib-resistant Bcr-Abl mutants to TKI inhibition. An engineered Abl SH2-binding fibronectin type III monobody inhibited Bcr-Abl kinase activity both in vitro and in primary CML cells, where it induced apoptosis. This work validates the SH2-kinase interface as an allosteric target for therapeutic intervention.

  4. Role of eukaryotic-like serine/threonine kinases in bacterial cell division and morphogenesis.

    Science.gov (United States)

    Manuse, Sylvie; Fleurie, Aurore; Zucchini, Laure; Lesterlin, Christian; Grangeasse, Christophe

    2016-01-01

    Bacteria possess a repertoire of versatile protein kinases modulating diverse aspects of their physiology by phosphorylating proteins on various amino acids including histidine, cysteine, aspartic acid, arginine, serine, threonine and tyrosine. One class of membrane serine/threonine protein kinases possesses a catalytic domain sharing a common fold with eukaryotic protein kinases and an extracellular mosaic domain found in bacteria only, named PASTA for 'Penicillin binding proteins And Serine/Threonine kinase Associated'. Over the last decade, evidence has been accumulating that these protein kinases are involved in cell division, morphogenesis and developmental processes in Firmicutes and Actinobacteria. However, observations differ from one species to another suggesting that a general mechanism of activation of their kinase activity is unlikely and that species-specific regulation of cell division is at play. In this review, we survey the latest research on the structural aspects and the cellular functions of bacterial serine/threonine kinases with PASTA motifs to illustrate the diversity of the regulatory mechanisms controlling bacterial cell division and morphogenesis.

  5. Radiation/Catalytic Augmented Combustion.

    Science.gov (United States)

    1980-09-01

    NATIO& NAk H(fJI At tl TANUAHTOb 19 A ~omm.81-0287 LVL RADIATION/CATALYTIC AUGMENTED COMBUST ION MOSHE LAVID CORPORATE RESEARCH-TECHNOLOGY FEASIBILITY...refinements as necessary. i. Perform cannular combustor experiments to Investigate ignition and flame attachment in flowing, liquid -fuel, unpremixed...stabilizer, with a sintered metal disk on the downstream side through which hot gases or products of partial fuel oxidation can be passed. Experimental

  6. Tyrosyl-DNA phosphodiesterase I catalytic mutants reveal an alternative nucleophile that can catalyze substrate cleavage.

    Science.gov (United States)

    Comeaux, Evan Q; Cuya, Selma M; Kojima, Kyoko; Jafari, Nauzanene; Wanzeck, Keith C; Mobley, James A; Bjornsti, Mary-Ann; van Waardenburg, Robert C A M

    2015-03-01

    Tyrosyl-DNA phosphodiesterase I (Tdp1) catalyzes the repair of 3'-DNA adducts, such as the 3'-phosphotyrosyl linkage of DNA topoisomerase I to DNA. Tdp1 contains two conserved catalytic histidines: a nucleophilic His (His(nuc)) that attacks DNA adducts to form a covalent 3'-phosphohistidyl intermediate and a general acid/base His (His(gab)), which resolves the Tdp1-DNA linkage. A His(nuc) to Ala mutant protein is reportedly inactive, whereas the autosomal recessive neurodegenerative disease SCAN1 has been attributed to the enhanced stability of the Tdp1-DNA intermediate induced by mutation of His(gab) to Arg. However, here we report that expression of the yeast His(nuc)Ala (H182A) mutant actually induced topoisomerase I-dependent cytotoxicity and further enhanced the cytotoxicity of Tdp1 His(gab) mutants, including H432N and the SCAN1-related H432R. Moreover, the His(nuc)Ala mutant was catalytically active in vitro, albeit at levels 85-fold less than that observed with wild type Tdp1. In contrast, the His(nuc)Phe mutant was catalytically inactive and suppressed His(gab) mutant-induced toxicity. These data suggest that the activity of another nucleophile when His(nuc) is replaced with residues containing a small side chain (Ala, Asn, and Gln), but not with a bulky side chain. Indeed, genetic, biochemical, and mass spectrometry analyses show that a highly conserved His, immediately N-terminal to His(nuc), can act as a nucleophile to catalyze the formation of a covalent Tdp1-DNA intermediate. These findings suggest that the flexibility of Tdp1 active site residues may impair the resolution of mutant Tdp1 covalent phosphohistidyl intermediates and provide the rationale for developing chemotherapeutics that stabilize the covalent Tdp1-DNA intermediate.

  7. Thermodynamics of catalytic nanoparticle morphology

    Science.gov (United States)

    Zwolak, Michael; Sharma, Renu; Lin, Pin Ann

    Metallic nanoparticles are an important class of industrial catalysts. The variability of their properties and the environment in which they act, from their chemical nature & surface modification to their dispersion and support, allows their performance to be optimized for many chemical processes useful in, e.g., energy applications and other areas. Their large surface area to volume ratio, as well as varying sizes and faceting, in particular, makes them an efficient source for catalytically active sites. These characteristics of nanoparticles - i.e., their morphology - can often display intriguing behavior as a catalytic process progresses. We develop a thermodynamic model of nanoparticle morphology, one that captures the competition of surface energy with other interactions, to predict structural changes during catalytic processes. Comparing the model to environmental transmission electron microscope images of nickel nanoparticles during carbon nanotube (and other product) growth demonstrates that nickel deformation in response to the nanotube growth is due to a favorable interaction with carbon. Moreover, this deformation is halted due to insufficient volume of the particles. We will discuss the factors that influence morphology and also how the model can be used to extract interaction strengths from experimental observations.

  8. Catalytic polarographic currents of oxidizers

    Energy Technology Data Exchange (ETDEWEB)

    Zajtsev, P.M.; Zhdanov, S.I.; Nikolaeva, T.D. (Vsesoyuznyj Nauchno-Issledovatel' skij Inst. Khimicheskikh Reaktivov i Osobo Chistykh Veshchestv, Moscow (USSR))

    1982-06-01

    The state of theory and practice of an important direction in polarography, i.e. catalytic currents of oxidizers-substrates that have found a wide application in the development of highly sensitive methods of determination of a large number of substrates, catalysts and polarographically nonactive ligands, is considered. Transition and some non-transition elements serve as catalysts of reactions that cause catalytic polarographic currents of substrates. Catalytic activity of an inorganic catalyst increases with the increase in the number of its d-orbit. Complex formation in most cases leads to the increase of catalyst activity, however, sometimes a reverse phenomenon takes place. For many catalysts the maximum activity is observed at pH values close to pK value of their hydrolysis. The properties of oxidizers-substrates is revealed by H/sub 2/O/sub 2/, ClO/sub 3//sup -/, BrO/sub 3//sup -/, IO/sub 3//sup -/, ClO/sub 4//sup -/, IO/sub 4//sup -/, NO/sub 2//sup -/, NO/sub 3//sup -/, NH/sub 2/OH, V(5), V(4), S/sub 2/O/sub 8//sup 2 -/, H/sub 2/SO/sub 4/, H/sub 2/C/sub 2/O/sub 4/, COHCOOH, alkenes compounds, organic halogen , sulfur- and amine-containing compounds.

  9. Preferential binding of allosteric modulators to active and inactive conformational states of metabotropic glutamate receptors

    Directory of Open Access Journals (Sweden)

    Klein-Seetharaman Judith

    2008-02-01

    Full Text Available Abstract Metabotropic glutamate receptors (mGluRs are G protein coupled receptors that play important roles in synaptic plasticity and other neuro-physiological and pathological processes. Allosteric mGluR ligands are particularly promising drug targets because of their modulatory effects – enhancing or suppressing the response of mGluRs to glutamate. The mechanism by which this modulation occurs is not known. Here, we propose the hypothesis that positive and negative modulators will differentially stabilize the active and inactive conformations of the receptors, respectively. To test this hypothesis, we have generated computational models of the transmembrane regions of different mGluR subtypes in two different conformations. The inactive conformation was modeled using the crystal structure of the inactive, dark state of rhodopsin as template and the active conformation was created based on a recent model of the light-activated state of rhodopsin. Ligands for which the nature of their allosteric effects on mGluRs is experimentally known were docked to the modeled mGluR structures using ArgusLab and Autodock softwares. We find that the allosteric ligand binding pockets of mGluRs are overlapping with the retinal binding pocket of rhodopsin, and that ligands have strong preferences for the active and inactive states depending on their modulatory nature. In 8 out of 14 cases (57%, the negative modulators bound the inactive conformations with significant preference using both docking programs, and 6 out of 9 cases (67%, the positive modulators bound the active conformations. Considering results by the individual programs only, even higher correlations were observed: 12/14 (86% and 8/9 (89% for ArgusLab and 10/14 (71% and 7/9 (78% for AutoDock. These findings strongly support the hypothesis that mGluR allosteric modulation occurs via stabilization of different conformations analogous to those identified in rhodopsin where they are induced by

  10. COURAGE AND FEAR IN THE CONTEXT OF OPPOSITION OF HUMAN ACTIVITY AND INACTIVITY: EXISTENTIAL ASPECT

    Directory of Open Access Journals (Sweden)

    Dmytro Yu. Snitko

    2013-12-01

    Full Text Available The purpose of the article is to analyse fear and courage in the history of philosophy in the context of opposi-tion of human activity and inactivity that may lead to a profound understanding of the essence, causes and existen-tial aspects of human activity and inactivity. The implementation of the objective assumes the solution of the follow-ing tasks: analysis of philosophical interpretation of fear and courage; investigation of the relationship of fear and courage with active and passive forms of human being; revelation of existential dialectic of human activity and inac-tivity through the opposition of fear and courage. Methodology. The application of phenomenological approach and other methods of existential philosophy enabled to discover the importance of fear and courage for human existence. Significant contribution to the importance of the investigation of the fear-courage opposition in the context of hu-man activity and inactivity was made by M. Heidegger who pointed to the main modes of human being - «authen-tic» and «inauthentic» in the context of human activity and passivity. The application of hermeneutic method made possible the reconstruction of the reflection of fear-courage opposition in the history of philosophy. Scientific nov-elty. For the first time the analysis of the fear-courage opposition in the context of human activity and inactivity was carried out. Due to the analysis the fundamental existential character of the fear and courage opposition and its es-sential relationship with active and passive forms of human being were justified. Conclusions. In the course of this research it was found out that fear is closely connected with passive modes of human being. If classical philosophy placed emphasis on courage and associated fear with human mind and conscious decision, non-classical philosophy of the XIX century and existentialism focused on existential and ontological character of fear, its fundamental mean

  11. The prevalence and correlates of physical inactivity among adults in Ho Chi Minh City

    Directory of Open Access Journals (Sweden)

    Phongsavan Philayrath

    2008-06-01

    Full Text Available Abstract Background Socioeconomic changes have led to profound changes in individuals' lifestyles, including the adoption of unhealthy food consumption patterns, prevalent tobacco use, alcohol abuse and physical inactivity, especially in large cities like Ho Chi Minh City (HCMC. The Stepwise Approach to Surveillance of Non-communicable Disease Risk Factors survey was conducted to identify physical activity patterns and factors associated with 'insufficient' levels of physical activity for health in adults in HCMC. Methods A cross-sectional survey was conducted in 2005 among 1906 adults aged 25–64 years using a probability proportional to size cluster sampling method to estimate the prevalence of non-communicable disease risk factors including physical inactivity. Data on socioeconomic status, health behaviours, and time spent in physical activity during work, commuting and leisure time were collected. Physical activity was measured using the validated Global Physical Activity Questionnaire (GPAQ. Responders were classified as 'sufficiently active' or 'insufficiently active' using the GPAQ protocol. Correlates of insufficient physical activity were identified using multivariable logistic regression. Results A high proportion of adults were physically inactive, with only 56.2% (95% CI = 52.1–60.4 aged 25–64 years in HCMC achieving the minimum recommendation of 'doing 30 minutes moderate-intensity physical activity for at least 5 days per week'. The main contributors to total physical activity among adults were from working and active commuting. Leisure-time physical activity represented a very small proportion (9.4% of individuals' total activity level. Some differences in the pattern of physical activity between men and women were noted, with insufficient activity levels decreasing with age among women, but not among men. Physical inactivity was positively associated with high income (OR = 1.77, 95% CI = 1.05–2.97 and high household

  12. Structural studies of Schistosoma mansoni adenylate kinases

    Energy Technology Data Exchange (ETDEWEB)

    Marques, I.A. [Universidade Federal de Goias (UFG), Goiania, GO (Brazil); Pereira, H.M.; Garrat, R.C. [Universidade de Sao Paulo (USP-SC), Sao Carlos, SP (Brazil)

    2012-07-01

    Full text: Parasitic diseases are a major cause of death in developing countries, however receive little or no attention from pharmaceutical companies for the development of novel therapies. In this respect, the Center for Structural Molecular Biology (CBME) of the Institute of Physics of Sao Carlos (IFSC / USP) has developed expertise in all stages of the development of active compounds against target enzymes from parasitic diseases. The present work focuses on the adenylate kinase enzymes (ADK's) from Schistosoma mansoni. These enzymes are widely distributed and catalyze the reaction of phosphoryl exchange between nucleotides in the reaction 2ADP to ATP + AMP, which is critical for the cells life cycle. Due to the particular property of the reaction catalyzed, the ADK's are recognized as reporters of the cells energetic state, translating small changes in the balance between ATP and ADP into a large change in concentration of AMP. The genome of S. mansoni was recently sequenced by the Sanger Center in England. On performing searches for genes encoding adenylate kinases we found two such genes. The corresponding gene products were named ADK1 (197 residues) and ADK2 (239 residues), and the two sequences share only 28 percent identity. Both have been cloned into the pET-28a(+)vector, expressed in E. coli and purified. Preliminary tests of activity have been performed only for ADK1 showing it to be catalytically active. Crystallization trials were performed for both proteins and thus far, crystals of ADK1 have been obtained which diffract to 2.05 at the LNLS beamline MX2 and the structure solved by molecular replacement. Understanding, at the atomic level, the function of these enzymes may help in the development of specific inhibitors and may provide tools for developing diagnostic tests for schistosomiasis. (author)

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

    Science.gov (United States)

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

    1994-02-15

    peak of c-Raf-1 kinase activity and immunoreactivity eluted at a predicted molecular mass of > 150 kDa, suggesting that active c-Raf-1 (but not inactive c-Raf-1) exists as a multimeric complex. This complex may not include p21ras, however, because immunoreactive p21ras was not identified in the active fractions.

  14. Etk/Bmx tyrosine kinase activates Pak1 and regulates tumorigenicity of breast cancer cells.

    Science.gov (United States)

    Bagheri-Yarmand, R; Mandal, M; Taludker, A H; Wang, R A; Vadlamudi, R K; Kung, H J; Kumar, R

    2001-08-03

    Etk/Bmx, a member of the Tec family of nonreceptor protein-tyrosine kinases, is characterized by an N-terminal pleckstrin homology domain and has been shown to be a downstream effector of phosphatidylinositol 3-kinase. P21-activated kinase 1 (Pak1), another well characterized effector of phosphatidylinositol 3-kinase, has been implicated in the progression of breast cancer cells. In this study, we characterized the role of Etk in mammary development and tumorigenesis and explored the functional interactions between Etk and Pak1. We report that Etk expression is developmentally regulated in the mammary gland. Using transient transfection, coimmunoprecipitation and glutathione S-transferase-pull down assays, we showed that Etk directly associates with Pak1 via its N-terminal pleckstrin homology domain and also phosphorylates Pak1 on tyrosine residues. The expression of wild-type Etk in a non-invasive human breast cancer MCF-7 cells significantly increased proliferation and anchorage-independent growth of epithelial cancer cells. Conversely, expression of kinase-inactive mutant Etk-KQ suppressed the proliferation, anchorage-independent growth, and tumorigenicity of human breast cancer MDA-MB435 cells. These results indicate that Pak1 is a target of Etk and that Etk controls the proliferation as well as the anchorage-independent and tumorigenic growth of mammary epithelial cancer cells.

  15. Protein Kinase A Subunit Balance Regulates Lipid Metabolism in Caenorhabditis elegans and Mammalian Adipocytes.

    Science.gov (United States)

    Lee, Jung Hyun; Han, Ji Seul; Kong, Jinuk; Ji, Yul; Lv, Xuchao; Lee, Junho; Li, Peng; Kim, Jae Bum

    2016-09-23

    Protein kinase A (PKA) is a cyclic AMP (cAMP)-dependent protein kinase composed of catalytic and regulatory subunits and involved in various physiological phenomena, including lipid metabolism. Here we demonstrated that the stoichiometric balance between catalytic and regulatory subunits is crucial for maintaining basal PKA activity and lipid homeostasis. To uncover the potential roles of each PKA subunit, Caenorhabditis elegans was used to investigate the effects of PKA subunit deficiency. In worms, suppression of PKA via RNAi resulted in severe phenotypes, including shortened life span, decreased egg laying, reduced locomotion, and altered lipid distribution. Similarly, in mammalian adipocytes, suppression of PKA regulatory subunits RIα and RIIβ via siRNAs potently stimulated PKA activity, leading to potentiated lipolysis without increasing cAMP levels. Nevertheless, insulin exerted anti-lipolytic effects and restored lipid droplet integrity by antagonizing PKA action. Together, these data implicate the importance of subunit stoichiometry as another regulatory mechanism of PKA activity and lipid metabolism.

  16. Molecular basis of the general base catalysis of an α/β-hydrolase catalytic triad.

    Science.gov (United States)

    Sun, Yueru; Yin, Shuhui; Feng, Yitao; Li, Jie; Zhou, Jiahai; Liu, Changdong; Zhu, Guang; Guo, Zhihong

    2014-05-30

    The serine-histidine-aspartate triad is well known for its covalent, nucleophilic catalysis in a diverse array of enzymatic transformations. Here we show that its nucleophilicity is shielded and its catalytic role is limited to being a specific general base by an open-closed conformational change in the catalysis of (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase (or MenH), a typical α/β-hydrolase fold enzyme in the vitamin K biosynthetic pathway. This enzyme is found to adopt an open conformation without a functional triad in its ligand-free form and a closed conformation with a fully functional catalytic triad in the presence of its reaction product. The open-to-closed conformational transition involves movement of half of the α-helical cap domain, which causes extensive structural changes in the α/β-domain and forces the side chain of the triad histidine to adopt an energetically disfavored gauche conformation to form the functional triad. NMR analysis shows that the inactive open conformation without a triad prevails in ligand-free solution and is converted to the closed conformation with a properly formed triad by the reaction product. Mutation of the residues crucial to this open-closed transition either greatly decreases or completely eliminates the enzyme activity, supporting an important catalytic role for the structural change. These findings suggest that the open-closed conformational change tightly couples formation of the catalytic triad to substrate binding to enhance the substrate specificities and simultaneously shield the nucleophilicity of the triad, thus allowing it to expand its catalytic power beyond the nucleophilic catalysis.

  17. New Insights on the Mechanism of the K+-Independent Activity of Crenarchaeota Pyruvate Kinases

    Science.gov (United States)

    De la Vega-Ruíz, Gustavo; Domínguez-Ramírez, Lenin; Riveros-Rosas, Héctor; Guerrero-Mendiola, Carlos; Torres-Larios, Alfredo; Hernández-Alcántara, Gloria; García-Trejo, José J.; Ramírez-Silva, Leticia

    2015-01-01

    Eukarya pyruvate kinases have glutamate at position 117 (numbered according to the rabbit muscle enzyme), whereas in Bacteria have either glutamate or lysine and in Archaea have other residues. Glutamate at this position makes pyruvate kinases K+-dependent, whereas lysine confers K+-independence because the positively charged residue substitutes for the monovalent cation charge. Interestingly, pyruvate kinases from two characterized Crenarchaeota exhibit K+-independent activity, despite having serine at the equivalent position. To better understand pyruvate kinase catalytic activity in the absence of K+ or an internal positive charge, the Thermofilum pendens pyruvate kinase (valine at the equivalent position) was characterized. The enzyme activity was K+-independent. The kinetic mechanism was random order with a rapid equilibrium, which is equal to the mechanism of the rabbit muscle enzyme in the presence of K+ or the mutant E117K in the absence of K+. Thus, the substrate binding order of the T. pendens enzyme was independent despite lacking an internal positive charge. Thermal stability studies of this enzyme showed two calorimetric transitions, one attributable to the A and C domains (Tm of 99.2°C), and the other (Tm of 105.2°C) associated with the B domain. In contrast, the rabbit muscle enzyme exhibits a single calorimetric transition (Tm of 65.2°C). The calorimetric and kinetic data indicate that the B domain of this hyperthermophilic enzyme is more stable than the rest of the protein with a conformation that induces the catalytic readiness of the enzyme. B domain interactions of pyruvate kinases that have been determined in Pyrobaculum aerophilum and modeled in T. pendens were compared with those of the rabbit muscle enzyme. The results show that intra- and interdomain interactions of the Crenarchaeota enzymes may account for their higher B domain stability. Thus the structural arrangement of the T. pendens pyruvate kinase could allow charge

  18. pkaPS: prediction of protein kinase A phosphorylation sites with the simplified kinase-substrate binding model

    Directory of Open Access Journals (Sweden)

    Schneider Georg

    2007-01-01

    Full Text Available Abstract Background Protein kinase A (cAMP-dependent kinase, PKA is a serine/threonine kinase, for which ca. 150 substrate proteins are known. Based on a refinement of the recognition motif using the available experimental data, we wished to apply the simplified substrate protein binding model for accurate prediction of PKA phosphorylation sites, an approach that was previously successful for the prediction of lipid posttranslational modifications and of the PTS1 peroxisomal translocation signal. Results Approximately 20 sequence positions flanking the phosphorylated residue on both sides have been found to be restricted in their sequence variability (region -18...+23 with the site at position 0. The conserved physical pattern can be rationalized in terms of a qualitative binding model with the catalytic cleft of the protein kinase A. Positions -6...+4 surrounding the phosphorylation site are influenced by direct interaction with the kinase in a varying degree. This sequence stretch is embedded in an intrinsically disordered region composed preferentially of hydrophilic residues with flexible backbone and small side chain. This knowledge has been incorporated into a simplified analytical model of productive binding of substrate proteins with PKA. Conclusion The scoring function of the pkaPS predictor can confidently discriminate PKA phosphorylation sites from serines/threonines with non-permissive sequence environments (sensitivity of ~96% at a specificity of ~94%. The tool "pkaPS" has been applied on the whole human proteome. Among new predicted PKA targets, there are entirely uncharacterized protein groups as well as apparently well-known families such as those of the ribosomal proteins L21e, L22 and L6. Availability The supplementary data as well as the prediction tool as WWW server are available at http://mendel.imp.univie.ac.at/sat/pkaPS. Reviewers Erik van Nimwegen (Biozentrum, University of Basel, Switzerland, Sandor Pongor (International

  19. Depressive symptoms are associated with physical inactivity in patients with type 2 diabetes. The DIAZOB Primary Care Diabetes study

    DEFF Research Database (Denmark)

    Koopmans, Berber; Pouwer, Francois; de Bie, Robert A

    2009-01-01

    BACKGROUND: Depression is a common complication of type 2 diabetes, associated with poor disease outcomes such as impaired glycaemic control, cardiovascular disease and increased mortality. The mechanisms behind these associations are unclear. Depression might contribute to poor disease outcomes...... through decreased physical activity. OBJECTIVE: To test whether type 2 diabetes patients with elevated depression scores are more often physically inactive. METHODS: Demographic features, clinical factors, level of physical inactivity and depressive symptoms were assessed in 2646 primary care patients...... with type 2 diabetes. Sequential multiple logistic regression analyses [odds ratio, 95% confidence interval (CI)] were performed to test the association between depressive symptoms and physical inactivity. RESULTS: About 48% of the respondents were physically inactive. Elevated depressive symptoms were...

  20. Effect of inactive yeast cell wall on growth performance, survival rate and immune parameters in Pacific White Shrimp (Litopenaeus vannamei

    Directory of Open Access Journals (Sweden)

    Rutchanee Chotikachinda

    2008-10-01

    Full Text Available Effects of dietary inactive yeast cell wall on growth performance, survival rate, and immune parameters in pacific white shrimp (Litopenaeus vannamei was investigated. Three dosages of inactive yeast cell wall (0, 1, and 2 g kg-1 were tested in three replicate groups of juvenile shrimps with an average initial weight of 7.15±0.05 g for four weeks. There was no significant difference in final weight, survival rate, specific growth rate, feed conversion ratio, feed intake, protein efficiency ratio, and apparent net protein utilization of each treatments. However, different levels of inactive yeast cell wall showed an effect on certain immune parameters (p<0.05. Total hemocyte counts, granular hemocyte count, and bacterial clearance were better in shrimp fed diets supplemented with 1 and 2 g kg-1 inactive yeast cell wall as compared with thecontrol group.

  1. Casein kinase-2 structure-function relationship

    DEFF Research Database (Denmark)

    Boldyreff, B; Meggio, F; Pinna, L A

    1992-01-01

    Nine mutants of human casein kinase-2 beta subunit have been created and assayed for their ability to assemble with the catalytic alpha subunit to give, at a 1:1 molar ratio, a fully competent CK-2 holoenzyme as judged by the following criteria: 1) the generation of an active heterotetrameric form......, consequently, stimulation of activity. This indicates that residue(s) located in the 171-209 sequence is essential for reconstitution. Also a four residue's N-terminal deletion (removing the autophosphorylation site) and single to quintuple substitutions of alanine for the acidic residues clustered in the 55......-70 sequence give rise to mutants that still assemble with the alpha subunit to give a tetrameric holoenzyme. However, in the case of the mutants A57,59, A63,64, A59-61,63,64 in vitro assembly with the CK-2 alpha subunit was not complete. There were also intermediate complexes, free alpha-subunit and beta...

  2. Administration of additional inactive iodide during radioiodine therapy for Graves' disease. Who might benefit?

    Energy Technology Data Exchange (ETDEWEB)

    Dietlein, M.; Moka, D.; Reinholz, U.; Schmidt, M.; Schomaecker, K.; Schicha, H.; Wellner, U. [Koeln Univ. (Germany). Dept. of Nuclear Medicine

    2007-07-01

    Aim: Graves' hyperthyroidism and antithyroid drugs empty the intrathyroid stores of hormones and iodine. The consequence is rapid {sup 131}I turnover and impending failure of radioiodine therapy. Can administration of additional inactive iodide improve 131I kinetics? Patients, methods: Fifteen consecutive patients, in whom the 48 h post-therapeutically calculated thyroid dose was between 150 and 249 Gy due to an unexpectedly short half-life, received 3 x 200 {mu}g inactive potassium-iodide ({sup 127}I) daily for 3 days (Group A), while 17 consecutive patients with a thyroid dose of = 250 Gy (Group B) served as the non-iodide group. 48 hours after {sup 131}I administration (M1) and 4 or 5 days later (M2) the following parameters were compared: effective {sup 131}I half-life, thyroid dose, total T3, total T4, {sup 131}I-activity in the T3- and T4-RIAs. Results: In Group A, the effective {sup 131}I half-life M1 before iodine (3.81 {+-} 0.93 days) was significantly (p <0.01) shorter than the effective {sup 131}I half-life M2 (4.65 {+-} 0.79 days). Effective {sup 131}I half-life M1 correlated with the benefit from inactive {sup 127}I (r = -0.79): Administration of {sup 127}I was beneficial in patients with an effective {sup 131}I half-life M1 of <3 or 4 days. Patients from Group A with high initial specific {sup 131}I activity of T3 and T4 showed lower specific {sup 131}I activity after addition of inactive iodine compared with patients from the same group with a lower initial specific {sup 131}I activity of T3 and T4 and compared with the patient group B who was given no additional inactive iodide. This correlation was mathematically described and reflected in the flatter gradient in Group A (y = 0.5195x + 0.8727 for {sup 131}I T3 and y = 1.0827x - 0.4444 for {sup 131}I T4) and steeper gradient for Group B (y = 0.6998x + 0.5417 for {sup 131}I T3 and y = 1.3191x - 0.2901 for {sup 131}I T4). Radioiodine therapy was successful in all 15 patients from Group A

  3. Physical inactivity prevalence and trends among Mexican adults: results from the National Health and Nutrition Survey (ENSANUT) 2006 and 2012

    OpenAIRE

    2013-01-01

    Background Lifestyles such as unhealthy diets and the lack of physical activity have been contributed to the increased prevalence of obesity. In 2012, the world health organization published the first global recommendation for physical activity and health. People who do not meet at least 150 minutes of moderate-to-vigorous physical activity are considered to be physically inactive. The prevalence of physical inactivity worldwide is 31%, however there is insufficient data from prevalence and t...

  4. Anthropometric measurements of students athletes in relation to physically inactive students

    Directory of Open Access Journals (Sweden)

    Namik Trtak

    2012-04-01

    Full Text Available Introduction: Anthropometry is a method of anthropology that refers to the measuring and testing the human body and to the relationship between the size of its individual parts.The task of anthropometry is as accurately as possible quantitatively characterize the morphological features of the human body.Measurements are made due to the anthropometric points which can be: fixed (standard on the site of prominence and virtual (change due to the bodyposition. Goals of research: To evaluate the impact of basketball on the growth and development of seventeen years old adolescents and prevention of deformities of the spinal column and chest.Methods: The study included 40 respondents. Criteria for inclusion: male respondents aged 17 years who played basketball for more than one year, male respondents aged 17 years who are physically inactive. Criteria for exclusion: female respondents, respondents who played basketball for less than one year, respondents who are engaged in some other sport professionally or recreationally, respondents younger and olderthan 17 years. In the study,there were made measurements of thorax scope in the axillary and mamilar level, measurements of body weight and height and measurements of Body mass index.Results of research: Out of 40 respondents 20 are basketball players and 20 physically inactive. Compared to the average value between the two groups of respondents certain differences were observed, which aremost noticeable in body weight (basketball players had more weigh about, 5 kg on average and height (basketball players are taller, about 7 cm on average. During the anthropometric measurements of thoraxdeformities of the spinal column have been observed which affect the deformation of the thorax. Of the 20 players one has a deformity of the spinal column, and out of the same number of physically inactive studentseven 12 have deformed spine.Conclusion: Basketball has a positive effect on the proper growth and development

  5. MAP Kinases in Immune Responses

    Institute of Scientific and Technical Information of China (English)

    YongliangZhang; ChenDong

    2005-01-01

    MAP kinases are evolutionarily conserved signaling regulators from budding yeast to mammals and play essential roles in both innate and adaptive immune responses. There are three main families of MAPKs in mammals. Each of them has its own activators, inactivators, substrates and scaffolds, which altogether form a fine signaling network in response to different extracellular or intracellular stimulation. In this review, we summarize recent advances in understanding of the regulation of MAP kinases and the roles of MAP kinases in innate and adaptive immune responses. Cellular & Molecular Immunology. 2005;2(1):20-27.

  6. MAP Kinases in Immune Responses

    Institute of Scientific and Technical Information of China (English)

    Yongliang Zhang; Chen Dong

    2005-01-01

    MAP kinases are evolutionarily conserved signaling regulators from budding yeast to mammals and play essential roles in both innate and adaptive immune responses. There are three main families of MAPKs in mammals. Each of them has its own activators, inactivators, substrates and scaffolds, which altogether form a fine signaling network in response to different extracellular or intracellular stimulation. In this review, we summarize recent advances in understanding of the regulation of MAP kinases and the roles of MAP kinases in innate and adaptive immune responses.

  7. Kinetics of heterogeneous catalytic reactions

    CERN Document Server

    Boudart, Michel

    2014-01-01

    This book is a critical account of the principles of the kinetics of heterogeneous catalytic reactions in the light of recent developments in surface science and catalysis science. Originally published in 1984. The Princeton Legacy Library uses the latest print-on-demand technology to again make available previously out-of-print books from the distinguished backlist of Princeton University Press. These paperback editions preserve the original texts of these important books while presenting them in durable paperback editions. The goal of the Princeton Legacy Library is to vastly increase acc

  8. Molecular catalytic coal liquid conversion

    Energy Technology Data Exchange (ETDEWEB)

    Stock, L.M.; Yang, Shiyong [Univ. of Chicago, IL (United States)

    1995-12-31

    This research, which is relevant to the development of new catalytic systems for the improvement of the quality of coal liquids by the addition of dihydrogen, is divided into two tasks. Task 1 centers on the activation of dihydrogen by molecular basic reagents such as hydroxide ion to convert it into a reactive adduct (OH{center_dot}H{sub 2}){sup {minus}} that can reduce organic molecules. Such species should be robust withstanding severe conditions and chemical poisons. Task 2 is focused on an entirely different approach that exploits molecular catalysts, derived from organometallic compounds that are capable of reducing monocyclic aromatic compounds under very mild conditions. Accomplishments and conclusions are discussed.

  9. Therapy of ovarian inactivity in postpartum Bulgarian Murrah buffaloes by PRID and Ovsynch estrus synchronization protocols

    Institute of Scientific and Technical Information of China (English)

    Stanimir Yotov; Anatoli Atanasov; Yordanka Ilieva

    2012-01-01

    Objective:The aim of the present study was to assess therapeutical effect of modified Ovsynch and PRID estrus synchronization protocols in Bulgarian Murrah buffalo with inactive ovaries during the low-breeding season. Methods:The study was carried out in 46 Bulgarian Murrah buffaloes with small inactive ovaries established by two consecutive transrectal ultrasonographies on Day 40 and 50 postpartum. At the start of the therapy the buffaloes were randomly divided into three groups. Group I (n=18) was treated by PRID-based protocol;Group II (n=18) was treated by Ovsynch based protocol and Group III (control;n=10) was injected intramuscular with saline at the same days as in the first two groups and fertile bull was introduced after that. The animals in the different groups were submitted to ultrasound examination at day of artificial insemination or bull introduction. Ovulation was determined 7 days post insemination by ultrasound. The pregnancy diagnosis was done 30 days after insemination. Mean diameter of the largest follicles at the start of therapy and the day of AI was registered. In the hormonal treated buffaloes estrus clinical sings, ovulation rate and pregnancy rate after AI were determined. In the control group pregnancy rate after spontaneous estrus was established. The mean diameter of the largest follicles determined on Days 40 and 50 after calving was not over 9 mm for all buffaloes. Results:At day of AI the average diameters of the preo-vulatory follicles in PRID and Ovsynch treated buffaloes were significantly (P<0.01) greater than these on Day 0. The cases of a clear uterine mucus discharge during the induced estrus were significant more (P<0.05) for Group I (94.4%) than Group II (66.7%). The pregnancy rate after AI (56.6%and 38.8%) in PRID and Ovsynch program was significant higher (P<0.05) than pregnancy rate after spontaneous estrus (10%) in the control group. Conclusions:the treatment of buffalo ovarian inactivity could start on Day 50

  10. The black hole - bulge mass relation in active and inactive galaxies

    OpenAIRE

    McLure, R. J.; Dunlop, J. S.

    2002-01-01

    New virial black-hole mass estimates are presented for a sample of 72 AGN covering three decades in optical luminosity. Using a model in which the AGN broad-line region (BLR) has a flattened geometry, we investigate the M_{bh)-L_{bulge} relation for a combined 90-object sample, consisting of the AGN plus a sample of 18 nearby inactive elliptical galaxies with dynamical black-hole mass measurements. It is found that, for all reasonable mass-to-light ratios, the M_{bh}-L_{bulge} relation is equ...

  11. Assessment of the Radiological Impact of the Inactive Uranium-Mill Tailings at Mexican Hat, Utah

    OpenAIRE

    Haywood, F. F.; Goldsmith, W. A.; Ellis, B. S.; Hubbard Jr., H. M.; Fox, W. F.; Shinpaugh, W. H.; Oak Ridge National Laboratory

    1980-01-01

    High surface soil concentrations of 226Ra and high above-ground measurements of gamma-ray intensity in the vicinity of the inactive uranium-mill tailings at Mexican Hat show both wind and water erosion of the tailings. The former mill area, occupied by a trade school at the time of this survey, shows a comparatively high level of contamination, probably from unprocessed ore on the surface of the ore storage area near the location of the former mill buildings. However, the estimated health e...

  12. Chronic fatigue syndrome in an ethnically diverse population: the influence of psychosocial adversity and physical inactivity

    Directory of Open Access Journals (Sweden)

    Wessely Simon

    2011-03-01

    Full Text Available Abstract Background Chronic fatigue syndrome (CFS is a complex multifactorial disorder. This paper reports the prevalence of chronic fatigue (CF and CFS in an ethnically diverse population sample and tests whether prevalence varies by social adversity, social support, physical inactivity, anxiety and depression. Methods Analysis of survey data linking the Health Survey for England (1998 and 1999 and the Ethnic Minority Psychiatric Illness Rates in the Community (EMPIRIC study undertaken in 2000. The study population comprised a national population sample of 4,281 people ages 16 to 74 years. CF and CFS were operationally defined on the basis of an interview in the EMPIRIC study, alongside questions about psychosocial risk factors. Previous illnesses were reported in the Health Survey for England during 1998 and 1999, as was physical inactivity. Results All ethnic minority groups had a higher prevalence of CFS than the White group. The lowest prevalence was 0.8% in the White group, and it was highest at 3.5% in the Pakistani group (odds ratio (OR, 4.1; 95% confidence interval (95% CI, 1.6 to 10.4. Anxiety (OR, 1.8; 95% CI, 1.4 to 2.2, depression (OR, 1.4; 95% CI, 1.1 to 1.8, physical inactivity (OR, 2.0; 95% CI, 1.1 to 3.8, social strain (OR, 1.24; 95% CI, 1.04 to 1.48 and negative aspects of social support (OR, 2.12; 95% CI, 1.4 to 3.3 were independent risk factors for CFS in the overall sample. Together these risk factors explained ethnic differences in the prevalence of CFS, but no single risk factor could explain a higher prevalence in all ethnic groups. Conclusions The prevalence of CFS, but not CF, varies by ethnic group. Anxiety, depression, physical inactivity, social strain and negative aspects of social support together accounted for prevalence differences of CFS in the overall sample.

  13. Physical inactivity affects skeletal muscle insulin signaling in a birth weight-dependent manner

    DEFF Research Database (Denmark)

    Mortensen, Brynjulf; Friedrichsen, Martin; Andersen, Nicoline Resen;

    2014-01-01

    AIMS: We investigated whether physical inactivity could unmask defects in insulin and AMPK signaling in low birth weight (LBW) subjects. METHODS: Twenty LBW and 20 normal birth weight (NBW) subjects were investigated using the euglycemic-hyperinsulinemic clamp with excision of skeletal muscle...... is not explained by impaired muscle insulin or AMPK signaling in subjects with or without LBW. Lower muscle insulin signaling in LBW subjects post bed rest despite similar degree of insulin resistance as seen in controls may to some extent support the idea that LBW subjects are at higher risk of developing type 2...

  14. An Internet-Based Physical Activity Intervention to Improve Quality of Life of Inactive Older Adults

    DEFF Research Database (Denmark)

    Broekhuizen, Karen; de Gelder, Jelle; Wijsman, Carolien A

    2016-01-01

    BACKGROUND: Increasing physical activity is a viable strategy for improving both the health and quality of life of older adults. OBJECTIVE: The aim of this study was to assess if an Internet-based intervention aimed to increase physical activity was effective in improving quality of life of inact......-based physical activity program was effective in improving quality of life in 60-70-year-olds after 3 months, particularly in participants that reached their individually targeted increase in daily physical activity. TRIAL REGISTRATION: Nederlands Trial Register: NTR 3045; http...

  15. Remedial action plan for stabilization of the inactive uranium mill tailings site at Monument Valley, Arizona

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1986-02-01

    This Remedial Action Plan (RAP) has been developed to serve a two-fold purpose. It presents the series of activities which are proposed by the U.S. Department of Energy (DOE) to accomplish long-term stabilization and control of radioactive materials at the inactive uranium processing site located near Monument Valley, Arizona It also serves to document the concurrence of both the Navajo Nation and the U.S. Nuclear Regulatory Commission (NRC) in the remedial action. This agreement, upon execution by DOE and the Navajo Nation and concurrence by NRC, becomes Appendix B of the Cooperative Agreement.

  16. Dynamic gadolinium-enhanced MR imaging in active and inactive immunoinflammatory gonarthritis

    DEFF Research Database (Denmark)

    Østergaard, Mikkel; Lorenzen, I; Henriksen, O

    1994-01-01

    examined 16 clinically active (CAG), 7 clinically inactive (CIG) and 4 healthy knees. The synovium of a preselected slice was outlined. Its area and relative signal intensity increase after gadopentetate dimeglumine on T1-SE and FLASH (at each time t) were calculated. The CAG knees showed a mean signal...... intensity increase on early dynamic FLASH images higher by far than the CIG knees, while no significant difference was found on spin-echo images obtained 5 to 15 min after contrast injection. The early signal enhancement probably reflects the perfusion and capillary permeability of the synovium. The area...

  17. Some Aspects of the Catalytic Organic Synthesis

    Institute of Scientific and Technical Information of China (English)

    Anil; K.Saikia

    2007-01-01

    1 Results Catalytic reactions are gaining importance due to its low cost, operational simplicity, high efficiency and selectivity. It is also getting much attention in green synthesis. Many useful organic reactions, including the acylation of alcohols and aldehydes, carbon-carbon, carbon-nitrogen, carbon-sulfur bond forming and oxidation reactions are carried out by catalyst. We are exploring the catalytic acylation of alcohols and aldehydes in a simple and efficient manner. Catalytic activation of unr...

  18. Structural basis for an inositol pyrophosphate kinase surmounting phosphate crowding

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Huanchen; Falck, J.R.; Hall, Traci M. Tanaka; Shears, Stephen B. (NIH); (UTSMC)

    2012-01-11

    Inositol pyrophosphates (such as IP7 and IP8) are multifunctional signaling molecules that regulate diverse cellular activities. Inositol pyrophosphates have 'high-energy' phosphoanhydride bonds, so their enzymatic synthesis requires that a substantial energy barrier to the transition state be overcome. Additionally, inositol pyrophosphate kinases can show stringent ligand specificity, despite the need to accommodate the steric bulk and intense electronegativity of nature's most concentrated three-dimensional array of phosphate groups. Here we examine how these catalytic challenges are met by describing the structure and reaction cycle of an inositol pyrophosphate kinase at the atomic level. We obtained crystal structures of the kinase domain of human PPIP5K2 complexed with nucleotide cofactors and either substrates, product or a MgF{sub 3}{sup -} transition-state mimic. We describe the enzyme's conformational dynamics, its unprecedented topological presentation of nucleotide and inositol phosphate, and the charge balance that facilitates partly associative in-line phosphoryl transfer.

  19. Cell fate regulation governed by a repurposed bacterial histidine kinase.

    Directory of Open Access Journals (Sweden)

    W Seth Childers

    2014-10-01

    Full Text Available One of the simplest organisms to divide asymmetrically is the bacterium Caulobacter crescentus. The DivL pseudo-histidine kinase, positioned at one cell pole, regulates cell-fate by controlling the activation of the global transcription factor CtrA via an interaction with the response regulator (RR DivK. DivL uniquely contains a tyrosine at the histidine phosphorylation site, and can achieve these regulatory functions in vivo without kinase activity. Determination of the DivL crystal structure and biochemical analysis of wild-type and site-specific DivL mutants revealed that the DivL PAS domains regulate binding specificity for DivK∼P over DivK, which is modulated by an allosteric intramolecular interaction between adjacent domains. We discovered that DivL's catalytic domains have been repurposed as a phosphospecific RR input sensor, thereby reversing the flow of information observed in conventional histidine kinase (HK-RR systems and coupling a complex network of signaling proteins for cell-fate regulation.

  20. Structural and functional characterization of human NAD kinase.

    Science.gov (United States)

    Lerner, F; Niere, M; Ludwig, A; Ziegler, M

    2001-10-19

    NADP is essential for biosynthetic pathways, energy, and signal transduction. Its synthesis is catalyzed by NAD kinase. Very little is known about the structure, function, and regulation of this enzyme from multicellular organisms. We identified a human NAD kinase cDNA and the corresponding gene using available database information. A cDNA was amplified from a human fibroblast cDNA library and functionally overexpressed in Escherichia coli. The obtained cDNA, slightly different from that deposited in the database, encodes a protein of 49 kDa. The gene is expressed in most human tissues, but not in skeletal muscle. Human NAD kinase differs considerably from that of prokaryotes by subunit molecular mass (49 kDa vs 30-35 kDa). The catalytically active homotetramer is highly selective for its substrates, NAD and ATP. It did not phosphorylate the nicotinic acid derivative of NAD (NAAD) suggesting that the potent calcium-mobilizing pyridine nucleotide NAADP is synthesized by an alternative route.

  1. Segmental helical motions and dynamical asymmetry modulate histidine kinase autophosphorylation.

    Directory of Open Access Journals (Sweden)

    Ariel E Mechaly

    2014-01-01

    Full Text Available Histidine kinases (HKs are dimeric receptors that participate in most adaptive responses to environmental changes in prokaryotes. Although it is well established that stimulus perception triggers autophosphorylation in many HKs, little is known on how the input signal propagates through the HAMP domain to control the transient interaction between the histidine-containing and ATP-binding domains during the catalytic reaction. Here we report crystal structures of the full cytoplasmic region of CpxA, a prototypical HK involved in Escherichia coli response to envelope stress. The structural ensemble, which includes the Michaelis complex, unveils HK activation as a highly dynamic process, in which HAMP modulates the segmental mobility of the central HK α-helices to promote a strong conformational and dynamical asymmetry that characterizes the kinase-active state. A mechanical model based on our structural and biochemical data provides insights into HAMP-mediated signal transduction, the autophosphorylation reaction mechanism, and the symmetry-dependent control of HK kinase/phosphatase functional states.

  2. Pkn9, a Ser/Thr protein kinase involved in the development of Myxococcus xanthus.

    Science.gov (United States)

    Hanlon, W A; Inouye, M; Inouye, S

    1997-02-01

    The Myxococcus xanthus gene, pkn9, encodes a protein that contains significant homology with eukaryotic Ser/Thr protein kinases. The pkn9 gene was singled out of a previously identified family of kinase genes by amplification techniques that displayed differences in kinase gene expression during selected periods of the M. xanthus life cycle. Pkn9 was constitutively expressed during vegetative growth and upregulated during the aggregation stage of early development. It consists of 589 amino acids, and its N-terminal 394 residues show 38% identity with both Pkn1 and Pkn2 of M. xanthus. This region also shows 29, 25 and 29% identify with myosin light-chain kinase, protein kinase C, and cAMP-dependent protein kinase, respectively. A 22-residue hydrophobic transmembrane domain separates the kinase domain from the 173-residue C-terminal domain that resides on the outside of the inner membrane. The C-terminal domain contains two sets of tandem repeats of 13 and 10 residues which have no known function. When expressed in Escherichia coli under the T7 promoter, Pkn9 was found to be phosphorylated on serine and threonine residues. Disruption of the pkn9 kinase catalytic subdomains I-III by the insertion of a kanamycin-resistance gene resulted in slightly delayed, smaller and more-crowded fruiting bodies, while spore formation was normal. Total deletion of the pkn9 gene caused severely reduced progression through development resulting in light loose mounds that become slightly more compact over time. Development progressed further at the centre than at the edge of the spot, and spore formation was significantly reduced. Two-dimensional gel analysis revealed that both the disruption and the deletion of pkn9 prevented the expression of five membrane proteins (KREP9-1-4). These results suggest that the loss of Pkn9 kinase activity caused altered fruiting-body formation, the absence of the KREP9 proteins in the membrane, and reduced spore production.

  3. Activation of brain B-Raf protein kinase by Rap1B small GTP-binding protein.

    Science.gov (United States)

    Ohtsuka, T; Shimizu, K; Yamamori, B; Kuroda, S; Takai, Y

    1996-01-19

    Rap1 small GTP-binding protein has the same amino acid sequence at its effector domain as that of Ras. Rap1 has been shown to antagonize the Ras functions, such as the Ras-induced transformation of NIH 3T3 cells and the Ras-induced activation of the c-Raf-1 protein kinase-dependent mitogen-activated protein (MAP) kinase cascade in Rat-1 cells, whereas we have shown that Rap1 as well as Ras stimulates DNA synthesis in Swiss 3T3 cells. We have established a cell-free assay system in which Ras activates bovine brain B-Raf protein kinase. Here we have used this assay system and examined the effect of Rap1 on the B-Raf activity to phosphorylate recombinant MAP kinase kinase (MEK). Recombinant Rap1B stimulated the activity of B-Raf, which was partially purified from bovine brain and immunoprecipitated by an anti-B-Raf antibody. The GTP-bound form was active, but the GDP-bound form was inactive. The fully post-translationally lipid-modified form was active, but the unmodified form was nearly inactive. The maximum B-Raf activity stimulated by Rap1B was nearly the same as that stimulated by Ki-Ras. Rap1B enhanced the Ki-Ras-stimulated B-Raf activity in an additive manner. These results indicate that not only Ras but also Rap1 is involved in the activation of the B-Raf-dependent MAP kinase cascade.

  4. Catalytic microrotor driven by geometrical asymmetry

    Science.gov (United States)

    Yang, Mingcheng; Ripoll, Marisol; Chen, Ke

    2015-02-01

    An asymmetric gear with homogeneous surface properties is, here, presented as a prototype to fabricate catalytic microrotors. The driving torque arises from the diffusiophoretic effect induced by the concentration gradients generated by catalytic chemical reactions at the gear surface. This torque produces a spontaneous and unidirectional rotation of the asymmetric gear. By means of mesoscopic simulations, we prove and characterize this scenario. The gear rotational velocity is determined by the gear-solvent interactions, the gear geometry, the solvent viscosity, and the catalytic reaction ratio. Our work presents a simple way to design self-propelled microrotors, alternative to existing catalytic bi-component, or thermophoretic ones.

  5. Nanostructured Catalytic Reactors for Air Purification Project

    Data.gov (United States)

    National Aeronautics and Space Administration — This SBIR Phase I project proposes the development of lightweight compact nanostructured catalytic reactors for air purification from toxic gaseous organic...

  6. Nanostructured Catalytic Reactors for Air Purification Project

    Data.gov (United States)

    National Aeronautics and Space Administration — This SBIR Phase II project proposes the development of lightweight compact nanostructured catalytic reactors for air purification from toxic gaseous organic...

  7. Identification of Novel Small Molecule Inhibitors of Oncogenic RET Kinase.

    Directory of Open Access Journals (Sweden)

    Marialuisa Moccia

    Full Text Available Oncogenic mutation of the RET receptor tyrosine kinase is observed in several human malignancies. Here, we describe three novel type II RET tyrosine kinase inhibitors (TKI, ALW-II-41-27, XMD15-44 and HG-6-63-01, that inhibit the cellular activity of oncogenic RET mutants at two digit nanomolar concentration. These three compounds shared a 3-trifluoromethyl-4-methylpiperazinephenyl pharmacophore that stabilizes the 'DFG-out' inactive conformation of RET activation loop. They blocked RET-mediated signaling and proliferation with an IC50 in the nM range in fibroblasts transformed by the RET/C634R and RET/M918T oncogenes. They also inhibited autophosphorylation of several additional oncogenic RET-derived point mutants and chimeric oncogenes. At a concentration of 10 nM, ALW-II-41-27, XMD15-44 and HG-6-63-01 inhibited RET kinase and signaling in human thyroid cancer cell lines carrying oncogenic RET alleles; they also inhibited proliferation of cancer, but not non-tumoral Nthy-ori-3-1, thyroid cells, with an IC50 in the nM range. The three compounds were capable of inhibiting the 'gatekeeper' V804M mutant which confers substantial resistance to established RET inhibitors. In conclusion, we have identified a type II TKI scaffold, shared by ALW-II-41-27, XMD15-44 and HG-6-63-01, that may be used as novel lead for the development of novel agents for the treatment of cancers harboring oncogenic activation of RET.

  8. Identification of Novel Small Molecule Inhibitors of Oncogenic RET Kinase.

    Science.gov (United States)

    Moccia, Marialuisa; Liu, Qingsong; Guida, Teresa; Federico, Giorgia; Brescia, Annalisa; Zhao, Zheng; Choi, Hwan Geun; Deng, Xianming; Tan, Li; Wang, Jinhua; Billaud, Marc; Gray, Nathanael S; Carlomagno, Francesca; Santoro, Massimo

    2015-01-01

    Oncogenic mutation of the RET receptor tyrosine kinase is observed in several human malignancies. Here, we describe three novel type II RET tyrosine kinase inhibitors (TKI), ALW-II-41-27, XMD15-44 and HG-6-63-01, that inhibit the cellular activity of oncogenic RET mutants at two digit nanomolar concentration. These three compounds shared a 3-trifluoromethyl-4-methylpiperazinephenyl pharmacophore that stabilizes the 'DFG-out' inactive conformation of RET activation loop. They blocked RET-mediated signaling and proliferation with an IC50 in the nM range in fibroblasts transformed by the RET/C634R and RET/M918T oncogenes. They also inhibited autophosphorylation of several additional oncogenic RET-derived point mutants and chimeric oncogenes. At a concentration of 10 nM, ALW-II-41-27, XMD15-44 and HG-6-63-01 inhibited RET kinase and signaling in human thyroid cancer cell lines carrying oncogenic RET alleles; they also inhibited proliferation of cancer, but not non-tumoral Nthy-ori-3-1, thyroid cells, with an IC50 in the nM range. The three compounds were capable of inhibiting the 'gatekeeper' V804M mutant which confers substantial resistance to established RET inhibitors. In conclusion, we have identified a type II TKI scaffold, shared by ALW-II-41-27, XMD15-44 and HG-6-63-01, that may be used as novel lead for the development of novel agents for the treatment of cancers harboring oncogenic activation of RET.

  9. Catalytic conversion of light alkanes

    Energy Technology Data Exchange (ETDEWEB)

    Lyons, J.E.

    1992-06-30

    The second Quarterly Report of 1992 on the Catalytic Conversion of Light Alkanes reviews the work done between April 1, 1992 and June 31, 1992 on the Cooperative Agreement. The mission of this work is to devise a new catalyst which can be used in a simple economic process to convert the light alkanes in natural gas to oxygenate products that can either be used as clean-burning, high octane liquid fuels, as fuel components or as precursors to liquid hydrocarbon uwspomdon fuel. During the past quarter we have continued to design, prepare, characterize and test novel catalysts for the mild selective reaction of light hydrocarbons with air or oxygen to produce alcohols directly. These catalysts are designed to form active metal oxo (MO) species and to be uniquely active for the homolytic cleavage of the carbon-hydrogen bonds in light alkanes producing intermediates which can form alcohols. We continue to investigate three molecular environments for the active catalytic species that we are trying to generate: electron-deficient macrocycles (PHASE I), polyoxometallates (PHASE II), and regular oxidic lattices including zeolites and related structures as well as other molecular surface structures having metal oxo groups (PHASE I).

  10. Catalytic reforming feed characterisation technique

    Energy Technology Data Exchange (ETDEWEB)

    Larraz Mora, R.; Arvelo Alvarez, R. [Univ. of La Laguna, Chemical Engineering Dept., La Laguna (Spain)

    2002-09-01

    The catalytic reforming of naphtha is one of the major refinery processes, designed to increase the octane number of naphtha or to produce aromatics. The naphtha used as catalytic reformer feedstock usually contains a mixture of paraffins, naphthenes, and aromatics in the carbon number range C{sub 6} to C{sub 10}. The detailed chemical composition of the feed is necessary to predict the aromatics and hydrogen production as well as the operation severity. The analysis of feed naphtha is usually reported in terms of its ASTM distillation curve and API or specific gravity. Since reforming reactions are described in terms of lumped chemical species (paraffins, naphthenes and aromatics), a feed characterisation technique should be useful in order to predict reforming operating conditions and detect feed quality changes. Unfortunately online analyzer applications as cromatography or recently introduced naphtha NMR [1] are scarce in most of refineries. This work proposes an algorithmic characterisation method focusing on its main steps description. The method could help on the subjects previously described, finally a calculation example is shown. (orig.)

  11. W55a Encodes a Novel Protein Kinase That Is Involved in Multiple Stress Responses

    Institute of Scientific and Technical Information of China (English)

    Zhao-Shi Xu; Li Liu; Zhi-Yong Ni; Pei Liu; Ming Chen; Lian-Cheng Li; Yao-Feng Chen; You-Zhi Ma

    2009-01-01

    Protein kinases play crucial roles In response to external environment stress signals. A putative protein kinase, W55a, belonging to SNF1-related protein kinase 2 (SnRK2) subfamily, was isolated from a cDNA library of drought-treated wheat seedlings. The entire length of W55a was obtained using rapid amplification of 5' cDNA ends (5'-RACE) and reverse transcription-polymerase chain reaction(RT-PCR). It contains a 1029-bp open reading frame (ORF) encoding 342 amino acids. The deduced amino acid sequence of W55a had eleven conserved catalytic subdomains and one Ser/Thr protein kinase active-site that characterize Ser/Thr protein kinases. Phylogenetic analysis showed that W55a was 90.38% homologous with rice SAPK1, a member of the SnRK2 family. Using nullisomic-tetrasomic and ditelocentric lines of Chinese Spring, W55a was located on chromosome 2BS. Expression pattern analysis revealed that W55a was upregulated by drought and salt, exogenous abscisic acid, salicylic acid, ethylene and methyl jasmonata, but was not responsive to cold stress. In addition, W55a transcripts were abundant in leaves, but not in roots or stems, under environmental stresses. Transgenic Arabidopsis plants overexprassing W55a exhibited higher tolerance to drought. Based on these findings, W55a encodes a novel dehydration-responsive protein kinase that is involved in multiple stress signal transductions.

  12. The cysteine-cluster motif of c-Yes, Lyn and FAK as a suppressive module for the kinases.

    Science.gov (United States)

    Rahman, Mohammad Aminur; Senga, Takeshi; Oo, Myat Lin; Hasegawa, Hitoki; Biswas, Md Helal Uddin; Mon, Naing Naing; Huang, Pengyu; Ito, Satoko; Yamamoto, Tadashi; Hamaguchi, Michinari

    2008-04-01

    The Src family of non-receptor protein tyrosine kinases plays a critical role in the progression of human cancers so that the development of its specific inhibitors is important as a therapeutic tool. We previously reported that cysteine residues in the cysteine-cluster (CC) motif of v-Src were critical for the kinase inactivation by the SH-alkylating agents such as N-(9-acridinyl) maleimide (NAM), whereas other cysteine residues were dispensable. We found similar CC-motifs in other Src-family kinases and a non-Src-family kinase, FAK. In this study, we explored the function of the CC-motif in Yes, Lyn and FAK. While Src has four cysteines in the CC-motif, c-Yes and Lyn have three and two of the four cysteines, respectively. Two conserved cysteines of the Src family kinases, corresponding to Cys487 and Cys498 of Src, were essential for the resistance to the inactivation of the kinase activity by NAM, whereas the first cysteine of c-Yes, which is absent in Lyn, was less important. FAK has similar CC-motifs with two cysteines and both cysteines were again essential for the resistance to the inactivation of the kinase activity by NAM. Taken together, modification of cysteine residues of the CC-motif causes a repressor effect on the catalytic activity of the Src family kinases and FAK.

  13. Ribosomal S6 kinase is activated as an early event in preemergence development of encysted embryos of Artemia salina.

    Science.gov (United States)

    Malarkey, K; Coker, K J; Sturgill, T W

    1998-01-15

    Dormant Artemia salina cysts contain desiccated gastrulae that are metabolically inactive, and physiologically arrested. Following rehydration, embryos resume development via alterations in protein expression, in the complete absence of cell division. In mammals, activation of p70 ribosomal S6 kinase (p70S6k) has been implicated in translational control, in particular the selective up-regulation of translation of mRNAs with polypyrimidine tracts at their 5' start sites. We therefore investigated ribosomal S6 kinase activity in preemergence development. We demonstrate that an S6 kinase activity is rapidly stimulated (within Artemia S6 kinase was inactivated by treatment with protein phosphatase 2A. Activation of S6 kinase activity was shown to be due to an enzymatic step(s), and not simply rehydration of stored, active enzyme. The temporal profile of activation of S6 kinase activity is compatible with a regulatory function for p70S6k in early preemergence development of encysted Artemia. These studies identify activated Artemia cysts as a system for biochemical studies of p70S6k regulation.

  14. Inactive corrinoid-compound significantly decreases in Spirulina platensis grown in a cobalt-deficient medium.

    Science.gov (United States)

    Watanabe, F; Miyamoto, E; Nakano, Y

    2001-11-01

    Spirulina platensis NIES-39 was grown under open culture system in the presence or absence of CoSO(4) (12 microg/L) and/or vitamin B(12) (10 microg/L) to confirm whether CoSO(4) and/or vitamin B(12) stimulate or are essential for growth of the algal cells and for accumulation of vitamin B(12). The addition of CoSO(4) and/or vitamin B(12) could not affect both cell growth and cell yield of the alga. The amount of corrinoid-compound was increased significantly by the addition of CoSO(4) but not by vitamin B(12). A C18 reversed-phase HPLC pattern of the Spirulina corrinoid-compound increased by the addition of CoSO(4) was identical to that of authentic pseudovitamin B(12), which is inactive for human. These results indicate that the algal cells grown in the absence of CoSO(4) are suitable for use of human health foods because the inactive corrinoid-compound can be reduced significantly.

  15. Epigenome changes in active and inactive Polycomb-group-controlled regions

    Science.gov (United States)

    Breiling, Achim; O'Neill, Laura P; D'Eliseo, Donatella; Turner, Bryan M; Orlando, Valerio

    2004-01-01

    The Polycomb group (PcG) of proteins conveys epigenetic inheritance of repressed transcriptional states. In Drosophila, the Polycomb repressive complex 1 (PRC1) maintains the silent state by inhibiting the transcription machinery and chromatin remodelling at core promoters. Using immunoprecipitation of in vivo formaldehyde-fixed chromatin in phenotypically diverse cultured cell lines, we have mapped PRC1 components, the histone methyl transferase (HMT) Enhancer of zeste (E(z)) and histone H3 modifications in active and inactive PcG-controlled regions. We show that PRC1 components are present in both cases, but at different levels. In particular, active target promoters are nearly devoid of E(z) and Polycomb. Moreover, repressed regions are trimethylated at lysines 9 and 27, suggesting that these histone modifications represent a mark for inactive PcG-controlled regions. These PcG-specific repressive marks are maintained by the action of the E(z) HMT, an enzyme that has an important role not only in establishing but also in maintaining PcG repression. PMID:15448640

  16. "Spatial Energetics": Integrating Data From GPS, Accelerometry, and GIS to Address Obesity and Inactivity.

    Science.gov (United States)

    James, Peter; Jankowska, Marta; Marx, Christine; Hart, Jaime E; Berrigan, David; Kerr, Jacqueline; Hurvitz, Philip M; Hipp, J Aaron; Laden, Francine

    2016-11-01

    To address the current obesity and inactivity epidemics, public health researchers have attempted to identify spatial factors that influence physical inactivity and obesity. Technologic and methodologic developments have led to a revolutionary ability to examine dynamic, high-resolution measures of temporally matched location and behavior data through GPS, accelerometry, and GIS. These advances allow the investigation of spatial energetics, high-spatiotemporal resolution data on location and time-matched energetics, to examine how environmental characteristics, space, and time are linked to activity-related health behaviors with far more robust and detailed data than in previous work. Although the transdisciplinary field of spatial energetics demonstrates promise to provide novel insights on how individuals and populations interact with their environment, there remain significant conceptual, technical, analytical, and ethical challenges stemming from the complex data streams that spatial energetics research generates. First, it is essential to better understand what spatial energetics data represent, the relevant spatial context of analysis for these data, and if spatial energetics can establish causality for development of spatially relevant interventions. Second, there are significant technical problems for analysis of voluminous and complex data that may require development of spatially aware scalable computational infrastructures. Third, the field must come to agreement on appropriate statistical methodologies to account for multiple observations per person. Finally, these challenges must be considered within the context of maintaining participant privacy and security. This article describes gaps in current practice and understanding and suggests solutions to move this promising area of research forward.

  17. Any link between sexual inactivity and treadle pump performance characteristics: The Malawi case

    Science.gov (United States)

    Joseph, Chidanti-Malunga; Yamikani, Malunga

    In mitigating the effects of climate change in Malawi, government promotes the use of low cost irrigation technologies to small-scale farmers, especially in wetlands where water is available. The treadle pump is one such technology. The pump is a manual water lifting device operated by feet. Although the technology has been widely accepted by small-scale farmers, there are documented reports that some farmers abandon the technology, preferring other technologies such as river diversion. One theory for the abandonment is that female farmers claim that the technology makes their male counterparts sexually inactive. This research seeks to find an explanation to the misconception. The study analyzed the physical characteristics of the treadle pump and its users. The results show that the technology is male-dominated (30% were females out of a sample of 40). The results also show that the technology is labor-intensive with very small discharge rates (an average of 0.78 l/s) achieved regardless of the BMI of the operator. With such small discharge rates, in order to fulfill irrigation requirements of a crop, the operator has to pump for long hours. This exercise makes men naturally tired, perhaps making them sexually inactive as well.

  18. Intestinal pseudo-obstruction in inactive systemic lupus erythematosus: An unusual finding

    Institute of Scientific and Technical Information of China (English)

    Giulia; Leonardi; Nicola; de; Bortoli; Massimo; Bellini; Maria; Gloria; Mumolo; Francesco; Costa; Angelo; Ricchiuti; Stefano; Bombardieri; Santino; Marchi

    2010-01-01

    Chronic intestinal pseudo-obstruction (CIP) is an infre-quent complication of an active systemic lupus erythema-tosus (SLE). We illustrate a case of SLE inactive-related CIP. A 51-year old female with inactive SLE (ECLAM score 2) was hospitalized with postprandial fullness, vomiting, abdominal bloating and abdominal pain. She had had no bowel movements for five days. Plain abdominal X-ray revealed multiple fluid levels and dilated small and large bowel loops with air-fluid levels. Intestinal contrast radiology detected dilated loops. CIP was diagnosed. The patient was treated with prokinetics, octreotide, claritromycin, rifaximin, azathioprine and tegaserod without any clinical improvement. Then methylprednisolone (500 mg iv daily) was started. After the first administration, the patient showed peristaltic movements. A bowel movement was reported after the second administration. A plain abdominal X-ray revealed no air-fluid levels. Steroid therapy was slowly reduced with complete resolution of the symptoms. The patient is still in a good clinical condition. SLE-related CIP is generally reported as a complication of an active disease. In our case, CIP was the only clinical demonstration of the SLE.

  19. Influence of obesity, physical inactivity, and weight cycling on chronic inflammation.

    Science.gov (United States)

    Strohacker, K; McFarlin, Brian K

    2010-01-01

    Obesity prevalence continues to rise due to excessive caloric intake and sedentary behavior. Weight loss can be achieved through diet and/or exercise, but maintenance of a reduced weight is rare and relapse is prevalent. Repeated periods of weight loss and regain have been termed "weight cycling." It has been speculated that weight cycling may further increase the elevated disease risk common with weight gain, obesity, and physical inactivity. Alterations in adipose tissue with weight cycling may create a more hypoxic environment; hypoxic adipose tissue secretes leptin, a stimulus for macrophage activation and accumulation within adipose tissue. Hypoxic adipocytes and macrophages release pro-inflammatory cytokines into circulation. Elevated body weight and adiposity are linked to cardiovascular disease and type 2 diabetes via an inflammatory mechanism. Thus, it is reasonable to speculate that weight cycling causes a more profound change in chronic inflammation than sustained weight gain. The purpose of this review is to explore inflammatory consequences associated with weight cycling as they are related to sustained weight gain, obesity, physical inactivity as well as relative disease risk.

  20. Detection of (Inactivity Periods in Human Body Motion Using Inertial Sensors: A Comparative Study

    Directory of Open Access Journals (Sweden)

    Miguel Damas

    2012-05-01

    Full Text Available Determination of (inactivity periods when monitoring human body motion is a mandatory preprocessing step in all human inertial navigation and position analysis applications. Distinction of (inactivity needs to be established in order to allow the system to recompute the calibration parameters of the inertial sensors as well as the Zero Velocity Updates (ZUPT of inertial navigation. The periodical recomputation of these parameters allows the application to maintain a constant degree of precision. This work presents a comparative study among different well known inertial magnitude-based detectors and proposes a new approach by applying spectrum-based detectors and memory-based detectors. A robust statistical comparison is carried out by the use of an accelerometer and angular rate signal synthesizer that mimics the output of accelerometers and gyroscopes when subjects are performing basic activities of daily life. Theoretical results are verified by testing the algorithms over signals gathered using an Inertial Measurement Unit (IMU. Detection accuracy rates of up to 97% are achieved.

  1. Multimotor transport in a system of active and inactive kinesin-1 motors.

    Science.gov (United States)

    Scharrel, Lara; Ma, Rui; Schneider, René; Jülicher, Frank; Diez, Stefan

    2014-07-15

    Long-range directional transport in cells is facilitated by microtubule-based motor proteins. One example is transport in a nerve cell, where small groups of motor proteins, such as kinesins and cytoplasmic dynein, work together to ensure the supply and clearance of cellular material along the axon. Defects in axonal transport have been linked to Alzheimer's and other neurodegenerative diseases. However, it is not known in detail how multimotor-based cargo transport is impaired if a fraction of the motors are defective. To mimic impaired multimotor transport in vitro, we performed gliding motility assays with varying fractions of active kinesin-1 motors and inactive kinesin-1 motor mutants. We found that impaired transport manifests in multiple motility regimes: 1), a fast-motility regime characterized by gliding at velocities close to the single-molecule velocity of the active motors; 2), a slow-motility regime characterized by gliding at close-to zero velocity or full stopping; and 3), a regime in which fast and slow motilities coexist. Notably, the transition from the fast to the slow regime occurred sharply at a threshold fraction of active motors. Based on single-motor parameters, we developed a stochastic model and a mean-field theoretical description that explain our experimental findings. Our results demonstrate that impaired multimotor transport mostly occurs in an either/or fashion: depending on the ratio of active to inactive motors, transport is either performed at close to full speed or is out of action.

  2. Why are Jupiter-family comets active and asteroids in cometary-like orbits inactive?

    CERN Document Server

    Gundlach, B

    2016-01-01

    Surveys in the visible and near-infrared spectral range have revealed the presence of low-albedo asteroids in cometary like orbits (ACOs). In contrast to Jupiter family comets (JFCs), ACOs are inactive, but possess similar orbital parameters. In this work, we discuss why ACOs are inactive, whereas JFCs show gas-driven dust activity, although both belong to the same class of primitive solar system bodies. We hypothesize that ACOs and JFCs have formed under the same physical conditions, namely by the gravitational collapse of ensembles of ice and dust aggregates. We use the memory effect of dust-aggregate layers under gravitational compression to discuss under which conditions the gas-driven dust activity of these bodies is possible. Owing to their smaller sizes, JFCs can sustain gas-driven dust activity much longer than the bigger ACOs, whose sub-surface regions possess an increased tensile strength, due to gravitational compression of the material. The increased tensile strength leads to the passivation again...

  3. Direct observation of the autophosphorylation of insulin receptor kinase by mass spectrometry

    Institute of Scientific and Technical Information of China (English)

    Zhi Li Li

    2009-01-01

    The catalytic and signaling activities of insulin receptor kinase(IRK)are regulated by the autophosphorylation of three tyrosine residues in a cytoplasmic protein-tyrosine kinase domain at Tyro 1158,Tyro 1162 and Tyro 1163.In this study,time-course of the auphosphorylation of the core kinase(residues 978-1283)from IRK was directly investigated by online electrospray ionization mass spectrometry.It is found that two tyrosine residues were phosphorylated in reaction time range of 30 min.This study implies that mass spectrometric technique must be a powerful tool to directly monitor the biological macromolecular modification and will also provide the information of the order and the mechanism of autophosphorylation at the tyrosine sites coupled with tandem mass spectrometric technique.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  5. Fine-Tuning of the RIG-I-Like Receptor/Interferon Regulatory Factor 3-Dependent Antiviral Innate Immune Response by the Glycogen Synthase Kinase 3/β-Catenin Pathway.

    Science.gov (United States)

    Khan, Kashif Aziz; Dô, Florence; Marineau, Alexandre; Doyon, Priscilla; Clément, Jean-François; Woodgett, James R; Doble, Bradley W; Servant, Marc J

    2015-09-01

    Induction of an antiviral innate immune response relies on pattern recognition receptors, including retinoic acid-inducible gene 1-like receptors (RLR), to detect invading pathogens, resulting in the activation of multiple latent transcription factors, including interferon regulatory factor 3 (IRF3). Upon sensing of viral RNA and DNA, IRF3 is phosphorylated and recruits coactivators to induce type I interferons (IFNs) and selected sets of IRF3-regulated IFN-stimulated genes (ISGs) such as those for ISG54 (Ifit2), ISG56 (Ifit1), and viperin (Rsad2). Here, we used wild-type, glycogen synthase kinase 3α knockout (GSK-3α(-/-)), GSK-3β(-/-), and GSK-3α/β double-knockout (DKO) embryonic stem (ES) cells, as well as GSK-3β(-/-) mouse embryonic fibroblast cells in which GSK-3α was knocked down to demonstrate that both isoforms of GSK-3, GSK-3α and GSK-3β, are required for this antiviral immune response. Moreover, the use of two selective small-molecule GSK-3 inhibitors (CHIR99021 and BIO-acetoxime) or ES cells reconstituted with the catalytically inactive versions of GSK-3 isoforms showed that GSK-3 activity is required for optimal induction of antiviral innate immunity. Mechanistically, GSK-3 isoform activation following Sendai virus infection results in phosphorylation of β-catenin at S33/S37/T41, promoting IRF3 DNA binding and activation of IRF3-regulated ISGs. This study identifies the role of a GSK-3/β-catenin axis in antiviral innate immunity.

  6. Unfolded protein response activates glycogen synthase kinase-3 via selective lysosomal degradation.

    Science.gov (United States)

    Nijholt, Diana A T; Nölle, Anna; van Haastert, Elise S; Edelijn, Hessel; Toonen, Ruud F; Hoozemans, Jeroen J M; Scheper, Wiep

    2013-07-01

    The unfolded protein response (UPR) is a stress response that is activated upon disturbed homeostasis in the endoplasmic reticulum. In Alzheimer's disease, as well as in other tauopathies, the UPR is activated in neurons that contain early tau pathology. A recent genome-wide association study identified genetic variation in a UPR transducer as a risk factor for tauopathy, supporting a functional connection between UPR activation and tau pathology. Here we show that UPR activation increases the activity of the major tau kinase glycogen synthase kinase (GSK)-3 in vitro via a selective removal of inactive GSK-3 phosphorylated at Ser(21/9). We demonstrate that this is mediated by the autophagy/lysosomal pathway. In brain tissue from patients with different tauopathies, lysosomal accumulations of pSer(21/9) GSK-3 are found in neurons with markers for UPR activation. Our data indicate that UPR activation increases the activity of GSK-3 by a novel mechanism, the lysosomal degradation of the inactive pSer(21/9) GSK-3. This may provide a functional explanation for the close association between UPR activation and early tau pathology in neurodegenerative diseases.

  7. Sulphate removal induces a major conformational change in Leishmania mexicana pyruvate kinase in the crystalline state.

    Science.gov (United States)

    Tulloch, Lindsay B; Morgan, Hugh P; Hannaert, Véronique; Michels, Paul A M; Fothergill-Gilmore, Linda A; Walkinshaw, Malcolm D

    2008-11-14

    We report X-ray structures of pyruvate kinase from Leishmania mexicana (LmPYK) that are trapped in different conformations. These, together with the previously reported structure of LmPYK in its inactive (T-state) conformation, allow comparisons of three different conformers of the same species of pyruvate kinase (PYK). Four new site point mutants showing the effects of side-chain alteration at subunit interfaces are also enzymatically characterised. The LmPYK tetramer crystals grown with ammonium sulphate as precipitant adopt an active-like conformation, with sulphate ions at the active and effector sites. The sulphates occupy positions similar to those of the phosphates of ligands bound to active (R-state) and constitutively active (nonallosteric) PYKs from several species, and provide insight into the structural roles of the phosphates of the substrates and effectors. Crystal soaking in sulphate-free buffers was found to induce major conformational changes in the tetramer. In particular, the unwinding of the Aalpha6' helix and the inward hinge movement of the B domain are coupled with a significant widening (4 A) of the tetramer caused by lateral movement of the C domains. The two new LmPYK structures and the activity studies of site point mutations described in this article are consistent with a developing picture of allosteric activity in which localised changes in protein flexibility govern the distribution of conformer families adopted by the tetramer in its active and inactive states.

  8. Multifunctional human transcriptional coactivator protein PC4 is a substrate of Aurora kinases and activates the Aurora enzymes.

    Science.gov (United States)

    Dhanasekaran, Karthigeyan; Kumari, Sujata; Boopathi, Ramachandran; Shima, Hiroki; Swaminathan, Amrutha; Bachu, Mahesh; Ranga, Udaykumar; Igarashi, Kazuhiko; Kundu, Tapas K

    2016-03-01

    Positive coactivator 4 (PC4), a human transcriptional coactivator, is involved in diverse processes like chromatin organization and transcription regulation. It is hyperphosphorylated during mitosis, with unknown significance. For the first time, we demonstrate the function of PC4 outside the nucleus upon nuclear envelope breakdown. A fraction of PC4 associates with Aurora A and Aurora B and undergoes phosphorylation, following which PC4 activates both Aurora A and B to sustain optimal kinase activity to maintain the phosphorylation gradient for the proper functioning of the mitotic machinery. This mitotic role is evident in PC4 knockdown cells where the defects are rescued only by the catalytically active Aurora kinases, but not the kinase-dead mutants. Similarly, the PC4 phosphodeficient mutant failed to rescue such defects. Hence, our observations establish a novel mitotic function of PC4 that might be dependent on Aurora kinase-mediated phosphorylation.

  9. Structure of Arabidopsis thaliana 5-methylthioribose Kinase Reveals a More Occluded Active Site Than its Bacterial Homolog

    Energy Technology Data Exchange (ETDEWEB)

    Ku,S.; Cornell, K.; Howell, P.

    2007-01-01

    Metabolic variations exist between the methionine salvage pathway of humans and a number of plants and microbial pathogens. 5-Methylthioribose (MTR) kinase is a key enzyme required for methionine salvage in plants and many bacteria. The absence of a mammalian homolog suggests that MTR kinase is a good target for the design of specific herbicides or antibiotics. The structure of Arabidopsis thaliana MTR kinase co-crystallized with ATP?S and MTR has been determined at 1.9 Angstroms resolution. The structure is similar to B. subtilis MTR kinase and has the same protein kinase fold observed in other evolutionarily related protein kinase-like phosphotransferases. The active site is comparable between the two enzymes with the DXE-motif coordinating the nucleotide-Mg, the D238 of the HGD catalytic loop polarizing the MTR O1 oxygen, and the RR-motif interacting with the substrate MTR. Unlike its bacterial homolog, however, the Gly-rich loop (G-loop) of A. thaliana MTR kinase has an extended conformation, which shields most of the active site from solvent, a feature that resembles eukaryotic protein kinases more than the bacterial enzyme. The G- and W-loops of A. thaliana and B. subtilis MTR kinase adopt different conformations despite high sequence similarity. The ATP?S analog was hydrolyzed during the co-crystallization procedure, resulting in ADP in the active site. This suggests that the A. thaliana enzyme, like its bacterial homolog, may have significant ATPase activity in the absence of MTR. The structure of A. thaliana MTR kinase provides a template for structure-based design of agrochemicals, particularly herbicides whose effectiveness could be regulated by nutrient levels. Features of the MTR binding site offer an opportunity for a simple organic salt of an MTR analog to specifically inhibit MTR kinase.

  10. Structure and inhibitor specificity of the PCTAIRE-family kinase CDK16.

    Science.gov (United States)

    Dixon-Clarke, Sarah E; Shehata, Saifeldin N; Krojer, Tobias; Sharpe, Timothy D; von Delft, Frank; Sakamoto, Kei; Bullock, Alex N

    2017-02-20

    CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family that has emerged as a key regulator of neurite outgrowth, vesicle trafficking and cancer cell proliferation. CDK16 is activated through binding to cyclin Y via a phosphorylation-dependent 14-3-3 interaction and has a unique consensus substrate phosphorylation motif compared with conventional CDKs. To elucidate the structure and inhibitor-binding properties of this atypical CDK, we screened the CDK16 kinase domain against different inhibitor libraries and determined the co-structures of identified hits. We discovered that the ATP-binding pocket of CDK16 can accommodate both type I and type II kinase inhibitors. The most potent CDK16 inhibitors revealed by cell-free and cell-based assays were the multitargeted cancer drugs dabrafenib and rebastinib. An inactive DFG-out binding conformation was confirmed by the first crystal structures of CDK16 in separate complexes with the inhibitors indirubin E804 and rebastinib, respectively. The structures revealed considerable conformational plasticity, suggesting that the isolated CDK16 kinase domain was relatively unstable in the absence of a cyclin partner. The unusual structural features and chemical scaffolds identified here hold promise for the development of more selective CDK16 inhibitors and provide opportunity to better characterise the role of CDK16 and its related CDK family members in various physiological and pathological contexts.

  11. Structure and inhibitor specificity of the PCTAIRE-family kinase CDK16

    Science.gov (United States)

    Dixon-Clarke, Sarah E.; Shehata, Saifeldin N.; Krojer, Tobias; Sharpe, Timothy D.; vonDelft, Frank; Sakamoto, Kei

    2017-01-01

    CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family that has emerged as a key regulator of neurite outgrowth, vesicle trafficking and cancer cell proliferation. CDK16 is activated through binding to cyclin Y via a phosphorylation-dependent 14-3-3 interaction and has a unique consensus substrate phosphorylation motif compared with conventional CDKs. To elucidate the structure and inhibitor-binding properties of this atypical CDK, we screened the CDK16 kinase domain against different inhibitor libraries and determined the co-structures of identified hits. We discovered that the ATP-binding pocket of CDK16 can accommodate both type I and type II kinase inhibitors. The most potent CDK16 inhibitors revealed by cell-free and cell-based assays were the multitargeted cancer drugs dabrafenib and rebastinib. An inactive DFG-out binding conformation was confirmed by the first crystal structures of CDK16 in separate complexes with the inhibitors indirubin E804 and rebastinib, respectively. The structures revealed considerable conformational plasticity, suggesting that the isolated CDK16 kinase domain was relatively unstable in the absence of a cyclin partner. The unusual structural features and chemical scaffolds identified here hold promise for the development of more selective CDK16 inhibitors and provide opportunity to better characterise the role of CDK16 and its related CDK family members in various physiological and pathological contexts. PMID:28057719

  12. Cancer metabolism meets systems biology: Pyruvate kinase isoform PKM2 is a metabolic master regulator

    Directory of Open Access Journals (Sweden)

    Fabian V Filipp

    2013-01-01

    Full Text Available Pyruvate kinase activity is controlled by a tightly woven regulatory network. The oncofetal isoform of pyruvate kinase (PKM2 is a master regulator of cancer metabolism. PKM2 engages in parallel, feed-forward, positive and negative feedback control contributing to cancer progression. Besides its metabolic role, non-metabolic functions of PKM2 as protein kinase and transcriptional coactivator for c-MYC and hypoxia-inducible factor 1-alpha are essential for epidermal growth factor receptor activation-induced tumorigenesis. These biochemical activities are controlled by a shift in the oligomeric state of PKM2 that includes acetylation, oxidation, phosphorylation, prolyl hydroxylation and sumoylation. Metabolically active PKM2 tetramer is allosterically regulated and responds to nutritional and stress signals. Metabolically inactive PKM2 dimer is imported into the nucleus and can function as protein kinase stimulating transcription. A systems biology approach to PKM2 at the genome, transcriptome, proteome, metabolome and fluxome level reveals how differences in biomolecular structure translate into a global rewiring of cancer metabolism. Cancer systems biology takes us beyond the Warburg effect, opening unprecedented therapeutic opportunities.

  13. Coordinate regulation of the mother centriole component nlp by nek2 and plk1 protein kinases.

    Science.gov (United States)

    Rapley, Joseph; Baxter, Joanne E; Blot, Joelle; Wattam, Samantha L; Casenghi, Martina; Meraldi, Patrick; Nigg, Erich A; Fry, Andrew M

    2005-02-01

    Mitotic entry requires a major reorganization of the microtubule cytoskeleton. Nlp, a centrosomal protein that binds gamma-tubulin, is a G(2)/M target of the Plk1 protein kinase. Here, we show that human Nlp and its Xenopus homologue, X-Nlp, are also phosphorylated by the cell cycle-regulated Nek2 kinase. X-Nlp is a 213-kDa mother centriole-specific protein, implicating it in microtubule anchoring. Although constant in abundance throughout the cell cycle, it is displaced from centrosomes upon mitotic entry. Overexpression of active Nek2 or Plk1 causes premature displacement of Nlp from interphase centrosomes. Active Nek2 is also capable of phosphorylating and displacing a mutant form of Nlp that lacks Plk1 phosphorylation sites. Importantly, kinase-inactive Nek2 interferes with Plk1-induced displacement of Nlp from interphase centrosomes and displacement of endogenous Nlp from mitotic spindle poles, while active Nek2 stimulates Plk1 phosphorylation of Nlp in vitro. Unlike Plk1, Nek2 does not prevent association of Nlp with gamma-tubulin. Together, these results provide the first example of a protein involved in microtubule organization that is coordinately regulated at the G(2)/M transition by two centrosomal kinases. We also propose that phosphorylation by Nek2 may prime Nlp for phosphorylation by Plk1.

  14. Electrochemical promotion of catalytic reactions

    Science.gov (United States)

    Imbihl, R.

    2010-05-01

    The electrochemical promotion of heterogeneously catalyzed reactions (EPOC) became feasible through the use of porous metal electrodes interfaced to a solid electrolyte. With the O 2- conducting yttrium stabilized zirconia (YSZ), the Na + conducting β″-Al 2O 3 (β-alumina), and several other types of solid electrolytes the EPOC effect has been demonstrated for about 100 reaction systems in studies conducted mainly in the mbar range. Surface science investigations showed that the physical basis for the EPOC effect lies in the electrochemically induced spillover of oxygen and alkali metal, respectively, onto the surface of the metal electrodes. For the catalytic promotion effect general concepts and mechanistic schemes were proposed but these concepts and schemes are largely speculative. Applying surface analytical tools to EPOC systems the proposed mechanistic schemes can be verified or invalidated. This report summarizes the progress which has been achieved in the mechanistic understanding of the EPOC effect.

  15. Catalytic Graphitization of Phenolic Resin

    Institute of Scientific and Technical Information of China (English)

    Mu Zhao; Huaihe Song

    2011-01-01

    The catalytic graphitization of thermal plastic phenolic-formaldehyde resin with the aid of ferric nitrate (FN) was studied in detail. The morphologies and structural features of the products including onion-like carbon nanoparticles and bamboo-shaped carbon nanotubes were investigated by transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), X-ray diffraction and Raman spectroscopy measurements. It was found that with the changes of loading content of FN and residence time at 1000℃, the products exhibited various morphologies. The TEM images showed that bamboo-shaped carbon nanotube consisted of tens of bamboo sticks and onion-like carbon nanoparticle was made up of quasi-spherically concentrically closed carbon nanocages.

  16. Unsteady processes in catalytic reactors

    Energy Technology Data Exchange (ETDEWEB)

    Matros, Yu.Sh.

    1985-01-01

    In recent years a realization has occurred that reaction and reactor dynamics must be considered when designing and operating catalytic reactors. In this book, the author has focussed on both the processes occurring on individual porous-catalyst particles as well as the phenomena displayed by collections of these particles in fixed-bed reactors. The major topics discussed include the effects of unsteady-state heat and mass transfer, the influence of inhomogeneities and stagnant regions in fixed beds, and reactor operation during forced cycling of operating conditions. Despite the title of the book, attention is also paid to the determination of the number and stability of fixed-bed steady states, with the aim of describing the possibility of controlling reactors at unstable steady states. However, this development is somewhat dated, given the recent literature on multiplicity phenomena and process control.

  17. Non-catalytic recuperative reformer

    Energy Technology Data Exchange (ETDEWEB)

    Khinkis, Mark J.; Kozlov, Aleksandr P.; Kurek, Harry

    2015-12-22

    A non-catalytic recuperative reformer has a flue gas flow path for conducting hot flue gas from a thermal process and a reforming mixture flow path for conducting a reforming mixture. At least a portion of the reforming mixture flow path is embedded in the flue gas flow path to permit heat transfer from the hot flue gas to the reforming mixture. The reforming mixture flow path contains substantially no material commonly used as a catalyst for reforming hydrocarbon fuel (e.g., nickel oxide, platinum group elements or rhenium), but instead the reforming mixture is reformed into a higher calorific fuel via reactions due to the heat transfer and residence time. In a preferred embodiment, extended surfaces of metal material such as stainless steel or metal alloy that are high in nickel content are included within at least a portion of the reforming mixture flow path.

  18. Selective catalytic oxidation of ammonia

    Energy Technology Data Exchange (ETDEWEB)

    Leppaelahti, J.; Koljonen, T. [VTT Energy, Espoo (Finland)

    1996-12-31

    In the combustion of fossil fuels, the principal source of nitrogen oxides is nitrogen bound in the fuel structure. In gasification, a large part of fuel nitrogen forms NH{sub 3}, which may form nitrogen oxides during gas combustion. If NH{sub 3} and other nitrogen species could be removed from hot gas, the NO emission could be considerably reduced. However, relatively little attention has been paid to finding new means of removing nitrogen compounds from the hot gasification gas. The possibility of selectively oxidizing NH{sub 3} to N{sub 2} in the hot gasification has been studied at VTT Energy. The largest NH{sub 3} reductions have been achieved by catalytic oxidation on aluminium oxides. (author) (4 refs.)

  19. Method of fabricating a catalytic structure

    Science.gov (United States)

    Rollins, Harry W.; Petkovic, Lucia M.; Ginosar, Daniel M.

    2009-09-22

    A precursor to a catalytic structure comprising zinc oxide and copper oxide. The zinc oxide has a sheet-like morphology or a spherical morphology and the copper oxide comprises particles of copper oxide. The copper oxide is reduced to copper, producing the catalytic structure. The catalytic structure is fabricated by a hydrothermal process. A reaction mixture comprising a zinc salt, a copper salt, a hydroxyl ion source, and a structure-directing agent is formed. The reaction mixture is heated under confined volume conditions to produce the precursor. The copper oxide in the precursor is reduced to copper. A method of hydrogenating a carbon oxide using the catalytic structure is also disclosed, as is a system that includes the catalytic structure.

  20. Structural properties of cyanase. Denaturation, renaturation, and role of sulfhydryls and oligomeric structure in catalytic activity.

    Science.gov (United States)

    Little, R M; Anderson, P M

    1987-07-25

    Cyanase is an inducible enzyme in Escherichia coli that catalyzes bicarbonate-dependent decomposition of cyanate to give ammonia and bicarbonate. The enzyme is composed of 8-10 identical subunits (Mr = 17,008). The objective of this study was to clarify some of the structural properties of cyanase for the purpose of understanding the relationship between oligomeric structure and catalytic activity. Circular dichroism studies showed that cyanase has a significant amount of alpha-helix and beta-sheet structure. The one sulfhydryl group per subunit does not react with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) unless cyanase is denatured. Denaturation is apparently complete in 10 M urea or 6 M guanidine hydrochloride, but is significantly reduced in 10 M urea by the presence of azide (analog of cyanate) and is incomplete in 8 M urea. Denatured cyanase could be renatured and reactivated (greater than 85%) by removal of denaturants. Reactivation was greatly facilitated by the presence of certain anions, particularly bicarbonate, and by high ionic strength and protein concentration. The catalytic activity of renatured cyanase was associated only with oligomer. Cyanase that had been denatured in the presence of DTNB to give a cyanase-DTNB derivative could also be renatured at 26 degrees C to give active cyanase-DTNB oligomer. The active oligomeric form of the cyanase-DTNB derivative could be converted reversibly to inactive dimer by lowering the temperature to 4 degrees C or by reduction of the ionic strength and removal of monoanions. These results provide evidence that free sulfhydryl groups are not required for catalytic activity and that catalytic activity may be dependent upon oligomeric structure.

  1. Saccharomyces cerevisiae DNA ligase IV supports imprecise end joining independently of its catalytic activity.

    Directory of Open Access Journals (Sweden)

    Kishore K Chiruvella

    2013-06-01

    Full Text Available DNA ligase IV (Dnl4 in budding yeast is a specialized ligase used in non-homologous end joining (NHEJ of DNA double-strand breaks (DSBs. Although point and truncation mutations arise in the human ligase IV syndrome, the roles of Dnl4 in DSB repair have mainly been examined using gene deletions. Here, Dnl4 catalytic point mutants were generated that were severely defective in auto-adenylation in vitro and NHEJ activity in vivo, despite being hyper-recruited to DSBs and supporting wild-type levels of Lif1 interaction and assembly of a Ku- and Lif1-containing complex at DSBs. Interestingly, residual levels of especially imprecise NHEJ were markedly higher in a deletion-based assay with Dnl4 catalytic mutants than with a gene deletion strain, suggesting a role of DSB-bound Dnl4 in supporting a mode of NHEJ catalyzed by a different ligase. Similarly, next generation sequencing of repair joints in a distinct single-DSB assay showed that dnl4-K466A mutation conferred a significantly different imprecise joining profile than wild-type Dnl4 and that such repair was rarely observed in the absence of Dnl4. Enrichment of DNA ligase I (Cdc9 in yeast at DSBs was observed in wild-type as well as dnl4 point mutant strains, with both Dnl4 and Cdc9 disappearing from DSBs upon 5' resection that was unimpeded by the presence of catalytically inactive Dnl4. These findings indicate that Dnl4 can promote mutagenic end joining independently of its catalytic activity, likely by a mechanism that involves Cdc9.

  2. Saccharomyces cerevisiae DNA ligase IV supports imprecise end joining independently of its catalytic activity.

    Science.gov (United States)

    Chiruvella, Kishore K; Liang, Zhuobin; Birkeland, Shanda R; Basrur, Venkatesha; Wilson, Thomas E

    2013-06-01

    DNA ligase IV (Dnl4 in budding yeast) is a specialized ligase used in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs). Although point and truncation mutations arise in the human ligase IV syndrome, the roles of Dnl4 in DSB repair have mainly been examined using gene deletions. Here, Dnl4 catalytic point mutants were generated that were severely defective in auto-adenylation in vitro and NHEJ activity in vivo, despite being hyper-recruited to DSBs and supporting wild-type levels of Lif1 interaction and assembly of a Ku- and Lif1-containing complex at DSBs. Interestingly, residual levels of especially imprecise NHEJ were markedly higher in a deletion-based assay with Dnl4 catalytic mutants than with a gene deletion strain, suggesting a role of DSB-bound Dnl4 in supporting a mode of NHEJ catalyzed by a different ligase. Similarly, next generation sequencing of repair joints in a distinct single-DSB assay showed that dnl4-K466A mutation conferred a significantly different imprecise joining profile than wild-type Dnl4 and that such repair was rarely observed in the absence of Dnl4. Enrichment of DNA ligase I (Cdc9 in yeast) at DSBs was observed in wild-type as well as dnl4 point mutant strains, with both Dnl4 and Cdc9 disappearing from DSBs upon 5' resection that was unimpeded by the presence of catalytically inactive Dnl4. These findings indicate that Dnl4 can promote mutagenic end joining independently of its catalytic activity, likely by a mechanism that involves Cdc9.

  3. Factors increasing the risk of inactivity among administrative, technical, and manual workers in Warszawa public institutions

    Directory of Open Access Journals (Sweden)

    Elżbieta Biernat

    2015-04-01

    Full Text Available Objectives: The research aims to assess the level of physical activity among administrative, technical, and manual workers employed in Warszawa public institutions and to analyze the factors that increase the risk of failing to meet World Health Organization (WHO recommendations. Material and Methods: The study comprised 373 employees of randomly selected institutions. A short version of International Physical Activity Questionnaire (IPAQ was applied. The correlation between the mean values of duration, days, MET-min/week of efforts, gender, and type of work was analyzed using the Tukey’s honest significant difference (HSD test, while the correlation between the level of physical activity and the socio-demographic characteristics was assessed with the Chi2 test. The strength of the relationship between socio-demographic characteristics and fulfilment of WHO standards was expressed by the odds ratio (OR and 95% confidence interval (95% CI. The significance level was p = 0.05. Results: High levels of physical activity were declared by 41.8% of the manual workers, 14.7% of the administration staff, and 7.3% of the technicians; 19%, 31.5% and 54.5%, respectively, reported low levels of physical activity. Factors determining the fulfilment of the WHO recommendations include: the nature of work (p = 0.003, education (p = 0.004, and income (p = 0.003. The risk of being inactive nearly doubles in the case of administration staff (31.5% and increases more than 4 times in the case of technicians (54.5%. Respondents with secondary school education (31.6% are exposed to a 3-fold higher risk of inactivity, while in respondents with higher education (37.2%, the level of the risk is 4-fold higher. Compared to those in the highest income group (23.4%, people who earn less (34.1% are inactive almost twice as often. Conclusions: Urgent intervention is necessary in all studied groups: increased energy expenditure for recreation and locomotion, educational offers

  4. Population-attributable causes of cancer in Korea: obesity and physical inactivity.

    Directory of Open Access Journals (Sweden)

    Sohee Park

    Full Text Available BACKGROUND: Changes in lifestyle including obesity epidemic and reduced physical activity influenced greatly to increase the cancer burden in Korea. The purpose of the current study was to perform a systematic assessment of cancers attributable to obesity and physical inactivity in Korea. METHODOLOGY/PRINCIPAL FINDINGS: Gender- and cancer site-specific population-attributable fractions (PAF were estimated using the prevalence of overweight and obesity in 1992-1995 from a large-scale prospective cohort study, the prevalence of low physical activity in 1989 from a Korean National Health Examination Survey, and pooled relative risk estimates from Korean epidemiological studies. The overall PAF was then estimated using 2009 national cancer incidence data from the Korea Central Cancer Registry. Excess body weight was responsible for 1,444 (1.5% and 2,004 (2.2% cancer cases among men and women, respectively, in 2009 in Korea. Among men, 6.8% of colorectal, 2.9% of pancreatic, and 16.0% of kidney cancer was attributable to excess body weight. In women, 6.6% of colorectal, 3.9% of pancreatic, 18.7% of kidney, 8.2% of postmenopausal breast, and 32.7% of endometrial cancer was attributable to excess body weight. Low leisure-time physical activity accounted for 8.8% of breast cancer, whereas the PAF for overall cancer was low (0.1% in men, 1.4% in women. Projections suggest that cancers attributable to obesity will increase by 40% in men and 16% in women by 2020. CONCLUSIONS/SIGNIFICANCE: With a significantly increasing overweight and physically inactive population, and increasing incidence of breast and colorectal cancers, Korea faces a large cancer burden attributable to these risk factors. Had the obese population of Korea remained stable, a large portion of obesity-related cancers could have been avoided. Efficient cancer prevention programs that aim to reduce obesity- and physical inactivity-related health problems are essential in Korea.

  5. Prevalence of physical inactivity and associated factors among adolescents from public schools in Uruguaiana, Rio Grande do Sul State, Brazil

    Directory of Open Access Journals (Sweden)

    Gabriel Gustavo Bergmann

    2013-11-01

    Full Text Available This study aimed to identify the prevalence of physical inactivity and associated factors in adolescents, using a cross-sectional design with a sample of 1,455 adolescents (741 females. Data were collected using a questionnaire consisting of socio-demographic, perceptual, and behavioral variables. Physical activity was estimated with the Physical Activity Questionnaire for Older Children and Adolescents. Prevalence of physical inactivity was 68% (95%CI: 65.6%-70.4%. The following variables remained associated with physical inactivity in the adjusted analysis (p < 0.05: living in an apartment, female gender, older adolescents, lower self-rated physical activity compared to peers, low perception of maternal physical activity, passive commuting to school, non-participation in physical education at school, non-enjoyment of physical education classes, and limited involvement in other types of physical exercise besides physical education at school. There were a high proportion of inactive adolescents. Strategies to prevent physical inactivity in adolescents should be elaborated with a central role for the school and family.

  6. Meta-analysis of three diabetes population studies: association of inactive ALDH2 genotype with maternal inheritance of diabetes.

    Science.gov (United States)

    Murata, C; Taniyama, M; Kuriyama, S; Muramatsu, T; Atsumi, Y; Matsuoka, K; Suzuki, Y

    2004-12-01

    To date, there have been three population studies that examined the association of mitochondrial aldehyde dehydrogenase 2 (ALDH2) genotype with inheritance of diabetes. Here, we summarize the results by meta-analysis. The study 1 consisted of 212 type 2 diabetics who did not have renal failure. The study 2 consisted of 73 type 2 diabetics who had renal failure. The study 3 consisted of 230 type 1 diabetics. In total, 515 subjects were examined for the association of ALDH2 genotype with inheritance of diabetes. Out of 515 subjects, 307 (60%) had active ALDH2 (ALDH2*1/ALDH2*1) and 208 (40%) had inactive ALDH2 (175 had ALDH2*1/ALDH2*2 and 33 had ALDH2*2/ALDH2*2). As for family history, 25 subjects (8.1%) in the active ALDH2 group had a diabetic mother, compared with 43 (20.6%) in the inactive ALDH2 group. Twenty-nine subjects (9.4%) in the active ALDH2 group had a diabetic father, compared with 14 (6.7%) in the inactive ALDH2 group. The percentage of diabetic mother was higher in the inactive ALDH2 group, the differences were statistically significant (P maternal trait of diabetes inheritance. In conclusion, meta-analysis using three diabetes population studies strongly confirmed the association between ALDH2 inactivity and maternal inheritance.

  7. Substrate-Specific Reorganization of the Conformational Ensemble of CSK Implicates Novel Modes of Kinase Function

    Science.gov (United States)

    Jamros, Michael A.; Oliveira, Leandro C.; Whitford, Paul C.; Onuchic, José N.; Adams, Joseph A.; Jennings, Patricia A.

    2012-01-01

    Protein kinases use ATP as a phosphoryl donor for the posttranslational modification of signaling targets. It is generally thought that the binding of this nucleotide induces conformational changes leading to closed, more compact forms of the kinase domain that ideally orient active-site residues for efficient catalysis. The kinase domain is oftentimes flanked by additional ligand binding domains that up- or down-regulate catalytic function. C-terminal Src kinase (Csk) is a multidomain tyrosine kinase that is up-regulated by N-terminal SH2 and SH3 domains. Although the X-ray structure of Csk suggests the enzyme is compact, X-ray scattering studies indicate that the enzyme possesses both compact and open conformational forms in solution. Here, we investigated whether interactions with the ATP analog AMP-PNP and ADP can shift the conformational ensemble of Csk in solution using a combination of small angle x-ray scattering and molecular dynamics simulations. We find that binding of AMP-PNP shifts the ensemble towards more extended rather than more compact conformations. Binding of ADP further shifts the ensemble towards extended conformations, including highly extended conformations not adopted by the apo protein, nor by the AMP-PNP bound protein. These ensembles indicate that any compaction of the kinase domain induced by nucleotide binding does not extend to the overall multi-domain architecture. Instead, assembly of an ATP-bound kinase domain generates further extended forms of Csk that may have relevance for kinase scaffolding and Src regulation in the cell. PMID:23028292

  8. Revolutionary systems for catalytic combustion and diesel catalytic particulate traps.

    Energy Technology Data Exchange (ETDEWEB)

    Stuecker, John Nicholas; Witze, Peter O.; Ferrizz, Robert Matthew; Cesarano, Joseph, III; Miller, James Edward

    2004-12-01

    This report is a summary of an LDRD project completed for the development of materials and structures conducive to advancing the state of the art for catalyst supports and diesel particulate traps. An ancillary development for bio-medical bone scaffolding was also realized. Traditionally, a low-pressure drop catalyst support, such as a ceramic honeycomb monolith, is used for catalytic reactions that require high flow rates of gases at high-temperatures. A drawback to the traditional honeycomb monoliths under these operating conditions is poor mass transfer to the catalyst surface in the straight-through channels. ''Robocasting'' is a unique process developed at Sandia National Laboratories that can be used to manufacture ceramic monoliths with alternative 3-dimensional geometries, providing tortuous pathways to increase mass transfer while maintaining low-pressure drops. These alternative 3-dimensional geometries may also provide a foundation for the development of self-regenerating supports capable of trapping and combusting soot particles from a diesel engine exhaust stream. This report describes the structures developed and characterizes the improved catalytic performance that can result. The results show that, relative to honeycomb monolith supports, considerable improvement in mass transfer efficiency is observed for robocast samples synthesized using an FCC-like geometry of alternating rods. Also, there is clearly a trade-off between enhanced mass transfer and increased pressure drop, which can be optimized depending on the particular demands of a given application. Practical applications include the combustion of natural gas for power generation, production of syngas, and hydrogen reforming reactions. The robocast lattice structures also show practicality for diesel particulate trapping. Preliminary results for trapping efficiency are reported as well as the development of electrically resistive lattices that can regenerate the structure

  9. Plant antimutagenic agents, 4. Isolation and structure elucidation of maesol, an inactive constituent of Maesa spp.

    Science.gov (United States)

    Wall, M E; Wani, M C; Gaetano, K; Manikumar, G; Taylor, H; McGivney, R

    1988-01-01

    Maesol, a novel dimeric phenol, was isolated from seeds of Maesa montana and Maesa indica. Maesol was shown to have the formula C28H42O4 with structure 1, a dimeric, symmetrical 1,12-bis(3,3'-dihydroxy-4,4'-dimethyl-5,5'-dimethoxyphenyl)dodecane. It is the first compound with such structure to be isolated from plant material. Structure elucidation was based largely on 1H- and 13C-nmr techniques and comparison with a known synthetic isomeric dimer 3. Although crude extracts showed strong inhibition of 2-aminoanthracene activity against Salmonella typhimurium (T-98), the pure compound was inactive when tested for inhibition of the mutagenic activity of several mutagens.

  10. Inactive experiments for advanced separation processes prior to high activity trials in ATALANTE

    Energy Technology Data Exchange (ETDEWEB)

    Duhamet, Jean; Lanoe, Jean-Yves; Rivalier, Patrick; Borda, Gilles [Commissariat a l' Energie Atomique (CEA), CEA/DEN/VRH/DTEC/SGCS, Centre de Marcoule - BP 17171, 302007 Bagnols-sur-Ceze cedex (France)

    2008-07-01

    Many trials have been performed in ATALANTE's shielded cells to demonstrate the technical feasibility of processes involving minor actinide separation. They required developments of new extractors as well as a step by step procedure have been used to lower the risks of malfunction during high active operation. The design of the extractors developed by Cea has included shielded cells restrictions, miniaturization to lower the quantity of high active material and wastes and the care for being representative of industrial equipment. After individual shake down inactive tests, with actual phases, each process experiment scheduled in ATALANTE has been tested at G1 Facility in Marcoule. The objective was to reproduce as much as possible all the equipment chosen for active tests. This procedure has demonstrated its efficiency to detect many problems that would have heavy impact if they have been discovered during active trials. It was also used for operators'training. (authors)

  11. Physical inactivity as a risk factor for diabetic retinopathy? A review.

    Science.gov (United States)

    Dirani, Mohamed; Crowston, Jonathan G; van Wijngaarden, Peter

    2014-08-01

    Physical inactivity and sedentary behaviour have been identified as modifiable risk factors for diabetes. However, little is known of the associations between physical activity, sedentary behaviour and diabetic retinopathy. The development of diabetic retinopathy is associated with longer duration of diabetes, elevated blood pressure and poor glycaemic control. However these factors only explain a proportion of the risk of retinopathy in individuals with diabetes. Several studies have suggested a protective role for physical activity in diabetic retinopathy. Other work has shown that the time spent watching television is independently associated with abnormal retinal vascular signs. Limitations of the existing studies, such as the absence of objective measures of physical activity, a lack of sedentary behaviour measures, the inclusion of only those with type 1 diabetes and a lack of longitudinal data, make it difficult to draw firm conclusions about the strength of these associations.

  12. Groundwater contamination from an inactive uranium mill tailings pile: 1. Application of a chemical mixing model

    Science.gov (United States)

    White, A. F.; Delany, J. M.; Narasimhan, T. N.; Smith, A.

    1984-11-01

    Low-pH process waters contained in a number of inactive and abandoned uranium mill tailings in the United States represent potential sources of radionuclide and trace metal contamination of groundwater. Detailed investigations at a typical site at Riverton, Wyoming, indicate that chemical transport occurs from initial dewatering of the tailings, downward infiltration due to precipitation, and groundwater intrusion into the base of the tailings pile. Except for elevated uranium and molybdenum concentrations, current radionuclide and trace metal transport is limited by the near-neutral pH conditions of the groundwater. Significant reactions include the dissolution of calcite, production of CO2, and precipitation of gypsum and the hydroxides of iron and aluminum. A geochemical mixing model employing the PHREEQE computer code is used to estimate current rates of the groundwater contamination by tailings water. A maximum mixing of 1.7% of pore water is a factor of 2 less than steady state estimates based on hydraulic parameters.

  13. Efficient production of transgenic chickens using self-inactive HIV-based lentiviral vectors

    Institute of Scientific and Technical Information of China (English)

    Shiyong XU; Yan SUN; Hongmei DING; Meng WANG; Yafei CAI; Jie CHEN; Honglin LIU

    2009-01-01

    We demonstrated the simple and effective production of transgenic chickens, in which the enhanced green fluorescence protein (EGFP) was expressed by using third-generation self-inactive HIV-based lentiviral vectors. In our experiments, lentiviruses were injected into 204 fertilized eggs, from which 30 ( 15% ) chickens were hatched. The exogenous gene was detected in the genomes of 16 out of 30 (53%) chickens. The green fluorescence signal was observed directly in various body parts, and was particularly significant in the testes. The transgenes were also found in the offspring of these chickens. The results indicate that HIV-based lentivirul vectors can be used to generate transgenic birds economically and effectively [Current Zoology 55 (5): 383 - 387,2009].

  14. Revegetation/rock cover for stabilization of inactive uranium mill tailings disposal sites

    Energy Technology Data Exchange (ETDEWEB)

    Beedlow, P.A.; McShane, M.C.; Cadwell, L.L.

    1982-07-01

    Pacific Northwest Laboratory is developing design and performance guidelines for surface stabilization of inactive uranium mill tailings. In this work, vegetation and rock covers are being evaluated for maintaining long-term integrity of impoundment systems. Methods are being developed to estimate erosion rates associated with rock and/or vegetation covers, and to determine the effects of surface treatments on soil moisture. Interactions between surface treatments and barriers (radon and biological) are being studied as well. The product will be a set of guidelines to aid in designing surface covers. This report presents the status of this program and a discussion of considerations pertinent to the application of surface covers to tailings. Test plots located in Grand Junction, Colorado and Waterflow, New Mexico are being used to study: (1) the interactions between vegetation and radon and biological barriers, (2) the effects of surface covers on soil moisture, and (3) the effects of rock covers on vegetation.

  15. Inactive dry yeast application on grapes modify Sauvignon Blanc wine aroma.

    Science.gov (United States)

    Šuklje, Katja; Antalick, Guillaume; Buica, Astrid; Coetzee, Zelmari A; Brand, Jeanne; Schmidtke, Leigh M; Vivier, Melané A

    2016-04-15

    This study investigated the potential to improve wine aroma by applying two inactive dry yeast products (IDYs) at the onset of ripening on Sauvignon Blanc grapes. Both products led to increased reduced glutathione concentrations in the grape juice and corresponding wines, as well as differences in individual higher alcohol acetates (HAAs) and ethyl esters of straight chain fatty acids (EEFAs) at the end of fermentation. After two months of storage, a significantly slower decrease of EEFAs and to a lesser extent of HAAs was found for wines made from grapes with IDY applications. These wines also resulted in significantly slower synthesis of ethyl esters of branched acids, whereas varietal thiols were altered in a product-specific manner. The modifications in the wine chemical composition were also sensorially corroborated. This study showed that vineyard additions of IDY products directly on the grapes at the onset of ripening have a subsequent benefit to the production and preservation of aroma in wines.

  16. Short term aerobic exercise alters the reinforcing value of food in inactive adults.

    Science.gov (United States)

    Panek, Leah M; Jones, Kelly R; Temple, Jennifer L

    2014-10-01

    Motivation to eat, or the reinforcing value of food, may be influenced by a number of factors, including physical activity. The purpose of these studies was to test the hypothesis that short-term moderate-vigorous intensity aerobic exercise would alter the reinforcing value of high (HED) and low (LED) energy density foods in inactive adults. The reinforcing value of LED and HED food was measured at baseline and again after two weeks of aerobic exercise. In Experiment 1, 41 participants were randomized to a no exercise condition or aerobic exercise for 3 days per week for two weeks. In Experiment 2, 76 participants were randomized to one of four aerobic exercise frequencies, 0, 1, 3, or 5 days per week for two weeks. In both experiments, exercise reduced the reinforcing value of HED food compared to baseline and to non-exercise controls. In Experiment 2, the 5 day group also showed a significant increase in the reinforcing value of LED food compared to baseline and other exercise frequencies. Liking of HED and LED foods and consumption of HED food were not affected by exercise treatment. Finally, in Experiment 2, the 5 day group reported consuming more energy outside of the laboratory than the other groups. Taken together, these data suggest, in inactive individuals, motivation to obtain HED and LED foods can be altered with a short-term moderate-vigorous intensity exercise intervention. Further research is needed to understand the cognitive and physiological processes involved in food choices paired with exercise.

  17. Maternal inflammation during late pregnancy is lower in physically active compared with inactive obese women.

    Science.gov (United States)

    Tinius, Rachel A; Cahill, Alison G; Strand, Eric A; Cade, W Todd

    2016-02-01

    The primary purpose of this study was to compare maternal plasma inflammation between physically active and inactive obese women during late pregnancy. The secondary purpose was to examine the relationships between maternal plasma inflammation and lipid metabolism and maternal and neonatal metabolic health in these women. A cross-sectional, observational study design was performed in 16 obese-inactive (OBI; means ± SD; age, 25.0 ± 4.8 years; prepregnancy body mass index (BMI), 36.3 ± 4.3 kg/m(2); body fat percentage in late gestation, 37.7% ± 3.5%) and 16 obese-active (OBA; age, 28.9 ± 4.8 years; prepregnancy BMI, 34.0 ± 3.7 kg/m(2); body fat in late gestation, 36.6% ± 3.8%) women during the third trimester of pregnancy. Maternal plasma inflammation (C -reactive protein (CRP)) and insulin resistance (Homeostatic Model Assessment-Insulin Resistance) were measured at rest. Plasma lipid concentration and metabolism (lipid oxidation and lipolysis) were measured at rest, during a 30-min bout of low-intensity (40% peak oxygen uptake) exercise, and during a resting recovery period using indirect calorimetry. Umbilical cord blood was collected for measurement of neonatal plasma insulin resistance, inflammation, and lipid concentration. Neonatal body composition was measured via air displacement plethysmography. Maternal plasma CRP concentration was significantly higher in OBI compared with OBA women (9.1 ± 4.0 mg/L vs. 6.3 ± 2.5 mg/L, p = 0.02). Maternal plasma CRP concentration was significantly associated with maternal lipolysis (r = 0.43, p = 0.02), baseline lipid oxidation rate (r = 0.39, p = 0.03), and baseline plasma free fatty acid concentration (r = 0.36, p = 0.04). In conclusion, maternal physical activity may reduce inflammation during pregnancy in obese women. Maternal lipid metabolism is related to systemic inflammation.

  18. Survey of active and inactive mines for possible use as in situ test facilities

    Energy Technology Data Exchange (ETDEWEB)

    1976-09-01

    A survey of active and inactive mines which might be useful for radioactive waste storage in situ test experiments was conducted. It was performed for Union Carbide Corporation, Nuclear Division, Office of Waste Isolation. The report covers available information gathered from literature, U.S. Bureau of Mines, the Mining Enforcement and Safety Agency, and a limited number of personal contacts with constructors or operators of facilities. This survey is preliminary in nature and the objective is to develop potential candidate facilities for in situ experiments which warrant further investigation. Included are descriptions of 244 facilities, with all the data about each one which was available within the time restraint of the study. These facility descriptions are additionally indexed by depth of mine, nature of the country rock, mineral mined, and type of entry. A total of 14 inactive mines and 34 active mines has been selected as those most worthy of further investigation for possible service as nuclear waste isolation test facilities. This investigation, being preliminary and having been performed in a very short time period, must be qualified, and the description of the qualification is presented in the body of this report. Qualifications deal primarily with the hazard of having omitted facilities and having incomplete data in some instances. Results indicate sedimentary rock mines of minerals of evaporite origin as a first ranking of preference for in situ testing, followed by other sedimentary rocks and then by mines producing minerals from any type rock where the mine is above the local water table. These are general rules and of course there can be exceptions to them.

  19. A method of active conformation search based on active and inactive analogues, and its application to allylamine antimycotics

    Institute of Scientific and Technical Information of China (English)

    张万年; 季海涛; 周有骏; 朱杰; 朱驹; 吕加国

    1999-01-01

    A new program ACSBAIA (Active Conformation Search Based on Active and Inactive Analogues) for determination of the active conformations was developed based on the rationales that specific functional groups of active analogues could reach and interact with the active site of target receptor by means of the change of conformations, but that of inactive analogues could not interact with the active site owing to conformational restriction. The program consisted of 4 sub-programs: conformation sampling system, active conformation constraint system, inactive conformation exclusion system, and activity prediction system. Pharmacophoric conformation of allylamine antimycotics was studied by this method. Activities of 2 analogues were predicted and tested. The results suggested that the method was scientific and practical. The application of this method was not restricted by the three-dimensional structural knowledge of target receptor. In the absence of structural information about the receptor, the method was

  20. Functional ability at age 75: is there an impact of physical inactivity from middle age to early old age?

    DEFF Research Database (Denmark)

    Christensen, Ulla; Støvring, N; Schultz-Larsen, K

    2006-01-01

    The aim of this study is to analyze the impact of physical inactivity from middle age to early old age on functional ability at age 75. Physical activity is measured both as cumulated activity from age 50 to 60 to 70 and at three separate points in time. Three hundred eighty-seven men and women......) and smoking, sex, school education, household composition, chronic disease at baseline and functional ability at age 70 as possible confounders. There was a strong association between physical inactivity at age 70 and disability at age 75. However, the analyses showed no effect of cumulated physical...... born in 1914 and living in seven municipalities in the western part of the County of Copenhagen were followed for 25 years with examinations in 1964, 1974, 1984 and 1989. Analyses were conducted with physical inactivity as an independent variable (accumulated and separately for each point in time...

  1. Topological entropy of catalytic sets: Hypercycles revisited

    Science.gov (United States)

    Sardanyés, Josep; Duarte, Jorge; Januário, Cristina; Martins, Nuno

    2012-02-01

    The dynamics of catalytic networks have been widely studied over the last decades because of their implications in several fields like prebiotic evolution, virology, neural networks, immunology or ecology. One of the most studied mathematical bodies for catalytic networks was initially formulated in the context of prebiotic evolution, by means of the hypercycle theory. The hypercycle is a set of self-replicating species able to catalyze other replicator species within a cyclic architecture. Hypercyclic organization might arise from a quasispecies as a way to increase the informational containt surpassing the so-called error threshold. The catalytic coupling between replicators makes all the species to behave like a single and coherent evolutionary multimolecular unit. The inherent nonlinearities of catalytic interactions are responsible for the emergence of several types of dynamics, among them, chaos. In this article we begin with a brief review of the hypercycle theory focusing on its evolutionary implications as well as on different dynamics associated to different types of small catalytic networks. Then we study the properties of chaotic hypercycles with error-prone replication with symbolic dynamics theory, characterizing, by means of the theory of topological Markov chains, the topological entropy and the periods of the orbits of unimodal-like iterated maps obtained from the strange attractor. We will focus our study on some key parameters responsible for the structure of the catalytic network: mutation rates, autocatalytic and cross-catalytic interactions.

  2. The allosteric regulation of pyruvate kinase.

    Science.gov (United States)

    Valentini, G; Chiarelli, L; Fortin, R; Speranza, M L; Galizzi, A; Mattevi, A

    2000-06-16

    Pyruvate kinase (PK) is critical for the regulation of the glycolytic pathway. The regulatory properties of Escherichia coli were investigated by mutating six charged residues involved in interdomain salt bridges (Arg(271), Arg(292), Asp(297), and Lys(413)) and in the binding of the allosteric activator (Lys(382) and Arg(431)). Arg(271) and Lys(413) are located at the interface between A and C domains within one subunit. The R271L and K413Q mutant enzymes exhibit altered kinetic properties. In K413Q, there is partial enzyme activation, whereas R271L is characterized by a bias toward the T-state in the allosteric equilibrium. In the T-state, Arg(292) and Asp(297) form an intersubunit salt bridge. The mutants R292D and D297R are totally inactive. The crystal structure of R292D reveals that the mutant enzyme retains the T-state quaternary structure. However, the mutation induces a reorganization of the interface with the creation of a network of interactions similar to that observed in the crystal structures of R-state yeast and M1 PK proteins. Furthermore, in the R292D structure, two loops that are part of the active site are disordered. The K382Q and R431E mutations were designed to probe the binding site for fructose 1, 6-bisphosphate, the allosteric activator. R431E exhibits only slight changes in the regulatory properties. Conversely, K382Q displays a highly altered responsiveness to the activator, suggesting that Lys(382) is involved in both activator binding and allosteric transition mechanism. Taken together, these results support the notion that domain interfaces are critical for the allosteric transition. They couple changes in the tertiary and quaternary structures to alterations in the geometry of the fructose 1, 6-bisphosphate and substrate binding sites. These site-directed mutagenesis data are discussed in the light of the molecular basis for the hereditary nonspherocytic hemolytic anemia, which is caused by mutations in human erythrocyte PK gene.

  3. The hydrophobic motif of ROCK2 requires association with the N-terminal extension for kinase activity.

    Science.gov (United States)

    Couzens, Amber L; Saridakis, Vivian; Scheid, Michael P

    2009-04-01

    ROCK (Rho-associated coiled-coil kinase) 2 is a member of the AGC kinase family that plays an essential role downstream of Rho in actin cytoskeleton assembly and contractility. The process of ROCK2 activation is complex and requires suppression of an autoinhibitory mechanism that is facilitated by Rho binding. ROCK2 harbours a C-terminal extension within the kinase domain that contains a hydrophobic cluster of phenylalanine and tyrosine residues surrounding a key threonine residue. In growth-factor-stimulated AGC kinases, the hydrophobic motif is important for the transition of the kinase from inactive to active complex and requires phosphorylation of the conserved serine/threonine residue. Less is understood about the contribution that the hydrophobic motif plays in the activation of ROCK, and the role of the hydrophobic motif threonine at position 405. In the present study, we show that this residue of ROCK is essential for substrate phosphorylation and kinase domain dimerization. However, in contrast with the growth-factor-activated AGC kinases, a phosphomimetic residue at position 405 was inhibitory for ROCK2 activity and dimerization. A soluble hydrophobic motif peptide allosterically activated ROCK2 In vitro, but not the equivalent peptide with Asp(405) substitution. Mechanistically, both ROCK2 activity and dimerization were dependent upon the interaction between Thr(405) of the hydrophobic motif and Asp(39) of the N-terminal extension. The reciprocal exchange of these residues was permissive for kinase activity, but dimerization was lost. These results support the rationale for development of small-molecule inhibitors designed to block ROCK activation by selectively interfering with hydrophobic motif-mediated activation-state transition and dimer formation.

  4. Molecular dynamics analysis of conserved hydrophobic and hydrophilic bond-interaction networks in ErbB family kinases.

    Science.gov (United States)

    Shih, Andrew J; Telesco, Shannon E; Choi, Sung-Hee; Lemmon, Mark A; Radhakrishnan, Ravi

    2011-06-01

    The EGFR (epidermal growth factor receptor)/ErbB/HER (human EGFR) family of kinases contains four homologous receptor tyrosine kinases that are important regulatory elements in key signalling pathways. To elucidate the atomistic mechanisms of dimerization-dependent activation in the ErbB family, we have performed molecular dynamics simulations of the intracellular kinase domains of three members of the ErbB family (those with known kinase activity), namely EGFR, ErbB2 (HER2) and ErbB4 (HER4), in different molecular contexts: monomer against dimer and wild-type against mutant. Using bioinformatics and fluctuation analyses of the molecular dynamics trajectories, we relate sequence similarities to correspondence of specific bond-interaction networks and collective dynamical modes. We find that in the active conformation of the ErbB kinases, key subdomain motions are co-ordinated through conserved hydrophilic interactions: activating bond-networks consisting of hydrogen bonds and salt bridges. The inactive conformations also demonstrate conserved bonding patterns (albeit less extensive) that sequester key residues and disrupt the activating bond network. Both conformational states have distinct hydrophobic advantages through context-specific hydrophobic interactions. We show that the functional (activating) asymmetric kinase dimer interface forces a corresponding change in the hydrophobic and hydrophilic interactions that characterize the inactivating bond network, resulting in motion of the αC-helix through allostery. Several of the clinically identified activating kinase mutations of EGFR act in a similar fashion to disrupt the inactivating bond network. The present molecular dynamics study reveals a fundamental difference in the sequence of events in EGFR activation compared with that described for the Src kinase Hck.

  5. Development of Catalytic Cooking Plates

    Energy Technology Data Exchange (ETDEWEB)

    Hjelm, Anna-Karin; Silversand, Fredrik [CATATOR AB, Lund (Sweden); Tena, Emmanuel; Berger, Marc [Gaz de France (France)

    2004-04-01

    Gas catalytic combustion for gas stoves or cooking plates (closed catalytic burner system with ceramic plates) is a very promising technique in terms of ease of cleaning, power modulation and emissions. Previous investigations show that wire mesh catalysts, prepared and supplied by Catator AB (CAT), seem to be very well suited for such applications. Beside significantly reducing the NOx-emissions, these catalysts offer important advantages such as good design flexibility, low pressure drop and high heat transfer capacity, where the latter leads to a quick thermal response. Prior to this project, Gaz de France (GdF) made a series of measurements with CAT's wire mesh catalysts in their gas cooking plates and compared the measured performance with similar results obtained with theirs cordierite monolith catalysts. Compared to the monolith catalyst, the wire mesh catalyst was found to enable very promising results with respect to both emission levels (<10 mg NO{sub x} /kWh, <5 mg CO/kWh) and life-time (>8000 h vs. 700 h at 200 kW/m{sup 2}). It was however established that the radiation and hence, the thermal efficiency of the cooking plate, was significantly less than is usually measured in combination with the monolith (15 % vs. 32 %). It was believed that the latter could be improved by developing new burner designs based on CAT's wire mesh concept. As a consequence, a collaboration project between GdF, CAT and the Swedish Gas Technology AB was created. This study reports on the design, the construction and the evaluation of new catalytic burners, based on CAT's wire mesh catalysts, used for the combustion of natural gas in gas cooking stoves. The evaluation of the burners was performed with respect to key factors such as thermal efficiency, emission quality and pressure drop, etc, by the use of theoretical simulations and experimental tests. Impacts of parameters such as the the wire mesh number, the wire mesh structure (planar or folded), the

  6. Sex differences in the physical inactivity and health-related quality of life relationship among rural adults

    Science.gov (United States)

    Hart, Peter D

    2016-01-01

    Background: The purpose of this study was to investigate the relationship between physical inactivity (PIA) and health-related quality of life (HRQOL) in rural adults and examine the extent to which sex differences exist in this relationship. Methods: A total of 5617 adults 18 years of age and older who indicated residing in a rural county was included in this analysis. PIA status was assessed by questions regarding recreational physical activity during the previous month. Five HRQOL measures (physical health, mental health, inactivity health, general health, & unhealthy days) were used as primary outcome variables. PIA and HRQOL prevalence estimates were computed with 95% CIs. Multiple logistic regression was used to obtain odds ratios (ORs) and 95% CIs adjusted for age, ethnicity, and income. Results: Physically inactive rural adults were significantly more likely to report poor HRQOL in all overall crude models with ORs ranging from 1.59 to 2.16. Additionally, sex-by-PIA interactions were significant across all crude HRQOL models with ORs ranging from 2.27 to 3.08 and 1.56 to 2.42 for women and men, respectively. Sex differences were maintained in fully adjusted models, except for mental health and inactivity health with ORs ranging from 1.80 to 2.58 and 1.41 to 1.79 for women and men, respectively. Conclusion: Results from this study show that PIA is a strong predictor of poor HRQOL even after controlling for confounding variables. Furthermore, physically inactive rural women appear more likely to report poor levels of HRQOL than physically inactive rural men.

  7. Metabolic profiles using (1)H-nuclear magnetic resonance spectroscopy in postpartum dairy cows with ovarian inactivity.

    Science.gov (United States)

    Xu, Chuchu; Xia, Cheng; Sun, Yuhang; Xiao, Xinhuan; Wang, Gang; Fan, Ziling; Shu, Shi; Zhang, Hongyou; Xu, Chuang; Yang, Wei

    2016-10-01

    To understand the differences in metabolic changes between cows with ovarian inactivity and estrus cows, we selected cows at 60-90 days postpartum from an intensive dairy farm. According to clinical manifestations, B-ultrasound scan, rectal examination, 10 cows were assigned to the estrus group (A) and 10 to the ovarian inactivity group (B). All plasma samples were analyzed by (1)H-nuclear magnetic resonance spectroscopy to compare plasma metabolomic profiles between the groups. We used multivariate pattern recognition to screen for different metabolites in plasma of anestrus cows. Compared with normal estrous cows, there were abnormalities in 12 kinds of metabolites in postpartum cows with ovarian inactivity (|r|> 0.602), including an increase in acetic acid (r = -0.817), citric acid (r = -0.767), and tyrosine (r = -0.714), and a decrease in low-density lipoprotein (r = 0.820), very low-density lipoprotein (r = 0.828), lipids (r = 0.769), alanine (r = 0.816), pyruvate (r = 0.721), creatine (r = 0.801), choline (r = 0.639), phosphorylcholine (r = 0.741), and glycerophosphorylcholine (r = 0.881). These metabolites were closely related to abnormality of glucose, amino acid, lipoprotein and choline metabolism, which may disturb the normal estrus. The decrease in plasma creatine and the increase in tyrosine were new changes for ovarian inactivity of postpartum cows. The decrease in plasma creatine and choline and the increase in tyrosine and p-hydroxyphenylalanine in cows with ovarian inactivity provide new directions for research on the mechanism of ovarian inactivity in cows.

  8. Sex differences in the physical inactivity and health-related quality of life relationship among rural adults

    Directory of Open Access Journals (Sweden)

    Peter D Hart

    2016-10-01

    Full Text Available Background: The purpose of this study was to investigate the relationship between physical inactivity (PIA and health-related quality of life (HRQOL in rural adults and examine the extent to which sex differences exist in this relationship. Methods: A total of 5617 adults 18 years of age and older who indicated residing in a rural county was included in this analysis. PIA status was assessed by questions regarding recreational physical activity during the previous month. Five HRQOL measures (physical health, mental health, inactivity health, general health, & unhealthy days were used as primary outcome variables. PIA and HRQOL prevalence estimates were computed with 95% CIs. Multiple logistic regression was used to obtain odds ratios (ORs and 95% CIs adjusted for age, ethnicity, and income. Results: Physically inactive rural adults were significantly more likely to report poor HRQOL in all overall crude models with ORs ranging from 1.59 to 2.16. Additionally, sex-by-PIA interactions were significant across all crude HRQOL models with ORs ranging from 2.27 to 3.08 and 1.56 to 2.42 for women and men, respectively. Sex differences were maintained in fully adjusted models, except for mental health and inactivity health with ORs ranging from 1.80 to 2.58 and 1.41 to 1.79 for women and men, respectively. Conclusion: Results from this study show that PIA is a strong predictor of poor HRQOL even after controlling for confounding variables. Furthermore, physically inactive rural women appear more likely to report poor levels of HRQOL than physically inactive rural men.

  9. Catalytic Wittig and aza-Wittig reactions

    Directory of Open Access Journals (Sweden)

    Zhiqi Lao

    2016-11-01

    Full Text Available This review surveys the literature regarding the development of catalytic versions of the Wittig and aza-Wittig reactions. The first section summarizes how arsenic and tellurium-based catalytic Wittig-type reaction systems were developed first due to the relatively easy reduction of the oxides involved. This is followed by a presentation of the current state of the art regarding phosphine-catalyzed Wittig reactions. The second section covers the field of related catalytic aza-Wittig reactions that are catalyzed by both phosphine oxides and phosphines.

  10. The diseasome of physical inactivity--and the role of myokines in muscle--fat cross talk

    DEFF Research Database (Denmark)

    Pedersen, Bente K

    2009-01-01

    Type 2 diabetes, cardiovascular diseases, colon cancer, breast cancer, dementia and depression constitute a cluster of diseases, which defines 'a diseasome of physical inactivity'. Both physical inactivity and abdominal adiposity, reflecting accumulation of visceral fat mass, are associated...... of exercise. The finding that muscles produce and release myokines provides a conceptual basis to understand the mechanisms whereby exercise influences metabolism and exerts anti-inflammatory effects. According to our theory, contracting skeletal muscles release myokines, which work in a hormone-like fashion......, exerting specific endocrine effects on visceral fat. Other myokines work locally within the muscle via paracrine mechanisms, exerting their effects on signalling pathways involved in fat oxidation....

  11. Essential role of PSM/SH2-B variants in insulin receptor catalytic activation and the resulting cellular responses.

    Science.gov (United States)

    Zhang, Manchao; Deng, Youping; Tandon, Ruchi; Bai, Cheng; Riedel, Heimo

    2008-01-01

    The positive regulatory role of PSM/SH2-B downstream of various mitogenic receptor tyrosine kinases or gene disruption experiments in mice support a role of PSM in the regulation of insulin action. Here, four alternative PSM splice variants and individual functional domains were compared for their role in the regulation of specific metabolic insulin responses. We found that individual PSM variants in 3T3-L1 adipocytes potentiated insulin-mediated glucose and amino acid transport, glycogenesis, lipogenesis, and key components in the metabolic insulin response including p70 S6 kinase, glycogen synthase, glycogen synthase kinase 3 (GSK3), Akt, Cbl, and IRS-1. Highest activity was consistently observed for PSM alpha, followed by beta, delta, and gamma with decreasing activity. In contrast, dominant-negative peptide mimetics of the PSM Pro-rich, pleckstrin homology (PH), or src homology 2 (SH2) domains inhibited any tested insulin response. Potentiation of the insulin response originated at the insulin receptor (IR) kinase level by PSM variant-specific regulation of the Km (ATP) whereas the Vmax remained unaffected. IR catalytic activation was inhibited by peptide mimetics of the PSM SH2 or dimerization domain (DD). Either peptide should disrupt the complex of a PSM dimer linked to IR via SH2 domains as proposed for PSM activation of tyrosine kinase JAK2. Either peptide abolished downstream insulin responses indistinguishable from PSM siRNA knockdown. Our results implicate an essential role of the PSM variants in the activation of the IR kinase and the resulting metabolic insulin response. PSM variants act as internal IR ligands that in addition to potentiating the insulin response stimulate IR catalytic activation even in the absence of insulin.

  12. Cystic fibrosis transmembrane regulator fragments with the Phe508 deletion exert a dual allosteric control over the master kinase CK2

    Science.gov (United States)

    Pagano, Mario A.; Marin, Oriano; Cozza, Giorgio; Sarno, Stefania; Meggio, Flavio; Treharne, Kate J.; Mehta, Anil; Pinna, Lorenzo A.

    2011-01-01

    Cystic fibrosis mostly follows a single Phe508 deletion in CFTR (cystic fibrosis transmembrane regulator) (CFTRΔF508), thereby causing premature fragmentation of the nascent protein with concomitant alterations of diverse cellular functions. We show that CK2, the most pleiotropic protein kinase, undergoes allosteric control of its different cellular forms in the presence of short CFTR peptides encompassing the Phe508 deletion: these CFTRΔF508 peptides drastically inhibit the isolated catalytic subunit (α) of the kinase and yet up-regulate the holoenzyme, composed of two catalytic and two non-catalytic (β) subunits. Remarkable agreement between in silico docking and our biochemical data point to different sites for the CFTRΔF508 peptide binding on isolated CK2α and on CK2β assembled into the holoenzyme, suggesting that CK2 targeting may be perturbed in cells expressing CFTRΔF508; this could shed light on some pleiotropic aspects of cystic fibrosis disease. PMID:19925455

  13. [Degradation of beta-naphthol by catalytic wet air oxidation].

    Science.gov (United States)

    Liu, Jie; Yu, Chao-Ying; Zhao, Pei-Qing; Chen, Ge-Xin

    2012-11-01

    A series of MnO(x)/nano-TiO2 catalysts were prepared and their application in degradation of beta-naphthol by catalytic wet air oxidation (CWAO) was investigated. The catalysts preparation conditions, reaction conditions and its stability were tested. The catalysts had been characterized by X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and temperature-programmed reduction (TPR) measurements. The results showed that the decrease of the COD removal for the degradation of beta-naphthol at high Mn loading was due to the aggregation of the highly dispersed Mn species and the formation of the correlated crystals. The decline of the COD removal at high calcination temperature was probably attributed to the weak electron transfer between Mn2O3 and MnO2 and the formation of the inactive Mn2O3. The COD removal had been falling slightly when the catalyst was used 6 times, and this was likely related to the decrease of the diffraction peaks. The catalyst had a high activity when the Mn loading (mass fraction) was 4% and the calcination temperature was 450 degrees C. The COD removal was up to 96.4% at 110 degrees C and 0.5 MPa with this catalyst. The COD removal of 92.4% could be obtained with the MnO(x)/nano-TiO2 catalyst was recycled 6 times. The Mn leaching at 50, 80, 110 and 150 degrees C were all less than 9.3 mg x L(-1) by means of Atomic Absorption Spectroscopy (AAS). The probable degradation pathway was proposed according to some publications.

  14. Catalytic Chemistry on Oxide Nanostructures

    Energy Technology Data Exchange (ETDEWEB)

    Asthagiri, Aravind; Dixon, David A.; Dohnalek, Zdenek; Kay, Bruce D.; Rodriquez, Jose A.; Rousseau, Roger J.; Stacchiola, Dario; Weaver, Jason F.

    2016-05-29

    Metal oxides represent one of the most important and widely employed materials in catalysis. Extreme variability of their chemistry provides a unique opportunity to tune their properties and to utilize them for the design of highly active and selective catalysts. For bulk oxides, this can be achieved by varying their stoichiometry, phase, exposed surface facets, defect, dopant densities and numerous other ways. Further, distinct properties from those of bulk oxides can be attained by restricting the oxide dimensionality and preparing them in the form of ultrathin films and nanoclusters as discussed throughout this book. In this chapter we focus on demonstrating such unique catalytic properties brought by the oxide nanoscaling. In the highlighted studies planar models are carefully designed to achieve minimal dispersion of structural motifs and to attain detailed mechanistic understanding of targeted chemical transformations. Detailed level of morphological and structural characterization necessary to achieve this goal is accomplished by employing both high-resolution imaging via scanning probe methods and ensemble-averaged surface sensitive spectroscopic methods. Three prototypical examples illustrating different properties of nanoscaled oxides in different classes of reactions are selected.

  15. Structural insight into selectivity and resistance profiles of ROS1 tyrosine kinase inhibitors.

    Science.gov (United States)

    Davare, Monika A; Vellore, Nadeem A; Wagner, Jacob P; Eide, Christopher A; Goodman, James R; Drilon, Alexander; Deininger, Michael W; O'Hare, Thomas; Druker, Brian J

    2015-09-29

    Oncogenic ROS1 fusion proteins are molecular drivers in multiple malignancies, including a subset of non-small cell lung cancer (NSCLC). The phylogenetic proximity of the ROS1 and anaplastic lymphoma kinase (ALK) catalytic domains led to the clinical repurposing of the Food and Drug Administration (FDA)-approved ALK inhibitor crizotinib as a ROS1 inhibitor. Despite the antitumor activity of crizotinib observed in both ROS1- and ALK-rearranged NSCLC patients, resistance due to acquisition of ROS1 or ALK kinase domain mutations has been observed clinically, spurring the development of second-generation inhibitors. Here, we profile the sensitivity and selectivity of seven ROS1 and/or ALK inhibitors at various levels of clinical development. In contrast to crizotinib's dual ROS1/ALK activity, cabozantinib (XL-184) and its structural analog foretinib (XL-880) demonstrate a striking selectivity for ROS1 over ALK. Molecular dynamics simulation studies reveal structural features that distinguish the ROS1 and ALK kinase domains and contribute to differences in binding site and kinase selectivity of the inhibitors tested. Cell-based resistance profiling studies demonstrate that the ROS1-selective inhibitors retain efficacy against the recently reported CD74-ROS1(G2032R) mutant whereas the dual ROS1/ALK inhibitors are ineffective. Taken together, inhibitor profiling and stringent characterization of the structure-function differences between the ROS1 and ALK kinase domains will facilitate future rational drug design for ROS1- and ALK-driven NSCLC and other malignancies.

  16. Critical role for hematopoietic cell kinase (Hck)-mediated phosphorylation of Gab1 and Gab2 docking proteins in interleukin 6-induced proliferation and survival of multiple myeloma cells.

    Science.gov (United States)

    Podar, Klaus; Mostoslavsky, Gustavo; Sattler, Martin; Tai, Yu-Tzu; Hayashi, Toshiaki; Catley, Laurence P; Hideshima, Teru; Mulligan, Richard C; Chauhan, Dharminder; Anderson, Kenneth C

    2004-05-14

    Interleukin-6 (LI-6) is a known growth and survival factor in multiple myeloma via activation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling cascade. In this report we show that Grb2-associated binder (Gab) family adapter proteins Gab1 and Gab2 are expressed by multiple myeloma cells; and that interleukin-6 induces their tyrosine phosphorylation and association with downstream signaling molecules. We further demonstrate that these events are Src family tyrosine kinase-dependent and specifically identify the role of hematopoietic cell kinase (Hck) as a new Gab family adapter protein kinase. Conversely, inhibition of Src family tyrosine kinases by the pyrazolopyrimidine PP2, as in kinase-inactive Hck mutants, significantly reduces IL-6-triggered activation of extracellular signal-regulated kinase and AKT-1, leading to significant reduction of multiple myeloma cell proliferation and survival. Taken together, these results delineate a key role for Hck-mediated phosphorylation of Gab1 and Gab2 docking proteins in IL-6-induced proliferation and survival of multiple myeloma cells and identify tyrosine kinases and downstream adapter proteins as potential new therapeutic targets in multiple myeloma.

  17. Reciprocal regulation of protein kinase and pyruvate kinase activities of pyruvate kinase M2 by growth signals.

    Science.gov (United States)

    Gao, Xueliang; Wang, Haizhen; Yang, Jenny J; Chen, Jing; Jie, Jiang; Li, Liangwei; Zhang, Yinwei; Liu, Zhi-Ren

    2013-05-31

    Pyruvate kinase isoform M2 (PKM2) is an enzyme-catalyzing conversion of phosphoenolpyruvate to pyruvate in the glycolysis pathway. It was demonstrated that PKM2 interacts with tyrosine phosphopeptide, and the interaction with the tyrosine phosphopeptide affects the pyruvate kinase activity of PKM2. Our experiments suggest that PKM2 is also an active protein kinase (Gao, X., Wang, H., Yang, J. J., Liu, X., and Liu, Z. R. (2012) Mol. Cell 45, 598-609). We report here that growth signals reciprocally regulate the pyruvate kinase and protein kinase activities of PKM2 by different mechanisms. On the one hand, growth signals induce protein tyrosine phosphorylations. The tyrosine-phosphorylated protein(s) regulates the conversion of pyruvate kinase and protein kinase of PKM2 by directly interacting with PKM2. Binding of the tyrosyl-phosphorylated proteins at the fructose 1,6-bisphosphate-binding site converts the tetrameric PKM2 to a dimer. On the other hand, growth stimulations also lead to PKM2 phosphorylation, which consequently regulates the conversion of protein kinase and pyruvate kinase activities. Growth factor stimulations significantly increase the dimer/tetramer PKM2 ratio in cells and consequently activate the protein kinase activity of PKM2. Our study suggests that the conversion between the pyruvate kinase and protein kinase activities of PKM2 may be an important mechanism mediating the effects of growth signals in promoting cell proliferation.

  18. Anticancer Alkaloid Lamellarins Inhibit Protein Kinases

    Directory of Open Access Journals (Sweden)

    Laurent Meijer

    2008-10-01

    Full Text Available Lamellarins, a family of hexacyclic pyrrole alkaloids originally isolated from marine invertebrates, display promising anti-tumor activity. They induce apoptotic cell death through multi-target mechanisms, including inhibition of topoisomerase I, interaction with DNA and direct effects on mitochondria. We here report that lamellarins inhibit several protein kinases relevant to cancer such as cyclin-dependent kinases, dualspecificity tyrosine phosphorylation activated kinase 1A, casein kinase 1, glycogen synthase kinase-3 and PIM-1. A good correlation is observed between the effects of lamellarins on protein kinases and their action on cell death, suggesting that inhibition of specific kinases may contribute to the cytotoxicity of lamellarins. Structure/activity relationship suggests several paths for the optimization of lamellarins as kinase inhibitors.

  19. Catalytic models developed through social work

    DEFF Research Database (Denmark)

    Jensen, Mogens

    2015-01-01

    The article develops the concept of catalytic processes in relation to social work with adolescents in an attempt to both reach a more nuanced understanding of social work and at the same time to develop the concept of catalytic processes in psychology. The social work is pedagogical treatment...... of adolescents placed in out-of-home care and is characterised using three situated cases as empirical data. Afterwards the concept of catalytic processes is briefly presented and then applied in an analysis of pedagogical treatment in the three cases. The result is a different conceptualisation of the social...... work with new possibilities of development of the work, but also suggestions for development of the concept of catalytic processes....

  20. Catalytic converters as a source of platinum

    Directory of Open Access Journals (Sweden)

    A. Fornalczyk

    2011-10-01

    Full Text Available The increase of Platinum Group Metals demand in automotive industry is connected with growing amount of cars equipped with the catalytic converters. The paper presents the review of available technologies during recycling process. The possibility of removing platinum from the used catalytic converters applying pyrometallurgical and hyrdometallurgical methods were also investigated. Metals such as Cu, Pb, Ca, Mg, Cd were used in the pyrometallurgical research (catalytic converter was melted with Cu, Pb and Ca or Mg and Cd vapours were blown through the whole carrier. In hydrometallurgical research catalytic converters was dissolved in aqua regia. Analysis of Pt contents in the carrier before and after the process was performed by means of atomic absorption spectroscopy. Obtained result were discussed.

  1. Catalytic Asymmetric Synthesis of Phosphine Boronates

    NARCIS (Netherlands)

    Hornillos, Valentin; Vila, Carlos; Otten, Edwin; Feringa, Ben L.

    2015-01-01

    The first catalytic enantioselective synthesis of ambiphilic phosphine boronate esters is presented. The asymmetric boration of ,-unsaturated phosphine oxides catalyzed by a copper bisphosphine complex affords optically active organoboronate esters that bear a vicinal phosphine oxide group in good y

  2. 5'-AMP-activated protein kinase activity and subunit expression in exercise-trained human skeletal muscle

    DEFF Research Database (Denmark)

    Nielsen, Jakob Nis; Mustard, Kirsty J.W.; Graham, Drew A.

    2002-01-01

    5'-AMP-activated protein kinase (AMPK) has been proposed to be a pivotal factor in cellular responses to both acute exercise and exercise training. To investigate whether protein levels and gene expression of catalytic (alpha(1), alpha(2)) and regulatory (beta(1), beta(2), gamma(1), gamma(2), gam...... muscle has increased alpha(1)-AMPK protein levels and blunted AMPK activation during exercise.......5'-AMP-activated protein kinase (AMPK) has been proposed to be a pivotal factor in cellular responses to both acute exercise and exercise training. To investigate whether protein levels and gene expression of catalytic (alpha(1), alpha(2)) and regulatory (beta(1), beta(2), gamma(1), gamma(2), gamma......(3)) AMPK subunits and exercise-induced AMPK activity are influenced by exercise training status, muscle biopsies were obtained from seven endurance exercise-trained and seven sedentary young healthy men. The alpha(1)- and alpha(2)-AMPK mRNA contents in trained subjects were both 117 +/- 2...

  3. Inhibitors of protein kinase C

    Institute of Scientific and Technical Information of China (English)

    LIU Shiying; JIANG Yuyang; CAO Jian; LIU Feng; MA Li; ZHAO Yufen

    2005-01-01

    Protein kinase catalyzes the transfer of the γ-phosphoryl group from ATP to the hydroxyl groups of protein side chains, which plays critical roles in signal transduction pathways by transmitting extracellular signals across the plasma membrane and nuclear membrane to the destination sites in the cytoplasm and the nucleus. Protein kinase C (PKC) is a superfamily of phospholipid-dependent Ser/Thr kinase. There are at least 12 isozymes in PKC family. They are distributed in different tissues and play different roles in physiological processes. On account of their concern with a variety of pathophysiologic states, such as cancer, inflammatory conditions, autoimmune disorder, and cardiac diseases, the inhibitors, which can inhibit the activity of PKC and the interaction of cytokine with receptor, and interfere signal transduction pathway, may be candidates of therapeutic drugs. Therefore, intense efforts have been made to develop specific protein kinase inhibitors as biological tools and therapeutic agents. This article reviews the recent development of some of PKC inhibitors based on their interaction with different conserved domains and different inhibition mechanisms.

  4. Deferasirox in pyruvate kinase deficiency

    OpenAIRE

    Deeren, Dries

    2008-01-01

    Deferasirox in pyruvate kinase deficiency phone: +32-51-237437 (Deeren, Dries) (Deeren, Dries) Department of Haematology, Heilig-Hartziekenhuis Roeselare-Menen vzw - Wilgenstraat 2 - B-8800 - Roeselare - BELGIUM (Deeren, Dries) BELGIUM Registration: 2008-09-10 Received: 2008-09-05 Accepted: 2008-09-10 ePublished: 2008-09-23

  5. Non-Viral Deoxyribonucleoside Kinases

    DEFF Research Database (Denmark)

    Christiansen, Louise Slot; Munch-Petersen, Birgitte; Knecht, Wolfgang

    2015-01-01

    Deoxyribonucleoside kinases (dNKs) phosphorylate deoxyribonucleosides to their corresponding monophosphate compounds. dNks also phosphorylate deoxyribonucleoside analogues that are used in the treatment of cancer or viral infections. The study of the mammalian dNKs has therefore always been of gr...

  6. Renal targeting of kinase inhibitors

    NARCIS (Netherlands)

    Dolman, M. E. M.; Fretz, M. M.; Segers, Gj. W.; Lacombe, M.; Prakash, J.; Storm, G.; Hennink, W. E.; Kok, R. J.

    2008-01-01

    Activation of proximal tubular cells by fibrotic and inflammatory mediators is an important hallmark of chronic kidney disease. We have developed a novel strategy to intervene in renal fibrosis, by means of locally delivered kinase inhibitors. Such compounds will display enhanced activity within tub

  7. Mechanisms for redox-regulation of protein kinase C

    Directory of Open Access Journals (Sweden)

    Susan F. Steinberg

    2015-06-01

    Full Text Available Protein kinase C (PKC is comprised of a family of signal-regulated enzymes that play pleiotropic roles in the control of many physiological and pathological responses. PKC isoforms are traditionally viewed as allosterically-activated enzymes that are recruited to membranes by growth factor receptor-generated lipid cofactors. An inherent assumption of this conventional model of PKC isoform activation is that PKCs act exclusively at membrane-delimited substrates and that PKC catalytic activity is an inherent property of each enzyme that is not altered by the activation process. This traditional model of PKC activation does not adequately explain the many well-documented actions of PKC enzymes in mitochondrial, nuclear, and cardiac sarcomeric (non-sarcolemmal subcellular compartments. Recent studies address this dilemma by identifying stimulus-specific differences in the mechanisms for PKC isoform activation during growth factor activation versus oxidative stress. This review discusses a number of noncanonical redox-triggered mechanisms that can alter the catalytic properties and subcellular compartmentation patterns of PKC enzymes. While some redox-activated mechanisms act at structural determinants that are common to all PKCs, the redox-dependent mechanism for PKCδ activation requires Src-dependent tyrosine phosphorylation of a unique phosphorylation motif on this enzyme and is isoform specific. Since oxidative stress contributes to pathogenesis of a wide range of clinical disorders, these stimulus specific differences in the controls and consequences of PKC activation have important implications for the design and evaluation of PKC-targeted therapeutics.

  8. Catalytic Radical Domino Reactions in Organic Synthesis

    Science.gov (United States)

    Sebren, Leanne J.; Devery, James J.; Stephenson, Corey R.J.

    2014-01-01

    Catalytic radical-based domino reactions represent important advances in synthetic organic chemistry. Their development benefits synthesis by providing atom- and step-economical methods to complex molecules. Intricate combinations of radical, cationic, anionic, oxidative/reductive, and transition metal mechanistic steps result in cyclizations, additions, fragmentations, ring-expansions, and rearrangements. This Perspective summarizes recent developments in the field of catalytic domino processes. PMID:24587964

  9. MOBILE COMPLEX FOR CATALYTIC THERMAL WASTE TREATMENT

    Directory of Open Access Journals (Sweden)

    Vedi V.E.

    2012-12-01

    Full Text Available The design and purpose of the basic units of the mobile waste processing complex “MPK” are described. Experimental data of catalytic purification of exhaust gases are presented. Experimental data on catalytic clearing of final gases of a designed mobile incinerator plant are shown. It is defined, that concentrating of parasitic bridging in waste gases of the complex are considerably smaller, rather than allowed by normative documents.

  10. Temperature Modulation of a Catalytic Gas Sensor

    OpenAIRE

    Eike Brauns; Eva Morsbach; Sebastian Kunz; Marcus Baeumer; Walter Lang

    2014-01-01

    The use of catalytic gas sensors usually offers low selectivity, only based on their different sensitivities for various gases due to their different heats of reaction. Furthermore, the identification of the gas present is not possible, which leads to possible misinterpretation of the sensor signals. The use of micro-machined catalytic gas sensors offers great advantages regarding the response time, which allows advanced analysis of the sensor response. By using temperature modulation, additi...

  11. Monoclonal Antibodies Against Xenopus Greatwall Kinase

    OpenAIRE

    WANG Ling; Fisher, Laura A.; Wahl, James K.; Peng, Aimin

    2011-01-01

    Mitosis is known to be regulated by protein kinases, including MPF, Plk1, Aurora kinases, and so on, which become active in M-phase and phosphorylate a wide range of substrates to control multiple aspects of mitotic entry, progression, and exit. Mechanistic investigations of these kinases not only provide key insights into cell cycle regulation, but also hold great promise for cancer therapy. Recent studies, largely in Xenopus, characterized a new mitotic kinase named Greatwall (Gwl) that pla...

  12. Radium-226 in vegetation and substrates at inactive uranium mill sites

    Energy Technology Data Exchange (ETDEWEB)

    Marple, M.L.

    1980-01-01

    Results of a study of the content of radium-226 in plants growing on inactive uranium mill tailings sites in the Four Corners Region of the southwestern United States and in plants grown under greenhouse conditions with minimal surficial contamination are reported. Field plant samples and associated substrates were analyzed from two carbonate tailings sites in the Grants Mineral Belt of New Mexico. Radium activities in air-cleaned samples ranged from 5 to 368 pCi/g (dry weight) depending on species and location: activities in plants growing on local soils averaged 1.0 pCi/g. The talings and local soils contain 140 to 1400 pCi/g and 2.1 pCi/g, respectively. An evaluation of cleaning methods on selected samples showed that from 17 to 79% of the radium activity measured in air-cleaned samples was due to surficial contamination, which varied with species and location. A survey of 18 inactive uranium mill sites in the Four Corners Region was performed. Radium activity in plant tissues from nine species ranged from 2 to 210 pCi/g on bare tailings and from 0.3 to 30 pCi/g on covered tailings The radium content in most of the soil overburdens on the covered tailings piles was 10 to 17 pCi/g. An experiment was performed to measure radium-226 uptake by two species grown on tailings covered with a shallow (5 cm) soil layer. A grass, Sporobolus airoides (alkali sacaton) and a shrub, Atriplex canescens (four-wing saltbush), were studied. The tailings were a mixture of sands and slimes from a carbonate pile. The tailings treatments were plants grown in a soil cover over tailings; the controls were plants grown only in soil. Three soil types, dune sand, clay loam, and loam, were used. The radium activity of the plant tissue from the tailings treatment compared to that of the appropriate control was 1 to 19 times greater for the grass and 4 to 27 times greater for the shrub.

  13. The crystal structure of the catalytic domain of a eukaryotic guanylate cyclase

    Directory of Open Access Journals (Sweden)

    Marletta Michael A

    2008-10-01

    Full Text Available Abstract Background Soluble guanylate cyclases generate cyclic GMP when bound to nitric oxide, thereby linking nitric oxide levels to the control of processes such as vascular homeostasis and neurotransmission. The guanylate cyclase catalytic module, for which no structure has been determined at present, is a class III nucleotide cyclase domain that is also found in mammalian membrane-bound guanylate and adenylate cyclases. Results We have determined the crystal structure of the catalytic domain of a soluble guanylate cyclase from the green algae Chlamydomonas reinhardtii at 2.55 Å resolution, and show that it is a dimeric molecule. Conclusion Comparison of the structure of the guanylate cyclase domain with the known structures of adenylate cyclases confirms the close similarity in architecture between these two enzymes, as expected from their sequence similarity. The comparison also suggests that the crystallized guanylate cyclase is in an inactive conformation, and the structure provides indications as to how activation might occur. We demonstrate that the two active sites in the dimer exhibit positive cooperativity, with a Hill coefficient of ~1.5. Positive cooperativity has also been observed in the homodimeric mammalian membrane-bound guanylate cyclases. The structure described here provides a reliable model for functional analysis of mammalian guanylate cyclases, which are closely related in sequence.

  14. Structure of the catalytic domain of the hepatitis C virus NS2-3 protease

    Energy Technology Data Exchange (ETDEWEB)

    Lorenz,I.; Marcotrigiano, J.; Dentzer, T.; Rice, C.

    2006-01-01

    Hepatitis C virus is a major global health problem affecting an estimated 170 million people worldwide. Chronic infection is common and can lead to cirrhosis and liver cancer. There is no vaccine available and current therapies have met with limited success. The viral RNA genome encodes a polyprotein that includes two proteases essential for virus replication. The NS2-3 protease mediates a single cleavage at the NS2/NS3 junction, whereas the NS3-4A protease cleaves at four downstream sites in the polyprotein. NS3-4A is characterized as a serine protease with a chymotrypsin-like fold, but the enzymatic mechanism of the NS2-3 protease remains unresolved. Here we report the crystal structure of the catalytic domain of the NS2-3 protease at 2.3 Angstroms resolution. The structure reveals a dimeric cysteine protease with two composite active sites. For each active site, the catalytic histidine and glutamate residues are contributed by one monomer, and the nucleophilic cysteine by the other. The carboxy-terminal residues remain coordinated in the two active sites, predicting an inactive post-cleavage form. Proteolysis through formation of a composite active site occurs in the context of the viral polyprotein expressed in mammalian cells. These features offer unexpected insights into polyprotein processing by hepatitis C virus and new opportunities for antiviral drug design.

  15. Emergent Catalytic Behavior of Self-Assembled Low Molecular Weight Peptide-Based Aggregates and Hydrogels.

    Science.gov (United States)

    Tena-Solsona, Marta; Nanda, Jayanta; Díaz-Oltra, Santiago; Chotera, Agata; Ashkenasy, Gonen; Escuder, Beatriu

    2016-05-04

    We report a series of short peptides possessing the sequence (FE)n or (EF)n and bearing l-proline at their N-terminus that self-assemble into high aspect ratio aggregates and hydrogels. We show that these aggregates are able to catalyze the aldol reaction, whereas non-aggregated analogues are catalytically inactive. We have undertaken an analysis of the results, considering the accessibility of catalytic sites, pKa value shifts, and the presence of hydrophobic pockets. We conclude that the presence of hydrophobic regions is indeed relevant for substrate solubilization, but that the active site accessibility is the key factor for the observed differences in reaction rates. The results presented here provide an example of the emergence of a new chemical property caused by self-assembly, and support the relevant role played by self-assembled peptides in prebiotic scenarios. In this sense, the reported systems can be seen as primitive aldolase I mimics, and have been successfully tested for the synthesis of simple carbohydrate precursors.

  16. Basal aurora kinase B activity is sufficient for histone H3 phosphorylation in prophase

    Directory of Open Access Journals (Sweden)

    Ly-Thuy-Tram Le

    2013-02-01

    Histone H3 phosphorylation is the hallmark of mitosis deposited by aurora kinase B. Benzo[e]pyridoindoles are a family of potent, broad, ATP-competitive aurora kinase inhibitors. However, benzo[e]pyridoindole C4 only inhibits histone H3 phosphorylation in prophase but not in metaphase. Under the C4 treatment, the cells enter into mitosis with dephosphorylated histone H3, assemble chromosomes normally and progress to metaphase, and then to anaphase. C4 also induces lagging chromosome in anaphase but we demonstrated that these chromosome compaction defects are not related to the absence of H3 phosphorylation in prophase. As a result of C4 action, mitosis lasts longer and the cell cycle is slowed down. We reproduced the mitotic defects with reduced concentrations of potent pan aurora kinase as well as with a specific aurora B ATP-competitive inhibitor; we therefore propose that histone H3 phosphorylation and anaphase chromosome compaction involve the basal activity of aurora kinase B. Our data suggest that aurora kinase B is progressively activated at mitosis entry and at anaphase onset. The full activation of aurora kinase B by its partners, in prometaphase, induces a shift in the catalytic domain of aurora B that modifies its affinity for ATP. These waves of activation/deactivation of aurora B correspond to different conformations of the chromosomal complex revealed by FRAP. The presence of lagging chromosomes may have deleterious consequences on the daughter cells and, unfortunately, the situation may be encountered in patients receiving treatment with aurora kinase inhibitors.

  17. Proteolytic cleavage of protein kinase Cmu upon induction of apoptosis in U937 cells.

    Science.gov (United States)

    Häussermann, S; Kittstein, W; Rincke, G; Johannes, F J; Marks, F; Gschwendt, M

    1999-12-03

    Treatment of U937 cells with various apoptosis-inducing agents, such as TNFalpha and beta-D-arabinofuranosylcytosine (ara-C) alone or in combination with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), bryostatin 1 or cycloheximide, causes proteolytic cleavage of protein kinase Cmu (PKCmu) between the regulatory and catalytic domain, generating a 62 kDa catalytic fragment of the kinase. The formation of this fragment is effectively suppressed by the caspase-3 inhibitor Z-DEVD-FMK. In accordance with these in vivo data, treatment of recombinant PKCmu with caspase-3 in vitro results also in the generation of a 62 kDa fragment (p62). Treatment of several aspartic acid to alanine mutants of PKCmu with caspase-3 resulted in an unexpected finding. PKCmu is not cleaved at one of the typical cleavage sites containing the motif DXXD but at the atypical site CQND378/S379. The respective fragment (amino acids 379-912) was expressed in bacteria as a GST fusion protein (GST-p62) and partially purified. In contrast to the intact kinase, the fragment does not respond to the activating cofactors TPA and phosphatidylserine and is thus unable to phosphorylate substrates effectively.

  18. Differential regulatory functions of three classes of phosphatidylinositol and phosphoinositide 3-kinases in autophagy.

    Science.gov (United States)

    Yu, Xinlei; Long, Yun Chau; Shen, Han-Ming

    2015-01-01

    Autophagy is an evolutionarily conserved and exquisitely regulated self-eating cellular process with important biological functions. Phosphatidylinositol 3-kinases (PtdIns3Ks) and phosphoinositide 3-kinases (PI3Ks) are involved in the autophagic process. Here we aim to recapitulate how 3 classes of these lipid kinases differentially regulate autophagy. Generally, activation of the class I PI3K suppresses autophagy, via the well-established PI3K-AKT-MTOR (mechanistic target of rapamycin) complex 1 (MTORC1) pathway. In contrast, the class III PtdIns3K catalytic subunit PIK3C3/Vps34 forms a protein complex with BECN1 and PIK3R4 and produces phosphatidylinositol 3-phosphate (PtdIns3P), which is required for the initiation and progression of autophagy. The class II enzyme emerged only recently as an alternative source of PtdIns3P and autophagic initiator. However, the orthodox paradigm is challenged by findings that the PIK3CB catalytic subunit of class I PI3K acts as a positive regulator of autophagy, and PIK3C3 was thought to be an amino acid sensor for MTOR, which curbs autophagy. At present, a number of PtdIns3K and PI3K inhibitors, including specific PIK3C3 inhibitors, have been developed for suppression of autophagy and for clinical applications in autophagy-related human diseases.

  19. NAD kinase controls animal NADP biosynthesis and is modulated via evolutionarily divergent calmodulin-dependent mechanisms.

    Science.gov (United States)

    Love, Nick R; Pollak, Nadine; Dölle, Christian; Niere, Marc; Chen, Yaoyao; Oliveri, Paola; Amaya, Enrique; Patel, Sandip; Ziegler, Mathias

    2015-02-03

    Nicotinamide adenine dinucleotide phosphate (NADP) is a critical cofactor during metabolism, calcium signaling, and oxidative defense, yet how animals regulate their NADP pools in vivo and how NADP-synthesizing enzymes are regulated have long remained unknown. Here we show that expression of Nadk, an NAD(+) kinase-encoding gene, governs NADP biosynthesis in vivo and is essential for development in Xenopus frog embryos. Unexpectedly, we found that embryonic Nadk expression is dynamic, showing cell type-specific up-regulation during both frog and sea urchin embryogenesis. We analyzed the NAD kinases (NADKs) of a variety of deuterostome animals, finding two conserved internal domains forming a catalytic core but a highly divergent N terminus. One type of N terminus (found in basal species such as the sea urchin) mediates direct catalytic activation of NADK by Ca(2+)/calmodulin (CaM), whereas the other (typical for vertebrates) is phosphorylated by a CaM kinase-dependent mechanism. This work indicates that animal NADKs govern NADP biosynthesis in vivo and are regulated by evolutionarily divergent and conserved CaM-dependent mechanisms.

  20. Crystal structure of inhibitor of ;#954;B kinase [beta

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Guozhou; Lo, Yu-Chih; Li, Qiubai; Napolitano, Gennaro; Wu, Xuefeng; Jiang, Xuliang; Dreano, Michel; Karin, Michael; Wu, Hao (Weill-Med); (Merck-Serono); (UCSD); (EMD Serono)

    2011-07-26

    Inhibitor of {kappa}B (I{kappa}B) kinase (IKK) phosphorylates I{kappa}B proteins, leading to their degradation and the liberation of nuclear factor {kappa}B for gene transcription. Here we report the crystal structure of IKK{beta} in complex with an inhibitor, at a resolution of 3.6 {angstrom}. The structure reveals a trimodular architecture comprising the kinase domain, a ubiquitin-like domain (ULD) and an elongated, {alpha}-helical scaffold/dimerization domain (SDD). Unexpectedly, the predicted leucine zipper and helix-loop-helix motifs do not form these structures but are part of the SDD. The ULD and SDD mediate a critical interaction with I{kappa}B{alpha} that restricts substrate specificity, and the ULD is also required for catalytic activity. The SDD mediates IKK{beta} dimerization, but dimerization per se is not important for maintaining IKK{beta} activity and instead is required for IKK{beta} activation. Other IKK family members, IKK{alpha}, TBK1 and IKK-i, may have a similar trimodular architecture and function.